WO2001060855A1

WO2001060855A1 - A novel human cell cycle control-related protein and a sequence encoding the same

Info

Publication number: WO2001060855A1
Application number: PCT/CN2001/000121
Authority: WO
Inventors: Jianren Gu; Shengli Yang
Original assignee: Shanghai Cancer Institute
Priority date: 2000-02-17
Filing date: 2001-02-12
Publication date: 2001-08-23
Also published as: CN1400977A; AU2001233587A1; CN1160370C

Abstract

The present invention discloses a novel human tumor-suppressing protein and a polynucleotide encoding the same, as well as a method of producing the polypeptide by recombinant technique. The present invention also discloses methods of using the polypeptide in treatment of various disease, such as tumor or the like. The present invention also discloses an antagonist against the polypeptide and the therapeutic use of the same. Also disclosed is the use of such polynucleotide encoding the novel human tumor-suppressing protein.

Description

Novel human cell cycle control related protein and its coding sequence Field of the invention

The present invention belongs to the field of biotechnology, and in particular, the present invention relates to a novel polynucleotide encoding a human cell cycle control related gene (crn-ike gen ', CLG) protein with cancer suppressing function, and the polynucleotide encoding Of peptides. The invention also relates to the use and preparation of such polynucleotides and polypeptides.

Background of the invention

Cell division and proliferation are one of the basic characteristics of cell life activity. During the growth and development of the human body and other multicellular organisms, the increase in the number of cells, the renewal of senescent and dead cells, and the continuation of life all need to be accomplished through cell proliferation. The cell proliferation cycle is the process by which cells achieve cell growth and proliferation through a series of intracellular events. There is a strict sequence between each event. Its regulatory mechanism involves multiple levels and multiple factors, not only involving growth factors and cyclic nucleosides. Acids, hormones, proto-oncogenes, etc., are more importantly controlled by the cell cycle regulatory system, such as cell division cycle genes, cyclins, cyclin-dependent kinases and their inhibitory proteins.

The regulation of the cell cycle is a core issue in the research of cell biology. It involves the orderly development of embryonic cells, the normal proliferation of body cells, the regeneration and differentiation of cells, and the aging and apoptosis of cells. Normal cell cycle must be strictly and orderly regulated. Abnormal cell cycle regulation will cause cells to mutate, deform or become cancerous.

In recent years, intensive research has been conducted on the cell cycle regulation system, which mainly includes cel l division cycle (cdc) genes, cyclin, and cyclin-dependent kinase (CDK) ) And cyclin-dependent kinase inhibitor (CKI). Based on model organisms such as yeast, drosophi la, and c. Elegans, many human cell cycle regulatory genes have been isolated. For example, Lieberman et al. Isolated a human cell cycle checkpoint control gene (Lieberman, HB, Hopkins, KM, et al. Proc. Natl. Acad. Sci. USA) based on the homology analysis method of yeast rad9 +. 93: 13890-13895; 1996) ₀

Cancer is one of the main diseases that endanger human health. In order to effectively treat and prevent tumors, people have paid more and more attention to gene therapy of tumors. Therefore, there is an urgent need in the art to develop and study human proteins and their agonists / inhibitors with cancer suppressing functions, especially proteins involved in cell cycle regulation.

Summary of invention

The object of the present invention is to provide a new class of human protein polypeptides with tumor suppressing function, as well as fragments, analogs and derivatives thereof.

Another object of the invention is to provide polynucleotides encoding these polypeptides.

Another object of the present invention is to provide a method for producing these polypeptides and the use of the polypeptide and coding sequence. In a first aspect of the present invention, there is provided a novel isolated protein polypeptide having a tumor suppressing function, which comprises a polypeptide having the amino acid sequence of SEQ ID NO: 2; or a conservative variant polypeptide thereof, or an active fragment thereof, or an activity thereof derivative. Preferably, the polypeptide is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

In a second aspect of the present invention, an isolated polynucleotide is provided, which comprises a nucleotide sequence that is at least 85% identical to a nucleotide sequence selected from the group consisting of: (A) a polynucleotide encoding the above-mentioned CLG protein polypeptide; (b) a polynucleotide complementary to the polynucleotide (a). Preferably, the polypeptide encoded by the polynucleotide has the amino acid sequence of SEQ ID NO: 2. More preferably, the sequence of the polynucleotide is selected from the group consisting of the coding region sequence of SEQ ID NO: 3 (22-2586 positions) or full-length sequences (1-2659 positions).

In a third aspect of the present invention, there are provided a vector containing the above polynucleotide, and a host cell transformed or transduced by the vector or a host cell directly transformed or transduced by the above polynucleotide.

In a fourth aspect of the present invention, a method for preparing a polypeptide having CLG protein activity is provided, which method comprises: (a) culturing the transformed or transduced host cell under conditions suitable for expressing the CLG protein; (b) ) A polypeptide having CLG protein activity is isolated from the culture.

In a fifth aspect of the present invention, an antibody that specifically binds to the CLG protein polypeptide described above is provided. Nucleic acid molecules that can be used for detection are also provided, which contain 10-800 consecutive nucleotides in the above-mentioned polynucleotides.

In a sixth aspect of the present invention, a pharmaceutical composition is provided, which contains a safe and effective amount of the CLG protein polypeptide of the present invention and a pharmaceutically acceptable carrier. These pharmaceutical compositions can treat diseases such as cancer and abnormal cell proliferation.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Figure 1 is the RNA expression profile of CLG.

Figure 2 shows the inhibitory effect of the full-length CLG cDNA on the colony formation of human hepatoma cell line S 7721. Which figure

2A is the vector control (pCMV-Scr ipt) with 56 colonies; Figure 2B is CLG (pCMV-Script / CLG) with only 3 colonies.

Figure 3 is a photograph of a paraffin section of tumor tissue formed by CLG-transfected S-deleted C-7721 cells inoculated in nude mice. Figure 3A shows necrotic foci, and more positive apoptotic cells can be seen; Figure 3B shows non-necrotic areas, and scattered apoptotic cells can be seen.

Figure 4 shows the "ladder" DNA electrophoresis map after CLG gene transfection. The lanes are: 1. Transfected empty vector; 2. Transfected p53; 3. Transfected CLG; 4. Untransfected control point; 5. Molecular weight standard.

Figure 5 SDS-PAGE electrophoresis of CLG protein expression. Among them, each lane is: 1. protein molecular weight standard; 2. ultrasonic supernatant of pET32a-CLG bacteria; 3. ultrasonic supernatant of pET32a bacteria; 4. ultrasonic precipitation of pET32a-CLG bacteria; 5. ultrasonic precipitation of pET32a bacteria. Detailed description of the invention

In the present invention, large-scale cDNA clones are used to transfect cancer cells. On the basis of obtaining a tumor suppressing effect, sequencing proves that they are new genes, and further obtains full-length cDNA clones. The DNA transfection test proves that the protein with tumor suppressing function of the present invention has the effect of inhibiting the formation of clones on cancer cells (liver cancer cells), and the inhibition rate is 50%.

In one example, a new gene with high homology to the cell cycle regulatory proteins of nematodes, Drosophila and yeast was isolated from the human placental cDNA library. It has 66% (557/837) identity to the cell cycle regulatory proteins of nematodes. Derived, has 41% (157/375) homology with the cell cycle regulatory protein (crooked neck, crn) of Drosophila, and 37 ° / with the cell cycle control protein of yeast. (228/599) Homology. According to bioinformatics, human CLG (crn-ike gene; original serial number is PP3898, GenBank accession number AF258567, registration date April 24, 2000) is a new human cell cycle-related gene. Preliminary functional studies found that CLG can inhibit the growth of human liver cancer cells SMMC-7721 in vitro; in situ apoptosis detection and DNA electrophoresis analysis of hepatocellular carcinoma S-cut C-7721 nude mice transplanted tumor tissues showed that CLG gene transfected with S-cut C-7721 cells can induce tumor cell apoptosis and inhibit tumor cell growth. In this article, "cell cycle control related gene proteins", "CLG protein", and "PP3898 protein" may Used interchangeably, they all refer to polypeptides having the amino acid sequence (SEQ ID NO: 2) of a human cell cycle control-related gene protein. They include cell cycle control related gene proteins with or without starting methionine.

As used herein, "isolated" refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in a natural state in a living cell are not separated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances coexisting in the natural state. of.

As used herein, "isolated CLG protein or polypeptide" means that a CLG protein polypeptide is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify CLG proteins using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of CLG protein can be analyzed by amino acid sequence.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide. The polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. The polypeptides of the invention may also include or exclude the initial methionine residue.

The invention also includes fragments, derivatives and analogs of the human CLG protein. As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially retains the same biological function or activity of the natural human CLG protein of the invention. A polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of a polypeptide, such as (Polyethylene glycol), a polypeptide formed by fusion, or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify the polypeptide or a protein sequence). According to the teachings herein, these fragments, derivatives, and analogs are within the scope of those skilled in the art.

The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding. Taking the CLG protein as an example, the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 3 or a degenerate variant. As used herein, "degenerate variant" refers in the present invention to a nucleic acid sequence that encodes a protein having SEQ ID NO: 2 but differs from the coding region sequence shown in SEQ ID NO: 3.

A polynucleotide encoding a mature polypeptide includes: a coding sequence that encodes only a mature polypeptide; a coding sequence that is a mature polypeptide

+ Various additional coding sequences; coding sequences of mature polypeptides (+ optional additional coding sequences) + non-coding sequences.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and / or non-coding sequences.

The present invention also relates to a variant of the above polynucleotide, which encodes a polypeptide having the same amino acid sequence as the present invention or fragments, analogs and derivatives of the polypeptide. Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .

The invention also relates to hybridizing to the sequence described above with at least 50%, preferably at least 70% 'between the two sequences. More preferably a polynucleotide that is at least 80% identical. The invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions. In the present invention, "strict conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 "C; or ( 2) A denaturant is added during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) only between two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%, and the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2 .

The invention also relates to a nucleic acid fragment that hybridizes to the sequence described above. As used herein, a "nucleic acid fragment" contains at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides. Nucleic acid fragments can be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding CLG proteins.

The DNA sequence of the present invention can be obtained by several methods. For example, DNA is isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous nucleotide sequences, and 2) antibody screening of expression libraries to detect cloned DNA fragments with common structural characteristics .

The specific DNA fragment sequence encoding CLG protein can also be obtained by: 1) isolating double-stranded DNA sequence from genomic DNA; 2) chemically synthesizing DNA sequence to obtain double-stranded DNA of desired polypeptide.

Of the methods mentioned above, genomic DNA isolation is the least commonly used. When the entire amino acid sequence of the desired polypeptide product is known, direct chemical synthesis of the DNA sequence is often the method of choice. If the entire sequence of the desired amino acid is unclear, direct chemical synthesis of the DNA sequence is not possible, and the method chosen is the isolation of the cDNA sequence. The standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for extracting mRNA, and kits are also commercially available (Qiagene). And the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When combined with polymerase reaction technology, even very small expression products can be cloned.

The genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to):

(1) DNA-DNA or DNA-RNA hybridization; (2) the occurrence or loss of the function of a marker gene; (3) determination of the level of CLG protein transcripts; (4) detection by immunological techniques or determination of biological activity Gene-expressed protein product. The above methods can be used alone or in combination.

In the method (1), the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and has a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2 kb, preferably within 1 kb. The probe used herein is generally a DNA sequence chemically synthesized based on the DNA sequence information of the gene of the present invention. The gene itself or a fragment of the present invention can of course be used as a probe. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

In the (4) method, the protein product of CLG protein gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).

A method for amplifying DNA / RNA using PCR technology (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE- rapid cDNA end amplification method) can be preferably used. The primers used for PCR can be based on the sequence information of the present invention disclosed herein. The information is appropriately selected and can be formed by a conventional method. The amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.

The nucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger t a). PNAS, 1977, 74: 5463-5467. Such nucleotide sequence determination can also be performed using a commercial sequencing kit or the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.

The present invention also relates to a vector comprising a polynucleotide of the present invention, a host cell genetically engineered using the vector or CLG protein coding sequence of the present invention, and a method for producing a polypeptide of the present invention by recombinant technology.

The polynucleotide sequences of the present invention can be used to express or produce recombinant CLG protein polypeptides by conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally there are the following steps:

(1) using the polynucleotide (or variant) encoding the human CLG protein of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

(2) host cells cultured in a suitable medium;

(3) Isolate and purify protein from culture medium or cells.

In the present invention, the human CLG protein polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors (Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes and translation control elements.

Methods known to those skilled in the art can be used to construct expression vectors containing human CLG protein-encoding DNA sequences and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al). The DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E. coli; the lambda phage PL promoter; eukaryotic promoters include the CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, anti Transcript virus LTRs and other promoters known to control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express a protein.

The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CH0, COS or Bowes melanoma cells.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if the enhancer sequence is inserted into the vector, Enhancing transcription. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, that act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.

Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote, such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl ₂ method. The steps used are well known in the art. Alternatively, MgCl ₂ is used. If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, and liposome packaging.

The obtained transformants can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used in the culture may be selected from various conventional mediums. Culture is carried out under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

The recombinant polypeptide in the above method may be coated intracellularly, extracellularly, or expressed on a cell membrane or secreted extracellularly. If necessary, the physical, chemical, and other properties can be used to separate and purify the recombinant protein by various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmosis, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

Recombinant human CLG proteins or polypeptides have many uses. These uses include (but are not limited to): direct use as a drug to treat diseases caused by hypofunction or loss of CLG protein, and to screen for antibodies, peptides, or other ligands that promote or fight CLG protein function. For example, antibodies can be used to activate or inhibit the function of human CLG proteins. Screening peptide libraries with expressed recombinant human CLG proteins can be used to find therapeutic peptides that can inhibit or stimulate the function of human CLG proteins.

The invention also provides methods for screening drugs to identify agents that increase (agonist) or suppress (antagonist) human CLG proteins. Agonists enhance human CLG proteins to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or membrane preparations expressing human CLG proteins can be cultured with labeled human CLG proteins in the presence of drugs. The ability of the drug to increase or block this interaction is then measured.

Antagonists of human CLG proteins include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of the human CLG protein can bind to the human CLG protein and eliminate its function, or inhibit the production of the human CLG protein, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions. Antagonists of human CLG proteins are useful in therapeutic applications.

When screening compounds that act as antagonists, CLG proteins can be added to bioanalytical assays to determine whether a compound is an antagonist by measuring the effect of the compound on the interaction between the CLG protein and its receptor. In the same manner as described above for the screening of compounds, it is possible to screen for receptor deletions and analogs that act as antagonists.

The polypeptide of the present invention can be directly used for treating diseases, for example, various malignant tumors, abnormal cell proliferation, and the like. The polypeptides of the present invention, and fragments, derivatives, analogs or their cells can be used as antigens to produce antibodies. These antibodies can be polyclonal or monoclonal antibodies. Polyclonal antibodies can be obtained by directly injecting the polypeptide Material method. Techniques for preparing monoclonal antibodies include hybridoma technology, triple tumor technology, human B-cell hybridoma technology, and EBV-hybridoma technology.

The polypeptides and antagonists of the present invention can be used in combination with a suitable pharmaceutical carrier. These carriers can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof. The composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.

The present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention. Along with these containers, there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders permit their administration on the human body by government agencies that produce, use, or sell them. In addition, the polypeptides of the invention can be used in combination with other therapeutic compounds.

The pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration. The CLG protein is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of CLG protein administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

The polynucleotide of the human CLG protein can also be used for a variety of therapeutic purposes. Gene therapy techniques can be used to treat abnormal cell proliferation, development, or metabolism caused by non-expressed CLG protein expression or abnormal / inactive CLG protein expression. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated CLG proteins to inhibit endogenous CLG protein activity. For example, a mutated CLG protein may be a shortened CLG protein lacking a signaling domain, and although it can bind to downstream substrates, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of CLG protein. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer CLG protein genes into cells. Methods for constructing a recombinant viral vector carrying the CLG protein gene can be found in existing literature (Sambr ₀₀ k, et al.). In addition, the recombinant human CLG protein gene can be packaged into liposomes and transferred into cells.

Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit human CLG protein mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation. Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleosides are connected by phosphorothioate or peptide bonds instead of phosphodiester bonds.

Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly into a tissue in vivo; or introducing the polynucleotide into a cell via a vector (such as a virus, phage, or plasmid) in vitro Then, the cells are transplanted into the body.

The invention also provides antibodies against human CLG protein epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries. Anti-human CLG protein antibodies can be used in immunohistochemical techniques to detect human CLG protein in biopsy specimens.

Monoclonal antibodies that bind to human CLG proteins can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis. The antibodies in the present invention can be used to treat or prevent diseases related to human CLG protein. Administration of an appropriate dose of antibody can stimulate or block the production or activity of human CLG protein.

Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, human CLG protein high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of the antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill human CLG protein-positive cells.

Polyclonal antibodies can be produced by immunizing animals such as rabbits, mice, and rats with human CLG proteins or peptides. A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like.

Human CLG protein monoclonal antibodies can be produced using hybridoma technology (Kohler and Ilstein. Nature, 1975, 256: 495-497). Chimeric antibodies that bind human constant regions to non-human-derived variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). The existing technology for producing single chain antibodies (U.S. Pat No. 4946778) can also be used to produce single chain antibodies against human CLG protein.

Polypeptide molecules capable of binding to human CLG protein can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the human CLG protein molecule must be labeled.

The invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human CLG protein. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of human CLG protein detected in the test can be used to explain the importance of human CLG protein in various diseases and to diagnose diseases in which CLG protein functions.

CLG protein polynucleotides are useful in the diagnosis and treatment of CLG protein-related diseases. In terms of diagnosis, the polynucleotide of CLG protein can be used to detect the expression of CLG protein or the abnormal expression of CLG protein in a disease state. For example, the CLG protein DNA sequence can be used to hybridize biopsy specimens to determine abnormal CLG protein expression. Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These technical methods are publicly available and mature technologies, and related kits are commercially available. Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues. CLG protein-specific primers for RNA-polymerase chain reaction (RT-PCR) amplification in vitro can also detect CLG protein transcripts.

Detection of mutations in the CLG protein gene can also be used to diagnose CLG protein-related diseases. CLG protein mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type CLG protein DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.

The sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) can be used to mark chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.

In short, PCR primers (preferably 15-35bp) are prepared from the cDNA, and the sequence can be mapped on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.

PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes. Use the hair A similar oligonucleotide primer can be similarly used to achieve sublocalization using a set of fragments from a specific chromosome or a large number of genomic clones. Other similar strategies that can be used for staining 'localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.

Fluorescent in situ hybridization (FISH) of cDNA clones with metaphase chromosomes allows precise chromosomal mapping in a single step. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergaraon Press, New York (1988 ).

Once the sequence is located at the exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with the gene map data. These data can be found in, for example, V. Mckusick, Mendel ian Inheri tance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.

Next, the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution Capacity and each 20kb corresponds to a gene).

The full-length nucleotide sequence of the CLG protein of the present invention or a fragment thereof can usually be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. For the PCR amplification method, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and a commercially available cDNA library or cDNA prepared according to conventional methods known to those skilled in the art The library is used as a template and the relevant sequences are amplified. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then stitch the amplified fragments together in the correct order.

Once the relevant sequence is obtained, it can be obtained in large quantities by recombination. This is usually done by cloning it into a vector, transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

In addition, relevant methods can also be synthesized by artificial synthesis, especially when the fragment length is short. In general, long sequences can be obtained by first synthesizing multiple small fragments and then concatenating them.

At present, the DNA sequence encoding the protein (or a fragment, or a derivative thereof) of the present invention can be completely synthesized by chemical synthesis. This DNA sequence can then be introduced into various DNA molecules (such as vectors) and cells in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

In addition, since the CLG protein of the present invention has a natural amino acid sequence derived from humans, it is expected to have higher activity and / or lower side effects when administered to humans compared to homologous proteins derived from other species ( (Eg, less or not immunogenic in humans). The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the examples are generally according to the conventional conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions. Suggested conditions.

Example 1: Obtaining a cDNA Gene Fragment and Inhibiting the Formation of Cancer Cell Clones

PP3898 (ie CLG) comes from the construction of a human placental cDNA library using conventional methods. Take 3, 6, and 10-month-old placental tissues, use Trizol reagent (GIBC0 BRL company) to extract total RNA according to the manufacturer's instructions, and use mRNA to purify the reagent Cassette (Pharmacia) for mRNA extraction. A pCMV-script TMXR cDNA library construction kit (Seratagene) was used to construct a cDNA library of the above-mentioned I RNA. The reverse transcriptase was changed to MMLV-RT- Superscript II (GIBC0 BRL), and the reverse transcription reaction was performed at 42 ° C. XL 10-Gold receptor cells were transformed to obtain a cDNA library with a lxlO ⁶ cfu / g cDNA titer. In the first round, cDNA clones were randomly selected, and thereafter, high-abundance cDNA clones and cDNA clones that had been shown to inhibit the growth of cancer cells were used as probes to hybridize and screen cDNA libraries, and weakly positive and negative clones were selected. Qiagen 96-well plate plasmid extraction kit was used to extract plasmid DNA according to the manufacturer's instructions. Plasmid DNA and empty vector were transfected into hepatocellular carcinoma cell line 7721 at the same time. After lOOng of DNA was precipitated and dried with alcohol, 6 μl Η _{20 was} added to dissolve the DNA, and the cells were transfected. Add 0.74μ1 liposome and 9.3μ1 serum-free culture solution to each DNA sample. After mixing, place at room temperature for 10 minutes. Add 150μ1 of serum-free culture solution to each tube, and add it to 7721 cells grown in 96-well plates in 3 wells. Leave at 37 ° C for 2 hours. Add 50μ1 of serum-free culture solution to each well for 37Ό for 24 hours. Replace 100 μl whole culture solution in each well at 37 ° C for 24 hours, and replace 100 μ1 whole culture solution with G418 for 24 to 48 hours at 37 ° C. While observing, change the culture solution with different concentrations of G418. After about 2-3 times, count until the colony is formed on the microscopy cells. It was found that PP3898 (that is, CLG) can inhibit the formation of cell clones (inhibition rate is 50% or more), and the results are shown in the following table.

Formation of cDNA clone-transfected cells (7721)

cDNA clone name Number of CDNA clones (three replicates) Number of empty vector clones (three replicates)

PP3898 (ie CLG) 2 0 0 33 34 38 The dideoxy termination method was used for cDNA clones, and the nucleotide sequence of one end of the clone was nearly 500bp on an ABI377 DNA automatic sequencer. After analysis, it was determined to be a new gene clone, and then completely sequenced. It was found that the CLG fragment was a non-full-length sequence, and the full-length cDNA clone was obtained by the RACE method. Example 2: RACE method to obtain full-length cDNA clone and RT-PCR method to obtain CLC gene

1. RACE method to obtain full-length cDNA clones

After analyzing the sequence of PP3898 cDNA clone, it was found that the gene was not complete. The SMART RACE cDNA amplification kit (Cat. No. K1811-1) from Clontech was used to design gene specific primers (shown in Table 2 below). Obtain full-length clones.

Table 2 CLG gene specific primers

Specifically, the following primers were used for the PP3898 clone:

Universal primer mix (UPM) Long 5 'CTAATACGACTCACTATAGGGCAAGCAGTGGTAACAACGCAGAGT3'

Nested Universal Primer (NUP) 5 'AAGCAGTGGTAACAACGCAGAGT 3'

pp3898-NB 5 'TCATCCAGCCGGTCACTTGACTTGA 3'

pp3898-B 5 'GCCACAGCTGGTAGTTGGACTTGCC 3'

The human placental tissue mRNA was used as the starting material, and the cDNA was obtained according to the instructions of Clontech's SMART RACE cDNA Amplification Kit (Catftl811-1). Then the first round of PCR was performed with UPM primers and gene-specific pp3898-B primers, and then NUP Primers and gene-specific pp3898-NB primers were subjected to a second round of PCR to obtain gene fragments.

The reaction conditions are as follows: 94 ° C for 1 minute, one cycle; 94 ° C for 30 seconds, 72 ° C for 4 minutes, 5 cycles; 94 ° C 30 seconds, 70 ° C 4 minutes, 5 cycles; 94 ° C 20 seconds, 65 ° C 30 seconds, 68 ° C 4 minutes, 27 cycles.

A SMART RACE reaction was performed to obtain a 5 'extension of the CLG gene, which was recombined to obtain a cDNA clone of the full-length CLG gene, as shown in SEQ ID NO: 1.

2. CLG gene obtained by RT-PCR

Synthesis of primers CLG- F (5 'ATGGTGGTGATGGCGCGACTCTCG 3', bp 22-45) and CLG-R (5 'GGTCAGTCTTCCTTCAGGCTCCC 3', bp 2569-2591), using RT-PCR method (where RT-PCR reaction conditions and The above SMART RACE reaction conditions are the same), so that the CLG gene containing the complete coding region is obtained, and the size of the RT-PCR product is 2570bp. Example 3: Sequence analysis of CLG cDNA clone

A: Nucleotide sequence (SEQ ID NO: 1) Length: 2659

GGTACCTGGG CATCCAGAAA AATGGTGGTG ATGGCGCGAC TCTCGCGGCC CGAGCGGCCG 60

GACCTTGTCT TCGAGGAAGA GGACCTCCCC TATGAGGAGG AAATCATGCG GAACCAATTC 120

TCTGTCAAAT GCTGGCTTCG CTACATCGAG TTCAAACAGG GCGCCCCGAA GCCCAGGCTC 180

AATCAGCTAT ACGAGCGGGC ACTCAAGCTG CTGCCCTGCA GCTACAAACT CTGGTACCGA 240

TACCTGAAGG CGCGTCGGGC ACAGGTGAAG CATCGCTGTG TGACCGACCC TGCCTATGAA 300

GATGTCAACA ACTGTCATGA GAGGGCCTTT GTGTTCATGC ACAAGATGCC TCGTCTGTGG 360

CTAGATTACT GCCAGTTCCT CATGGACCAG GGGCGCGTCA CACACACCCG CCGCACCTTC 420

GACCGTGCCC TCCGGGCACT GCCCATCACG CAGCACTCTC GAATTTGGCC CCTGTATCTG 480

CGCTTCCTGC GCTCACACCC ACTGCCTGAG ACAGCTGTGC GAGGCTATCG GCGCTTCCTC 540

AAGCTGAGTC CTGAGAGTGC AGAGGAGTAC ATTGAGTACC TCAAGTCAAG TGACCGGCTG 600

GATGAGGCCG CCCAGCGCCT GGCCACCGTG GTGAACGACG AGCGTTTCGT GTCTAAGGCC 660

GGCAAGTCCA ACTACCAGCT GTGGCACGAG CTGTGCGACC TCATCTCCCA GAATCCGGAC 720

AAGGTACAGT CCCTCAATGT GGACGCCATC ATCCGCGGGG GCCTCACCCG CTTCACCGAC 780

CAGCTGGGCA AGCTCTGGTG TTCTCTCGCC GACTACTACA TCCGCAGCGG CCATTTCGAG 840

AAGGCTCGGG ACGTGTACGA GGAGGCCATC CGGACAGTGA TGACCGTGCG GGACTTCACA 900

CAGGTGTTTG ACAGCTACGC CCAGTTCGAG GAGAGCATGA TCGCTGCAAA GATGGAGACC 960

GCCTCGGAGC TGGGGCGCGA GGAGGAGGAT GATGTGGACC TGGAGCTGCG CCTGGCCCGC 1020

TTCGAGCAGC TCATCAGCCG GCGGCCCCTG CTCCTCAACA GCGTCTTGCT GCGCCAAAAC 1080

CCACACCACG TGCACGAGTG GCACAAGCGT GTCGCCCTGC ACCAGGGCCG CCCCCGGGAG 1140

ATCATCAACA CCTACACAGA GGCTGTGCAG ACGGTGGACC CCTTCAAGGC CACAGGCAAG 1200

CCCCACACTC TGTGGGTGGC GTTTGCCAAG TTTTATGAGG ACAACGGACA GCTGGACGAT 1260

GCCCGTGTCA TCCTGGAGAA GGCCACCAAG GTGAACTTCA AGCAGGTGGA TGACCTGGCA 1320

AGCGTGTGGT GTCAGTGCGG AGAGCTGGAG CTCCGACACG AGAACTACGA TGAGGCCTTG 1380

CGGCTGCTGC GAAAGGCCAC GGCGCTGCCT GCCCGCCGGG CCGAGTACTT TGATGGTTCA 1440

GAGCCCGTGC AGAACCGCGT GTACAAGTCA CTGAAGGTCT GGTCCATGCT CGCCGACCTG 1500

GAGGAGAGCC TCGGCACCTT CCAGTCCACC AAGGCCGTGT ACGACCGCAT CCTGGACCTG 1560

CGTATCGCAA CACCCCAGAT CGTCATCAAC TATGCCATGT TCCTGGAGGA GCACAAGTAC 1620

TTCGAGGAGA CCTTCAAGGC GTACGAGCGC GGCATCTCGC TGTTCAAGTG GCCCAACGTG 1680

TCCGACATCT GGAGCACCTA CCTGACCAAA TTCATTGCCC GCTATGGGGG CCGCAAGCTG 1740

GAGCGGGCAC GGGACCTGTT TGAACAGGCT CTGGACGGCT GCCCCCCAAA ATATGCCAAG 1800

ACCTTGTACC TGCTGTACGC ACAGCTGGAG GAGGAGTGGG GCCTGGCCCG GCATGCCATG 1860

GCCGTGTACG AGCGTGCCAC CAGGGCCGTG GAGCCCGCCC AGCAGTATGA CATGTTCAAC 1920

ATCTACATCA AGCGGGCGGC CGAGATCTAT GGGGTCACCC ACACCCGCGG CATCTACCAG 1980

AAGGCCATTG AGGTGCTGTC GGACGAGCAC GCGCGTGAGA TGTGCCTGCG GTTTGCAGAC 2040

ATGGAGTGCA AGCTCGGGGA GATTGACCGC GCCCGGGCCA TCTACAGCTT CTGCTCCCAG 2100

ATCTGTGACC CCCGGACGAC CGGCGCGTTC TGGCAGACGT GGAAGGACTT TGAGGTCCGG 2160

CATGGCAATG AGGACACCAT CAAGGAAATG CTGCGTATCC GGCGCAGCGT GCAGGCCACG 2220

TACAACACGC AGGTCAACTT CATGGCCTCG CAGATGCTCA AGGTCTCGGG CAGTGCCACG 2280

GGCACCGTGT CTGACCTGGC CCCTGGGCAG AGTGGCATGG ACGACATGAA GCTGCTGGAA 2340

CAGCGGGCAG AGCAGCTGGC GGCTGAGGCG GAGCGTGACC AGCCCTTGCG CGCCCAGAGC 2400

AAGATCCTGT TCGTGAGGAG TGACGCCTCC CGGGAGGAGC TGGCAGAGCT GGCACAGCAG 2460 GTCAACCCCG AGGAGATCCA GCTGGGCGAG GACGAGGACG AGGACGAGAT GGACCTGGAG 2520 CCCAACGAGG TTCGGCTGGA GCAGCAGAGC GTGCCAGCCG CAGTGTTTGG GAGCCTGAAG 2580 GAAGACTGAC CCGTCCCTCC CCCCTCCCCA CCCCCTCCCC AATACAGCTA CGTTTAAAAAAAAA 2

B: amino acid sequence (SEQ I [NO: 2) length: 85

1 MVVMARLSRP ERPDLVFEEE DLPYEEEIMR NQFSVKCWLR YIEFKQGAPK

51 PRLNQLYERA LKLLPCSYKL WYRYLKARRA QVKH CVTDP AYEDVNNCHE

101 RAFVF HK P RLWLDYCQFL MDQGRVTHTR RTFDRALRAL PITQHSRIWP

151 LYLRFLRSHP LPETAVRGYR RFLKLSPESA EEYIEYLKSS DRLDEAAQRL

201 ATVVNDERFV SKAGKSNYQL WHELCDLISQ NPDKVQSLNV DAI IRGGLTR

251 FTDQLGKLWC SLADYYIRSG HFEKARDVYE EAIRTVMTVR DFTQVFDSYA

301 QFEESMIAAK METASELGRE EEDDVDLELR LARFEQLISR RPLLLNSVLL

351 RQNPHHVHEW HKRVALHQGR PREI INTYTE AVQTVDPFKA TGKPHTLWVA

401 FAKFYEDNGQ LDDARVILEK ATKVNFKQVD DLASVWCQCG ELELRHENYD

451 EALRLLRKAT ALPARRAEYF DGSEPVQNRV YKSLKVWSML ADLEESLGTF

501 QSTKAVYDRI LDLRIATPQI VINYAMFLEE HKYFEESFKA YERGISLFKW

551 PNVSDIWSTY LTKFIARYGG RKLERARDLF EQALDGCPPK YAKTLYLLYA

601 QLEEEWGLAR HAMAVYERAT RAVEPAQQYD MFNIYIKRAA EIYGVTHTRG

651 IYQKAIEVLS DEHAREMCLR FADMECKLGE IDRARAIYSF CSQICDPRTT

701 GAFWQTWKDF EVRHGNEDTI EMLRIRRSV QATYNTQVNF MASQMLKVSG

751 SATGTVSDLA PGQSGMDDMK LLEQRAEQLA AEAERDQPLR AQSKILFVRS

801 DASREELAEL AQQVNPEEIQ LGEDEDEDEM DLEPNEVRLE QQSVPAAVFG

851 SLKED

C. Nucleotide and amino acid combination sequence: (SEQ ID NO: 3) Cloning number: CLG (ie PP3898) Start coding: 22 ATG Stop coding: 2589 TGA Protein molecular weight: 100004. 44

1 GGT ACC TGG GCA TCC AGA AAA ATG GTG GTG ATG GCG CGA CTC TCG CGG 48

1 Met Val Val Met Al a Arg Leu Ser Arg 9

49 CCC GAG CGG CCG GAC CTT GTC TTC GAG GAA GAG GAC CTC CCC TAT GAG 96

10 Pro Glu Arg Pro Asp Leu Val Phe Glu Glu Glu Asp Leu Pro Tyr Gl u 25

97 GAG GAA ATC ATG CGG AAC CAA TTC TCT GTC AAA TGC TGG CTT CGC TAC 144

26 Glu Glu l i e Met Arg Asn Gin Phe Ser Val Lys Cys Trp Leu Arg Tyr 41

145 ATC GAG TTC AAA CAG GGC GCC CCG AAG CCC AGG CTC AAT CAG CTA TAC 192

42 l ie Glu Phe Lys Gin Gly Ala Pro Lys Pro Arg Leu Asn Gin Leu Tyr 57

193 GAG CGG GCA CTC AAG CTG CTG CCC TGC AGC TAC AAA CTC TGG TAC CGA 240

58 Glu Arg Ala Leu Lys Leu Leu Pro Cys Ser Tyr Lys Leu Trp Tyr Arg 73

241 TAC CTG AAG GCG CGT CGG GCA CAG GTG AAG CAT CGC TGT GTG ACC GAC 288

74 Tyr Leu Lys Ala Arg Arg Ala Gin Val Lys Hi s Arg Cys Val Thr Asp 89

289 CCT GCC TAT GAA GAT GTC AAC AAC TGT CAT GAG AGG GCC TTT GTG TTC 336

90 Pro Ala Tyr Glu Asp Val Asn Asn Cys Hi s Glu Arg Ala Phe Val Phe 105

337 ATG CAC AAG ATG CCT CGT CTG TGG CTA GAT TAC TGC CAG TTC CTC ATG 384

106 Met Hi s Lys Met Pro Arg Leu Trp Leu Asp Tyr Cys Gin Phe Leu Met 121

385 GAC CAG GGG CGC GTC ACA CAC ACC CGC CGC ACC TTC GAC CGT GCC CTC 432

122 Asp Gin Gl y Arg Val Thr His Thr Arg Arg Thr Phe Asp Arg Al a Leu 137

433 CGG GCA CTG CCC ATC ACG CAG CAC TCT CGA ATT TGG CCC CTG TAT CTG 480

138 Arg Ala Leu Pro l ie Thr Gin Hi s Ser Arg l ie Trp Pro Leu Tyr Leu 153

481 CGC TTC CTG CGC TCA CAC CCA CTG CCT GAG ACA GCT GTG CGA GGC TAT 528

154 Arg Phe Leu Arg Ser Hi s Pro Leu Pro Glu Thr Ala Val Arg Gly Tyr 169

529 CGG CGC TTC CTC AAG CTG AGT CCT GAG AGT GCA GAG GAG TAC ATT GAG 576

170 Arg Arg Phe Leu Lys Leu Ser Pro Glu Ser Ala Glu Glu Tyr l i e Glu 185

577 TAC CTC AAG TCA AGT GAC CGG CTG GAT GAG GCC GCC CAG CGC CTG GCC 624

186 Tyr Leu Lys Ser Ser Asp Arg Leu Asp Glu Ala Al a Gin Arg Leu Ala 201 --

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iriOO / IOMD / IDd SS809 / I0 OAV 586 Gly Cys Pro Pro Lys Tyr Ala Lys Thr Leu Tyr Leu Leu Tyr Ala Gin 601

1825 CTG GAG GAG GAG TGG GGC CTG GCC CGG CAT GCC ATG GCC GTG TAC GAG 1872

602 Leu Glu Glu Glu Trp Gly Leu Ala Arg His Ala Met Ala Val Tyr Glu 617

1873 CGT GCC ACC AGG GCC GTG GAG CCC GCC CAG CAG TAT GAC ATG TTC AAC 1920

618 Arg Ala Thr Arg Ala Val Glu Pro Ala Gin Gin Tyr Asp Met Phe Asn 633

1921 ATC TAC ATC AAG CGG GCG GCC GAG ATC TAT GGG GTC ACC CAC ACC CGC 1968

634 l ie Tyr l ie Lys Arg Ala Ala Glu l ie Tyr Gly Val Thr Hi s Thr Arg 649

1969 GGC ATC TAC CAG AAG GCC ATT GAG GTG CTG TCG GAC GAG CAC GCG CGT 2016

650 Gly l ie Tyr Gin Lys Ala l ie Glu Val Leu Ser Asp Glu His Ala Arg 665

2017 GAG ATG TGC CTG CGG TTT GCA GAC ATG GAG TGC AAG CTC GGG GAG ATT 2064

666 Glu Met Cys Leu Arg Phe Ala Asp Met Glu Cys Lys Leu Gly Glu l ie 681

2065 GAC CGC GCC CGG GCC ATC TAC AGC TTC TGC TCC CAG ATC TGT GAC CCC 2112

682 Asp Arg Ala Arg Ala l ie Tyr Ser Phe Cys Ser Gin l ie Cys Asp Pro 697

2113 CGG ACG ACC GGC GCG TTC TGG CAG ACG TGG AAG GAC TTT GAG GTC CGG 2160

698 Arg Thr Thr Gly Ala Phe Trp Gin Thr Trp Lys Asp Phe Glu Val Arg 713

2161 CAT GGC AAT GAG GAC ACC ATC AAG GAA ATG CTG CGT ATC CGG CGC AGC 2208

714 His Gly Asn Glu Asp Thr l ie Lys Glu Met Leu Arg l ie Arg Arg Ser 729

2209 GTG CAG GCC ACG TAC AAC ACG CAG GTC AAC TTC ATG GCC TCG CAG ATG 2256

730 Val Gin Ala Thr Tyr Asn Thr Gin Val Asn Phe Met Ala Ser Gin Met 745

2257 CTC AAG GTC TCG GGC AGT GCC ACG GGC ACC GTG TCT GAC CTG GCC CCT 2304

746 Leu Lys Val Ser Gly Ser Ala Thr Gly Thr Val Ser Asp Leu Al a Pro 761

2305 GGG CAG AGT GGC ATG GAC GAC ATG AAG CTG CTG GAA CAG CGG GCA GAG 2352

762 Gly Gin Ser Gly Met Asp Asp Met Lys Leu Leu Glu Gin Arg Ala Glu 777

2353 CAG CTG GCG GCT GAG GCG GAG CGT GAC CAG CCC TTG CGC GCC CAG AGC 2400

778 Gin Leu Ala Ala Glu Ala Glu Arg Asp Gin Pro Leu Arg Al a Gin Ser 793

2401 AAG ATC CTG TTC GTG AGG AGT GAC GCC TCC CGG GAG GAG CTG GCA GAG 2448

794 Lys l ie Leu Phe Val Arg Ser Asp Ala Ser Arg Glu Glu Leu Ala Glu 809

2449 CTG GCA CAG CAG GTC AAC CCC GAG GAG ATC CAG CTG GGC GAG GAC GAG 2496

810 Leu Ala Gin Gin Val Asn Pro Glu Glu l ie Gin Leu Gl y Glu Asp Glu 825

2497 GAC GAG GAC GAG ATG GAC CTG GAG CCC AAC GAG GTT CGG CTG GAG CAG 2544

826 Asp Glu Asp Glu Met Asp Leu Glu Pro Asn Glu Val Arg Leu Glu Gin 841

2545 CAG AGC GTG CCA GCC GCA GTG TTT GGG AGC CTG AAG GAA GAC TGA CCC 2592

842 Gin Ser Val Pro Ala Ala Val Phe Gly Ser Leu Lys Glu Asp *** 856

2593 GTC CCT CCC CCC TCC CCA CCC CCT CCC CAA TAC AGC TAC GTT TGT AAA 2640

2641 AAA AAA AAA AAA AAA AAA AAA A 2659 Example 4: Comparison of homology and structural analysis

Using Blast software, the homology analysis of the amino acid sequence of CLG was found to be highly homologous with the cell cycle regulatory proteins of Drosophila, nematodes and yeast: 41% with the cell cycle regulatory proteins (crooked neck, crn) of Drosophila ( 157/375). It has 66% (557/837) homology with the cell cycle regulatory protein of nematodes and 37% (228/599) homology with the cell cycle control protein of yeast.

Query = CLG Sbjct = crn (Drosophila)

crn gene length = 702

Score = 69.9 bits (168), Expected = 6e-l l

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VV3ai0aH3n into 3Λ into a3m¾d Gu II human ΜΛΜΙ 丄丄 3 <naHSrU3I humanΊ Ι ΗΟ 丄 AcHSS SH: ao.fqs VV30 ++ a 1Α3 + λ + 3 ++ d ++ Ml + HMA H + 13dl HS Id + Α MIH H5l + d1 + H

6ΐ vv3aiaassMiA3iA33VS3dsi) nHaaAoaAVj, 3didHsraaiAidAiiHSHOJ.icnvM 8ει fU lVyaHAHai3inDM¾IWA3DADIMIMdramaW13H3AiaDl ++ S aid3¾NAIISaa S8 + H + + + + + + + + + + +

ε ΐ Bacteria dia leg asoairaci :) human en belly dra leg ν touch :) leg Acm into vdQ :) intestine ΛΟlan sz i aanQ 8 A IAHAMlMASHHdAVIViiHAnJWO VdS NiilMHaiAHGMD ASldNHIiaaaiWAag 9Z

ΜΊ 人 person MIMAS + 1VM3A + ++ d + ¾ + IM M3 AS Na + I + 3H + d + 03

LI V lAaA l ASDcm lVaHAlO IHdMdVOO— Md3IAaiM3¾ASdONHWI333Acna3: ^ ^8η δ

Znε8 / Zΐ = ϋ¾-'(% 99) ZC8 / Z99 = ¾' capsule '(% L) L2S / 6Z =

0 = 1 weight '(ZZOZ) s iq Z6L = ^ f 9 lavadosaoHsiDwg ε99 ^^ fqs 1 ++ 0+ s + +

6SZ laSAlOIVSDSAMlW 5 ^ L

Z9 JMVdSNMAMAHOlHHnMHA— l0HV131301VA3JaiAVMig3dWaiad00AV13dIV 96 ^: l. "QS

V N Λ + + I ++ H +++ + 9 AHdia + + M 0 + IV

m DSVWdN-AGJ.NAlV0ASMaiHlW3¾Iia3N9HHA3Ja Ml0MHV0IIHdaDlDSDJSAIV 989: ^ aanQ f6f avaaiaDn 3i3VJ¾— MiA33dDH3iJ + 3 + A +++ + a + AD +

989 HVaai3Dl¾33WaVdai3W3HVH3aS1A3IV¾5AI0aiHlAOAiawa {IAI dKaA0GV 929: ^ ^9η δ

9 £ WDIV 101V¾HVHOl3M3HI3dOVAnMlMSJIilO¾Hdn3131) iAl5aia3Vag S8G ^^ fqS

+ V + + V MV 3+ 3 δνλΊΊ + Ί M d +1 +++ H H3

9Z9 daAVMivaaAAVHVHMViDMaa --- 3ΐονλΠλΐι¾— vA¾dd3oaivi53diaava3iia us

Z8 £ V3133; HV into NIAH a, dA SIV, 3 people;; 3ν3Ι1, Nao Tongtong;

1+ + person Jft + N SI Η3λ + ++ 3+ λ +

0Z9 DDAHV IJ¾nAlS iaSANdM¾JlSI0H3AV ilS33JAMH331iMVANIAIi5dlV 9IS ^^ "δ

ZZZ dNVVA35HA5AMH) lSA— ΙΛα3Ι0νΗα0ΑΜ¾3Η¾Ιλν¾ίΠ3δΙΗα ΙάΙ LίλΙ \ LZ ^^ fqS

+++ people)! + +3+ V + ¾ + ++ a dl V +

9TS imaiiaaAAVMSiidioisaaiaviKSMAjns ^ AAH OAdHsoadAavHavcn-viv ^ H g ^-: ^ "5

OLZ into iiMVHara¾to33dHVdvi: na33uaa d3A--va3dAHMS Hi 3HS33dHVd¾iAi ziz: + v a + + a ++ + a d A V + H + M + + 9+ g j + vd + M

99t- naiVHaA 3Hin31H9D53MA-SViaaAC) id AMIV¾miAMVaai50NaaAJMVdVA 86C 3ΐιδ Query: 498 GTFQSTKAVYDRILDLRIATPQIVINYAMFLEEHKYFEESFKAYERGISLFKWPNVSDIW 557

GT + S + VYD ++++ LR + A + PQ +++ NYAMFLEE ++ YFE + F + AYE + GI + LFKWP V DIW Sbjct: 502 GTVESCRKVYDKMIELRVASPQMIMNYAMFLEENEYFELAFQAYEKGIALFKWPGVFDIW 561

Query: 558 STYLTKFIARYGGRKLERARDLFEQALDGCPPKYAKTLYLLYAQLEEEWGLARHAMAVYE 617

+ TYL KFI RYGG + KLERARDLFEQ L + CPP + AK ++ LLYA + LEEE GLARHA +++ Y Sbjct: 562 NTYLVKFIKRYGGKKLERARDLFEQCLENCPPTHA YIFLLYAKLEEEHGLARHALSIYN 621

Query: 618 RATRAVEPAQQYYFNIYIKRAAEIYGVTHTRGIYQKAIEVLSDEHAREMCLRFADMECK 677

RA V + A + M + NIYIK + E + YG + R I +++ AI L ++ + R M LR + A + E Sbjct: 622 RACSGVDRADMHSMYNIYIKKVQEMYGIAQCRPIFERAISELPEDKSRAMSLRYAQLETT 681

Query: 678 LGEID ARAIYSFCSQICDPRTTGAFWQTWKDFEVRHGNEDTIKEMLRI RSVQATYNTQ 737

+ GEIDRARAIY + ++ I DP + FW TWK + FEV HGNE T +++ MLR + RRSV + A + YN Sbjct: 682 VGEIDRARAIYAHAAEISDPKVHVKFWDTWKNFEVAHGNEATVRDMLRVRRSVEASYNVN 741

Query: 738 VNFMASQM-LKVSGSATGTVSDLAPGQSGMDDMKXXXXXXXXXXXXXXXDQPLRAQSK I L 796

V + QM + A T + P S + D + Q + I

Sbjct: 742 VTLTSVQMRVDAERKAQETTTSSNPMDS-LDQQQQQPSDGAGSIT QVSMNKGNIS 795

Query: 797 FVRSDASREELAELAQQVNPEEIQLGXXXXXXXXXXXXXXVRLEQQSVPAAVFGSLK 853

FVR + + NP + EI L + + + VPA + FG + LK

Sbjct: 796 FVRGAG --- KTVQQNTTENPDEIDLDEDDDDEEDDGGDADISV— KVVPAQIFGNL 847

Query = CLG Sbjct = putative cell cycle regulatory protein (yeast)

> Putative cell cycle regulatory length = 674

Score = 78.4 bits (190), Expected = le-13

Identity = 125/599 (20%), Similarity = 228/599 (37%) Gap = 115/599 (19%)

Query: 24 YEEEIMRNQFSVKCWLRYIEFKQGAPK-PRLNQLYERALKLLPCSYKLWYRYLKARRAQV 82

+ E + I RN + ++ W + RY +++ + R ++ ERAL + LW + Y ++ ++

Sbjct: 59 FEDAIRRNRLAMGHWMRYGQWELDQ EFARARSVFERALDVDSTYIPLWLKYIEC --- EM 115

Query: 83 KHRCVTDPAYEDVNNCHE AFVFMHKMPRLWLDYCQFLMDQGRVTHTRRTFDRALRALPI 142

K + R + N + RA + ++ + LW Y G + T + F + R L + P

Sbjct: 116 KNRNINH ARNLFDRAVTQLPRVDKLWYK Y V YMEEMLGN I TGCRQ VFERWLKWEP- 169

Query: 143 TQHSRIWPLYLRFLRSHPLPETAVRGYRRFLKLSPESAEEYIEYLKSSDRLDEAAQRLAT 202

W Y + R + E A Y RF + + PE ++ + + + AA

Sbjct: 170 — DENCWMSYIRMERRYHENERARGIYERFVVVHPE-VTNWLRWARFEEECGNAA 221

Query: 203 VVNDERFVSKAGKSNYQLWHELCDLISQNPDKVQSLNVDAI IRGGLTRFTDQLGKLWCSL 262

N + V + DA + + L + + +

Sbjct: 222 NVRQVYLAAIDALGQEFLNE RFFIAF 247

Query: 263 ADYYIRSGHFEKARDVYEEAIRTVMTVRDFTQVFDSYAQFEESMIAAKMETASXXXXXXX 322

A + IR + E + AR +++ AI M +++ Y FE +

Sbjct: 248 AKFEIRQKEYERARTIFKYAI-DFMPRSKSMELYKEYTHFEKQF 290

Query: 323 XXXXXXXXXXXFEQLISRRPLLLNSVLLRQNPHHVHEWHKRVALHQ— GRPREI INTYTE 380

E + + L LL + + P + W + L + G I TY + Sbjct: 291 GDHLGVESTVLDK LQYEKLLKDSPYDYDTWLDLLKLEES AGD INT I RETYEK 344

Query: 381 AVQTVDPF---KATGKPHTLWVAFAKFYE-DNGQLDDARV I LEKATKVNFKQVDDLAS VW 436

A + V A + + W + + F E D + D AR + ++ A K + + A + W

Sbjct: 345 AIAKVPEVVEKNAWRRYVYIWLNYCLFEEIDVKDVDDRARKVYQEALKLIPHKKFTFAKLW 404

Query: 437 CQCGELELRHENYDEALRLLRKATAL--_ PARRAEYFDGSEPVQN—— VY 481

ELR D A + L + A + P Y + + ++ R +

Sbjct: 405 LMY AMFELRQRK I DV ARKTLGRALGMCPKPKLFRGY I EFED A I KQFDRCR I L YEKW I L YD 464

Query: 482 -KSLKVWSMLADLEESLGTFQSTKAVYDRILDLRI-ATPQIVIN-YAMFLEEHKYFEESF 538

++ WA LE LG + A + Y + ++ I TP ++ VYFE + ++ Sbjct: 465 PEACAPWLG YAALETKLGDSDRARALYNLAVNQP I LETPELVWKA YI DFEFEEME YGKAR 524 Query: 539 KAYERGISLFKWPNVSDIWSTYLTKFIARYGGRKLE RARDLFEQAL 584

Y ++ L P + V + w IA E RAR ++ FE AL

Sbjct: 525 SIYQQ-LLRTAPHVK-VWISf ANFEIAHLEDDDEEPPNEEVASPTAVVRARNVFENAL 580 The amino acid sequence & structure analysis of CLG protein was found to contain a crn consensus sequence similar to the crn protein of Drosophila (crn consensus). The crn consensus sequence of this fruit fly is similar to yeast The TPR motif in the related gene protein. It is now known that the gene family with TPR motif in yeast is a series of cell cycle control genes, such as CDC16, CDC23, nuc2 +, and bimA. In addition, members of the TPR gene family in yeast include the negative regulator SSN6 of the sucrose-inducible gene, the negative regulator SKI3 of yeast killer toxin and the mitochondrial membrane protein MAS70 related to protein input. The CLG gene is the same as these Drosophila and yeast genes. This conserved motif is repeated in series in straight lines in the protein sequence of _CLG . It appears repeatedly and has commonality.

Conserved sequence of era protein contained in CLG gene

1-VKCWLRY I EFKQGAPK-PRLNQLYERALKLLPCS (35-67) 13

2- PRLWLDYCQFLMDQGRVTHTRRTFDRALRALPIT (110-143) 10

： 1 1: I ： 1 ：：：： 1: ：： 1 1 1 ： I I

3- SRIWPLYLRFLRSHPLPETAVRGYRRFLKLSPES (146-179) 9

：: 1 I I I ：： I ： 1: 1 1: 1 ： I ：

4- GKLWCSLADYYIRSGHFEKARDVYEEAIRTVMTV (256-289) 9

5- TQVFDSYAQFEESMIAAKMETASELGREEEDD— (293-324) 7

6- VDLELRLARFEQLISRRPLLLNSVLLRQNPHHVH (325-358) 7

I I :: 1 1 1 1: 1:

7- HTLWVAFAKFYEDNGQLDDARVILEKATKVNFKQ (395-428) 11

I I: 1: 1: 1 I :: 1 I I I 1: 1

8- LKVWSMLADLEESLGTFQSTKAVYDRILDLRIAT (484-517) 9

： 1: 1 I ： 1 1 I ：: ：: ：! ： 1 1 ：：

9- PQIVINYA FLEEHKYFEESFKAYERGISLFKWP (518-551) 9

10- KTLYLLYAQLEEEWGLARHAMAVYERATRAVEPA (593-626) 10

1: 1: 1 1: 1: 1 1: 1: 1! 1 1

11-YDMFNIYIKRAAEIYGVTHTRGIYQKAIEVLSDE (629-662) 6

::: 1 1 1: 1: 1: 1 I

12- REMCLRFADMECKLGEIDRARAIYSFCSQICDPR (665-698) 11

VKLWIKYARFEELLKEIDRAREIYERALEFLPRD crn consensus sequence

AEA FGLGHIYEKLGDLEKALDAFQKALELDPNN TP motif Example 5: RNA expression profile of CLG in human tissue

Using CLG as a probe, hybridization was performed with mRNA patches (Clontech) of various human tissues. The results are shown in Figure 1. CLG was found in human heart (H), brain (B), placenta (P), lung (Lu), liver (Li), muscle (SM), kidney (K), and pancreas (Pa) tissues. It is widely expressed, and the expression level is relatively uniform, in which the transcript size in human brain, placenta, lung, liver, kidney and pancreas is about 2. 6kb, which is the same as the full-length cDNA size of CLG obtained in Example 2. Ok, a transcript of about 3. Okb appears in the heart and muscle tissue. Example 6: Inhibitory effect of CLG on colony formation of human liver cancer cells

The CLG gene containing the complete coding region obtained by RT-PCR (Example 2) was cloned into the pT-Adv vector (Clontech) using AdvanTAge ™ PCR Cloning reagent (Clontech, Cat #: K1901- 1), and then digested with EcoR I The CLG gene fragment containing the complete coding region was recovered and subcloned into the eukaryotic cell expression vector pCMV-

Script (Stratagene, Cattt: 212220) to obtain the plasmid pCMV-Script / CLG. The plasmid DNA was extracted using the Qiagen plasmid extraction kit for transfection.

Take 5μ _§ CLG plasmid DNA (the vector is pCMV-Script), add serum-free and antibiotic-free DMEM to 600μ1, and blank control with sterile rai ll iQ water instead of plasmid DNA. Another 25μ1 liposome Lipof ectamine (GIBC0 BRL) was added, and serum-free antibiotic DMEM was added to 600μ1. The above plasmid DNA and liposome were mixed, gently shaken, and left at room temperature for 30-45 minutes, and then added serum-free and antibiotic-free DMEM 1800μ1, the total volume is 3ml. The transfection complex was added 3ml grown in SMMC- 7721 6cm cell culture dish (1-2χ 10 ⁵ cells per dish I), 37

After 6 hours of incubation, the whole medium was replaced with 24 hours later, and the whole medium was replaced with G418. About two weeks, colonies appeared, and crystal violet stained, and the number of colored colonies was observed.

The results showed that SMMC-7721 cells transfected with CLG full-length cDNA significantly inhibited colony formation. After CLG transfection

S-C-7721 cells formed only 3 colonies, while S-band C-7721 cells transfected with empty vectors formed 56 colonies. The CLG gene had a significant inhibitory effect on human liver cancer cell line Sili C-7721 in vitro (Figure 2A and 2B). Example 7: Tumor formation experiments and apoptosis detection of CLG gene-transformed cells

In the same way as in Example 6, human liver cancer cells SMMC-7721 were transfected with CLG gene, and then the transformed cells and control cells not transfected with CLG gene were expanded and cultured. Nude mice were subcutaneously inoculated to observe the effect of CLG gene on tumorigenesis. The experiment was divided into a CLG transfection group and a control group. Each nude mouse was inoculated with 2 × 10 ⁶ cells. The observation period was 6 weeks. Table 3 shows the tumor formation results.

Results of SMMC-7721 cell tumor formation experiment

Group tumor weight (g) Mean tumor weight (g) T test results

1 2 3 4 5 6

Contrast 0. 24 0. 11 0. 15 0. 16 0. 12 0. 18 0. 16 P <0 · 05

CLG 0. 14 0. 07 0. 01 0. 03 0. 10 0. 07 0. 07 In tumor formation experiments, 6 experimental nude mice were inoculated with CLG-transfected SSA C-7721 cells, and the average tumors formed were The tumor weight was 0.07 grams, and the tumor weight of the 6 control cells inoculated with SSA C-7721 cells was 0.16 grams. The average paired two samples were analyzed by T test, and there was a significant difference between the two. Difference, ρ <0.05, tumor inhibition rate was 50%.

CLG-transfected S-band C-7721 cells were inoculated with tumor tissues formed in nude mice and used as paraffin sections. In situ detection of apoptosis by Tunel method (Roche), blue-violet positive apoptotic cells were seen, among which necrosis There were more positive apoptotic cells in the focus (shown in Figure 3A), and scattered apoptotic cells were seen in the non-necrotic area (shown in Figure 3B).

-80ΌFrozen tumor-forming tissue was crushed, digested with a DNA extraction solution containing RNase and proteinase K, digested at 50 ° C for 2 hours, and then extracted twice with phenol, the DNA was precipitated with alcohol, and 5% agarose was coagulated. Gel electrophoresis analysis to see if there is a "trapezoid" DNA band. 5% 琼斯 The tumor tissue DNA formed by SLI C-7721 cells transfected with the full-length CLG gene Liposomal gel electrophoresis analysis showed obvious "trapezoidal" DNA bands, similar to the electrophoretic behavior of tumor tissue DNA formed by SMMC-7721 cells transfected with p53. The tumor tissue DNA formed by untransfected S-band C-7721 cells and the tumor tissue DNA formed by SMMC-7721 cells transfected with pCMV-Script empty vector plasmid DNA showed only a single large DNA band. "DNA band. It was shown that the full-length cDNA of CLG gene could induce SMMC-7721 cells to undergo apoptosis after transfection. Example 8: Protein expression of the CLG gene

Based on the full-length CLG cDNA sequence, primer 3 (5 'TCGGAATTCGTGGTGATGGCGCGACTT 3', 5 'end contains EcoR I site) and primer 4 (5' TCTAAGCTTTCAGTCTTCCTTCAGGCT 3 ', 5' end contains Hind III site), PCR amplification of CLG Coding region sequence. The PCR reaction conditions were: 94 ° C, denaturation for 5 min; then denaturation at 94 ° C for 1 min, annealing at 55 ° C for 1 min, extension at 72 ° C for 2 min, and performing 40 cycles; finally, extension at 72 ° C for 10 min. After the reaction product is detected by electrophoresis, it is recovered by enzyme digestion. The fragment of the CLG coding region recovered by PCR amplification and digestion was inserted into the EcoR I-Hind III site of the vector pET32a (Novagen) to obtain a pET32a-CLG recombinant plasmid.

The constructed recombinant plasmid was transformed into E. coli BL21 (DE3), single colonies were picked and inoculated in LB medium (containing ampicillin 50mg / L), cultured at 37 ° C overnight, 2% inoculation amount was transferred to 5ml LB medium, Incubate until OD600≤0.5, then transfer to 150ml LB (containing ampicillin 50mg / L) by 1%. When OD600 = 0.5-1.0, induce by IPTG (ImM). Continue incubating for about 3 hours. Centrifuge to collect The bacterial cells, ultrasonically disrupted the bacterial cells, and after high-speed centrifugation, it was found that CLG protein with high expression appeared in both the supernatant and the pellet (Figure 5). The molecular weight of CLG protein is 100kDa, which is consistent with the predicted molecular weight.

Claims

Claim

An isolated human CLG protein, characterized in that it comprises: a polypeptide having the amino acid sequence of SEQ ID NO: 2; or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof.

The polypeptide according to claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

3. An isolated polynucleotide, characterized in that it comprises a nucleotide sequence that is at least 85% identical to a nucleotide sequence selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide according to claims 1 and 2;

(b) A polynucleotide complementary to the polynucleotide (a).

The polynucleotide according to claim 3, wherein the polypeptide encoded by the polynucleotide has SEQ ID

Amino acid sequence of NO: 2.

The polynucleotide according to claim 3, wherein the sequence of the polynucleotide is selected from the group consisting of the coding region sequence at positions 22-2258 in SEQ ID NO: 3, or the entire position at positions 1-2659 Long sequence.

6. A vector comprising the polynucleotide according to claim 3.

7. A genetically engineered host cell, characterized in that it is a host cell selected from the group consisting of:

(a) a host cell transformed or transduced with the vector of claim 6;

(b) a host cell transformed or transduced with the polynucleotide of claim 3.

A method for preparing a polypeptide having human CLG protein activity, comprising: (a) culturing the host cell according to claim 7 under conditions suitable for expressing human CLG protein;

(b) A polypeptide having human CLG protein activity is isolated from the culture.

An antibody capable of specifically binding to the human CLG protein according to claim 1.

10. A pharmaceutical composition, characterized in that it contains a safe and effective amount of the polypeptide of claim 1 and a pharmaceutically acceptable carrier.