CN1710069A - Adenylosuccinate synthetase sample molecule and its coding sequence and use - Google Patents

Abstract

The invention provides a new adenylosuccinate synthetase like molecule (adenylosuccinatesynthetase like 1)-ADSSL1 protein, a polynucleotide encoding the ADSSL1 protein and a method for producing the ADSSL1 protein through recombinant technology. The invention also discloses the application of the polynucleotide encoding the ADSSL1 protein. ADSSL1 protein has the function of catalyzing the formation of adenosuccinate.

Description

Adenylosuccinate synthase-like molecule and its coding sequence and use

technical field

The invention belongs to the field of biotechnology and medicine, in particular, the invention relates to a new polynucleotide encoding human adenylosuccinate synthetase like 1 (adenylosuccinate synthetase like 1, referred to as "ADSSL1"), and the polynucleoside Acid-encoded polypeptides. The present invention also relates to the use and preparation of such polynucleotides and polypeptides. The polypeptide of the present invention is a novel adenosuccinate synthetase molecule.

Background technique

Enzymes are a class of biological macromolecules with catalytic activity and special spatial conformation in biological cells, which play an important role in maintaining normal intracellular substance metabolism and regulating cell functions. As the mapping and sequencing of the human genome is about to be completed, searching for and discovering new enzyme molecules, exploring their gene expression and regulation, and studying the relationship between enzymatic activity changes and disease occurrence have become the most popular topics in modern biochemical research. The regulation of enzyme activity involves many aspects, among which some enzyme inhibitors naturally present in organisms play an important role.

Adenosuccinate synthetase (adenylosuccinate synthetase, AdSS, EC 6.3.4.4)) can catalyze GTP, IMP, L-Asp to generate adenylosuccinate. This enzyme is highly conserved. Enzymes from different sources include bacteria and Mammals have high homology (40%-60%). There are two isoenzymes of adenosuccinate synthase in vertebrates. The isoelectric points of these two enzymes are different, and their tissue distribution and dynamics are different. The academic nature is also different. The acid-type enzyme (AdSS2, also known as non-muscle type) in the liver has an isoelectric point of about 8.9, which is related to the de novo synthesis of purine and is the first key enzyme in the reaction; in muscle tissue (skeletal muscle, cardiac muscle, etc.) The alkaline-type enzyme (AdSS1, also known as muscle-type) distributed in the middle isoelectric point is about 6, which is related to glycolysis through the purine cycle.

Purine metabolizing enzymes, including AdSS, play important roles in cellular function and organ development. It has been found that AdSS1 enzyme activity is significantly reduced in muscle disorders, including congenital myopathy, exercise intolerance and progressive systemic sclerosis, while in cardiomegaly AdSS1 enzyme activity was significantly increased. Defects in AdSS are closely related to hyperuricemia status, and genetically deficient AdSS may lead to certain types of congenital gout. The immunosuppressant azathioprine, which can inhibit purine metabolizing enzymes such as AdSS, can alleviate childhood acute leukemia and prolong the survival of kidney transplantation, so it has been used in the treatment of malignant hematological tumors, rheumatism, solid organ transplantation and inflammatory bowel disease. AdSS is also the target of some antibiotics, including hadacidin and alanosine. Inhibition of AdSS activity by alanosine can exert antiviral and antitumor effects. Later, it was discovered that these enzymes may be used as biomarkers of malignant tumors. Therefore, the study of AdSS at the level of enzyme activity and gene regulation is helpful for the development of specific tumor chemotherapy and the development of specific purine synthesis inhibitors, especially for the prevention of transplant rejection and muscle disorders, malignant hematological tumors Treatment.

Studies have shown that AdSS is related to various life activities. Therefore, it is of great significance to study and develop new adenylylsuccinate synthases for diagnostic and therapeutic purposes.

Contents of the invention

The object of the present invention is to provide a new human adenylosuccinate synthetase, i.e. adenylosuccinate synthetase like 1 (adenylosuccinate synthetase like 1, referred to as "ADSSL1") and its fragments, analogs and derivatives .

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

Another object of the present invention is to provide methods for producing these polypeptides and uses of the polypeptides and coding sequences.

In the first aspect of the present invention, a novel isolated ADSSL1 polypeptide is provided, which includes: a polypeptide having an amino acid sequence of SEQ ID NO: 2, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof.

Preferably, the polypeptide is selected from the group consisting of:

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

(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 has the function of catalyzing the formation of adenosuccinic acid, a polypeptide derived from (a).

More preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.

In a second aspect of the present invention, polynucleotides encoding isolated polypeptides are provided, the polynucleotides comprising a nucleotide sequence having at least 70 degrees to a nucleotide sequence selected from the group consisting of % identity: (a) polynucleotide encoding the above-mentioned human ADSSL1 polypeptide; and (b) polynucleotide complementary to polynucleotide (a). Preferably, the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO:2. More preferably, the sequence of the polynucleotide is one selected from the following group: (a) having the sequence of positions 60-1430 in SEQ ID NO: 1; (b) having positions 1-1738 in SEQ ID NO: 1 the sequence of.

In the third aspect of the present invention, there are provided vectors containing the above-mentioned polynucleotides, and host cells transformed or transduced by the vectors or host cells directly transformed or transduced by the above-mentioned polynucleotides.

In the fourth aspect of the present invention, a method for preparing a polypeptide having human ADSSL1 protein activity is provided, the method comprising: (a) cultivating the above-mentioned transformed or transduced host cells under conditions suitable for expressing human ADSSL1 protein; ( b) isolating a polypeptide having human ADSSL1 protein activity from the culture.

In the fifth aspect of the present invention, an antibody specifically binding to the above-mentioned human ADSSL1 polypeptide is provided.

In the sixth aspect of the present invention, compounds that mimic, promote and antagonize the activity of human ADSSL1 polypeptide, and compounds that inhibit the expression of human ADSSL1 polypeptide are provided. Methods of screening and/or preparing these compounds are also provided. Preferably, the compound is the antisense sequence of the coding sequence of human ADSSL1 polypeptide or a fragment thereof.

In the seventh aspect of the present invention, there is provided a method for detecting whether there is ADSSL1 protein in a sample, which includes: contacting the sample with a specific antibody for the ADSSL1 protein, and observing whether an antibody complex is formed, and the formation of an antibody complex means that the ADSSL1 protein is present in the sample. ADSSL1 protein is present.

In the eighth aspect of the present invention, there is provided a method for detecting a disease or susceptibility to a disease associated with abnormal expression of a human ADSSL1 polypeptide, the method comprising: detecting whether there is a mutation in the nucleic acid sequence encoding the polypeptide.

In the ninth aspect of the present invention, uses of the polypeptides and coding sequences of the present invention are provided. For example, the polypeptide of the present invention can be used to screen for agonists that promote the activity of the human ADSSL1 polypeptide, or to screen for antagonists that inhibit the activity of the human ADSSL1 polypeptide, or to identify peptide fingerprints. The coding sequence of the human ADSSL1 protein or its fragments of the present invention can be used as primers for PCR amplification reactions, or as probes for hybridization reactions, or for making gene chips or microarrays.

In the tenth aspect of the present invention, a composition is provided, which contains 0.001-99.99% of the ADSSL1 polypeptide of the present invention and an acceptable carrier. A preferred composition is a pharmaceutical composition, which contains a safe and effective amount of the human ADSSL1 polypeptide of the present invention or its agonist, antagonist and a pharmaceutically acceptable carrier. These pharmaceutical compositions can respectively treat diseases such as congenital myopathy, muscle exercise intolerance, progressive systemic sclerosis, cardiac hypertrophy, hyperuricemia, muscle disorders, malignant blood tumors and transplant rejection.

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 shows the nucleotide sequence (SEQ ID NO: 1) of human AdSSL1, wherein the ORF is located at positions 60-1430.

Figure 2 shows the amino acid sequence of human AdSSL1 (SEQ ID NO: 2).

Figure 3 shows the RT-PCR expression analysis of human AdSSL1 in different cells. Tip AdSSL1 expression in solid tumors and hematological tumor cells. Wherein, Marker is a molecular weight standard.

Figure 4 shows the results of Northern blot analysis of human AdSSL1 in normal human tissues. The results suggest that AdSSL1 is predominantly expressed in muscle tissues such as skeletal muscle and cardiac muscle.

Figure 5 shows the Western analysis of the expression of human AdSSL1 eukaryotic expression vector in eukaryotic cells. Wherein, lane 1: COS-7 cells transfected with eukaryotic expression vector of human AdSSL1; lane 2: COS-7 cells transfected with blank control vector.

Fig. 6 shows the results of the enzyme activity detection test of human AdSSL1. *P＜0.01

Detailed ways

Through in-depth and extensive research, the present inventors isolated a novel alkaline adenosuccinate synthase, AdSSL1, for the first time from a cDNA library of human bone marrow stromal cells. AdSSL1 has high homology with known basic AdSS derived from various species, with an isoelectric point of 8.76, is predominantly expressed in muscle tissue, and has a typical AdSS enzyme activity that catalyzes the formation of adenosuccinate . This indicates that AdSSL1 molecule may be a new type of basic adenylyl succinate synthase, and similar to other AdSSs, it plays an important regulatory role in the development of cells and tissues, and may play an important role in anti-tumor, anti-transplant rejection, anti- It has important development and application value in immunodiagnosis and immunotherapy in multiple fields such as muscular system disorders and anti-inflammatory response.

In the present invention, the term "ADSSL1 protein", "ADSSL1 polypeptide" or "adenylate succinate synthase-like molecule ADSSL1" can be used interchangeably, and all refer to the human adenylate succinate synthase-like molecule ADSSL1 amino acid sequence ( The protein or polypeptide of SEQ ID NO: 2). They include the adenosuccinate synthase-like molecule ADSSL1 with or without the starting methionine.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state .

As used herein, "isolated ADSSL1 protein or polypeptide" means that the ADSSL1 polypeptide 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 ADSSL1 protein using standard protein purification techniques. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels. The purity of the ADSSL1 polypeptide can be analyzed by amino acid sequence.

The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. Polypeptides of the present invention may be naturally purified, or chemically synthesized, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated. Polypeptides of the invention may or may not include an initial methionine residue.

The present invention also includes fragments, derivatives and analogs of human ADSSL1 protein. As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially maintains the same biological function or activity of the natural human ADSSL1 protein of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with Formation of fusion proteins of antigen IgG fragments). Such 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 ADSSL1 polypeptide" refers to a polypeptide having the sequence of SEQ ID NO: 2 having human ADSSL1 protein activity. The term also includes variant forms of the sequence of SEQ ID NO: 2 that have the same function as the human ADSSL1 protein. These variations include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the human ADSSL1 protein.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with human ADSSL1 DNA under high or low stringency conditions , and the polypeptide or protein obtained by using the antiserum against human ADSSL1 polypeptide. The present invention also provides other polypeptides, such as fusion proteins (such as fusion proteins formed with GST) comprising human ADSSL1 polypeptide or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes soluble fragments of human ADSSL1 polypeptides. Typically, the fragment has at least about 10 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 80 contiguous amino acids of the human ADSSL1 polypeptide sequence. 100 consecutive amino acids.

The invention also provides analogs of human ADSSL1 protein or polypeptide. The difference between these analogs and the natural human ADSSL1 polypeptide may be the difference in amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

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

In the present invention, "human ADSSL1 protein 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 Three amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1.

Table 1 initial residue representative replacement preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Leu

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

The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present 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 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 altering the function of the polypeptide it encodes .

The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO:2.

The present invention also relates to nucleic acid fragments that hybridize to the above-mentioned sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides in length, more preferably at least 50 nucleotides in length, most preferably at least 100 nucleotides in length. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and/or isolate polynucleotides encoding ADSSL1 protein.

The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

The full-length human ADSSL1 nucleotide sequence or its fragments of the present invention 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, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

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 encoding the protein of the present invention (or its fragment, or its derivative) 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. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

A method of amplifying DNA/RNA using the PCR technique (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. Especially when it is difficult to obtain full-length cDNA from the library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used, and the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, And can be synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector or ADSSL1 protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

The polynucleotide sequences of the present invention can be used to express or produce recombinant ADSSL1 polypeptides by conventional recombinant DNA techniques (Science, 1984; 224:1431). Generally speaking, there are the following steps:

(1). Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the human ADSSL1 polypeptide of the present invention, or with a recombinant expression vector containing the polynucleotide;

(2). Host cells cultured in a suitable medium;

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

In the present invention, the human ADSSL1 polynucleotide sequence can be inserted into the recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmid, phage, 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: T7-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells (Lee and Nathans, J Bio Chem.263:3521, 1988) and vectors derived from baculovirus expressed in insect cells. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements.

Methods well known to those skilled in the art can be used to construct an expression vector containing the human ADSSL1 coding DNA sequence 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). Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, reverse LTRs of transcription viruses and other promoters known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

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

Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

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.

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

Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. 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 of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The recombinant human ADSSL1 protein or polypeptide has many uses. These uses include (but are not limited to): directly treating diseases caused by ADSSL1 protein function hypofunction or loss as a drug, and for screening antibodies, polypeptides or other ligands that promote or resist the ADSSL1 protein function. Screening the polypeptide library with the expressed recombinant human ADSSL1 protein can be used to find therapeutically valuable polypeptide molecules that can inhibit or stimulate the function of the human ADSSL1 protein.

On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies, specific for polypeptides encoded by human ADSSL1 DNA or its fragments. Here, "specificity" means that the antibody can bind to human ADSSL1 gene product or fragment. Preferably, it refers to those antibodies that can bind to human ADSSL1 gene products or fragments but do not recognize and bind to other irrelevant antigen molecules. Antibodies in the present invention include those molecules capable of binding and inhibiting human ADSSL1 protein, as well as those antibodies that do not affect the function of human ADSSL1 protein. The invention also includes antibodies that bind to modified or unmodified forms of the human ADSSL1 gene product.

The present invention includes not only complete monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules ( Ladner et al., US Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have the binding specificity of a murine antibody but retain portions of the antibody from humans.

Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified human ADSSL1 gene product, or an antigenic fragment thereof, can be administered to an animal to induce polyclonal antibody production. Similarly, cells expressing human ADSSL1 protein or antigenic fragments thereof can be used to immunize animals to produce antibodies. Antibodies of the invention may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur.J. Immunol. 6:511, 1976; Kohler et al., Eur.J. Immunol . 6: 292, 1976; Hammerling et al., In Mon. C. lonal Antibodies and T Cell Hybridomas , Elsevier, NY, 1981). The antibodies of the present invention include antibodies capable of blocking the function of human ADSSL1 protein and antibodies that do not affect the function of human ADSSL1 protein. Various antibodies of the present invention can be obtained by conventional immunization techniques using fragments or functional regions of the human ADSSL1 gene product. These fragments or functional regions can be prepared using recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to unmodified forms of the human ADSSL1 gene product can be produced by immunizing animals with gene products produced in prokaryotic cells (e.g., E. coli); antibodies that bind to post-translationally modified forms (such as glycosylated or phosphorylated Proteins or polypeptides), which can be obtained by immunizing animals with gene products produced in eukaryotic cells (such as yeast or insect cells).

Antibodies against human ADSSL1 protein can be used in immunohistochemical techniques to detect human ADSSL1 protein in biopsy specimens.

The antibody of the present invention can be used to treat or prevent diseases related to human ADSSL1 protein. Administration of appropriate doses of antibodies can stimulate or block the production or activity of human ADSSL1 protein.

Antibodies can also be used to design immunotoxins to target a particular part of the body. For example, a monoclonal antibody with high affinity to human ADSSL1 protein can be covalently combined with bacterial or plant toxins (such as diphtheria toxin, ricin, rhododine, etc.). A common method is to use a sulfhydryl cross-linking agent such as SPDP to attack the amino group of the antibody, and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill cells positive for human ADSSL1 protein.

For the production of polyclonal antibodies, human ADSSL1 protein or polypeptide can be used to immunize animals, such as rabbits, mice, rats, etc. Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like.

By using the protein of the present invention, substances that interact with the ADSSL1 protein, such as receptors, inhibitors, agonists or antagonists, can be screened out through various conventional screening methods.

When the protein of the present invention and its antibody, inhibitor, agonist, antagonist or receptor are administered (administered) therapeutically, various effects can be provided. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

The polypeptide of the present invention can be directly used in the treatment of diseases, for example, in the treatment of muscle disorders, malignant hematological tumors and transplant rejection. When using the ADSSL1 protein of the present invention, other therapeutic agents, such as TNF, can also be used simultaneously.

The present invention also provides a pharmaceutical composition, which contains a safe and effective amount (such as 0.01-99.99%) of the ADSSL1 polypeptide of the present invention or its agonist, antagonist, 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 1 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, a safe and effective amount of ADSSL1 protein or its antagonist, agonist is administered to the mammal, wherein the safe and effective amount is usually at least about 10 μg/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is about 10 micrograms/kg body weight to about 1 mg/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.

Polynucleotides of human ADSSL1 protein can also be used for various therapeutic purposes. Gene therapy technology can be used to treat abnormalities in cell proliferation, development or metabolism due to non-expression of ADSSL1 protein or expression of abnormal/inactive ADSSL1 protein. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated ADSSL1 protein to inhibit the activity of endogenous ADSSL1 protein. Expression vectors derived from viruses such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer the ADSSL1 gene into cells. The method for constructing a recombinant viral vector carrying the ADSSL1 gene can be found in existing literature (Sambrook, et al.). In addition, the recombinant human ADSSL1 gene can be packaged into liposomes and then transferred into cells.

Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit human ADSSL1 mRNA are also within the scope of the invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA to perform an endonucleic cut. Antisense RNA, DNA and ribozyme can be obtained by any existing RNA or DNA synthesis technology, such as solid-phase phosphoamide chemical synthesis of oligonucleotides, which has been widely used. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of the DNA sequence encoding the RNA. This DNA sequence has been integrated into the vector downstream of the RNA polymerase promoter. In order to increase the stability of nucleic acid molecules, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the connection between ribonucleosides should use phosphothioester bonds or peptide bonds instead of phosphodiester bonds.

The methods for introducing polynucleotides into tissues or cells include: directly injecting polynucleotides into tissues in the body; or first introducing polynucleotides into cells in vitro through vectors (such as viruses, phages, or plasmids, etc.) , and then transplant the cells into the body, etc.

The polypeptide molecule capable of binding to human ADSSL1 protein can be obtained by screening a random polypeptide library composed of various possible combinations of amino acids bound to solid phases. During screening, the human ADSSL1 protein molecule must be labeled.

The invention also relates to a diagnostic test method for quantitative and localized detection of human ADSSL1 protein level. These assays are well known in the art and include FISH assays and radioimmunoassays. The human ADSSL1 protein level detected in the test can be used to explain the importance of the human ADSSL1 protein in various diseases and to diagnose diseases in which the ADSSL1 protein plays a role.

A method for detecting the presence of ADSSL1 protein in a sample is to use a specific antibody for the ADSSL1 protein for detection, which includes: contacting the sample with an antibody specific for the ADSSL1 protein; observing whether an antibody complex is formed, which means ADSSL1 protein was present in the sample.

The polynucleotide of ADSSL1 protein can be used for the diagnosis and treatment of diseases related to ADSSL1 protein. In terms of diagnosis, the polynucleotide of ADSSL1 protein can be used to detect the expression of ADSSL1 protein or the abnormal expression of ADSSL1 protein in a disease state. For example, the DNA sequence of ADSSL1 can be used for hybridization of biopsy specimens to determine the abnormal expression of ADSSL1 protein. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These technical methods are all open and mature technologies, and relevant kits are available from commercial sources. Part or all of the polynucleotides of the present invention can be immobilized as probes on microarrays or DNA chips (also known as "gene chips") for analysis of differential expression of genes in tissues and gene diagnosis. RNA-polymerase chain reaction (RT-PCR) in vitro amplification with ADSSL1 protein-specific primers can also detect the transcript of ADSSL1 protein.

Detection of mutations in the ADSSL1 gene can also be used to diagnose diseases associated with the ADSSL1 protein. The form of ADSSL1 protein mutation includes point mutation, translocation, deletion, recombination and any other abnormality compared with the normal wild-type ADSSL1 DNA sequence. Mutations can be detected using established techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene has a mutation.

The sequences of the invention are also valuable for chromosome identification. In short, PCR primers (preferably 15-35bp) are prepared according to the cDNA of the ADSSL1 protein of the present invention, and the sequence can be positioned on the chromosome. These primers were then used for PCR screening of somatic heterozygous cells containing individual human chromosomes. Only those cells heterozygous for the human gene corresponding to the primer will produce an amplified fragment.

Once a sequence has been mapped to an exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with gene map data. These data can be found, for example, in V. Mckusick, Mendelian Inheritance in Man (available online through Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between the gene and the disease that has been mapped to the chromosomal region.

In one example of the present invention, an isolated polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is provided. The polynucleotides of the present invention were isolated from a human bone marrow stromal cell cDNA library. Its sequence is shown in SEQ ID NO: 1, and the polynucleotide sequence that it comprises is 1738 bases in full length, and its open reading frame is positioned at 60-1430 positions, and the human AdSSL1 protein (SEQ ID NO :2). The AdSSL1 protein belongs to adenylosuccinate synthase, which is an important enzyme in purine metabolism and plays an important role in cell function, tissue development and self-stabilization, and thus has great application prospects.

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's instructions suggested conditions.

Embodiment 1: the cloning of human AdSSL1 cDNA

Total RNA of human bone marrow stromal cells was extracted with Trizol reagent (Life Technologies). Then, poly(A) mRNA was isolated from total RNA. After the poly(A) mRNA was reverse-transcribed to form cDNA, the cDNA fragment was directional inserted into the multiple cloning site of the vector with the SuperScriptII cloning kit (Life Technologies), and the commonly used Escherichia coli DH5α bacteria were transformed to form a cDNA plasmid library. The 5' ends of randomly selected clones were sequenced by the dideoxy method. Comparing the determined cDNA sequence with the existing public DNA sequence database, 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 is bidirectionally determined by synthesizing a series of primers. Computer analysis showed that the full-length cDNA contained in the clone was a new cDNA sequence (as shown in SEQ ID NO: 1 and Figure 1), encoding a new protein (as shown in SEQ ID NO: 2 and Figure 2). The protein is named human adenylosuccinate synthase-like molecule, and its coding gene is named human adenylylsuccinate synthase-like molecule gene.

Sequence SEQ ID NO: 1 is 1738bp in full length, including a 5' non-coding region of 59 bp and a 3' non-coding region of 305 bp, encoding a polypeptide of 457 amino acids. Theoretically calculated molecular weight of unglycosylated mature molecule is about 50.2kD. Structural analysis shows that the 32-455 position of the AdSSL1 protein contains an AdSS structural signal (AdSS signature), and the 42-48 position and 362-365 position contain a GTP-binding domain (GTP-binding domain), suggesting that AdSSL1 belongs to the synthesis of adenosuccinate enzyme.

BLAST analysis showed that it was different from known genes, and showed high homology with mouse adenylosuccinate synthase 1. In addition, AdSSL1 and other AdSS molecules such as human AdSS, mouse AdSS2, rat AdSS and yeast AdSS protein also show a certain degree of homology.

Embodiment 2: Carry out the cell expression analysis of human AdSSL1 by RT-PCR method

The total RNA of dendritic cells derived from corresponding cell lines in the logarithmic growth phase, human peripheral blood mononuclear cells and human peripheral blood mononuclear cells stimulated by LPS for different times was extracted with Trizol reagent, and 5 μg of total cellular RNA was mixed with 1 μg Oligo- dT ₁₂ - ₁₈ mixed for reverse transcription. The reverse transcription system is 20 μl, and 80 μl ddH ₂ O is added after the reaction for dilution. The primers for PCR amplification of AdSSL1 are as follows: sense primer 5'-ATGTCGGGGACCCGAGCC-3' (SEQ ID NO: 3), antisense primer 5'-CTAAAACAGCTGGATCAT-3' (SEQ ID NO: 4), and β-actin as a positive control . The PCR reaction volume was 50 μl, which contained 10 μl of reverse transcription template, 0.5 mM primers, 0.2 mM dNTP and 1 U rTaq DNA polymerase (Takara), and the amplification parameters were 95°C for 15 seconds, 57°C for 30 seconds, 72°C for 30 seconds, 28 After two cycles, the PCR products were preliminarily confirmed by 1.5% agarose gel electrophoresis. The result of DNA sequence analysis showed that the coding DNA sequence of the PCR product was completely identical to 60-1430 shown in SEQ ID NO:1.

The RT-PCR results are shown in Figure 3. AdSSL1 mRNA was expressed in all solid tumor cells and hematological tumor cell lines tested, and was highly expressed in tumor cells such as KG-1, SMMC7721, MCF-7 and CaoV-3.

Northern blot analysis of embodiment 3 human AdSSL1

Perform Northern blotting according to the following routine method: put the filter membrane to be tested in 10ml of hybridization solution preheated at 68°C, and pre-hybridize at 68°C for 30 minutes in a hybridization oven (Bellco); put the labeled cDNA probe at 95-100 Denature at ℃ for 2-5 minutes, place on ice to cool quickly, add hybridization solution (cDNA probe final concentration is 2-10ng/ml or 1-2× ¹⁰⁶ cpm/ml), mix well, and hybridize at 68℃ for 2 hours . After the hybridization, the filter membrane was washed several times with 2×SSC and 0.05% SDS at room temperature, followed by shaking and washing for 30-40 minutes, during which the washing solution was changed several times. Then rinse with 0.1×SSC and 0.1% SDS at 50° C. for 20 to 40 minutes with shaking. Finally, the filter membrane was wrapped with plastic cling film, and exposed to X-ray film at -70°C for 24 to 48 hours.

The results of Northern blot hybridization are shown in Figure 4: Northern blot shows that human AdSSL1 mRNA is a 2.2kb band in normal human tissues, which is highly expressed in muscle tissues such as skeletal muscle and heart, and very low in other tissues. This indicates that human AdSSL1 is a molecule predominantly expressed in muscle tissue.

Example 4 Recombinant expression of human AdSSL1

In this example, the reverse-transcribed human bone marrow stromal cell cDNA was used as a template, and PCR oligonucleotide primers with the following sequences at the 5' and 3' ends were used to amplify to obtain human AdSSL1 DNA as an insert.

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

5'-CAGGATCCATGTCGGGGACCCGAGCC-3' (SEQ ID NO: 5)

The primer contains a restriction endonuclease cutting site of BamHI, followed by a partial coding sequence of a translation initiator and human AdSSL1 after the cutting site;

The 3' end primer sequence is:

5'-GTGAATTC CTAAAACAGCTGGATCAT-3' (SEQ ID NO: 6)

The primer contains the restriction endonuclease restriction site of EcoRI, the translation terminator and the partial coding sequence of human AdSSL1.

After the obtained PCR product was purified, it was digested with BamHI-EcoRI and then recombined with the plasmid pGEM-3ZF (purchased from Promega Company) according to conventional methods and transformed into competent Escherichia coli BL21, and the positive clones were picked and identified, purified and sequenced (ABI The company's 377 sequencer, BigDye Terminator kit, PE company). The human AdSSL1 cDNA BamHI-EcoR I fragment of the correct sequence was cloned into the expression vector pGEX-2T (Pharmacia Company) to form the vector pGEX-2T-AdSSL1, and then transformed into Escherichia coli BL21. Positive clones were identified by digestion with BamHI-EcoRI, and the products were analyzed by 0.8% agarose gel electrophoresis. It was confirmed by sequencing that the designed AdSSL1 coding sequence had been inserted.

Pick the positive Escherichia coli BL21 clone expressing AdSSL1 and inoculate it in 100ml 2×YTA medium, culture at 37°C with shaking at 300rpm for 12-15hr, dilute 1:10 in the preheated 2×YTA medium and continue shaking culture for 1.5hr, add 100mM IPTG After reaching 0.1mM, induce at 30°C for 2-6hr, centrifuge at 5,000g at 4°C for 10min to remove the supernatant, place on ice and wash with 50ml 1×PBS (0.14M NaCl, 2.7mM KCl, 10.1mM Na ₂ HPO ₄ , 1.8mM KH ₂ PO ₄ , pH 7.3) resuspended, ultrasonic (B. Braun Labsonic U) crushed, then added 20% Triton X-100 to 1% shake gently for 30min, then centrifuged at 12000g 4°C for 10min, the supernatant was filtered with a 0.8μm filter membrane, After passing through 1ml of 50% glutathione Sepharose 4B chromatography column and fully washing with 1×PBS, add 500ul glutathione elution buffer (10mM glutathione, 50mM Tris-HCl, pH 8.0) and let stand at room temperature for 30 Minutes later, the eluate was collected, and the elution was repeated 2-3 times to obtain the human AdSSL1-GST fusion protein. The molecular weight of the fusion protein was consistent with the predicted value.

Example 5: Generation of anti-human AdSSL1 antibody

The recombinant human AdSSL1 fusion protein obtained in Example 4 was used to immunize animals to produce antibodies, the specific method is as follows. The recombinant molecules are separated by chromatography for further use. It can also be separated by SDS-PAGE gel electrophoresis, and the electrophoresis bands are excised from the gel and emulsified with an equal volume of complete Freund's adjuvant. Mice were injected intraperitoneally with 50-100 [mu]g/0.2 ml emulsified protein. Fourteen days later, mice were boosted by intraperitoneal injection of the same antigen emulsified with incomplete Freund's adjuvant at a dose of 50-100 µg/0.2 ml. Give booster immunizations at least three times every 14 days. The specific reactivity of the obtained antiserum was assessed by its ability to precipitate the translation product of the human AdSSL1 gene in vitro.

As a result, it was found that the antibody can specifically bind to the protein of the present invention.

Example 6: Construction and gene transfection of human AdSSL1 eukaryotic expression vector

In this example, the full-length plasmid DNA in Example 1 was used as a template, and PCR oligonucleotide primers with the following sequences at the 5' and 3' ends were used to amplify to obtain the full-length coding region DNA of human AdSSL1 as an insert fragment . The PCR reaction parameters were 95°C for 15 seconds, 58°C for 30 seconds, 72°C for 30 seconds, and 72°C for 10 minutes after 20 cycles.

The upstream primer is 5'-AC GAA TTC ATGTCGGGGACCCGAGCC-3' (the 5' end contains the EcoR I site and the start code of the human AdSSL1 coding region) (SEQ ID NO: 7), and the downstream primer is 5'-T GAA GCT TAA CAGCTG GAT CAT-3' (5' end contains Hind III site) (SEQ ID NO: 8).

After purification, the PCR product was directly recombined with pGEM-T vector (Promega Company) and transformed into competent Escherichia coli DH5α according to conventional methods. White clones were picked for identification, purification and sequencing (sequencing primers were T7 and SP6). The EcoR I-Hind III fragment with the correct sequence was purified and cloned into the expression vector pcDNA3.1/Myc-His(-)B (Invitrogen). After positive clones were identified by enzyme digestion, the recombinants were designated as pcDNA-human AdSSL1. It was confirmed by sequencing that the designed human AdSSL1 coding sequence had been inserted.

Human AdSSL1 eukaryotic expression vector plasmid DNA and control plasmid pcDNA3.1/Myc-His(-)B were co-transfected into COS-7 Vero cells with LipofectAMINE reagent (Invitrogen), and detected 48 hours after transfection.

Embodiment 7: Western detection of human AdSSL1 eukaryotic expression product

48 hours after transfection, the cells (5×10 ⁶ cells/sample) were collected, washed once with PBS, and the whole protein was extracted with T-PER tissue protein extraction reagent (Pierce Company), and the specific operation was performed according to the kit instructions. Then use the BCA method (Pierce company) to measure the protein concentration. After adjusting to a consistent level, every 20 μl of the sample was mixed with 4 μl of 6 times loading buffer (100mmol/L Tris-HCL, 200mmol/L DTT, 4% SDS, 0.2% bromophenol blue , 20% glycerol, pH 6.8) and mix well, boil in a water bath at 100°C for 5 minutes, perform 12% SDS-PAGE electrophoresis, and then transfer the protein in polyacrylamide to nitrocellulose membrane at 4°C with a constant voltage of 100V On the top, Ponceau stained and marked the direction, marking the position of the Marker. Block at room temperature for 4 hours (10% TBST solution of skimmed milk powder), dilute mouse anti-His tag antibody in skimmed milk powder solution at 1:1000, react with nitrocellulose film at room temperature for 2 hours, then use TBST (0.05% Tween20 in TBS solution) was washed 3 times for 10 minutes each time. Then add the corresponding secondary antibody (1:2000 dilution) labeled with horseradish peroxidase (HRP), incubate at room temperature for 60 minutes, wash three times with TBST, each time for 10 minutes, and add the Western blot fluorescence detection reagent (Cell Signaling Company) Mix A solution and B solution in equal volumes, incubate at room temperature for 1 minute, shake dry, press film, expose and develop.

The results are shown in Figure 5. The expression of human AdSSL1 recombinant protein with His tag can be detected in COS-7 African green monkey kidney cells transfected with human AdSSL1 eukaryotic expression vector, and the molecular weight is about 52Kd, which is consistent with the calculated molecular weight of human AdSSL1. No expression was detected in the cells transfected with the control plasmid pcDNA3.1/Myc-His(-)B.

Example 8: Detection of adenylosuccinate synthetase activity of human AdSSL1

Using the full-length human AdSSL1 recombinant protein recombinantly expressed in Example 4, an AdSS enzyme activity detection system was established, and the detection buffer (20 mM Hepes, pH 8.0) contained 10 mM MgCl ₂ , 60 μM GTP, 150 μM L-Asp, and 25 mM L-Asp. After adding recombinant AdSSL1 protein or GST protein as a control, the reaction was started. Under the conditions of 25°C and 280nm, human AdSSL1 protein catalyzed the formation of adenylate succinate (ε ₂₈₀ 11.7mM ^-1 .cm ^-1 ) to reflect the adenosine glucosuccinate synthase activity.

The results are shown in Figure 6. The recombinant full-length human AdSSL1 protein has a moderate level of adenylosuccinate synthase activity of 0.2 μmol/min/mg protein, while the control GST protein does not have this enzyme activity. It is suggested that human AdSSL1 protein has typical adenosuccinate synthetase activity.

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

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<223> Primer

<400>5

caggatccat gtcggggacc cgagcc 26

<210>6

<211>26

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>6

gtgaattcct aaaacagctg gatcat 26

<210>7

<211>26

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>7

acgaattcat gtcggggacc cgagcc 26

<210>8

<211>22

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> primer

<400>8

tgaagcttaa cagctggatc at 22

Claims (10)

1. An isolated human ADSSL1 polypeptide, characterized in that the polypeptide is selected from the group consisting of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 2; (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 has the function of catalyzing the formation of adenosuccinic acid, and a polypeptide derived from (a). 2. The polypeptide according to claim 1, wherein the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO:2. 3. An isolated polynucleotide, characterized in that it is selected from the group consisting of: (a) polynucleotide encoding polypeptide as claimed in claim 1; (b) A polynucleotide complementary to polynucleotide (a). 4. The polynucleotide of claim 3, wherein the polynucleotide encodes a polypeptide having an amino acid sequence shown in SEQ ID NO:2. 5. The polynucleotide of claim 3, wherein the sequence of the polynucleotide is selected from one of the following groups: (a) having the sequence of positions 60-1430 in SEQ ID NO: 1; (b) has the sequence of positions 1-1738 in SEQ ID NO:1. 6. A vector comprising the polynucleotide according to claim 3. 7. A genetically engineered host cell, characterized in that it contains the vector according to claim 6. 8. A method for preparing a polypeptide, characterized in that the method comprises: (a) cultivating the host cell according to claim 7 under conditions suitable for expression; (b) Isolating the human ADSSL1 protein polypeptide from the culture. 9. An antibody capable of specifically binding to the human ADSSL1 polypeptide according to claim 1. 10. A composition, characterized in that it contains 0.001-99.99% of the polypeptide according to claim 1 and an acceptable carrier.

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