CN1952165A - Use of pp3774 gene in inhibiting cell growth - Google Patents

Abstract

The expression profile of the gene pp3774 (human Aph2, hAph2) and the function of inhibiting cell growth of the present invention relate to new tumor-related genes, especially the functional application of the pp3774 gene. The pp3774 gene can inhibit cell growth, induce cell apoptosis, affect the cell cycle, and combine with the known gene JAB1, so the pp3774 gene has potential as a drug screening target for screening drugs (substances that promote or inhibit the combination of pp3774 and JAB1) use.

Description

Application of pp3774 gene in inhibiting cell growth

technical field

The invention relates to novel tumor-related genes, in particular to the functional application of the pp3774 gene. The pp3774 gene can inhibit cell growth, induce cell apoptosis, affect the cell cycle, and combine with the known gene JAB1, so the pp3774 gene has potential as a drug screening target for screening drugs (substances that promote or inhibit the combination of pp3774 and JAB1) use.

Background technique

With the completion of the Human Genome Project and the continuous advancement of science and technology, the "post-genome era" with the main content of research on the function of genes (especially disease-causing genes) has come. Although genes related to various functions have been cloned successively at home and abroad, and their preliminary biological functions and mechanisms of action have been studied, the inventors have discovered and cloned 372 new genes based on large-scale functional screening based on cell growth. Genes [Wan et al., PNAS (2004) 101(44): 15724-15729].

The pp3774 gene is a new gene cloned from the human placenta cDNA library using this functional screening technology platform. The gene is about 1747bp in length, located on chromosome 10q24, contains 10 exons, and encodes 361 amino acids. The cDNA sequence of this gene was registered in GeneBank on April 18, 2000 (Accession No. AF258563).

The pp3774 gene shares 96% with the Aph2 gene [Li et al., J Biol Chem 277 (2002) 28870-28876] that was cloned from the mouse liver cDNA library by Li et al. The homology of Aph2 gene is about 1817bp.

Currently, although the sequences of numerous genes are known, their functions are not known or understood. The pp3774 gene is one of the genes whose function is unknown.

Based on the fact that many physiological reactions and diseases are related to the functions of genes or proteins, in order to fully understand various genes, it is urgent to determine the exact functions of various genes with unknown functions.

Contents of the invention

The purpose of the present invention is to provide the exact function of a function unknown gene pp3774 gene. The research of the present invention shows that the pp3774 gene can inhibit cell growth, affect cell cycle, regulate cell apoptosis and interact with pp3774-JAB1.

Another object of the present invention is to provide the application of pp3774 gene based on the above function.

In the first aspect of the present invention, a kind of method of screening pp3774 accelerator is provided, it comprises steps:

(a) in the test group, adding the candidate candidate and pp3774 to the cell culture, and detecting the proliferation of the cells;

(b) comparing the proliferation of cells in the step (a) test group with the proliferation of cells in the control group, wherein the control group includes:

Group (i): cell cultures to which no candidate candidate was added, nor pp3774 added;

Group (ii): cell cultures supplemented with candidate candidates but without pp3774; and

Group (iii): cell cultures to which the candidate candidate was not added but to which pp3774 was added;

Wherein, if the proliferation of the cells in the test group is statistically lower (preferably significantly lower) than the control group (iii) and there is no significant difference between the cell proliferation of the control group (i) and the control group (ii), then it is indicated This candidate is a pp3774 enhancer.

The sequence of the pp3774 gene described therein is shown in SEQ ID NO: 1.

In a preferred example, the cells are cancer cells, more preferably liver cancer cells.

In another aspect of the present invention, a method for screening pp3774 and JAB1 combined promoters is provided, comprising the steps of:

(a) in the test group, add the candidate to be screened to the culture that combines pp3774 and JAB1, and detect the combination of pp3774 and JAB1;

(b) compare the binding of pp3774 and JAB1 in the test group of step (a) with the binding of pp3774 and JAB1 in the control group to which no candidate was added, if the binding of pp3774 and JAB1 in the test group is statistically higher than (preferably significantly higher than) the control, indicating that the candidate is a compound that promotes the binding of pp3774 and JAB1.

In another aspect of the present invention, a method for screening pp3774 and JAB1 combined inhibitors is provided, comprising the steps of:

(a) in the test group, add the candidate to be screened to the culture that combines pp3774 and JAB1, and detect the combination of pp3774 and JAB1;

(b) compare the binding of pp3774 and JAB1 in the test group of step (a) to the binding of pp3774 and JAB1 in the control group to which no candidate was added, if the binding of pp3774 and JAB1 in the test group is statistically lower than (preferably significantly lower than) the control, indicating that the candidate is a compound that inhibits the binding of pp3774 and JAB1.

In another aspect of the present invention, a use of pp3774 gene or protein is provided, including (a) preparing a composition for inhibiting cancer cell growth; or (b) preparing a composition for regulating cell apoptosis.

In a preferred embodiment, the composition is a pharmaceutical composition, wherein the composition contains 0.001-99.9wt% pp3774 protein, calculated by the total weight of the composition.

In another preferred embodiment, the composition also contains a promoter of pp3774 protein.

The present invention provides a clear function of the pp3774 gene: it can inhibit cell growth, induce cell apoptosis, affect the cell cycle, and combine with the known gene JAB1, and use the pp3774 gene as a target for screening drugs (promoting or inhibiting pp3774 and JAB1 Combined substances), etc., provide its rich application prospects.

Description of drawings

Figure 1 shows the localization of pp3774-GFP fusion protein in cells.

Figure 2 shows the gel electrophoresis results of RT-PCR amplification products of pp3774 mRNA in various tumor cell lines.

Figure 3 shows the results of immunohistochemical detection of human liver cancer and adjacent tissues.

Figure 4 shows the results of apoptosis analysis of SMMC-7721 and COS-7 cells.

Figure 5 shows the construction of pp3774 gene and JAB1 deletion mutants and the results of in vitro protein binding test (Pull down).

Fig. 6 shows the results of pp3774 gene deletion pp3774-Δ4 and JAB1 protein and in vivo co-immunoprecipitation assay. In the figure, vector is an empty vector.

Detailed ways

The inventors have found through extensive and in-depth research that the pp3774 gene can inhibit cell growth, induce cell apoptosis, affect the cell cycle, and combine with the known gene JAB1, so the pp3774 gene has the potential to be used as a drug screening target for screening drugs (promoting or substances that inhibit the combination of pp3774 and JAB1) and other potential uses. On this basis, the present invention has been accomplished.

Specifically, the experiments of the present invention show that the pp3774 protein is mainly located in the endoplasmic reticulum of cells, and the results of bioinformatics analysis show that the gene has 96% homology with the mouse Aph2 gene at the amino acid level; the previous research of the inventors The results also show that the gene has different levels of expression in normal human tissues, specifically in skeletal muscle and cardiac muscle, followed by placenta, pancreas and liver, moderate in kidney and brain, and low expression in lung tissue, indicating that the protein is an evolutionarily conserved gene.

In the present invention, the terms "pp3774 protein", "pp3774 polypeptide" or "tumor-associated protein pp3774" are used interchangeably, and all refer to a protein or Polypeptides. These include pp3774 polypeptides with or without the initial methionine.

In the present invention, the term "pp3774 gene", "pp3774 polynucleotide" or "tumor-associated gene pp3774" can be used interchangeably, and all refer to a gene having a human pp3774 nucleotide sequence (SEQ ID NO: 1 or AF258563 (gi: 10834671 ) nucleic acid sequence.

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 pp3774 protein or polypeptide" means that the pp3774 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 pp3774 protein using standard protein purification techniques. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels. The purity of the pp3774 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 pp3774 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 pp3774 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 pp3774 polypeptide" refers to a polypeptide having the sequence of SEQ ID NO: 2 [AAG23766 (gi: 10834672)] having human pp3774 protein activity. The term also includes variant forms of the sequence of SEQ ID NO: 2 [AAG23766 (gi: 10834672)] having the same function as the human pp3774 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 pp3774 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 pp3774 DNA under high or low stringency conditions , and the polypeptide or protein obtained by using antiserum against human pp3774 polypeptide. The present invention also provides other polypeptides, such as a fusion protein comprising a human pp3774 polypeptide or a fragment thereof (such as the fusion protein shown in SEQ ID NO: 3). In addition to nearly full-length polypeptides, the present invention also includes soluble fragments of human pp3774 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 pp3774 polypeptide sequence. 100 consecutive amino acids.

The present invention also provides analogs of human pp3774 protein or polypeptide. The difference between these analogs and the natural human pp3774 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 pp3774 protein conservative variant polypeptide" refers to at most 10, preferably at most 8, more preferably Up to 5, optimally up to 3 amino acids are replaced by amino acids of similar or closely related 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 sequence of the coding region encoding the mature polypeptide can be the same as the sequence of the coding region shown in SEQ ID NO: 1 or a degenerate variant.

Polynucleotides encoding the mature polypeptide of SEQ ID NO: 2 [AAG23766 (gi: 10834672)] include: 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 sequences.

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. The nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and/or isolate the polynucleotide encoding the pp3774 protein.

The full-length human pp3774 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. Primers for PCR can be appropriately selected based on 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 pp3774 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 pp3774 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 pp3774 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 pp3774 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. 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 pp3774 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 Clo1ing, 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.

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 pp3774 protein or polypeptide has many uses. These uses include (but are not limited to): for screening promoters, ligands or other small molecular compounds that promote the function of pp3774 protein. For example, screening a polypeptide library with expressed recombinant human pp3774 protein can be used to find therapeutically valuable polypeptide molecules that can stimulate the function of human pp3774 protein.

In a preferred example, a method for screening pp3774 protein promoters is provided, which includes the steps of:

(a) in the test group, adding the candidate candidate and pp3774 to the cell culture, and detecting the proliferation of the cells;

(b) comparing the proliferation of cells in the test group in step (a) with the proliferation of cells in the control group, wherein the control group includes (i) group: neither the candidate candidate nor the addition of Cell cultures of pp3774; (ii) group: cell cultures added with the candidate candidate but without pp3774; (iii) cell cultures without the candidate candidate but added pp3774;

Wherein, if the proliferation of the cells in the test group is statistically lower (preferably significantly lower) than the control group (iii) and there is no significant difference between the cell proliferation of the control group (i) and the control group (ii), then it is indicated This candidate is a pp3774 enhancer.

On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies, specific to human pp3774 DNA or polypeptides encoded by its fragments. Here, "specificity" means that the antibody can bind to human pp3774 gene product or fragment. Preferably, it refers to those antibodies that can bind to human pp3774 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 pp3774 protein, as well as those antibodies that do not affect the function of human pp3774 protein. Also included in the invention are antibodies that bind to modified or unmodified forms of the human pp3774 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 pp3774 gene product, or an antigenic fragment thereof, can be administered to an animal to induce polyclonal antibody production. Similarly, cells expressing human pp3774 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 Monoclonal Antibodies and T Cell Hybridomas , Elsevier, NY, 1981). The antibodies of the present invention include antibodies capable of blocking the function of human pp3774 protein and antibodies that do not affect the function of human pp3774 protein. Various types of antibodies of the present invention can be obtained by conventional immunization techniques using fragments or functional regions of the human pp3774 gene product. These fragments or functional regions can be prepared using recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to the unmodified form of the human pp3774 gene product can be produced by immunizing animals with the gene product produced in prokaryotic cells (e.g., E. coli); antibodies that bind to the post-translationally modified form (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 pp3774 protein can be used in immunohistochemical techniques to detect human pp3774 protein in biopsy specimens.

The antibody of the present invention can be used to treat or prevent diseases related to human pp3774 protein. Administration of appropriate doses of antibodies can stimulate or block the production or activity of human pp3774 protein.

Antibodies can also be used to design immunotoxins to target a particular part of the body. For example, monoclonal antibodies with high affinity to human pp3774 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 pp3774 protein.

For the production of polyclonal antibodies, human pp3774 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.

Using the protein of the present invention, through various conventional screening methods, substances that interact with the pp3774 protein, such as receptors, agonists or promoters, can be screened out.

The protein of the present invention and its antibody, agonist or promoter, etc., when administered (administered) therapeutically, can provide desired effects (such as inhibition of tumor cell growth). 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 present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the pp3774 polypeptide of the present invention or its agonist or accelerator 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 pp3374 polypeptide of the present invention can also be used together with other therapeutic agents (such as cisplatin, 5-FU, etc.).

When the pharmaceutical composition is used, a safe and effective amount of pp3774 protein or its agonist or accelerator is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/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.

The polypeptide molecule capable of binding to human pp3774 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 pp3774 protein molecule must be labeled.

The invention also relates to a diagnostic test method for quantitative and localized detection of human pp3774 protein level. These assays are well known in the art and include FISH assays and radioimmunoassays. The human pp3774 protein level detected in the test can be used to explain the importance of the human pp3774 protein in various diseases and to diagnose diseases in which the pp3774 protein plays a role.

A method for detecting the presence of pp3774 protein in a sample is to use a specific antibody for pp3774 protein for detection, which includes: contacting the sample with an antibody specific for pp3774 protein; observing whether an antibody complex is formed, which means that the antibody complex is formed The pp3774 protein was present in the sample.

The polynucleotide of pp3774 protein can be used for diagnosis and treatment of diseases related to pp3774 protein. In terms of diagnosis, the polynucleotide of pp3774 protein can be used to detect the expression of pp3774 protein or the abnormal expression of pp3774 protein in a disease state. For example, the pp3774 DNA sequence can be used for hybridization of biopsy specimens to determine the abnormal expression of pp3774 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 pp3774 protein-specific primers can also detect the transcript of pp3774 protein.

Detection of mutations in the pp3774 gene can also be used to diagnose pp3774 protein-related diseases. The mutated forms of pp3774 protein include point mutations, translocations, deletions, recombinations, and any other abnormalities compared with the normal wild-type pp3774 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 main advantages of the present invention are:

The pp3774 gene has clear functions: it can inhibit cell growth, induce cell apoptosis, affect the cell cycle, and combine with the known gene JAB1, so the pp3774 gene can be used as a target for screening drugs (substances that promote or inhibit the combination of pp3774 and JAB1 ) and other potential uses.

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 is usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (NewYork: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions.

Example 1

Cellular localization of pp3774 protein

Utilize specific primers containing restriction enzyme cutting sites to amplify the coding region of the pp3774 gene by the method of PCR, and load the eukaryotic expression vector EGFP-N1 (purchased from Clontech Company) with EcoRI/BamHI restriction sites, so that It can express the pp3774 fusion protein with a GFP tag at the C-terminus, which is verified by enzyme digestion and sequencing, and is constructed into a pp3774-EGFP-N1 fusion expression vector, which is transfected into SMMC-7721 cells by lipofection and observed by fluorescence Cellular localization of PP3774-GFP fusion protein.

The subcellular localization was done by transfecting live COS-7 cells with pp3774 gene and adding endoplasmic reticulum-specific fluorescent tracer ER-Tracker Blue-White DPX and then incubating with fluorescence microscope and ISIS image analysis system.

The results showed that the empty vector tagged protein GFP was evenly distributed in the nucleus and cytoplasm, while the pp3774-EGFP-N1 fusion expression vector was transfected into SMMC-7721 cells, and the PP3774-GFP fusion protein was localized in the cytoplasm by fluorescence observation (Figure 1a, b). Colocalization with the ER-specific fluorescent tracer ER-TrackerBlue-White DPX showed that the PP3774 protein was mainly localized in the ER (Fig. 1c-e).

Example 2

Semi-quantitative reverse transcription-polymerase chain reaction (Semi-Q RT-PCR)

a) Use Trizol reagent to extract human liver cancer cell lines SMMC-7721, BEL-7402, MHCC-LM3, MHCC-97L, Huh-7, Hep3B, HepG2 and human immortalized liver cells L-02 in good growth state, and other tumors Cell lines such as gallbladder cancer cell line GBC-SD, melanoma cell line A375, cervical cancer cell line HeLa, leukemia cell line K562, HL60, and human liver cancer tissue and paracancerous tissue total RNA were quantified and tested for purity by UV spectrophotometer, and Take a small amount of total RNA for denaturing gel electrophoresis to check whether the RNA is degraded;

b) Mix 9 μl (5 μg) of total cellular RNA treated with DNase I with 1 μl of Oligo dT, heat at 72°C for 5 minutes, and immediately place on ice for 10 minutes;

c) Add 8 μl reverse transcription mixture (10×RT Buffer 2 μl, 25mM MgCl ₂ 2 μl, 10mM dNTP mixture 1 μl, 0.1mM DTT 2 μl, RNase inhibitor 1 μl);

d) After heating at 42°C for 5 minutes on the PCR instrument, add 1 μl (50U) SuperScript II reverse transcriptase, and react at 42°C for 50 minutes;

e) Heating at 70°C for 15 minutes to terminate the reaction and place in an ice bath;

f) Add 1 μl of RNase H, 37°C for 20 minutes, PCR mixture: take 1 μl of each reverse transcription product, 0.5 μl of 10 mM dNTP, 2 μl of 25 mM MgCl ₂ , 0.1 μl of 5U/μl Taq polymerase, 2.5 μl of 10× reaction buffer, 10 pmol of primers RTF, RTR, reaction volume 25 μl,

The reaction conditions are: 94°C for 5min; 94°C for 30s, 58°C for 30s, 72°C for 45s, 25 cycles; 72°C for 10min to terminate the reaction;

g) PCR amplifies the housekeeping gene β-actin as a criterion for testing whether the reverse transcription is successful;

h Take 5 μl of PCR products respectively for 1.5% agarose gel electrophoresis, and the gel imaging analysis system measures the optical density value of each amplified band, and compares the optical density of each target DNA amplified fragment with that of the internal reference β-actin amplified The ratio (e/c) of the optical density of the augmented fragment was used as a semi-quantitative index of its mRNA expression level, and statistical analysis was performed.

The primer sequences are:

pp3774 RTF: 5'-CTGGCTGGTAGACAACGTGA-3' (20bp) (SEQ ID NO: 3)

pp3774 RTR: 5'-GAAAGGAGAAGGTGGGTGGT-3' (20bp) (SEQ ID NO: 4)

The size of the reverse transcription amplification product is 521bp;

Upstream of β-actin: 5'GGACTTCGAGCAAGAGATGG-3'(20bp) (SEQ IDNO: 5)

Downstream of β-actin: 5′-AGCACTGTGTTGGCGTACAG-3′ (20bp) (SEQ ID NO: 6)

The size of the reverse transcription amplification product was 234bp.

The results showed that the expression of pp3774 mRNA in 7 liver cancer cell lines was highly expressed in BEL-7402, Huh-7, MHCC-97L and HepG2, and moderately expressed in SMMC-7721, MHCC-LM3 and immortalized human liver cell line L-02. expression in Hep3B, but low expression in Hep3B (Fig. 2); while in 18 pairs of liver cancer and adjacent tissues, the expression in 13 cases was higher than that in adjacent tissues, in 1 case, it was basically the same as that in adjacent tissues, and in 4 cases, it was higher than that in cancerous tissues. Beside, the results of paired t-test analysis showed that the overall rate was higher in para-cancer than in cancer (P<0.05).

Example 3

Immunocytochemistry and Immunohistochemical Detection of Liver Cancer Tissue Microarray

a) All monolayer cell culture sheets used for immunocytochemical detection and tissue samples used for immunohistochemical detection were fixed with 10% neutral buffered formalin;

b) Tissue samples were embedded in paraffin, serially sectioned with a thickness of 5 μm, and stained with HE for general histopathological observation, pathological grading of liver cancer tissues and positioning before preparation of liver cancer tissue chips, including 214 pairs of human liver cancer and paracancerous tissues, 18 cases The tissue chip preparation method of liver cirrhosis tissue and 10 cases of normal liver tissue is completely in accordance with the technical platform established by our laboratory;

c) Use rabbit anti-pp3774 polyclonal antiserum to isolate IgG as the primary antibody, apply EnvisionSystem-HRP (rabbit), DAB color development, Mayer's hematoxylin counterstaining nuclei, and perform immunocytochemical and immunohistochemical detection of the gene in 7 strains Liver cancer cell lines SMMC-7721, BEL-7402, MHCC-LM3, MHCC-97L, Huh-7, Hep3B, HepG2, gallbladder cancer cell line GBC-SD, melanoma cell line A375, cervical cancer cell line HeLa, leukemia cell line Expression differences of K562, HL60 and human normal liver tissue, liver cirrhosis, paracancerous liver and liver cancer tissue.

The results showed that the expression of wild-type pp3774 protein was very low in seven hepatoma cell lines. pp3774 protein was highly expressed in 10 cases of normal liver tissue and 18 cases of liver cirrhosis. In 214 cases of liver cancer-paracancer specimens, 194 cases of paracancerous tissues were higher than cancerous tissues, accounting for 90.65% (194/214); 8 cases of paracancerous tissues and liver cancer tissues had no significant difference in expression, accounting for 3.74% (8/214); In 12 cases, cancer was higher than paracancerous tissue, accounting for 5.61% (12/214); overall, the positive reaction of normal liver, cirrhotic liver, and paracancerous liver tissue was significantly higher than that of liver cancer tissue (Fig. 3a-p). The positive cells in the tissues adjacent to liver cancer mainly include hepatocytes, bile duct epithelial cells, and some vascular endothelium; the positive cells in liver cancer tissues are mainly liver cancer cells. The expression of the protein pp3774 encoded by this gene in liver cancer and paracancerous tissues was significantly higher than that in liver cancer tissues, which was consistent with the characteristics of tumor suppressor candidate genes.

Example 4

Detection of apoptosis in pp3774 gene transfected cells

Apoptosis analysis of SMMC-7721 cells transiently transfected with pp3774-HA fusion gene and empty vector pcDNA-HA.

The results showed that although the transfection rate of pp3774-HA fusion gene was generally slightly lower than that of empty vector pcDNA-HA transfected cells, the apoptosis rate of pp3774 gene transfected cells was about 10% higher than that of the empty vector group (10.5%) (FIG. 4), indicating that pp3774 gene inhibits cell growth and induces apoptosis is one of the reasons.

Example 5

Interaction between pp3774 and JAB1 in vitro and in vivo

The present inventors used yeast two-hybrid method to search for proteins that can interact with PP3774 from human placenta library and fetal brain cDNA library with pp3774 as bait protein, and obtained 6 positive clones. After sequencing, it was found that all 6 positive clones encoded The same protein JAB1. The full length of JAB1 is 1510bp, encoding 334 amino acids.

In order to determine the key region of the interaction between pp3774 and JAB1, different deletions of pp3774 and JAB1 were constructed, and the interaction between pp3774 and JAB1 deletions was detected by yeast two-hybrid system. It was found that the zf-DHHC functional domain of pp3774 is the key region for the interaction between pp3774 and JAB1, and the carboxy-terminal of JAB1 is indispensable for the interaction between JAB1 and pp3774 (Fig. 5A).

In order to confirm the interaction between pp3774 and JAB1, an in vitro pull-down experiment was also performed.

Interaction between pp3774 and JAB1: Cloning different PP3774 deletions and PP3774 full-length genes into pGBKT7 vector, and co-transfected into Y187 cells with pACT2-JAB1, and different JAB1 deletions were cloned into pACT2 vector, and pGBKT7- 3774 was co-transfected into Y187 cells, and the interaction between the two was determined by detecting the expression of reporter genes His3 and LacZ.

Figure 5B shows that different pp3774 deletions containing zf-DHHC functional domains and pp3774 full-length genes can be induced to express in vitro, and JAB1 can be pulled-down by the above-mentioned pp3774 deletions and pp3774 full-length genes, thus confirming the relationship between pp3774 and JAB1 in vitro interactions between them (Fig. 5C).

The results in Fig. 5 show that the key region of pp3774 interacting with JAB1 exists at amino acid 100-150, and this region is exactly where the zf-DHHC functional domain is located. The 284-334 amino acids at the carboxyl terminal of JAB1 are indispensable for the interaction between JAB1 and pp3774.

To confirm the interaction of pp3774 with JAB1 in vivo, co-precipitation of pp3774 with JAB1 in vivo was performed. COS-7 cells were co-transfected with GFP-tagged pp3774 plasmid and JAB1 plasmid. The cells were lysed 36 hours after transfection, and the total protein was extracted. Precipitated with anti-GFP and anti-JAB1 antibodies, respectively, and detected by western-blot.

The results in Fig. 6 show that pp3774 and pp3774-Δ4 can be co-precipitated by JAB1, respectively, and JAB1 can also be co-precipitated by pp3774 and pp3774-Δ4. In order to test the specificity of the precipitation, it was also detected whether there was an interaction between the GFP protein and JAB1, and it was found that GFP could not be co-precipitated by JAB1, thus illustrating the specificity of the interaction between pp3774 and JAB1.

Example 6

Screening for pp3774 enhancers

In the test group, add the candidates to be selected and pp3774 to the SMMC-7721 cell culture, and detect the proliferation of the cells;

At the same time, the following three control groups were established including:

(a) SMMC-7721 cell cultures to which no candidate for selection nor pp3774 was added;

(b) SMMC-7721 cell cultures added with candidate candidate but without pp3774;

(c) SMMC-7721 cell cultures to which the candidate candidate was not added but to which pp3774 was added;

The proliferation of cells in the test group is compared with the proliferation of cells in the control group, and the proliferation of cells between the control groups is compared at the same time.

result:

(a) is the blank control of normal cell growth; (b) shows the individual promoting or inhibiting effect of the candidate candidate on the cell; (c) is the individual inhibiting effect of pp3774 protein on cell growth; the control group (b) cell proliferation If it is higher than (a), then add the candidate to be selected as a cell growth promoter, if it is lower than (a), then it is a cell growth inhibitor; if the cells in the test group in step (1) proliferate higher than the control group (c) , indicating that the candidate to be selected is an antagonist of the pp3774 protein; if the cell proliferation in the test group in step (1) is lower than that in the control group (c), it indicates that the candidate to be selected is a synergistic promoter of the pp3774 protein.

Several candidate substances were screened, and one candidate was found to promote the growth inhibitory effect of pp3774 protein on SMMC-7721 cells.

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 Cancer Institute

<120> Application of pp3774 gene in inhibiting cell growth

<130>058127

<160>6

<170>PatentIn version 3.1

<210>1

<211>1747

<212>DNA

<213> Homo sapiens (homo sapiens)

<220>

<221> CDS

<222>(158)..(1243)

<223>

<400>1

ccgggtccgc tgcctggcgc tgcgggcggc gggccatggt ggtttggatt gagccgggcc 60

cggccggggc gccgagtcgg aggggtggc agtgagcggc ggcagaggct acggggctcg 120

gtttggctga ctggggagtc ggcaggcggc aggaacc atg cga ggc cag cgg agc 175

MET ARG GLY GLN ARG Serg Ser

1 5

ctg ctg ctg ggc ccg gcc cgc ctc tgc ctc cgc ctc ctt ctg ctg ctg 223

Leu Leu Leu Gly Pro Ala Arg Leu Cys Leu Arg Leu Leu Leu Leu Leu

10 15 20

ggt tac agg cgc cgc tgt cca cct cta ctc cgg ggt cta gta cag cgc 271

Gly Tyr Arg Arg Arg Cys Pro Pro Leu Leu Arg Gly Leu Val Gln Arg

25 30 35

tgg cgc tac ggc aag gtc tgc ctg cgc tcc ctg ctc tac aac tcc ttt 319

Trp Arg Tyr Gly Lys Val Cys Leu Arg Ser Leu Leu Tyr Asn Ser Phe

40 45 50

ggg ggc agt gac acc gct gtt gat gct gcc ttt gag cct gtc tac tgg 367

Gly Gly Ser Asp Thr Ala Val Asp Ala Ala Phe Glu Pro Val Tyr Trp

55 60 65 70

ctg gta gac aac gtg atc cgc tgg ttt gga gtg gtg ttc gtg gtc ctg 415

Leu Val Asp Asn Val Ile Arg Trp Phe Gly Val Val Phe Val Val Leu

75 80 85

gtg atc gtg ctg aca ggc tcc att gta gct atc gcc tac ctg tgt gtc 463

Val Ile Val Leu Thr Gly Ser Ile Val Ala Ile Ala Tyr Leu Cys Val

90 95 100

ctg cct ctc atc ctc cga acc tac tca gtg cca cga ctc tgc tgg cat 511

Leu Pro Leu Ile Leu Arg Thr Tyr Ser Val Pro Arg Leu Cys Trp His

105 110 115

ttc ttc tat agc cac tgg aat ctg atc ctg att gtc ttc cac tac tac 559

Phe Phe Tyr Ser His Trp Asn Leu Ile Leu Ile Val Phe His Tyr Tyr

120 125 130

cag gcc atc acc act ccg cct ggg tac cca ccc cag ggc agg aat gat 607

Gln Ala Ile Thr Thr Pro Pro Gly Tyr Pro Pro Gln Gly Arg Asn Asp

135 140 145 150

atc gcc acc gtc tcc atc tgt aag aag tgc att tac ccc aag cca gcc 655

Ile Ala Thr Val Ser Ile Cys Lys Lys Cys Ile Tyr Pro Lys Pro Ala

155 160 165

cga aca cac cac tgc agc atc tgc aac agg tgt gtg ctg aag atg gat 703

Arg Thr His His Cys Ser Ile Cys Asn Arg Cys Val Leu Lys Met Asp

170 175 180

cac cac tgc ccc tgg cta aac aat tgt gtg ggc cac tat aac cat cgg 751

His His Cys Pro Trp Leu Asn Asn Cys Val Gly His Tyr Asn His Arg

185 190 195

tacttc ttc tct ttc tgc ttt ttc atg act ctg ggc tgt gtc tac tgc 799

Tyr Phe Phe Ser Phe Cys Phe Phe Met Thr Leu Gly Cys Val Tyr Cys

200 205 210

agc tat gga agt tgg gac ctt ttc cgg gag gct tat gct gcc att gag 847

Ser Tyr Gly Ser Trp Asp Leu Phe Arg Glu Ala Tyr Ala Ala Ile Glu

215 220 225 230

act tat cac cag acc cca cca ccc acc ttc tcc ttt cga gaa agg atg 895

Thr Tyr His Gln Thr Pro Pro Pro Thr Phe Ser Phe Arg Glu Arg Met

235 240 245

act cac aag agt ctt gtc tac ctc tgg ttc ctg tgc agt tct gtg gca 943

Thr His Lys Ser Leu Val Tyr Leu Trp Phe Leu Cys Ser Ser Val Ala

250 255 260

ctt gcc ctg ggt gcc cta act gta tgg cat gct gtt ctc atc agt cga 991

Leu Ala Leu Gly Ala Leu Thr Val Trp His Ala Val Leu Ile Ser Arg

265 270 275

ggt gag act agc atc gaa agg cac atc aac aag aag gag aga cgt cgg 1039

Gly Glu Thr Ser lle Glu Arg His Ile Asn Lys Lys Glu Arg Arg Arg

280 285 290

cta cag gcc aag ggc aga gta ttt agg aat cct tac aac tac ggc tgc 1087

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

295 300 305 310

ttg gac aac tgg aag gta ttc ctg ggt gtg gat aca gga agg cac tgg 1135

Leu Asp Asn Trp Lys Val Phe Leu Gly Val Asp Thr Gly Arg His Trp

315 320 325

ctt act cgg gtg ctc tta cct tct agt cac ttg ccc cat ggg aat gga 1183

Leu Thr Arg Val Leu Leu Pro Ser Ser His Leu Pro His Gly Asn Gly

330 335 340

atg agc tgg gag ccc cct ccc tgg gtg act gct cac tca gcc tct gtg 1231

Met Ser Trp Glu Pro Pro Pro Trp Val Thr Ala His Ser Ala Ser Val

345 350 355

atg gca gtg tga gctggactgt gtcagccacg actcgagcac tcattctgct 1283

Met Ala Val

360

ccctatgtta tttcaagggc ctccaagggc agcttttctc agaatccttg atcaaaaaga 1343

gccagtgggc ctgccttagg gtaccatgca ggacaattca aggaccagcc tttttaccac 1403

tgcagaagaa agacacaatg tggagaaatc ttaggactga catcccttta ctcaggcaaa 1463

cagaagttcc aacccccagac taggggtcag gcagctagct acctaccttg cccagtgctg 1523

acccggacct cctccaggat acagcactgg agttggccac cacctcttct acttgctgtc 1583

tgaaaaaaca cctgactagt acagctgaga tcttggcttc tcaacagggc aaagatacca 1643

ggcctgctgc tgaggtcact gccacttctc acatgctgct taagggagca caaataaagg 1703

tattcgattt ttaaaaaaaa aaaaaaaaaa aaaaaaaaa aaaa 1747

<210>2

<211>361

<212>PRT

<213> Homo sapiens (homo sapiens)

<400>2

Met Arg Gly Gln Arg Ser Leu Leu Leu Gly Pro Ala Arg Leu Cys Leu

1 5 10 15

Arg Leu Leu Leu Leu Leu Leu Gly Tyr Arg Arg Arg Cys Pro Pro Leu Leu

20 25 30

Arg Gly Leu Val Gln Arg Trp Arg Tyr Gly Lys Val Cys Leu Arg Ser

35 40 45

Leu Leu Tyr Asn Ser Phe Gly Gly Ser Asp Thr Ala Val Asp Ala Ala

50 55 60

Phe Glu Pro Val Tyr Trp Leu Val Asp Asn Val Ile Arg Trp Phe Gly

65 70 75 80

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

85 90 95

Ile Ala Tyr Leu Cys Val Leu Pro Leu Ile Leu Arg Thr Tyr Ser Val

100 105 110

Pro Arg Leu Cys Trp His Phe Phe Tyr Ser His Trp Asn Leu Ile Leu

115 120 125

Ile Val Phe His Tyr Tyr Gln Ala Ile Thr Thr Pro Pro Gly Tyr Pro

130 135 140

Pro Gln Gly Arg Asn Asp Ile Ala Thr Val Ser Ile Cys Lys Lys Cys

145 150 155 160

Ile Tyr Pro Lys Pro Ala Arg Thr His His Cys Ser Ile Cys Asn Arg

165 170 175

Cys Val Leu Lys Met Asp His His Cys Pro Trp Leu Asn Asn Cys Val

180 185 190

Gly His Tyr Asn His Arg Tyr Phe Phe Ser Phe Cys Phe Phe Met Thr

195 200 205

Leu Gly Cys Val Tyr Cys Ser Tyr Gly Ser Trp Asp Leu Phe Arg Glu

210 215 220

Ala Tyr Ala Ala Ile Glu Thr Tyr His Gln Thr Pro Pro Pro Thr Phe

225 230 235 240

Ser Phe Arg Glu Arg Met Thr His Lys Ser Leu Val Tyr Leu Trp Phe

245 250 255

Leu Cys Ser Ser Val Ala Leu Ala Leu Gly Ala Leu Thr Val Trp His

260 265 270

Ala Val Leu Ile Ser Arg Gly Glu Thr Ser Ile Glu Arg His Ile Asn

275 280 285

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

290 295 300

Pro Tyr Asn Tyr Gly Cys Leu Asp Asn Trp Lys Val Phe Leu Gly Val

305 310 315 320

Asp Thr Gly Arg His Trp Leu Thr Arg Val Leu Leu Pro Ser Ser His

325 330 335

Leu Pro His Gly Asn Gly Met Ser Trp Glu Pro Pro Pro Trp Val Thr

340 345 350

Ala His Ser Ala Ser Val Met Ala Val

355 360

<210>3

<211>20

<212>DNA

<213> oligonucleotide

<400>3

ctggctggta gacaacgtga 20

<210>4

<211>20

<212>DNA

<213> oligonucleotide

<400>4

gaaaggagaa ggtgggtggt 20

<210>5

<211>20

<212>DNA

<213> oligonucleotide

<400>5

ggacttcgag caagagatgg 20

<210>6

<211>20

<212>DNA

<213> oligonucleotide

<400>6

agcactgtgt tggcgtacag 20

Claims (10)

1. a method for screening pp3774 accelerator, is characterized in that, comprises steps: (a) in the test group, adding the candidate candidate and pp3774 to the cell culture, and detecting the proliferation of the cells; (b) comparing the proliferation of cells in the step (a) test group with the proliferation of cells in the control group, wherein the control group includes: Group (i): cell cultures to which no candidate candidate was added, nor pp3774 added; Group (ii): cell cultures supplemented with candidate candidates but without pp3774; and Group (iii): cell cultures to which the candidate candidate was not added but to which pp3774 was added; Wherein, if the proliferation of the cells in the test group is statistically lower (preferably significantly lower) than the control group (iii) and there is no significant difference between the cell proliferation of the control group (i) and the control group (ii), then it is indicated This candidate is a pp3774 enhancer. 2. The method according to claim 1, wherein the sequence of the pp3774 gene is as shown in SEQID NO: 1. 3. The method of claim 1, wherein said cell is a cancer cell. 4. The method according to claim 1 or 3, wherein said cells are liver cancer cells. 5. A method for screening a promoter combining pp3774 and JAB1, characterized in that it comprises the steps of: (a) in the test group, add the candidate to be screened to the culture that combines pp3774 and JAB1, and detect the combination of pp3774 and JAB1; (b) compare the binding of pp3774 and JAB1 in the test group of step (a) with the binding of pp3774 and JAB1 in the control group to which no candidate was added, if the binding of pp3774 and JAB1 in the test group is statistically higher than (preferably significantly higher than) the control, indicating that the candidate is a compound that promotes the binding of pp3774 and JAB1. 6. A method for screening an inhibitor combining pp3774 and JAB1, characterized in that it comprises the steps of: (a) in the test group, add the candidate to be screened to the culture that combines pp3774 and JAB1, and detect the combination of pp3774 and JAB1; (b) compare the binding of pp3774 and JAB1 in the test group of step (a) to the binding of pp3774 and JAB1 in the control group to which no candidate was added, if the binding of pp3774 and JAB1 in the test group is statistically lower than (preferably significantly lower than) the control, indicating that the candidate is a compound that inhibits the binding of pp3774 and JAB1. 7. A use of pp3774 gene or protein, characterized in that it is used for (a) preparing a composition for inhibiting the growth of cancer cells; or (b) preparing a composition for regulating cell apoptosis. 8. The use according to claim 7, wherein said composition is a pharmaceutical composition. 9. The use according to claim 8, characterized in that the composition contains 0.001-99.9 wt% pp3774 protein, calculated by the total weight of the composition. 10. The use according to claim 8, characterized in that, said composition also contains a promoter of pp3774 protein.

Images