CN100335620C - Human cholesteryl ester synthetase-2b and its coding sequence - Google Patents

Abstract

The present invention provides human cholesteryl ester synthase-2b (ACAT2b) cDNA sequence and a human cholesteryl ester synthase amino acid sequence encoded therein, constructs and vectors containing the sequence, and their application. The nucleotide sequence of human ACAT2b cDNA is about 1.5kb long, encoding 502 amino acid sequences. The human ACAT2b cDNA and the human cholesteryl ester synthase encoded by the present invention can be used for the research and screening of drugs for the treatment or auxiliary treatment of hypercholesterolemia, atherosclerosis and the like.

Description

Human cholesteryl ester synthase-2b and its coding sequence

field of invention

The invention relates to the technical fields of biochemistry, molecular biology, genetic engineering and DNA recombination. More specifically, it relates to human cholesteryl ester synthase-2b (ACAT2b) and its coding sequence and their applications.

Background technique

Human cholesteryl ester synthase, namely human acyl-coenzyme A: cholesterol acyltransferase (ACAT, Acyl-coenzyme A: cholesterol acyltransferase, EC2.3.1.26), is the only enzyme in human cells that catalyzes the formation of cholesterol ester from long-chain fatty acids and free cholesterol . ACAT plays an important physiological function in the living body, mainly involved in the absorption, transportation and storage of cholesterol and its esters in the living body, and is one of the key enzymes to maintain the metabolic balance of cholesterol and its esters in the body. A large number of studies at home and abroad have shown that it synthesizes cholesteryl esters with high activity in macrophages, leading to the excessive accumulation of cholesteryl esters in the cells to form foam cells, causing early atherosclerosis (AS) lesions; its activity can affect nerve cell membranes. directly regulates the production of Aβ protein, which is related to Alzheimer's disease (AD); its activity changes in liver and intestinal cells can directly affect the synthesis of cholesterol esters, the absorption of cholesterol, and the assembly of lipoproteins, until it affects cholesterol High blood levels and blood transport are directly related to hypercholesterolemia; and long-term lack of dietary cholesterol absorption can also cause serious diseases, such as children and senile dementia. Therefore, it is an extremely important drug target protein for diseases related to cholesterol metabolism, and ACAT has been taken as the main target protein for the development of anti-atherosclerosis drugs in the world. However, ACAT is an endoplasmic reticulum membrane protein with extremely low content. Even though many laboratories or companies in the world have invested a lot of human and material resources in research, so far they have not been able to purify its natural enzyme protein. Therefore, it is very important to carry out the work at the gene level for the in-depth study of the mechanism, structure and function of ACAT and its relationship with related diseases, and it is the only feasible way at present.

Two different ACAT genes encoding ACAT1 and ACAT2 are currently found in mammals. In 1993, the Chang TY laboratory of the Dartmouth School of Medicine in the United States cloned for the first time the human ACAT1 cDNA (4011bp) with active functions, and its open reading frame encoded a sequence of 550 amino acids (Chang TY et al., 1993, J.Biol.Chem. , 268:20747-20755). In 1996, the R.V.Farese laboratory of the University of California, San Francisco successfully carried out the mouse ACAT1 gene knockout experiment, and found that there was still a strong ACAT enzyme activity in the liver and small intestine, so it was speculated that there was another form of ACAT1. ACAT. Starting from the computer analysis of the ACAT conserved sequence, in 1998, three laboratories cloned the African green monkey (Anderson RA et al., 1998, J.Biol.Chem., 273:26747-26754), mouse (Cases S et al. ., 1998, J.Biol.Chem., 273:26755-26764) and the ACAT2 cDNA of people (Oelkers P et al., 1998, J.Biol.Chem., 273:26765-26771). Human ACAT2 cDNA is about 2040bp long, and its open reading frame encodes 522 amino acids. ACAT1 is expressed in almost all tissues, while ACAT2 is only highly expressed in the liver and small intestine. Therefore, ACAT2 is considered to be mainly involved in the absorption of dietary cholesterol and the assembly and secretion of ApoB-containing apolipoprotein, which is directly related to the blood level and blood transport of cholesterol.

In 1999, the inventor's laboratory and Chang TY's laboratory jointly published the DNA structure organization of the human ACAT1 genome, and found that the human ACAT1 mRNA sequences came from two different chromosomes (Li Bo-Liang et al., J.Biol.Chem. , 1999, 274:11060-11071). At the same time, the inventors carried out studies on the DNA structure organization of the human ACAT2 genome. In liver and intestinal cells, the inventors found that in addition to being identical to the ACAT2 cDNA (systematic named ACAT2a cDNA) published by foreign counterparts, there is another form of ACAT2b cDNA. Multiple forms of ACAT2 exist, suggesting the importance and complexity of this gene's function.

With the improvement of Chinese people's living standards and changes in dietary structure, cardiovascular diseases and neurodegenerative diseases have increasingly become important killers that threaten people's lives and health. A large number of epidemiological and molecular studies have proved that these diseases are related to biological Cholesterol levels are closely related. Because ACAT plays a key role in the cholesterol balance in the body and the pathogenesis of atherosclerosis, the screening of specific inhibitors of ACAT has become a hot research topic for some pharmaceutical companies. Since the natural form of ACAT protein has not been successfully purified, it is still difficult to find specific and effective inhibitors of ACAT. More and more scientists and large pharmaceutical companies at home and abroad have participated in the research in this field. In particular, scientific research experts who study cardiovascular diseases hope to screen and invent cardiovascular disease drugs targeting anti-atherosclerosis ACAT. The latest literature believes that ACAT2, which is specifically expressed in liver and intestinal cells, is involved in the absorption of exogenous cholesterol and the assembly of lipoproteins, which directly affects the level of cholesterol in the blood. Drugs that target ACAT2 will control the cholesterol level of the human body while at the same time Therefore, the research on ACAT2 has attracted more and more attention.

The ACAT2b discovered by the inventors is of great significance for elucidating the important functions of ACAT and revealing the molecular mechanisms of hypercholesterolemia and atherosclerosis. This breakthrough in basic research will soon lead to its applied research, and Rapidly promotes the invention of drugs for cholesterol-related diseases such as cardiovascular diseases. However, at present, the amino acid sequence of human ACAT2b cDNA and its encoded human cholesteryl ester synthase has not been obtained.

Therefore, there is an urgent need in the art to develop human ACAT2b cDNA and its encoded human cholesteryl ester synthase.

Contents of the invention

The purpose of the present invention is to provide a new human cholesteryl ester synthase 2b and its coding sequence.

Another object of the present invention is to provide methods for producing these human cholesteryl ester synthases and their coding sequences and their uses.

The first aspect of the present invention provides a human cholesteryl ester synthase-2b, the protein is selected from the following group:

(a) A protein having the amino acid sequence of SEQ ID NO: 2, or a conservative variant thereof, or an active fragment thereof, or an active derivative thereof.

(b) The amino acid sequence of SEQ ID NO: 2 is formed by substitution, deletion or addition of one or more amino acid residues, and a protein derived from (a) having the function of human cholesteryl ester synthase-2b.

In a preferred example, the human cholesteryl ester synthase-2b (ACAT2b) has the amino acid sequence shown in SEQ ID NO:2.

In a second aspect of the present invention, an isolated polynucleotide is provided, said polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(a) a polynucleotide encoding the above protein;

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

Preferably, the polynucleotide encodes a protein having the amino acid sequence shown in SEQ ID NO:2.

Preferably, the sequence of the polynucleotide comprises one selected from the following group:

(a) the sequence of positions 13-1521 in SEQ ID NO: 1;

(b) The sequence of positions 1-1522 in SEQ ID NO:1.

More preferably, the sequence of the polynucleotide has one selected from the following group:

(a) the sequence of positions 13-1521 in SEQ ID NO: 1;

(b) The sequence of positions 1-1522 in SEQ ID NO:1.

The third aspect of the present invention provides a vector containing the above polynucleotide. Preferably, the polynucleotide comprises the sequence of positions 13-1521 in SEQ ID NO:1.

The fourth aspect of the present invention provides a host cell comprising the above-mentioned vector. Preferably, said host cell is a mammalian cell. More preferably, the host cells are mouse, rat or human cells.

The fifth aspect of the present invention provides a method for preparing a protein having human cholesteryl ester synthase-2b activity, characterized in that the method comprises:

(a) cultivating the above-mentioned host cells under conditions suitable for expressing human cholesteryl ester synthase-2b;

(b) Isolating a protein having human cholesteryl ester synthase-2b activity from the culture.

The sixth aspect of the present invention provides an antibody capable of specifically binding to the above-mentioned human cholesteryl ester synthase-2b.

The seventh aspect of the present invention provides a pharmaceutical composition, which contains a safe and effective amount of the above-mentioned human cholesteryl ester synthase-2b, or the above-mentioned polynucleotide, and a pharmaceutically acceptable carrier.

The eighth aspect of the present invention provides the use of the human cholesteryl ester synthase-2b in screening or preparing drugs for treating cholesterol-related diseases.

The ninth aspect of the present invention provides the use of the above polynucleotides in screening or preparing drugs for treating cholesterol-related diseases.

The tenth aspect of the present invention provides a method for screening drugs for the treatment of cholesterol-related diseases, the method comprising the steps of:

(a) Under conditions suitable for growth, culture host cells, the host cells contain the above expression vector, the expression vector contains the above polynucleotide and an exogenous promoter operably linked to the polynucleotide, wherein The candidate substance is added to the culture medium or lysate or protein preparation of the first group of host cells, and the candidate substance is not added to the culture medium or lysate or protein preparation of the second group of host cells;

(b) Determining the activity of cholesteryl ester synthase-2b in the first group and the second group of host cells or lysates or prepared proteins, wherein the enzyme activity of the first group of host cells or lysates or prepared proteins is higher and lower than that of the first group of host cells or lysates or prepared proteins The second group means that the candidate substance is a drug for treating cholesterol-related diseases.

Other aspects of the invention will be apparent to those skilled in the art from the technical disclosure herein.

Description of drawings

The following drawings are used to illustrate specific embodiments of the present invention, but not to limit the scope of the present invention defined by the claims.

Figure 1: Human ACAT2b cDNA sequence is the polynucleotide coding sequence of human cholesteryl ester synthase 2b.

Figure 2: Amino acid sequence of human cholesteryl ester synthase 2b.

Figure 3: Recombinant expression vector containing human ACAT2b coding sequence and western blot analysis:

A. Schematic diagram of the construction of the recombinant expression vector of the human ACAT2b coding sequence;

B. Western blot analysis of human cholesteryl ester synthase 2b.

Figure 4: Activity analysis of human cholesteryl ester synthase expressed by human ACAT2b gene.

Detailed ways

After extensive and in-depth research, the present inventor isolated and cloned human ACAT2b cDNA for the first time, and analyzed it to determine the amino acid sequence of a human cholesteryl ester synthase encoded by the cDNA, and constructed a recombinant expression vector of human ACAT2b cDNA. Expressed in mammalian cells to analyze the enzymatic activity of human ACAT2b. The present invention has been accomplished on this basis.

As used herein, the terms "cholesteryl ester synthase-2b", "ACAT2b" are used interchangeably to refer to an enzyme having the amino acid sequence of ACAT2b (Figure 2); the terms "cholesteryl ester synthase-2b cDNA", "ACAT2b cDNA"" Cholesteryl ester synthase-2b gene " " ACAT2b gene " can be used interchangeably, refers to the polynucleotide sequence (Fig. 1, SEQ NO:1) that has the aminoacid sequence of coding ACAT2b.

In the present invention, "cholesteryl ester synthase-2b conservative variant polypeptide" means that compared with the amino acid sequence of SEQ ID NO: 2, there are at most 10, preferably at most 8, more preferably at most 5, and most preferably at most Preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the 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 that encodes a protein with SEQ ID NO: 2, but differs from the sequence of the coding region shown in SEQ ID NO: 1.

A 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 optional additional coding sequences) and non-coding sequence.

The present invention also relates to polynucleotide variants of the above cDNA. These polynucleotide variants may 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, which may be a substitution, deletion or insertion of one or more nucleotides without substantially changing the function of the cDNA.

The present invention also relates to nucleic acid fragments that hybridize to the above-mentioned sequences. As used herein, a "nucleic acid fragment" has a length of at least 10 nucleotides, preferably at least 50 nucleotides, more preferably at least 100 nucleotides, and most preferably at least 200 nucleotides. Full length of cholesteryl ester synthase-2b cDNA. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to determine and/or clone the nucleotide sequence of the cDNA of the present invention.

The full-length nucleotide sequence of the human ACAT2b cDNA of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, and cDNA libraries derived from human cells or cDNA libraries containing human chromosome cells can be used as templates to amplify to obtain relevant sequences.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them. At present, the DNA sequence of the gene (or its fragment, or its derivative) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.

The method of amplifying DNA/RNA using the PCR technique (Saiki RK et al., Science, 1985, 230: 1350-1354) is preferably used to obtain the cDNA of the present invention.

The present invention also relates to a construct or vector comprising the gene of the present invention, a host cell transformed or transduced with the construct or vector, and a method for producing ATAC2b through recombinant technology.

The exogenous promoters applicable to the present invention are not particularly limited, and almost all exogenous promoters can be used in the present invention. Examples of representative exogenous promoters include (but are not limited to): various recombinant cloned promoters, such as SV40 promoter, CMV promoter, etc., and various artificially synthesized promoters, etc. It should be noted that foreign promoters also include promoters derived from the host itself. For example, for some genetic diseases (such as diseases caused by the high activity or low activity of a certain promoter), the relevant promoter can be isolated from the patient itself, and then connected to the cholesteryl ester synthase-2bcDNA sequence of the present invention , forming a construct containing an exogenous promoter, and then using the construct for screening drugs for treating cholesterol-related diseases or for gene therapy.

An example of a construct is the exogenous promoter linked to the cholesteryl ester synthase-2b cDNA. As for the positional relationship between cholesteryl ester synthase-2b cDNA and the exogenous promoter, the cDNA sequence should be located downstream of the exogenous promoter.

In the present invention, the nucleotide sequence of human cholesteryl ester synthase-2b cDNA contained can preferably be inserted into a vector, such as an expression vector. The term "vector" refers to bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art. Vectors applicable in the present invention include but are not limited to: expression vectors, cloning vectors, such as vectors suitable for prokaryotic (such as bacteria such as Escherichia coli), eukaryotic (such as yeast), insect cells, plant cells, and mammalian cells. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. In the expression vector, in addition to containing the origin of replication and cholesteryl ester synthase-2b cDNA, it can also contain exogenous promoters and other control elements such as transcription and translation.

Methods well known to those skilled in the art can be used to construct expression vectors containing cholesteryl ester synthase-2b cDNA and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). The DNA sequence can be effectively linked with the foreign promoter to be expressed to direct the synthesis of the cDNA and the corresponding mRNA.

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 for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

The vector comprising the appropriate sequence of human cholesteryl ester synthase-2b cDNA and an appropriate promoter or control sequence can be used to transform appropriate host cells so that it can express human cholesteryl ester synthase-2b.

The host cell may 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: Escherichia coli, Streptomyces spp; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, or Bowes melanoma cells animal cells, etc. Mammalian cells such as mouse, rat and human cells are preferred.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. Some transformation methods employed include, but are not limited to: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformants can be cultured by conventional methods to express ACAT2b. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth and expression of the host cells. The above-mentioned recombinant polypeptides can be expressed in cells, on cell membranes, or secreted outside cells.

The present invention also includes polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies, specific to ACAT2b DNA or polypeptides encoded by its fragments. Here, "specificity" means that the antibody can bind to ACAT2b gene product or fragment. The invention also includes immunologically active antibody fragments, such as Fab' or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; or chimeric antibodies, such as those that have the binding specificity of a murine antibody but retain the portion of the antibody from a human Antibody. Antibodies of the present invention can be prepared by various techniques known to those skilled in the art.

Antibodies against ACAT2b can be used in immunohistochemical techniques to detect ACAT2b in biopsy specimens. Using the protein of the present invention, substances that interact with ACAT2b, such as inhibitors, agonists or antagonists, can be screened out through various conventional screening methods.

The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of ACAT2b of the present invention, its encoding nucleic acid, and/or antisense nucleic acid, 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 match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 0.01 microgram/kg body weight to about 5 mg/kg body weight per day. In addition, the polypeptides of the invention can also be used with other therapeutic agents.

When using the pharmaceutical composition, the safe and effective amount of ACAT2b or its antagonist, agonist is administered to mammals, wherein the safe and effective amount is usually at least about 0.01 micrograms/kg body weight, and in most cases no more than about 10 mg /kg body weight, preferably the dose is about 0.01 μg/kg body weight to about 100 μg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

The invention also relates to a diagnostic test method for quantitative and localized detection of ACAT2b level. These assays are well known in the art and include FISH assays and radioimmunoassays. The level of ACAT2b detected in the test can be used to explain the importance of ACAT2b in various diseases and to diagnose diseases caused by the absence or increase of ACAT2b.

A method for detecting the presence of ACAT2b in a sample is to use an ACAT2b-specific antibody for detection, which includes: contacting the sample with an ACAT2b-specific antibody; observing whether an antibody complex is formed, and the formation of an antibody complex indicates the presence of ACAT2b in the sample .

In one embodiment of the present invention, the human ACAT2b cDNA sequence was obtained by PCR amplification, and it was found that the 5'-region of the cDNA sequence has an ATG translation initiation codon and the 3'-region has a TAG translation termination codon, indicating that This is the gene cDNA sequence with a typical translation reading frame, which encodes a human cholesteryl ester synthase ACAT2b with a sequence of 502 amino acids.

In another embodiment, the present invention transfects the expression vector of human ACAT2b cDNA into mammalian cells for expression, and analyzes the cholesteryl ester synthase protein expressed by human ACAT2b cDNA, and simultaneously uses Cell-free and Intact cell two system methods to determine Cholesteryl ester synthase activity, indicating that the transfected cells expressing human ACAT2b has cholesterol ester synthase activity, but it is much lower than the positive control activity, showing that it is extremely important for the diagnosis, treatment and drug research of cholesterol metabolism-related diseases such as hypercholesterolesteremia value.

The cholesteryl ester synthase-2b cDNA of the present invention has broad application prospects. ACAT-2b is of great significance both for basic theoretical research and for the diagnosis, treatment and drug screening of cholesterol-related diseases (including atherosclerosis, Alzheimer's disease, etc.). The present invention makes it possible to change the expression activity of ACAT in a specific period and in a specific tissue, so as to provide another possible way for the treatment of diseases related to cholesterol metabolism (such as atherosclerosis, etc.), and the present invention can be used in mammalian cells Expresses ACAT-2b.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions.

Cloning and identification of embodiment 1 people's ACAT2b cDNA

In order to obtain the cDNA sequence containing human ACAT2b, design synthetic primers, the sequence is as follows:

C41: 5'GGAGACCGCACCATGGAG3'

C42: 5'TGTCTAGACCTCTAGGTATGGCAGGAC3'

C21: 5'GGAAGAGTATCAGCATGACG3'

Using two pairs of primers C41 and C42, C41 and C21 respectively, the cDNA products of HepG2 and Caco-2 cells were amplified by RT-PCR, the PCR products were cloned into T-Easy Vector (purchased from Promega), sequenced and analyzed, and found It has a cDNA sequence encoding 502 amino acids of human cholesteryl ester synthase, named ACAT2b gene sequence.

The measured nucleotide sequence of human ACAT2b cDNA is shown in Figure 1 and SEQ ID NO:1.

Example 2 Human ACAT2b cDNA reading frame and its encoded amino acid sequence analysis

According to the ACAT2b cDNA sequence obtained by sequencing, the analysis found that the 5'-region of the cDNA sequence has an ATG translation initiation codon, and the 3'-region has a TAG translation termination codon, indicating that this is a gene cDNA sequence with a typical translation reading frame. It encodes a human cholesteryl ester synthase of 502 amino acid sequences, namely ACAT2b (Figure 2), which is 20 amino acid residues less than the human ACAT2a reported by foreign counterparts.

The construction of embodiment 3 people ACAT2b cDNA eukaryotic expression vector

In order to determine the protein expression and enzyme activity analysis of human ACAT2b cDNA, first use the total RNA prepared from human intestinal cell line Caco-2, and obtain the coding region of ACAT2 cDNA sequence published in the literature by RT-PCR amplification as a positive control; Then, the obtained positive control and sample cDNA were respectively inserted between EcoRV and XbaI of pcDNA3 (purchased from Invitrogene Company), located at the downstream of the CMV promoter, to obtain corresponding eukaryotic expression vectors: pcDNA3-A2 and pcDNA3-A2b ( See Figure 3 top line plot).

The protein analysis of embodiment 4 cell culture, DNA transfection and human ACAT2b cDNA expression

An ACAT-deficient CHO cell line AC29 was used and cultured in F12 medium (containing 10% FBS, 100 μg/ml streptomycin, 100 μg/ml penicillin) at 95% humidity, 5% CO ₂ , and 37°C. The expression analysis of human ACAT2b cDNA-encoded protein was determined by transfection experiment, and the method used was calcium-phosphate co-precipitation method (Calcium-phosphate transfection, Liu J et al.1997). Three plasmid DNAs, pcDNA3 (Negative control), pcDNA3-A2 (Positive control) and pcDNA3-A2b, were transfected into ACAT-deficient CHO cell line AC29 by calcium phosphate method for expression study.

The specific experimental process is as follows: the day before transfection, the cells were inoculated into a 60mm dish, the number of cells per dish was 500,000/5ml, and the cell abundance reached 30-50% at the time of transfection. Replace the medium with fresh medium 3 hours before transfection. Prepare calcium phosphate precipitated plasmid DNA, including 12 μg sample plasmid DNA (per dish), then add DNA/calcium phosphate precipitated solution to the culture medium dropwise, and transfect AC29 cells at 37°C. After 10 hours, the cells were washed twice with PBS, and the growth was resumed in fresh medium. Transfected cells were collected after 48 hours.

The method of collecting cells to measure protein expression was as follows: cultured transfected cells were washed twice with cold PBS, and 100 μl of cell lysate (10% SDS, 50 mM Tris-HCl pH6.8, 1 mM EDTA, 25 mM DTT) was added to dissolve cell proteins. The cell lysate protein solution was sheared several times with a 18-gauge syringe needle to break the viscous chromosomal DNA. The determination of protein content was carried out according to Lowry's method with a slight modification. SDS-PAGE analysis was carried out according to the Laemmli method, and the gel concentration was 12%. After the electrophoresis is completed, 4 layers of filter paper soaked in the electrotransfer solution-nitrocellulose membrane-polyacrylamide gel rinsed with the electrotransfer buffer-4 layers of filter paper soaked in the electrotransfer solution were laid on the electrophoresis gel transfer instrument from bottom to top, Set current 1mA/ ^cm2 to transfer for 30 minutes. After the transferred nitrocellulose membrane was blocked with 5% skim milk powdered TBST buffer for 30 minutes, the diluted DM54ACAT2 antibody was added to cover the membrane, and shaken overnight at 4°C or at room temperature for 2 hours. Wash the membrane 3 times with TBST buffer. Then add diluted horseradish peroxidase-labeled goat anti-rabbit IgG to cover the membrane, room temperature for 1 hour, wash the membrane three times with TBST buffer, and wash the membrane twice with TBS buffer. Fluorescent development was performed according to the method provided by the ECL Western Blot Detection Kit (Santa Cruz Biotechnology Inc. Company), and the results are shown at the bottom of Figure 3.

Example 5 Cell culture, DNA transfection and enzyme activity analysis of human ACAT2b cDNA expression protein

The cell culture and DNA transfection experiments were basically the same as those in Example 4 above, and then the Cell-free and IntactCell systems were used to measure the cholesterol ester synthase activity.

The analysis of the Cell free system to measure the activity of cholesterol ester synthase refers to the method reported in the literature (Chang CCYet al., 1995, J. Biol. Chem. 270: 29532-29540), lysing the transfected cells, adding cholesterol and ³ H-labeled oil Acyl-CoA substrate, measuring the enzymatic activity of ACAT in microsomes. Specifically, add 100 μl of 1 mM Tris (pH 7.8) containing 1 mM EDTA to the transfected AC29 cells washed twice with cold PBS, collect the cell lysate after 5 minutes on ice, take 15 μl of 2M KCl, 7.5 μl of 10% CHAPS, 15μl cell lysate and reaction substrate reacted at 37°C for 10 minutes, then added 4ml CHCl3:MeOH (2:1) and mixed to stop the reaction, then added 10μl oleoyl cholesterol, mixed, added 1ml of water, centrifuged, discarded water phase, dried, added 60 μl ethyl acetate, thin-layer chromatography, collected cholesterol lipids, isotope counting, and Western blot to correct the expression of ACAT2b. According to this method, ACAT2b has cholesteryl ester synthase activity, but it is much lower than that of the positive control (see FIG. 4A ).

The analysis of the Intact Cell system to measure the activity of cholesteryl ester synthase refers to the method reported in the literature (Chang CCYet al., 1986, Biochemistry, 25: 1700-1705; Cadigan KM et al., 1988, J.Biol.Chem., 263: 274 -82) proceed. The brief method is to add ³ H-labeled oleic acid to the transfected AC29 cells and incubate them for 3-4 hours, then collect the cells and lyse them, and finally measure the cholesterol lipid synthesis (CE synthesis) in the cells, and correct the expression of ACAT2b by Western blot . According to this method, ACAT2b has cholesteryl ester synthase activity, which is also much lower than that of the positive control (see FIG. 4B ).

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Sequence Listing

<110> Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

<120> Human cholesteryl ester synthase-2b and its coding sequence

<130>031115b

<160>5

<170>PatentIn version 3.1

<210>1

<211>1522

<212>DNA

<213> Homo sapiens

<220>

<221> CDS

<222>(13)..(1521)

<223>

<400>1

ggagaccgca cc atg gag cca ggc ggg gcc cgt ctg cgt ctg cag agg aca 51

           Met Glu Pro Gly Gly Ala Arg Leu Arg Leu Gln Arg Thr

1 5 10

gaa ggg ctg gga ggg gag cgg gag cgc caa ccc tgt gga gat gga aac 99

Glu Gly Leu Gly Gly Glu Arg Glu Arg Gln Pro Cys Gly Asp Gly Asn

15 20 25

act gag acg cac aga gcc ccg gac ttg gta caa tgg acc cga cac atg 147

Thr Glu Thr His Arg Ala Pro Asp Leu Val Gln Trp Thr Arg His Met

30 35 40 45

gag gct gtg aag gca caa ttg ctg gag caa gcg cag gga caa ctg agg 195

Glu Ala Val Lys Ala Gln Leu Leu Glu Gln Ala Gln Gly Gln Leu Arg

50 55 60

gag ctg ctg gat cgg gcc atg cgg gag gct ata caa tcc tac cca tca 243

Glu Leu Leu Asp Arg Ala Met Arg Glu Ala Ile Gln Ser Tyr Pro Ser

65 70 75

caa gac aaa cct ctg ccc cca cct ccc cca ggt tcc ttg agc agt gag 291

Gln Asp Lys Pro Leu Pro Pro Pro Pro Pro Gly Ser Leu Ser Ser Glu

80 85 90

ctg atg gag gtg cag cat ttc cgc acc atc tac cac atg ttc atc gct 339

Leu Met Glu Val Gln His Phe Arg Thr Ile Tyr His Met Phe Ile Ala

95 100 105

ggc ctg tgt gtc ttc atc atc agc acc ctg gcc atc gac ttc att gat 387

Gly Leu Cys Val Phe Ile Ile Ser Thr Leu Ala Ile Asp Phe Ile Asp

110 115 120 125

gag ggc agg ctg ctg ctg gag ttt gac cta ctg atc ttc agc ttc gga 435

Glu Gly Arg Leu Leu Leu Glu Phe Asp Leu Leu Ile Phe Ser Phe Gly

130 135 140

cag ctg cca ttg gcg ctg gtg acc tgg gtg ccc atg ttt ctg tcc acc 483

Gln Leu Pro Leu Ala Leu Val Thr Trp Val Pro Met Phe Leu Ser Thr

145 150 155

ctg ttg gcg ccg tac caa gcc cta cgg ctg tgg gcc agg ggc acc tgg 531

Leu Leu Ala Pro Tyr Gln Ala Leu Arg Leu Trp Ala Arg Gly Thr Trp

160 165 170

acg cag gcg acg ggc ctg ggc tgt gcg ctg cta gcc gcc cac gcc gtg 579

Thr Gln Ala Thr Gly Leu Gly Cys Ala Leu Leu Ala Ala His Ala Val

175 180 185

gtg ctc tgc gcg ctg ccg gtc cac gtg gcc gtg gag cat cag ctc ccg 627

Val Leu Cys Ala Leu Pro Val His Val Ala Val Glu His Gln Leu Pro

190 195 200 205

ccg gcc tcc cgt tgt gtc ctg gtc ttc gag cag ggt agg ttc ctg atg 675

Pro Ala Ser Arg Cys Val Leu Val Phe Glu Gln Gly Arg Phe Leu Met

210 215 220

aaa agc tac tcc ttc ctg aga gag gct gtg cct ggg acc ctt cgt gcc 723

Lys Ser Tyr Ser Phe Leu Arg Glu Ala Val Pro Gly Thr Leu Arg Ala

225 230 235

aga cga ggt gag ggg atc cag gcc ccc agt ttc tcc agc tac ctc tac 771

Arg Arg Gly Glu Gly Ile Gln Ala Pro Ser Phe Ser Ser Tyr Leu Tyr

240 245 250

ttc ctc ttc tgc cca aca ctc atc tac agg gag act tac cct agg acg 819

Phe Leu Phe Cys Pro Thr Leu Ile Tyr Arg Glu Thr Tyr Pro Arg Thr

255 260 265

ccc tat gtc agg tgg aat tat gtg gcc aag aac ttt gcc cag gcc ctg 867

Pro Tyr Val Arg Trp Asn Tyr Val Ala Lys Asn Phe Ala Gln Ala Leu

270 275 280 285

gga tgt gtg ctc tat gcc tgc ttc atc ctg ggc cgc ctc tgt gtt cct 915

Gly Cys Val Leu Tyr Ala Cys Phe Ile Leu Gly Arg Leu Cys Val Pro

290 295 300

gtc ttt gcc aac atg agc cga gag ccc ttc agc acc cgt gcc ctg gtg 963

Val Phe Ala Asn Met Ser Arg Glu Pro Phe Ser Thr Arg Ala Leu Val

305 310 315

ctc tct atc ctg cac gcc acg ttg cca ggc atc ttc atg ctg ctg ctc 1011

Leu Ser Ile Leu His Ala Thr Leu Pro Gly Ile Phe Met Leu Leu Leu

320 325 330

atc ttc ttt gcc ttc ctc cat tgc tgg ctc aac gcc ttt gcc gag atg 1059

Ile Phe Phe Ala Phe Leu His Cys Trp Leu Asn Ala Phe Ala Glu Met

335 340 345

cta cga ttt gga gac agg atg ttc tac cgg gac tgg tgg aac tca acg 1107

Leu Arg Phe Gly Asp Arg Met Phe Tyr Arg Asp Trp Trp Asn Ser Thr

350 355 360 365

tcc ttc tcc aac tac tac cgc act tgg aac gtg gtg gtc cat gac tgg 1155

Ser Phe Ser Asn Tyr Tyr Arg Thr Trp Asn Val Val Val His Asp Trp

370 375 380

ctg tac agc tac gtg tat cag gat ggg ctg cgg ctc ctt ggt gcc cgg 1203

Leu Tyr Ser Tyr Val Tyr Gln Asp Gly Leu Arg Leu Leu Gly Ala Arg

385 390 395

gcc cga ggg gta gcc atg ctg ggt gtg ttc ctg gtc tcc gca gtg gcc 1251

Ala Arg Gly Val Ala Met Leu Gly Val Phe Leu Val Ser Ala Val Ala

400 405 410

cat gag tat atc ttc tgc ttc gtc ctg ggg ttc ttc tat ccc gtc atg 1299

His Glu Tyr Ile Phe Cys Phe Val Leu Gly Phe Phe Tyr Pro Val Met

415 420 425

ctg ata ctc ttc ctt gtc att gga gga atg ttg aac ttc atg atg cat 1347

Leu Ile Leu Phe Leu Val Ile Gly Gly Met Leu Asn Phe Met Met His

430 435 440 445

gac cag cgc acc ggc ccg gca tgg aac gtg ctg atg tgg acc atg ctg 1395

Asp Gln Arg Thr Gly Pro Ala Trp Asn Val Leu Met Trp Thr Met Leu

450 455 460

ttt cta ggc cag gga atc cag gtc agc ctg tac tgc cag gag tgg tac 1443

Phe Leu Gly Gln Gly Ile Gln Val Ser Leu Tyr Cys Gln Glu Trp Tyr

465 470 475

gca cgg cgg cac tgc ccc tta ccc cag gca act ttc tgg ggg ctg gtg 1491

Ala Arg Arg His Cys Pro Leu Pro Gln Ala Thr Phe Trp Gly Leu Val

480 485 490

aca cct cga tct tgg tcc tgc cat acc tag a 1522

Thr Pro Arg Ser Trp Ser Cys His Thr

495 500

<210>2

<211>502

<212>PRT

<213> Homo sapiens

<400>2

Met Glu Pro Gly Gly Ala Arg Leu Arg Leu Gln Arg Thr Glu Gly Leu

1 5 10 15

Gly Gly Glu Arg Glu Arg Gln Pro Cys Gly Asp Gly Asn Thr Glu Thr

20 25 30

His Arg Ala Pro Asp Leu Val Gln Trp Thr Arg His Met Glu Ala Val

35 40 45

Lys Ala Gln Leu Leu Glu Gln Ala Gln Gly Gln Leu Arg Glu Leu Leu

50 55 60

Asp Arg Ala Met Arg Glu Ala Ile Gln Ser Tyr Pro Ser Gln Asp Lys

65 70 75 80

Pro Leu Pro Pro Pro Pro Pro Gly Ser Leu Ser Ser Ser Glu Leu Met Glu

85 90 95

Val Gln His Phe Arg Thr Ile Tyr His Met Phe Ile Ala Gly Leu Cys

100 105 110

Val Phe Ile Ile Ser Thr Leu Ala Ile Asp Phe Ile Asp Glu Gly Arg

115 120 125

Leu Leu Leu Glu Phe Asp Leu Leu Ile Phe Ser Phe Gly Gln Leu Pro

130 135 140

Leu Ala Leu Val Thr Trp Val Pro Met Phe Leu Ser Thr Leu Leu Ala

145 150 155 160

Pro Tyr Gln Ala Leu Arg Leu Trp Ala Arg Gly Thr Trp Thr Gln Ala

165 170 175

Thr Gly Leu Gly Cys Ala Leu Leu Ala Ala His Ala Val Val Leu Cys

180 185 190

Ala Leu Pro Val His Val Ala Val Glu His Gln Leu Pro Pro Ala Ser

195 200 205

Arg Cys Val Leu Val Phe Glu Gln Gly Arg Phe Leu Met Lys Ser Tyr

210 215 220

Ser Phe Leu Arg Glu Ala Val Pro Gly Thr Leu Arg Ala Arg Arg Gly

225 230 235 240

Glu Gly Ile Gln Ala Pro Ser Phe Ser Ser Tyr Leu Tyr Phe Leu Phe

245 250 255

Cys Pro Thr Leu Ile Tyr Arg Glu Thr Tyr Pro Arg Thr Pro Tyr Val

260 265 270

Arg Trp Asn Tyr Val Ala Lys Asn Phe Ala Gln Ala Leu Gly Cys Val

275 280 285

Leu Tyr Ala Cys Phe Ile Leu Gly Arg Leu Cys Val Pro Val Phe Ala

290 295 300

Asn Met Ser Arg Glu Pro Phe Ser Thr Arg Ala Leu Val Leu Ser Ile

305 310 315 320

Leu His Ala Thr Leu Pro Gly Ile Phe Met Leu Leu Leu Ile Phe Phe

325 330 335

Ala Phe Leu His Cys Trp Leu Asn Ala Phe Ala Glu Met Leu Arg Phe

340 345 350

Gly Asp Arg Met Phe Tyr Arg Asp Trp Trp Asn Ser Thr Ser Phe Ser

355 360 365

Asn Tyr Tyr Arg Thr Trp Asn Val Val Val His Asp Trp Leu Tyr Ser

370 375 380

Tyr Val Tyr Gln Asp Gly Leu Arg Leu Leu Gly Ala Arg Ala Arg Gly

385 390 395 400

Val Ala Met Leu Gly Val Phe Leu Val Ser Ala Val Ala His Glu Tyr

405 410 415

Ile Phe Cys Phe Val Leu Gly Phe Phe Tyr Pro Val Met Leu Ile Leu

420 425 430

Phe Leu Val Ile Gly Gly Met Leu Asn Phe Met Met His Asp Gln Arg

435 440 445

Thr Gly Pro Ala Trp Asn Val Leu Met Trp Thr Met Leu Phe Leu Gly

450 455 460

Gln Gly Ile Gln Val Ser Leu Tyr Cys Gln Glu Trp Tyr Ala Arg Arg

465 470 475 480

His Cys Pro Leu Pro Gln Ala Thr Phe Trp Gly Leu Val Thr Pro Arg

485 490 495

Ser Trp Ser Cys His Thr

500

<210>3

<211>18

<212>DNA

<213> Synthetic

<220>

<221>misc_feature

<222>(1)..(18)

<223> Primer C41

<400>3

ggagaccgca ccatggag 18

<210>4

<211>27

<212>DNA

<213> Synthetic

<220>

<221>misc_feature

<222>(1)..(27)

<223> Primer C42

<400>4

tgtctagacc tctaggtatg gcaggac 27

<210>5

<211>20

<212>DNA

<213> Synthetic

<220>

<221>misc_feature

<222>(1)..(20)

<223> Primer C21

<400>5

ggaagagtat cagcatgacg 20

Claims (7)

1. an isolated human cholesteryl ester synthase-2b, characterized in that the amino acid sequence of the protein is as shown in SEQ ID NO: 2. 2. An isolated polynucleotide, characterized in that the sequence of the polynucleotide is selected from the group consisting of: (a) the sequence of positions 13-1521 in SEQ ID NO: 1; (b) The sequence of positions 1-1522 in SEQ ID NO:1. 3. A vector comprising the polynucleotide according to claim 2. 4. A host cell, characterized in that it contains the vector according to claim 3. 5. The host cell according to claim 4, wherein said host cell is a mammalian cell. 6. The host cell according to claim 5, wherein the host cell is a mouse, rat or human cell. 7. A method for preparing a protein with human cholesteryl ester synthase-2b activity, characterized in that the method comprises: (a) cultivating the host cell according to claim 4 under conditions suitable for expressing human cholesteryl ester synthase-2b; (b) Isolating a protein having human cholesteryl ester synthase-2b activity from the culture.

Images