WO2009030088A1 - Function and use of a new human calcium/calmodulin-dependent protein kinase ii inhibitor - Google Patents

Abstract

The present invention relates to a new human calcium/calmodulin-dependent protein kinase II inhibitor a (CaM-KIINa). The present invention also discloses the use of CaM-KIINa and the encoding sequence thereof. The protein of the present invention is a calcium/calmodulin-dependent protein kinase II inhibitor a, which is expressed at a low level in low differentiated malignant colon adenocarcinoma and inhibits the growth of colon adenocarcinoma cells obviously.

Description

 Function and use of novel human calcium/calmodulin-dependent protein kinase II inhibitory protein

 The present invention belongs to the fields of biotechnology and medicine, and in particular, the present invention relates to a human calcium/Calmodulin-Dependent Protein Kinase II Inhibitor a (CaM-KIINa) and a coding sequence thereof. . Human CaMKIINa is highly expressed in normal and highly differentiated colon adenocarcinoma and has an inhibitory effect on the growth of colon adenocarcinoma cells. Background technique

 Calcium ion is an important second messenger and plays an important role in cell growth and differentiation.

Ca ²⁺ /CaM phosphorylates a variety of enzymes, of which calcium/calmodulin-dependent protein kinase II (CaMKII) is an important target molecule. CaMKII is a multifunctional serine/threonine protein kinase widely distributed in various organ tissues, mainly in nervous tissues. It is especially high in the brain and can reach 1-2% of total protein. It can phosphorylate more than 40 protein molecules with important physiological functions in vivo including enzymes, ion channels, transcription factors, etc.; CaMKII can regulate the metabolism of carbohydrates, amino acids, fats, synthesis and release of neurotransmitters, transcription factors Transcription, cytoskeletal construction, cell cycle progression, DNA damage repair, etc. Physiological and pathological processes. In recent years, some studies have suggested that CaMKII plays an important role in the growth and differentiation of cells. It is also believed that CaMKII is an essential molecule for the smooth progression of the cell cycle.

The discovery and application of CaMKII inhibitors is an effective way to study the physiological functions of CaMKII. CaMKII inhibitors are mainly divided into three categories: inhibitors of chemical synthesis (KN-62, KN-93, KN-92), biosynthesis inhibitory polypeptides (AIP), and inhibitory proteins endogenously expressed by cells. The mechanism of action of chemical inhibitors is mainly through the binding of Ca ²⁺ /CaM binding sites, preventing the binding of Ca ²⁺ /CaM to CaMKII, making CaMKII inactivated by phosphorylation, and specifically inhibiting the activity of CaMKII. . The endogenous inhibitory protein has a structure similar to the CaMKII self-regulating region, which interacts with the catalytic domain to block the binding of CaMKII and its phosphorylated substrate. Inhibition of CaMKII activity blocks the phosphorylation of its substrate by CaMKII or regulates the transcription of certain genes, leading to abnormal cell functions such as cell cycle regulation and DNA damage repair, affecting the normal growth process of cells. It has been reported that chemical CaMKII inhibitors can induce apoptosis, necrosis and growth retardation in tumor cells, and this aspect can provide new strategies and directions for clinical tumor treatment.

In addition, the currently known inhibitory proteins of four kinds of cells have different tissue distribution in normal individuals, and their potential functions may also be different. Rat CaMKIINoc is specifically distributed in the brain, while rat CaMKIINP has specific high expression in both brain and testis. Human CaMKIINP is in the heart, skeletal muscle, Liver, kidney, small intestine, placenta, etc. are expressed. Human CaMKIINoc has high expression in brain, kidney, small intestine and colon, and low expression in placenta, heart, pancreas, spleen and testis, but no expression in skeletal muscle. Differences in endogenous CaMKII inhibitory protein expression profiles suggest that these proteins may play different roles in different tissues and organs. However, the specific expression of CaMKII inhibitory protein and the state of cell growth have not been reported so far, which is the relationship between tumor cell growth.

 Since CaMKII inhibitory proteins play an important role in the regulation of cellular activity, there is an urgent need in the art to develop new CaMKII inhibitory proteins and their applications. Summary of the invention

 The object of the present invention is to provide a novel human calcium/calmodulin-dependent protein kinase II inhibitory protein.

CaMKIINa protein as well as fragments, analogs and derivatives thereof.

 Another object of the invention is to provide the use of a CaMKIINa polypeptide and its coding sequence. In a first aspect of the invention, there is provided a use of a human calcium/calmodulin-dependent protein kinase II inhibitory protein a, CaM-KIINa, or a coding sequence thereof, for use in the preparation of a composition for inhibiting colonic adenocarcinoma cells .

 In another preferred embodiment, the composition is also useful for treating colonic adenocarcinoma.

 In another preferred embodiment, the human CaMKIINa protein is selected from the group consisting of:

 (a) a polypeptide of the amino acid sequence of SEQ ID NO: 2;

 (b) a polypeptide derived from (a) having the amino acid sequence of SEQ ID NO: 2 formed by substitution, deletion or addition of one or more amino acid residues, and having an inhibitory function against growth of colon adenocarcinoma cells;

(c) A fusion protein of a CaMKIINa protein comprising the amino acid sequence of SEQ ID NO: 2 and having an inhibitory function on the growth of colonic adenocarcinoma cells.

 In another preferred embodiment, the coding sequence is a nucleotide sequence encoding the human CaMKIINa protein of the above 0), (b) or (c). More preferably, the coding sequence is selected from the group consisting of:

 (i) the sequence of positions 401-637 in SEQ ID NO: 1; or

 (ii) the sequence of position 1-860 in SEQ ID NO: 1.

 In another preferred embodiment, the composition is a pharmaceutical composition.

 In another preferred embodiment, CaM-KIINa or a coding sequence thereof is also used to prepare a reagent or kit for detecting the degree of differentiation of colon adenocarcinoma.

In a second aspect of the invention, there is provided a method of screening for an agonist of a CaM-KIINa polypeptide, comprising the steps of: (a) providing a test group and a control group, wherein the control group is a culture system of a tumor cell expressing a CaMKIINa polypeptide or a culture system of a tumor cell to which a CaMKIINa polypeptide is added, the test group is added with a test substance a culture system of a tumor cell expressing a CaMKIINa polypeptide, or a culture system of a tumor cell to which a test substance and a CaMKIINa polypeptide are added;

 (b) observing the growth of the tumor cells in the test group and comparing with the growth of the tumor cells of the control group;

 Among them, the growth rate of the tumor cells in the test group was smaller than that of the control group, indicating that the test substance was an agonist of the CaM-KIINa polypeptide.

 In another preferred embodiment, the tumor cell is a colon adenocarcinoma cell.

 In another preferred embodiment, the method further comprises the steps of: (c) screening the obtained agonist of the CaM-KIINa polypeptide to further test its ability to inhibit tumor cell growth, thereby selecting a potential treatment for inhibiting tumor cell growth. Agent.

 In a third aspect of the invention, a method for inhibiting the growth of colonic adenocarcinoma cells in vitro is provided, comprising the steps of: adding a human calcium/calmodulin-dependent protein kinase II inhibitory protein a, ie, CaM, to a colonic adenocarcinoma cell culture system -KIINa o

 In a fourth aspect of the invention, there is provided a use of a human calcium/calmodulin-dependent protein kinase II inhibitory protein a, CaM-KIINa or a coding sequence thereof, for use in the preparation of a reagent for detecting the degree of differentiation of colon adenocarcinoma Or kit.

 In another preferred embodiment, the reagent is an antibody, a primer or a probe (such as a primer pair that specifically amplifies a CaM-KIINa gene or transcript or a probe that specifically binds to a CaM-KIINa gene or transcript) ;).

 In another preferred embodiment, the kit comprises an antibody that specifically binds to a CaM-KIINa polypeptide, a primer pair that specifically amplifies a CaM-KIINa gene or transcript, or specifically binds to a CaM-KIINa gene or transcription. Probe for the object. Other aspects of the invention will be apparent to those skilled in the art from this disclosure. DRAWINGS

 The following drawings are used to illustrate the specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the appended claims.

 Figure 1 shows the Western blot identification of anti-CaMKIINa antibody specificity.

Figure 2 shows the eukaryotic recombinant expression vector of human CaMKIINa transfected into human tumor cells expressing CaMKIINa Western blot analysis of the protein.

 Figure 3 shows that human CaMKIINa protein overexpression of the present invention inhibits tumor cell proliferation.

 Figure 4 shows the inhibitory effect of CaMKIINoc protein on tumor growth in vivo. detailed description

 After extensive and intensive research, a novel human calcium/calmodulin-dependent protein kinase II inhibitor (CaM-KIINa;) was first discovered. CaM-KIINa has an inhibitory effect on the growth of colonic adenocarcinoma cells, and thus can be used as a candidate substance and target for the treatment of colon adenocarcinoma. In addition, CaM-KIINa is highly expressed in normal colon tissues and highly differentiated colon adenocarcinoma tissues, and is highly expressed in malignant colon adenocarcinoma tissues with low degree of differentiation, and thus can be used as an auxiliary for indicating the degree of differentiation of colon adenocarcinoma. The marker is detected.

Specifically, the CaMKIINa protein is selectively highly expressed in normal colon tissues and colon cancer adenocarcinoma tissues with a higher degree of differentiation, and is less expressed in a less differentiated malignant colon adenocarcinoma tissue. CaMKIIN _α inhibits the growth of LoVo colonic adenocarcinoma cells in vitro, and can significantly inhibit the growth of LoVo tumors inoculated subcutaneously in nude mice. Selective expression of CaMKIINa in colon adenocarcinoma and overexpression of CaMKIINa inhibit colon cell growth of colon adenocarcinoma. These experiments CaMKIINa can be used as a target for the treatment of colon adenocarcinoma.

CaMKIINa peptide

 In the present invention, the terms "CaM-KIINa protein", "CaM-KIINa protein" or "calcium ion/calmodulin-dependent protein kinase II inhibitory protein a" are used interchangeably and all have human calcium ions/calcium. A protein or polypeptide that regulates a protein kinase II inhibitory protein a amino acid sequence (SEQ ID NO: 2). They include CaM-KIINa polypeptides with or without the initial methionine.

 As used herein, "isolated" means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .

 As used herein, "isolated CaM-KIINa protein or polypeptide" means that the CaM-KIINa protein is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify the CaM-KIINa protein using standard protein purification techniques. A substantially pure polypeptide produces a single major band on a non-reducing polyacrylamide gel.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. This hair The polypeptide may be a naturally purified product, or a chemically synthesized product, or produced by recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells; The polypeptide of the invention may be glycosylated, or may be non-glycosylated, depending on the host used in the recombinant production protocol. Polypeptides of the invention may also or may not include an initial methionine residue.

 The invention also encompasses fragments, derivatives and analogs of the human CaM-KIINa 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 native human CaM-KIINa protein of the invention. The polypeptide fragment, derivative or analog of the present invention may be a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues;), and such substituted amino acid residues may Is not also 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 the polypeptide, such as poly Ethylene glycol;) a polypeptide formed by fusion, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or A fusion protein for the formation of an antigenic IgG fragment;). These fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

 In the present invention, the term "human CaM-KIINa protein" means having the activity of human CaM-KIINa protein.

A polypeptide of the sequence of SEQ ID NO. The term also encompasses variant forms of the sequence of SEQ ID NO. 2 that have the same function as the human CaM-KIINa protein. These variants include, but are not limited to, several (usually 1-20, more preferably 1-10, optimally 1-5) amino acid deletions, insertions and/or substitutions, and at the C-terminus and / or N-terminal addition of one or several (; usually less than 20, preferably less than 10, more preferably less than 5) amino acids. For example, in the field, similar or similar amino acids When substituted, it usually does not change the function of the protein. For example, the addition of one or several amino acids at the C-terminus and / or N-terminus usually does not alter the function of the protein. The term also includes active fragments of the human CaM-KIINa protein. And active derivatives.

 Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA encoded by DNA that hybridizes to human CaM-KIINa DNA under high or low stringency conditions Protein, and polypeptide or protein obtained using an antiserum against human CaM-KIINa protein. The invention also provides other polypeptides, such as fusion proteins comprising the human CaM-KIINa protein or a fragment thereof. In addition to the nearly full length polypeptide, the present invention also encompasses soluble fragments of the human CaM-KIINa protein. Typically, the fragment has at least about 10 contiguous amino acids of the human CaM-KIINa protein sequence, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, and most preferably at least about 70 contiguous amino acids.

The invention also provides analogs of the human CaM-KIINa protein or polypeptide. The difference between these analogs and the native human CaM-KIINa protein may be a difference in amino acid sequence or a modification that does not affect the sequence. Formal differences, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by irradiation or exposure to a mutagen, or by site-directed mutagenesis or other techniques known to molecular biology. Analogs also include analogs having residues other than the native L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, Υ-amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.

 Modifications (usually without altering the primary structure) include chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides that have been modified to enhance their resistance to proteolytic properties or to optimize solubility properties.

 In the present invention, "human CaM-KIINa protein conservative variant polypeptide" means up to 10, preferably up to 8, more preferably up to 5, optimally compared to the amino acid sequence of SEQ ID NO: 2. Up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide.

CaM-KIINa coding sequence

 As used herein, the term "CaM-KIINa coding sequence" refers to a nucleotide sequence that encodes a CaM-KIINa protein, or an active fragment thereof, a fusion protein, a derivative, and the like.

 The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1, or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence which encodes a protein having SEQ ID NO: 2 but differs from the coding region sequence shown in SEQ ID NO: 1.

 Polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (; and optionally an additional coding sequence) And non-coding sequences.

 The term "polynucleotide encoding a polypeptide" may be a polynucleotide comprising the polypeptide, or may be a polynucleotide further comprising additional coding and/or non-coding sequences.

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

 The invention also relates to hybridization to the sequences described above and having at least 50% between the two sequences, preferably at least

70%, more preferably at least 80% identical polynucleotide. The invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions. In the present invention, "stringent conditions" means: (1) hybridization and elution at a lower ionic strength and higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 ° C _; or (2) hybridization Adding a denaturant such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) at least 90% identity between the two sequences More preferably, hybridization occurs more than 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide of SEQ ID NO: 2.

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

 The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity. The human CaM-KIINa full-length nucleotide sequence of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method. For PCR amplification, primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art. The library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.

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

 The invention also relates to vectors comprising the polynucleotides of the invention, and host cells genetically engineered using the vector of the invention or the CaM-KIINa protein coding sequence, and methods of producing the polypeptides of the invention by recombinant techniques.

 The polynucleotide sequence of the present invention can be used to express or produce a recombinant CaM-KIINa protein by conventional recombinant DNA techniques. Generally there are the following steps:

(1) using the polynucleotide (or variant) of the human CaM-KIINa protein of the present invention, or containing the same A recombinant expression vector for nucleotides transforms or transduces a suitable host cell;

 (2) a host cell cultured in a suitable medium;

 (3) Separating and purifying proteins from the culture medium or cells. An agonist of CaMKIINa polypeptide

 The invention also relates to agonists of the CaMKIINa polypeptide. As used herein, an agonist of a CaMKIINa polypeptide refers to a substance that enhances, or enhances, the activity or expression of a CaMKIINa polypeptide. The agonist can be used to promote the inhibition of the growth of colonic adenocarcinoma cells by the CaMKIINa polypeptide. Antibody and detection method for CaMKIINa polypeptide

 The present invention also encompasses polyclonal antibodies and monoclonal antibodies, particularly monoclonal antibodies, which are specific for human CaM-KI INa DNA or a polypeptide encoded by the fragment thereof. Here, "specificity" means that an antibody binds to a human CaM-KI INa gene product or fragment. Preferably, those antibodies which bind to a human CaM-KI INa gene product or fragment but which do not recognize and bind to other non-related antigen molecules.

 Antibodies against human CaM-KI INa protein can be used in immunohistochemistry to detect human CaM-KI INa protein in biopsy specimens.

 The invention also relates to a diagnostic test method for quantifying and localizing the level of human CaM-KI INa protein. These assays are well known in the art and include FISH assays and radioimmunoassays. The person tested in the test

The CaM-KI INa protein level can be used to explain the importance of the human CaM-KI INa protein in various diseases and the disease for diagnosing the action of the CaM-KI INa protein.

 A method for detecting the presence or absence of a CaM-KIINa protein in a sample is detected by using a specific antibody of the CaM-KIINa protein, which comprises: contacting a sample with a specific antibody against a CaM-KIINa protein; observing whether an antibody complex is formed, forming The antibody complex indicates the presence of the CaM-KIINa protein in the sample. This test can be used as an auxiliary indicator to indicate the degree of differentiation of colon adenocarcinoma. Pharmaceutical composition and method of administration

The CaMKIINa polypeptide of the present invention or an agonist thereof or the like can be used for inhibiting the growth of tumor cells (especially colonic adenocarcinoma cells) when administered therapeutically (administered;). Generally, these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary. The formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration. In the present invention, the CaMKIINa polypeptide or its agonist can be directly used for the treatment of diseases, for example, for the treatment of diseases such as tumors (such as colon adenocarcinoma; When the CaMKIINa protein of the present invention and/or its agonist is used, other therapeutic agents can also be used simultaneously.

 The invention also provides a pharmaceutical composition comprising a safe and effective amount of a CaMKIINa polypeptide of the invention and/or an agonist thereof, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the present invention can be formulated in the form of injections, for example It can be prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by a conventional method. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably used. Manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 1 microgram per kilogram of body weight per day to about 5 milligrams per kilogram of body weight.

 When a pharmaceutical composition is used, a safe and effective amount of the CaMKIINa protein or agonist thereof is administered to the mammal, wherein the safe and effective amount is usually at least about 1 microgram per kilogram of body weight, and in most cases no more than about 8 milligrams per minute. In kilograms of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician. The main advantages of the invention are:

 (a) Based on the ability of CaMKIINa polypeptide to inhibit the growth of colonic adenocarcinoma cells, CaMKIINa can be used as an effective tumor therapeutic target and can be used to screen therapeutic agents (caMKIINa agonists) for treating tumors by interacting with CaMKIINa.

 (b) Based on the correlation between CaMKIINa and differentiation of colon adenocarcinoma cells, CaMKIINa can be used as an auxiliary detection marker for indicating the degree of differentiation of colon adenocarcinoma. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Example 1

 Cloning of human CaM-KIINa cDNA

Human dendritic cells total RNA was extracted with Trizol reagent (Life Technologies). Then, from Poly(A) mRNA was isolated from total RNA. After the poly(A) mRNA was reverse-transcribed to form a cDNA, the cDNA fragment was inserted into the multiple cloning site of the vector by SuperScriptll cloning kit (purchased from Gibco), and DH5oc bacteria were transformed to form a cDNA plasmid library. The sequence of the 5' end of the randomly selected clone was determined by the dideoxy method. The determined cDNA sequence was compared with an existing public DNA sequence database, and it was found that the DNA sequence of one cDNA clone was a new full-length cDNA. The DNA sequence contained in the new clone was bidirectionally determined by synthesizing a series of primers. Computer analysis indicated that the full-length cDNA contained in the clone was a new cDNA sequence (shown as SEQ ID NO: 1) encoding a novel protein (shown as SEQ ID NO: 2; This protein was named as Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor a (CaM-KIINa), and its coding gene was named human calcium ion/calmodulin-dependent. The protein kinase II inhibitory protein a gene, the CaM-KIINa gene.

 The sequence SEQ ID NO: 1 is 860 bp in full length (SEQ ID ΝΟ: 1) and comprises a 400 bp 5' non-coding region and a 223 bp 3' non-coding region encoding a polypeptide of 78 amino acids (SEQ ID NO: 2). Example 2: Cloning of the coding sequence of human CaMKIINa protein by RT-PCR

 PCR amplification of CaMKIINa primers using normal human brain cDNA (Clontech) as template: sense primer 5'- ATGTCGGAGGTGCTGCCCT-3' (SEQ ID NO: 3), antisense primer 5'-TTAG AC ACC AGG AGG TGC CTT G-3' (SEQ ID NO: 4), with β-actin as a positive control. The PCR reaction volume was 50μ1, including reverse transcription template 10μ1, 0.5mM primer, 0.2mM dNTP and 1U rTaq DNA polymerase (Takara), and the amplification parameters were 94°C for 15 seconds, 58°C for 30 seconds, and 72°. C 30 seconds, after 25 cycles, the PCR product was initially confirmed by 1.5% agarose gel electrophoresis.

 The results of DNA sequence analysis indicated that the DNA sequence of the PCR product was identical to that of 401-637 shown in SEQ ID NO: 1. Example 3: Recombinant expression of human CaMKIINa protein

 In this example, the PCR product obtained in Example 2 was used as a template, and amplified with PCR primers at the 5' and 3' ends of the sequence as follows to obtain human CaMKIINa DNA as an insert.

 The 5' oligonucleotide primer sequence used in the PCR reaction is:

 5'- CG GGA TCC ATC AGT GAA GAG CTC CAG AGA A -3' (SEQ ID NO: 5) The 3' primer sequence is:

5'-CG GAA TTC TCA TGC CTC CCT AAA ATA TGT A -3 '(SEQ ID NO: 6) The obtained PCR product was purified and digested with BamH I-EcoR I and then with plasmid pGEM-3ZF. The method was recombined and transformed into competent E. coli DH5a, and the positive clones were identified and purified and sequenced (ABI 377 sequencer, BigDye Terminator kit, PE company). The correct sequence of human CaMKIINa cDNA BamH I-EcoR I was cloned into the expression vector pGEX-2T (Pharmacia), and then transformed into E. coli DH5a. The digested products were analyzed by 0.8% agarose gel electrophoresis. It was confirmed by sequencing that the complete CaMKIINa coding sequence has been inserted.

 The positive DH5a clone expressing CaMKIINa was inoculated in 100 ml of 2xYTA medium, and cultured at 37 ° C for 30-15 rpm for shaking for 12-15 hr, 1:10 diluted in pre-warmed 2xYTA medium, and cultured for 1.5 hr, shaking force 100 mM IPTG to O. lmM After 30 °C induction for 2-6 hr, 5,000 g at 4 ° C, centrifuge for 10 min to remove the supernatant, and resuspend on ice with 50 ml of lxPBS (0.14 M NaCl, 2.7 mM KC1, 10. lmM. 2HPO4, 1.8 mM KH2PO4, pH 7.3). Ultrasound (B. Braun Labsonic U) was crushed and then added with 20% Triton X-100 to 1% for 30 min, then centrifuged at 12,000 g for 10 min at 4 ° C. The supernatant was filtered through a 0.8 μηι filter and passed through 1 ml of 50% glutathione. Glycyrrhiza Sepharose 4B column, lxPBS was washed thoroughly, and then added to 500 ul of glutathione elution buffer (10 mM glutathione, 50 mM Tris-HCl, pH 8.0), allowed to stand at room temperature for 30 minutes, and then the eluate was collected. , Repeat elution 2-3 times to obtain human CaMKIINa protein.

 The GMKIINa protein was obtained by excision of GST by thrombin (thrombin XSigma), and the molecular weight was about 8 kD.

 The N-amino acid sequence analysis by Edams hydrolysis confirmed that the N-terminal sequence was consistent with the N-terminal sequence shown in SEQ ID NO: 2. Example 4: Production of polyclonal antibody against human CaMKIINa protein

 The recombinant human CaMKIINa protein obtained in Example 3 was used to immunize an animal to produce an antibody, and the specific method was as follows. The recombinant molecules are separated by chromatography and used. Separation can also be performed by SDS-PAGE gel electrophoresis, and the electrophoresis band is excised from the gel and emulsified with an equal volume of complete Freund's adjuvant. Mice were intraperitoneally injected with 50-100 g/0.2 ml of emulsified protein. After 14 days, mice were intraperitoneally injected with a dose of 50-100 μg/0.2 ml with the same antigen emulsified with incomplete Freund's adjuvant to boost the immunization. A booster immunization is performed every 14 days, at least three times. The specific reactivity of the obtained antiserum was evaluated by its ability to precipitate the translation product of the human CaMKIINa gene in vitro. As a result, it was found that the antibody specifically binds to the protein of the present invention (Fig. 1). Example 5: Immunohistochemical analysis of human CaMKIINa protein expression

Immunohistochemical analysis was performed using a tissue microarray (Xi'an Superying Company;) containing normal adult colon tissue and colonic adenocarcinoma tissues of different degrees of differentiation. The dyeing process is in accordance with conventional procedures. Color development according to avidin-biotin The peroxidase complex (ABC) method was performed using a Vectastain Elite ABC kit (; Vector company;). The simple procedure is as follows: First, the tissue chip is baked in a 60-degree incubator for 30 minutes, and then dewaxed and hydrated with xylene and ethanol. The antigen was repaired at a high temperature and pressure of 0.01 M CB (pH 6.0). The endogenous peroxidase was blocked with 0.3% H 2 O 2 , the non-specific site was blocked with non-immune sheep serum, and the endogenous biotin was blocked with a biotin blocking reagent. The anti-CaMKIINa antibody (1:40) obtained in Example 5 was then diluted with the primary antibody dilution in the kit and incubated with the sections overnight at 4 °C. The negative control was replaced with PBS instead of the primary antibody, and the rest of the conditions were the same. After washing with PBST (containing 0.1% Tween 20 in PBS), the HRP-conjugated secondary antibody solution was added dropwise, and allowed to stand at room temperature for 30 min. After washing, it was incubated with ABC Elite reagent for 30 min at room temperature and rinsed with PBS. Observed under the microscope.

 Tissue microarray immunohistochemistry showed that the normal colon tissue highly expressed CaMKIINa protein, and the positive rate was more than 95%. In the colon adenocarcinoma tissues of different degrees of differentiation, the positive rate of grade I (highly differentiated;) colon adenocarcinoma was about 87%, and the positive rate of grade 11 (medium differentiation) colon adenocarcinoma was about 75%. grade III (Low-differentiation;) Colonic adenocarcinoma tissue positive rate was only 28% (Table 1). Table 1: Expression of CaMKIINa in normal colon tissues and different graded colon adenocarcinoma tissues

 Classification Number of samples (X) Number of positive samples Positive rate (%;) Normal colon tissue 23 21 91.3

 Gradel colon adenocarcinoma 15 13 86.67*

 Gradell Colon Adenocarcinoma 41 31 75.61*

 Gradelll colon adenocarcinoma 18 27.78**

*Compared with normal group, P<0.05

 **Compared with the normal group, <0.001 results suggest that CaMKIINa protein is selectively expressed in normal and differentiated colon and colon adenocarcinoma tissues, and the lower the degree of differentiation, the lower the expression. Example 6: Construction of human CaMKIINa eukaryotic expression vector and transfection of eukaryotic gene

 In this example, the PCR product obtained in Example 2 was used as a template, and amplified with PCR primers at the 5' and 3' ends of the sequence below to obtain human CaMKIINa DNA as an insert.

 The 5' oligonucleotide primer sequence used in the PCR reaction is:

 5'- AC GAA TTC ATG TCG GAG GTG CTG CCC T -3' (SEQ ID NO: 7)

The 3' primer sequence is: 5'- T GAA GCT TAC ACC AGG AGG TGC CTT G -3' (SEQ ID NO: 8) The obtained PCR product was purified and digested with EcoR I-Hind III and then eukaryotic expression vector plasmid pcDNA3.1/myc -His (-) B (Invitrogen) was recombined and transformed into competent E. coli DH5a by conventional methods, and positive clones were picked for restriction enzyme digestion, purified and sequenced (ABI's Model 377 Sequencer, BigDye Terminator Kit, PE) the company). It was confirmed by sequencing that the complete CaMKIINa coding sequence has been inserted.

The CaMKIINa eukaryotic expression plasmid DNA was transfected into human colon adenocarcinoma LoVo cells by liposome LipofectAMINE (invitrogen); pcDNA3.1 plasmid vector was used as a mock transfection control. Follow the instructions. The main steps are as follows: The plasmid DNA to be transfected is mixed with liposome LipofectAMINE in a certain ratio and allowed to react at room temperature for 45 minutes; at 60-80% confluent in LoVo cells grown in 6-well cell culture plate, with OPTI-MEM After washing the serum medium (Invitrogen) twice, add the plasmid DNA-liposome mixture, incubate at 37 ° C 5% CO _{2 for} 6-8 hours, add an equal volume of normal medium containing 20% serum, continue to culture 6 Replace the fresh medium after an hour. Transient expression was collected 48 hours after transfection and subjected to Western blot analysis to detect transfection effects. Example 7: Western blot detection

 LoVo cells transiently transfected with expression of CaMKIINa protein in Example 7 were lysed with cell lysate (Cell Signaling). The supernatant was taken at 13,000 rpm x lOmin by centrifugation at 4 ° C, and protein quantification was performed using a BCA protein detection kit (; PIERCE). The protein samples were subjected to SDS-PAGE followed by a constant voltage of 100 V at 4 ° C to a nitrocellulose membrane (Schleicher & Schuell), which was stained with Ponceau and marked in size and orientation. Block at room temperature for 2 hours (5% of TBST solution of skimmed milk powder was diluted with blocking solution, and incubated for 1 hour at room temperature. TBST (0.05% Tween 20 in TBS solution) was washed for 15 minutes, 3 times, and the secondary antibody was diluted with blocking solution. Incubate for 2 hours at room temperature. Wash TBST for 15 minutes, 3 times, wash with TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl) for 15 minutes, then add chemiluminescent substrate (Pierce) for 1 min, and seal the membrane quickly. And auto-development. The primary antibody used for Western blot detection was the anti-CaMKIINa antibody obtained in Example 5. The secondary antibody was HRP-labeled anti-rabbit IgG (Cell Signaling).

 The results showed that the expression product of human CaMKIINa was detected in cells transfected with human CaMKIINa eukaryotic expression vector 48 hours after transfection, with a molecular weight of 11 kD (including the 3kD myc-his tag sequence, Figure 2). Example 8: MTT assay for cell proliferation

LoVo cells overexpressing the CaMKIINa protein in Example 6 in the logarithmic growth phase were seeded in a 96-well flat-bottomed plate, and each of the three wells was placed and cultured at 37 ° C, 5% CO ₂ . On the day of testing, add 10% MTT (5mg/ml, Sigma) medium 100μ1, continue to incubate for 4 hours, carefully aspirate the supernatant and then add 150μ1/well DMSO, incubate at 37 °C for 10 minutes to dissolve intracellular forasia (foraiazan), using enzyme-linked The instrument measures the OD value at a wavelength of 570 nm.

The results showed that the OD value of LoVo cells overexpressing CaMKIINa protein was significantly lower than that of untransfected LoVo cells and mock-transfected LoVo cells (P < 0.01). The results showed that overexpression of CaMKIINa protein in vitro can inhibit colon adenocarcinoma tumors. The cells proliferated in vitro (Fig. 3). Example 9: Establishment of a human colon cancer xenograft model and observation of CaMKIINa anti-tumor effect Under sterile experimental conditions, nude mice (Balb/c nude mice, male, 4-5 weeks old, Chinese Academy of Sciences;) LoVo cells grown in the right side of the left forelimb were injected subcutaneously with 2×10 ⁶ cells/nose in nude mice, and local visceral lesions were observed to observe the tumor growth. The nodules usually appear after 3-4 days, that is, the establishment of the transplanted tumor model.

 Nude mice with a tumor size of 3-4 mm were selected from the mice modeled as LoVo cell xenografts and randomly divided into three groups of 6 rats: (1) CaMKIIN (group X: 20 g of CaMKIIN injected intratumorally) Oc eukaryotic recombinant plasmid; (2) empty vector pcDNA3.1/myc-His (-) B mock control group: intratumor injection of 20 g of empty vector plasmid; (3) PBS control group: intratumoral injection 2 (^1 PBS; Immediately after the injection of each group, the plasmid DNA was introduced into the tumor tissue by the «« vivo electroporation electric pulse (300V, 960 μΡ) gene introduction method (ΒΤΧ 830 electroporator).

The tumor growth was observed dynamically during the experiment. The maximum diameter a and the transverse b were measured with vernier calipers every three days. The tumor volume and tumor volume inhibition rate were calculated according to the formula: mean tumor volume (CV) = l/6x0xb) _; tumor volume suppression Rate (R) = l - mean tumor volume of the experimental group / mean tumor volume of the control group x l00%.

 Table 2: Inhibition rate of CaMKIIN on tumors transplanted into LoVo cells in vivo

Mean tumor volume (cm ³ )

 Time (day) inhibition rate (%)

 CaMKIINa simulation control

 0 0.075 0.075 0

 5 0. 14 0. 162 14.6*

 10 0.252 0.364 30.8*

 15 0.289 0.424 33. 1 *

20 0.298 0.449 33.6*

The results showed that the growth of tumors in the CaMKIINa group was significantly inhibited compared with the control group (Ρ<0.05χ图) 4); There was no significant difference between the mock control group and the PBS control group. Calculation of tumor inhibition rates at various time points (Table 2) showed that the tumor inhibition rates at 5, 10, 15 and 20 days were 14.6%, 30.8%, 33.1% and 33.6%, respectively. Therefore, the expression of CaMKIINoc in tumor tissues can significantly inhibit the growth of human colon cancer cell LoVo in nude mice. Example 10: Preparation of a pharmaceutical composition

 In a conventional mixing method, 1 mg of CaMKIINa prepared in Example 3 and 200 ml of physiological saline for injection were placed in an injection solution, and dispensed in a 10 ml vial. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it is to be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

Rights request

1. Use of human calcium/calmodulin-dependent protein kinase II inhibitory protein a, i.e., CaM-KIINa or a coding sequence thereof, for use in the preparation of a composition for inhibiting colonic adenocarcinoma cells.

 2. The use according to claim 1, wherein the composition is further for treating colonic adenocarcinoma.

3. The use according to claim 1, wherein the human CaMKIINa protein is selected from the group consisting of:

(a) a polypeptide of the amino acid sequence of SEQ ID NO: 2;

 (b) a polypeptide derived from (a) having the amino acid sequence of SEQ ID NO: 2 substituted, deleted or added by one or more amino acid residues, and having an inhibitory function against colonic adenocarcinoma growth;

 (c) a fusion protein of a CaMKIINa protein, which comprises the amino acid sequence of SEQ ID NO: 2 and has an inhibitory function on the growth of colonic adenocarcinoma cells,

 Alternatively, the coding sequence is selected from the group consisting of:

 (i) the sequence of positions 401-637 in SEQ ID NO: 1; or

 (ii) the sequence of position 1-860 in SEQ ID NO: 1.

 4. Use according to claim 1 wherein the composition is a pharmaceutical composition.

 5. The use according to claim 1, characterized in that CaM-KIINa or a coding sequence thereof is also used for the preparation of a reagent or kit for detecting the degree of differentiation of colon adenocarcinoma.

 6. A method of screening an agonist of a CaM-KIINa polypeptide, comprising the steps of:

(a) providing a test group and a control group, wherein the control group is a culture system of a tumor cell expressing a CaMKIINa polypeptide or a culture system of a tumor cell to which a CaMKIINa polypeptide is added, and the test group is added with a test substance a culture system of a tumor cell expressing a CaMKIINa polypeptide, or a culture system of a tumor cell to which a test substance and a CaMKIINa polypeptide are added;

 (b) observing the growth of the tumor cells in the test group and comparing with the growth of the tumor cells of the control group;

 Wherein, the growth rate of the tumor cells in the test group is smaller than that of the control group, indicating that the test substance is

An agonist of the CaM-KIINa polypeptide.

 7. The method according to claim 6, wherein the tumor cells are colonic adenocarcinoma cells.

8. The method according to claim 6, wherein the method further comprises the step of: (c) screening the obtained agonist of the CaM-KIINa polypeptide to further test its ability to inhibit tumor cell growth, thereby selecting A potential therapeutic agent that inhibits the growth of tumor cells.

 A method for inhibiting growth of colonic adenocarcinoma cells in vitro, comprising the steps of: adding a human calcium/calmodulin-dependent protein kinase II inhibitory protein a, CaM-KIINa, to a colon adenocarcinoma cell culture system.

 10. Use of a human calcium/calmodulin-dependent protein kinase II inhibitory protein a, i.e., CaM-KIINa or a coding sequence thereof, for use in the preparation of a reagent or kit for detecting the degree of differentiation of colon adenocarcinoma.