CN1986568A - INF-alpha expression relevant protein and its coding gene - Google Patents

Abstract

The invention discloses a protein related to TNF-α expression, its coding gene and application. The protein related to the expression of TNF-α is the following (a) or (b) protein: (a) a protein consisting of the amino acid residue sequence of sequence 2 in the sequence listing; (b) the protein of sequence 2 in the sequence listing A protein derived from (a) whose amino acid residue sequence has undergone substitution and/or deletion and/or addition of one or several amino acid residues and is related to the expression of TNF-α. The coding gene of the protein related to TNF-α expression can be used for screening and/or preparing drugs for promoting or inhibiting sperm formation, screening and/or preparing drugs for resisting kidney inflammation, and for establishing transgenic mouse models and/or drug screening Model.

Description

A protein related to TNF-α expression and its coding gene

technical field

The invention relates to a protein related to TNF-α expression and its coding gene.

Background technique

TNF was reported to have anti-tumor activity a century ago, so it was called tumor necrosis factor, but it was not until 1984 that human TNF-α was cloned and recombined (Sugarman B.J.et al., 1985, Science, 230 :943-945), since then the research on TNF-α and its receptors has developed rapidly. The researchers found that TNF-α not only caused the apoptosis of specific tumor cells, but also mediated the inflammatory response and regulated immune function. The abnormal expression or continuous activation of TNF is an important sign in the pathological process of many human diseases, including sepsis, diabetes, chronic nephritis, cardiovascular disease, cancer, osteoporosis, transplant rejection and various autoimmune diseases ( Hotamisligil G.S. et al, 1993, Science, 259:87-91; Chen G. and Goeddel D, 2002, Science;). Therefore, TNF-α is currently receiving more attention as an inflammatory cytokine (Eugene E. et al., 2004, Cell, 116: 491-497). Also thus, multiple TNF-based inhibitors or anti-inflammatory drugs came into being (He, et al.Science, 2005, 310:1022-1025; Christopher.A.et al., 2006Nature Reviews Drug Discovery, 5: 864-876).

The kidney is a very important regulatory organ in the human body. It can secrete kidney hormones, such as renin, erythropoietin, calmodulin, and prostaglandins. In 2005, researchers discovered a new protein in the human kidney, that is, renalase protein, which is a FAD-dependent monoamine oxidase secreted by the kidney and circulates in the blood. Further research results show that renalase has the function of regulating heart function and blood pressure, and is considered to be a promising drug that is currently expected to be used clinically. Although the protein is secreted by the kidney, the protein content in the blood of patients with advanced kidney disease is significantly reduced (Xu, J, 2005, The Journal of Clinical Investigation, 115, 5: 1275-1280; Friedrich C., 2005, Cell Metabolism 1, 6:358-60). Homology and structural analysis revealed that renalase is a novel FAD-dependent amino oxidase with only a few amino acid similarities to known monoamine oxidases (MAO-A and MAO-B) and with different specificity substrates substances and antagonists. Both MAO-A and MAO-B are mitochondrial enzymes, anchored on the outer membrane of mitochondria through the C-terminus, and mainly metabolize catecholamines in cells, while renalase is secreted into the blood by the kidneys, circulates in the blood, and regulates the production of catecholamines in the body. Metabolism, thereby regulating the heart function and blood pressure of the human body.

The results of in vitro studies show that human renalase has a strong ability to metabolize dopamine, followed by epinephrine and norepinephrine. After injecting rats with purified human renalase protein expressed by Escherichia coli, it was found that the myocardial contractility, heart rate and blood pressure of the rats were all significantly reduced. And because renalase is a secreted protein, its activity can be detected in blood, and it is a protein naturally existing in blood (Xu, J, 2005, The Journal of Clinical Investigation, 115, 5: 1275-1280), indicating that renalase It will have a broad application prospect in clinic, and will have a huge impact on the treatment of kidney disease and cardiovascular disease.

Contents of the invention

The object of the present invention is to provide a protein related to the expression of tumor necrosis factor-alpha (TNF-alpha) and its coding gene.

The protein associated with the expression of TNF-α provided by the present invention is called mMAO-C, which is derived from the genus Mus musculus (Mus musculus), and is the protein of the following (a) or (b):

(a) a protein consisting of the amino acid residue sequence of Sequence 2 in the Sequence Listing;

(b) Substitution and/or deletion and/or addition of one or several amino acid residues to the amino acid residue sequence of Sequence 2 in the Sequence Listing and a protein derived from (a) related to the expression of TNF-α.

Wherein, Sequence 2 in the sequence listing consists of 342 amino acid residues. Amino acid residues 1-17 from the amino terminal of sequence 2 are signal peptide sequences, amino acid residues 1-172 from the amino terminal of sequence 2 are FAD binding regions, and amino acids 3-332 from the amino terminal of sequence 2 Residues are amino oxidase (Amino oxidase) domain.

In order to facilitate the purification and detection of mMAO-C in (a), tags shown in Table 1 can be attached to the N-terminal or C-terminal of the protein consisting of the amino acid residue sequence of Sequence 2 in the Sequence Listing.

Table 1. Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His-tag 2-10 (usually 6) HHHHHH Flag-tag 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 11 MASMQKLISEEDL HA-tag 9 YPYDVPDYA GFP fusion 238  MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

The mMAO-C in (b) above can be synthesized artificially, or its coding gene can be synthesized first, and then biologically expressed according to the following method. The coding gene of mMAO-C in (b) above can be obtained by deleting the codon of one or several amino acid residues in the DNA sequence of Sequence 1 in the sequence listing, and/or carrying out one or several base pairs of missense mutation, and/or link the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end.

The above-mentioned protein coding gene (mMAO-C) related to the expression of TNF-α also belongs to the protection scope of the present invention.

The nucleotide sequence of the protein-coding gene related to the expression of TNF-α is preferably a polynucleotide encoding the protein of sequence 2 in the sequence listing.

The coding sequence (ORF) of the protein coding gene related to the expression of TNF-α is the nucleotide sequence consisting of the 1st-1029th deoxynucleotides from the 5' end of the sequence 1 in the sequence listing.

The cDNA gene of the protein related to the expression of TNF-α can specifically be the gene of the following 1) or 2):

1) DNA sequence of sequence 1 in the sequence listing;

2) A nucleotide sequence that hybridizes to the DNA sequence defined in Sequence 1 in the Sequence Listing under stringent conditions.

The stringent conditions may be hybridization at 65° C. in a solution of 0.1×SSPE (or 0.1×SSC), 0.1% SDS, and the solution is used to wash the membrane.

Sequence 1 in the sequence listing is composed of 1038 deoxynucleotides, and the nucleotide sequence at positions 1-1029 from the 5' end is a coding sequence, which encodes the amino acid residue sequence having the sequence 2 in the sequence listing.

The genome sequence of mMAO-C is located on chromosome 19 of the mouse (19C1, 33.2M-33.9M), and its genome sequence number is NW_000143 or NT_039687 or NW_001030643. The coding region has 7 exons, encoding 342aa, and human The homology of the coding region of MAO-C gene and its protein sequence was 81.7% and 72.2%, respectively. The 7 exons of its coding region include 254,518bp, and the positions on the genome Contig (NT_039687) are: 331910-331792 (exon 1), 331025-330918 (exon 2), 329897-329752 ( exon 3), 322246-322205 (exon 4), 142486-142313 (exon 5), 108337-108158 (exon 6), 78115-77952 (exon 7).

Recombinant expression vectors, engineering bacteria and transgenic cell lines containing the above genes also belong to the protection scope of the present invention.

The mMAO-C of (a) or (b) of the present invention can be expressed by Escherichia coli and mammalian cells, such as cloning the coding sequence of the mMAO-C of (a) or (b) into a prokaryotic expression vector, by transferring into the large intestine Bacterial strain expression; the coding sequence of mMAO-C in (a) or (b) is constructed on a eukaryotic expression vector, and expressed by transfecting animal cells.

The results of bioinformatics analysis showed that MAO-C widely exists in higher animals and has a high degree of homology, indicating that it is a highly conserved gene in evolution, and most protein transcript sequences contain signal peptides. The results of the tissue distribution study showed that the expression distribution of the mMAO-C gene in different tissues of the mouse had obvious differences. The gene was expressed in a large amount in the kidney and testis, and there was also a certain amount of expression in the heart, brain, liver, and embryonic tissues. The mouse mMAO-C of the present invention is highly expressed in the kidney and testis, and is expected to be applied in the analysis of reproductive mechanisms and drugs for promoting or inhibiting sperm formation. The mMAO-C of the present invention can be injected into nephropathy model mice to detect the effect of mMAO-C as a drug, and can also be used to construct a mouse research model as an experimental platform for research on the mechanism of MAO-C and potential clinical application. Since mMAO-C is highly expressed in the testis, it indicates that this gene may be used in the analysis of reproductive mechanisms and drugs that promote or inhibit sperm formation. The mMAO-C of the present invention lays the foundation for the research of antibody preparation, protein interaction and gene regulation mechanism, and further provides a theoretical basis for clinical application. Another important discovery in the present invention is that mMAO-C has the function of significantly inhibiting the expression of endogenous inflammatory factor TNF-α in embryonic kidney cells 293T, which is the first evidence and an important breakthrough of mMAO-C in the study of the mechanism of action. It shows that MAO-C protein can improve the anti-inflammation ability of kidney by inhibiting the expression of TNF-α, which indicates that MAO-C protein will play an important role in the treatment of nephritis.

Description of drawings

Fig. 1 is the RT-PCR amplification result of mMAO-C of the present invention and the enzyme digestion identification result cloned into the pCDNA3.1 vector.

Fig. 2 shows the chromosomal location and protein structure of mMAO-C of the present invention.

Fig. 3 is a comparison of the phylogenetic tree of mMAO-C of the present invention and other species of MAO-C family members.

Figure 4 is the expression diagram of mMAO-C and GST fusion protein of the present invention in Escherichia coli, L-MAO-C means mMAO-C with signal peptide, S-mMAO-C means mMAO-C without signal peptide.

Fig. 5 is the result of RT-PCR tissue distribution detection of mMAO-C of the present invention.

Fig. 6 is a graph showing the expression of mMAO-C gene and GFP fusion protein in embryonic kidney cell 293T of the present invention.

Fig. 7 shows the effect of the overexpression of mMAO-C gene of the present invention on the expression of TNF-α in embryonic kidney cell 293T.

Detailed ways

Embodiment 1, the acquisition of mMAO-C gene

Using the NCBI BLASTn tool (http://www.ncbi.nih.gov/BLAST/) with the human gene sequence (BC005364.1) as a template, BLAST obtained a mouse cDNA sequence fragment with high homology, and then used http: //www.cbs.dtu.dk/services/SignalP/ website and biological software such as Bioedit for gene information analysis. The mouse cDNA sequence fragments with high homology obtained by searching. Compared with the protein sequence of other species, it has high homology, but lacks a peptide at the N-terminus. According to the sequence comparison results, mouse transcript 1 (XM_619582.2) was selected as the reference sequence, and primers were designed according to the conserved reading frames of other species. The upstream primers were JF1M5'CG A GA ATT C AG ATGTCC CGG GTA CTG GTC G 3 (the underlined sequence is the EcoRI recognition site), and the downstream primer is JR15' CCG CTC GAG CAC AGA CAC TAA ATA TAA CGC 3' (the underlined sequence is the XhoI recognition site). Using the total RNA extracted from the kidneys of Kunming mice (Beijing Experimental Animal Research Center) as a template, the target fragment was amplified with the One Step RNA PCR Kit (AMV) from TaKaRa Company. The PCR amplification conditions were as follows: first 94°C for 4min; then 94°C for 45sec, 65°C for 45sec, 72°C for 50sec, 30 cycles; finally 72°C for 10min. A specific 1038bp fragment was amplified by RT-PCR (A in Figure 1, 1 is the DNA molecular weight standard, and 2 is the RT-PCR product). The fragment was cloned into the pGEM-T-Easy (Promega Corporation) vector, and sequenced after identification by enzyme digestion. Three samples were sequenced in duplicate. Sequencing results show that this sequence has a reading frame similar to the MAO-C gene of other species, the coding sequence is 1029bp, encoding 342 amino acid residues (sequence 2 in the sequence listing), and the coding region of the human MAO-C gene The homology of their protein sequences is 81.7% and 72.2%, respectively. According to prediction ( http://www.ebi.ac.uk/InterProScan/ ), amino acid residues 1-17 at the amino terminal of Sequence 2 are the signal peptide sequence, and amino acid residues 1-172 are the FAD binding region , the amino acid residues 3-332 are the amino oxidase (Amino oxidase) domain (Figure 2). The predicted isoelectric point is 7.93 and the molecular weight is 37.6kDa (http://cn.expasy.org/cgi-bin/pi_tool).

The sequence of the cDNA gene of mMAO-C is sequence 1 in the sequence listing.

The genome sequence of mMAO-C is located on chromosome 19 of the mouse (19C1, 33.2M-33.9M), and its genome sequence number is NW_000143 or NT_039687 or NW_001030643. The coding region has 7 exons, encoding 342aa, and human The homology of the coding region of MAO-C gene and its protein sequence is 81.7% and 72.2% respectively (Fig. 3). The 7 exons of its coding region include 254,518bp, and the positions on the genome Contig (NT_039687) are: 331910-331792 (exon 1), 331025-330918 (exon 2), 329897-329752 ( exon 3), 322246-322205 (exon 4), 142486-142313 (exon 5), 108337-108158 (exon 6), 78115-77952 (exon 7).

Using mouse and human MAO-C BLAST NCBI GenBank, it was found that MAO-C widely exists in different animal species, and MAO-C has high homology among different species, which is in line with the evolutionary taxonomic relationship.

Embodiment 2, expression of mMAO-C gene in escherichia coli

According to the method of Example 1, the mMAO-C gene of the mouse (the nucleotide fragment containing the signal peptide and the nucleotide fragment not containing the coding sequence of the signal peptide) was obtained. Digest and recover the target fragment through gel, connect to pGEX-4T-2 (Amersham Pharmacia) which has been digested by EcoR I and Xho I with T ₄ DNA ligase, and be identified by EcoR I and Xho I double digestion Construct the correct recombinant vector pGEX-4T-2-mMAO-C (from the 1st-1037th nucleotide sequence at the 5' end of sequence 1) containing the signal peptide and the coding sequence fragment (from sequence 1) that does not contain the signal peptide 127-1037th nucleotide sequence at the 5' end) recombinant vector pGEX-4T-2-ΔmMAO-C. The pGEX-4T-2-mMAO-C and pGEX-4T-2-ΔmMAO-C were respectively transformed into Escherichia coli JM109, and positive clones were screened with ampicillin (50 μg/mL). Then pGEX-4T-2-mMAO-C and pGEX-4T-2-ΔmMAO-C plasmids were transformed into Escherichia coli BL21 strain, cultivated overnight at 37°C, and then inoculated in 6ml containing ampicillin (50μg/ mL) in LB test tubes, cultivated to OD600=0.6, added IPTG to a final concentration of 0.1mM, cultured with shaking at 37°C, and induced protein expression. Before induction and after induction, 200μl and 100μl of bacterial liquid were taken, respectively, and removed after centrifugation. To the supernatant, add 50 μl deionized water and 50 μl 2-fold protein loading buffer night, and take 25 μl of the mixture for SDS-PAGE electrophoresis analysis. The results showed that the correct GST-mMAO-C and GST-ΔmMAO-C fusion proteins were expressed, with sizes of about 64kDa and 60kDa, respectively. L-mMAO-C in lanes 2 and 3 in Figure 4 refers to the GST-mMAO-C fusion protein with a signal peptide, including the expression products before (lane 2) and after induction (lane 3) by IPTG. S-mMAO-C in lanes 5 and 6 refers to the GST-ΔmMAO-C fusion protein without signal peptide, including the expression products before (lane 5) and after induction (lane 6) by IPTG.

Embodiment 3, the expression distribution of mMAO-C in mouse tissue

After the mice (same as in Example 1) were put to death, their different tissues were quickly taken out, and the whole embryos of these tissues and the 12.5-day mice (same as in Example 1) were ground in liquid nitrogen respectively, and after being powdered, they were mixed according to Trizol ( Invitrogen) reagent operating method was used to extract tissue total RNA, the concentration of RNA was measured with a spectrophotometer, and its integrity was detected by 1.2% agarose gel electrophoresis. Semi-quantitative RT-PCR was carried out using the same amount of total RNA from different tissues of the mouse as a template. The results are shown in Figure 5, which indicated that the expression of mMAO-C gene was the largest in the mouse kidney and testis, and in the mouse heart, liver and There is also a certain expression in the embryo, indicating that the gene is expressed from the mouse embryonic stage to the adult stage, and it is a gene that plays an important role in reproduction and development.

Embodiment 4, the expression of mMAO-C gene in 293T eukaryotic cell

The whole protein coding sequence of mMAO-C containing the signal peptide (from the 1st-1037th nucleotide sequence at the 5' end of sequence 1) was digested with EcoRI and XhoI from pGEX-4T-2-mMAO-C and gel After purification, it was cloned into the pEGFP-N1 vector (BD Biosciences Clontech) treated with two enzymes, EcoRI and SalI, and identified by restriction enzyme digestion with EcoRI and BamHI, and a recombinant plasmid pEGFP-N1 containing the coding sequence fragment of the signal peptide was constructed. -mMAO-C plasmid, then the pEGFP-N1-mMAO-C plasmid was transfected into 293T cells according to the conventional cell transfection method, the cells were harvested and lysed, and the supernatant after centrifugation was used as the primary antibody (Santa Cruz ) for Western Blot detection, as shown in Figure 6, the correct protein expression product was obtained, and the arrows indicated GFP and the fusion protein GFP-mMAO-C of GFP and mMAO-C. Lane 1 is the hybridization result of GFP antibody as the primary antibody with GFP protein, and lane 2 is the result of hybridization with GFP-mMAO-C fusion protein with GFP antibody as the primary antibody. The results in Figure 6 further show that there is a large amount of GFP protein expression in the cytoplasmic lysate, which is consistent with previous reports, because the GFP protein is located in the cytoplasm and is non-secreted, and when combined with mMAO-C with signal peptide After the protein was constructed into a fusion protein, the amount of the GFP fusion protein detected in the cytoplasmic lysate was significantly reduced, indicating that part of the protein had been secreted out of the cell.

Example 5. Effect of overexpression of mMAO-C gene on expression of TNF-α in embryonic kidney cell 293T.

The whole protein coding sequence of mMAO-C containing the signal peptide (from the 1st-1037th nucleotide sequence at the 5' end of sequence 1) was digested with EcoRI and XhoI and gel-purified, and then cloned into pcDNA3.1-Myc ( +) between the EcoRI and XhoI restriction sites of the vector (Invitrogen), use EcoRI and XhoI to carry out enzyme digestion identification (B in Figure 1), and construct a recombinant pcDNA3.1-Myc-mMAO-C plasmid (containing signal peptide coding sequence fragment), and then transfect 293T cells with pcDNA3.1-Myc(+) (control group) and pcDNA3.1-Myc-mMAO-C plasmids according to the conventional cell transfection method, and carry out 12 hours after transfection Treat with LPS (lipopolysaccharide endotoxin, 30 μg/ml), collect the cells after 12 hours, and lyse them, and use rabbit TNF-α and β-actin antibodies as the primary antibodies (Santa Cruz) to detect the supernatant by Western Blot respectively , the result is shown in Figure 7. In Figure 7, swimming lanes 1 and 2 are 293T cells transfected with pcDNA3.1-Myc (+) empty plasmid (as a control), and swimming lanes 3 and 4 are 293T cells transfected with pcDNA3.1-Myc-mMAO-C plasmid, Among them, lanes 2 and 4 were treated with LPS. The upper figure is the hybridization result of TNF-α antibody as the primary antibody, and the lower figure is the hybridization result of β-actin antibody as the primary antibody, as a control of the amount of sample loaded. The results showed that the expression of endogenous TNF-α in 293T cells transfected with mMAO-C gene was significantly reduced, indicating that the mMAO-C expression product has the function of inhibiting the expression of endogenous inflammatory factor TNF-α protein, which is a very important Because TNF-α plays an important role in regulating inflammatory response and immune function, the abnormal expression of TNF-α is closely related to the occurrence of many diseases. When 293T cells transfected with different plasmids were treated with LPS, the expression of TNF-α in the same group did not change, indicating that 293T cells did not respond to LPS, which was consistent with previous reports.

sequence listing

<160>2

<210>1

<211>1038

<212>DNA

<213> Mice belong to Mus musculus (Mus musculus)

<400>1

atgtcccggg tactggtcgt gggcgctggg ctaaccggaa gtctgtgtgc cgcgctgctg 60

aggaaggaaa taaccgcccc cctgtacctc gccctgtggg acaaggctgg ggatataggg 120

ggaagaatga ttactgccag cagtcctcat aatccccgat gcacagctga cttgggagct 180

cagtacatca cctgctctcc tcatttatgtc aaagagcacc aaaattttta tgaggaactg 240

ttagctcatg gaattttgaa gcctctgaca tcccccattg aaggaatgaa agggaaggaa 300

ggagattgca actttgtggc acctcaagga ttttcttcag ttatcaagta ctacttgaaa 360

aagtcaggtg cagaagtctc cctcaagcac tgtgtgactc agatccacct gaaagataac 420

aagtgggaag tctccacaga cactggctct gctgagcagt ttgaccttgt catcctcacc 480

atgccagctc ctcagattct ggaacttcaa ggtgacattg tgaacttaat tagtgaacgc 540

cagaggggagc aactgaaatc tgtgagctac tcctctcgct atgctctggg cctcttttat 600

gaagtaggca tgaagattgg tgtcccttgg tcctgccgct acctcagcag tcacccctgc 660

atatgcttca tctccatga taataagaag cgcaatatag agtcatcaga atgtggtcca 720

tccgtggtga tccaaaccac tgtcccattt gagttcaac acttggaggc cagtgaggcg 780

gatgtgcaga agttaatgat ccagcaattg gaaaccattc tgccgggttt gcctcagcca 840

gttgctacca tatgccataa atggacatat tcacaggtta caagctcagt ttccgacaga 900

cctggtcaga tgactcttca tctcaagcct ttcctggtgt gcggagggga tggatttact 960

cactccaact tcaatggctg catctcctct gccctgagtg tcatgaaagt tttaaagcgt 1020

tatatttagt gtctgtgc 1038

<210>2

<211>342

<212>PRT

<213> Mice belong to Mus musculus (Mus musculus)

<400>2

Met Ser Arg Val Leu Val Val Gly Ala Gly Leu Thr Gly Ser Leu Cys

1 5 10 15

Ala Ala Leu Leu Arg Lys Glu Ile Thr Ala Pro Leu Tyr Leu Ala Leu

20 25 30

Trp Asp Lys Ala Gly Asp Ile Gly Gly Arg Met Ile Thr Ala Ser Ser

35 40 45

Pro His Asn Pro Arg Cys Thr Ala Asp Leu Gly Ala Gln Tyr Ile Thr

50 55 60

Cys Ser Pro His Tyr Val Lys Glu His Gln Asn Phe Tyr Glu Glu Leu

65 70 75 80

Leu Ala His Gly Ile Leu Lys Pro Leu Thr Ser Pro Ile Glu Gly Met

85 90 95

Lys Gly Lys Glu Gly Asp Cys Asn Phe Val Ala Pro Gln Gly Phe Ser

100 105 110

Ser Val Ile Lys Tyr Tyr Leu Lys Lys Ser Gly Ala Glu Val Ser Leu

115 120 125

Lys His Cys Val Thr Gln Ile His Leu Lys Asp Asn Lys Trp Glu Val

130 135 140

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

145 150 155 160

Met Pro Ala Pro Gln Ile Leu Glu Leu Gln Gly Asp Ile Val Asn Leu

165 170 175

Ile Ser Glu Arg Gln Arg Glu Gln Leu Lys Ser Val Ser Tyr Ser Ser

180 185 190

Arg Tyr Ala Leu Gly Leu Phe Tyr Glu Val Gly Met Lys Ile Gly Val

195 200 205

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

210 215 220

Ser Ile Asp Asn Lys Lys Arg Asn Ile Glu Ser Ser Ser Glu Cys Gly Pro

225 230 235 240

Ser Val Val Ile Gln Thr Thr Val Pro Phe Gly Val Gln His Leu Glu

245 250 255

Ala Ser Glu Ala Asp Val Gln Lys Leu Met Ile Gln Gln Leu Glu Thr

260 265 270

Ile Leu Pro Gly Leu Pro Gln Pro Val Ala Thr Ile Cys His Lys Trp

275 280 285

Thr Tyr Ser Gln Val Thr Ser Ser Ser Val Ser Asp Arg Pro Gly Gln Met

290 295 300

Thr Leu His Leu Lys Pro Phe Leu Val Cys Gly Gly Asp Gly Phe Thr

305 310 315 320

His Ser Asn Phe Asn Gly Cys Ile Ser Ser Ala Leu Ser Val Met Lys

325 330 335

Val Leu Lys Arg Tyr Ile

340

Claims (10)

1. A protein related to TNF-α expression, which is the protein in (a) or (b): (a) a protein consisting of the amino acid residue sequence of Sequence 2 in the Sequence Listing; (b) Substitution and/or deletion and/or addition of one or several amino acid residues to the amino acid residue sequence of Sequence 2 in the Sequence Listing and a protein derived from (a) related to the expression of TNF-α. 2. The gene encoding the protein related to TNF-α expression as claimed in claim 1. 3. The protein coding gene related to TNF-α expression according to claim 2, characterized in that: the nucleotide sequence of the protein coding gene related to TNF-α expression is sequence 2 in the coding sequence list protein polynucleotides. 4. The protein-encoding gene related to TNF-α expression according to claim 3, characterized in that: the coding sequence of the protein-encoding gene related to TNF-α expression is from the 5' of sequence 1 in the sequence listing The nucleotide sequence consisting of 1-1029 deoxynucleotides at the end. 5. The gene encoding the protein related to TNF-α expression according to claim 4, characterized in that: the cDNA gene of the protein related to TNF-α expression is the gene of the following 1) or 2): 1) DNA sequence of sequence 1 in the sequence listing; 2) A nucleotide sequence that hybridizes to the DNA sequence defined in Sequence 1 in the Sequence Listing under stringent conditions. 6. A recombinant expression vector containing the gene of any one of claims 2-5. 7. The transgenic cell line and engineering bacteria containing any one of the genes of claims 2-5. 8. Use of the gene according to any one of claims 2-5 in screening and/or preparing drugs for promoting or inhibiting sperm formation. 9. The use of the gene according to any one of claims 2-5 in establishing a transgenic mouse model and/or a drug screening model. 10. The use of the gene according to any one of claims 2-5 in screening and/or preparing anti-renal inflammation drugs.

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