NL2038217A

NL2038217A - Application of Chicken TRIM45 Truncated Recombinant Protein or Polyclonal Antibody Thereof

Info

Publication number: NL2038217A
Application number: NL2038217A
Authority: NL
Inventors: Wang Jiaxing; Bai Hao; Chang Guobin; Ping Yuyu; Huang Xuan; Chen Shihao
Original assignee: Univ Yangzhou
Priority date: 2023-11-22
Filing date: 2024-07-13
Publication date: 2025-06-02
Also published as: CN117821469B; CN117821469A

Abstract

Disclosed is an application of chicken TRIM45 truncated recombinant protein or polyclonal antibody thereof. The invention discloses an application of chicken TRIM45 gene, recombinant vector, truncated recombinant protein or polyclonal antibody thereof in preparing anti-avian leukaemia virus related drugs, where the accession number of chicken TRIM45 gene Genbank is XM_0 70 7211. The truncated recombinant protein of chicken TRIM45 constructed by the invention avoids the defect of full-length expression stability, and meanwhile, the immune effect is more stable and the antibody level is higher. At the same time, the eukaryotic expression vector 10 and polyclonal antibody prepared by the invention can be applied to the research on the mechanism of chicken TRIM45' s regulation of ALV-J replication, which has far-reaching significance and makes up for the blank of chicken TRIM45 antibody preparation and research; It provides a new idea for the mechanism study of anti-ALV-J infection and immune regulation in chickens.

Description

Application of Chicken TRIM45 Truncated Recombinant Protein or Polyclonal Antibody

Thereof

TECHNICAL FIELD

The invention relates to the application of chicken TRIM45 truncated recombinant protein or polyclonal antibody thereof, belonging to the technical field of bioengineering.

BACKGROUND

Tripartite motif (TRIM) protein is a large class of molecules with three conserved domains, namely RING, B-box and Coiled-coil, at the N-terminal, which is widely found in most eukaryotes.

The proportion of TRIM family members directly involved in immune regulation is very high, and it is considered that the number of TRIM genes in different species is related to the complex immune system. At present, nearly 80 TRIM family proteins have been found in humans, while about 40 TRIM proteins have been found in chickens. Studies have shown that TRIM proteins have various functions, but most of them are involved in regulating natural immune response, apoptosis, resistance to virus infection and so on. On the one hand, TRIM protein mostly belongs to interferon stimulating gene, which plays an important role in natural immune regulation. On the other hand, TRIM protein can directly interact with virus proteins and participate in the regulation of virus replication as a host factor.

Avian leukaemia virus (ALVs) is a kind of retrovirus, and its infection can lead to tumour proliferation of chicken hematopoietic and liver tissues. According to the difference of virus host and envelope glycoprotein, ALVs can be divided into 11 subgroups of A-K, among which subgroup J avian white disease virus (ALV-J) caused great losses to poultry industry after it was introduced into China. At present, there is no vaccine or drug for ALVs (including ALV-J), and more research is needed to explore the target of genes or antibody drugs against ALVs infection.

TRIM45, as a member of the tripartite motif protein family, is different from other TRIM family proteins in that its N-terminal structure contains three motifs, namely RING, B-BOX and CC, while its C-terminal only has FLMN domain. Previous studies have shown that the FLMN domain of

TRIM45 can target RAKC1 (activated kinase C), negatively regulate MAPK signalling pathway and inhibit the growth of tumour cells, which indicates that TRIM45 has anti-tumour effect. Another report shows that TRIM45 has E3 ubiquitin ligase activity, which can inhibit the proliferation of nerve cells and promote the apoptosis of cancer cells. It has also been reported that TRIM45 gene is highly expressed in tumour tissues infected by bovine leukaemia virus. However, at present, there is no report on the biological characteristics and functions of chicken TRIM45, and the relationship between TRIM45 and avian viral infections such as avian leukaemia needs to be clarified. In addition, the preparation of chicken TRIM45 antibody is still blank, and there is no research on the application of chicken TRIM45 polyclonal antibody.

SUMMARY

Purpose of the invention: the purpose of the present invention is to provide an application of chicken TRIM45 gene, recombinant vector, truncated recombinant protein or polyclonal antibody thereof in preparing drugs related to avian leukaemia.

Technical scheme: the invention provides an application of chicken TRIM45 gene, recombinant vector, truncated recombinant protein or polyclonal antibody thereof in preparing drugs related to avian leukaemia, and the Genbank accession number of the chicken TRIM45 gene is XM_04070 7211.

Further, the recombinant vector is obtained by inserting a gene sequence encoding a chicken TRIM45 truncated into a pColdl vector, where the gene sequence encoding the chicken

TRIM45 truncated is shown in SEQ ID No.2 and the amino acid sequence is shown in SEQ ID

No.1.

Further, the truncated recombinant protein is obtained by transforming and expressing a chicken TRIM45 recombinant vector.

Further, the antibody is prepared by immunizing animals with chicken TRIM45 truncated recombinant protein.

Further, the preparation method of the chicken TRIM45 truncated recombinant protein comprises the following steps: (1) carrying out protein sequence analysis on chicken TRIM45; (2) designing a specific primer pair for the gene sequence encoding the truncated chicken

TRIM45, and connecting the primer pair to the plasmid pColdl after PCR amplification to obtain a pColdl-His-TRIM45 recombinant plasmid, and transforming the pColdI-His-TRIM45 recombinant plasmid into competent cells; the gene sequence of the truncated chicken TRIM45 is shown in

SEQ ID No.2; (3) selecting the positive clone obtained in the step (2) and culturing it in the LB medium containing ampicillin, and then inducing the expression of TRIM45 protein through IPTG of isopropyl-p-D-thiogalactoside, and extracting the protein to obtain the chicken TRIM45 truncated recombinant protein.

Further, the protein sequence analysis in step (1) includes protein signal peptide prediction of chicken TRIM45, hydrophobicity analysis, transmembrane domain analysis, disorder region prediction, TRIM45 protein comparison of different species and phylogenetic tree construction.

Specifically, the SignalP tool is used to predict the signal peptide; the hydrophobicity of

TRIMA45 is analysed by Expasy tool. Using TMHMM tool to analyse the transmembrane domain;

Using NOVOPRO tool to predict the disorder region of TRIM45 protein; using Mega tool to compare TRIM45 proteins of different species and construct phylogenetic tree.

Further, the nucleotide sequence of the specific primer pair mentioned in step (2) is shown in SEQ ID No.3-4.

Further, in step (2), the truncated sequence of chicken TRIM45 and the insertion sites of plasmid pCold! are Ndel and Xhol.

Further, the competent cell in step (2) is BL21{DE3).

Further, the preparation method of the polyclonal antibody comprises the following steps: (1) chicken TRIM45 truncated recombinant protein is purified by Ni column chromatography, and then the protein is renatured; (2) Immunizing animals with purified protein and renatured protein to obtain serum containing chicken TRIM45 polyclonal antibody.

Further, the animal used for animal immunization in step (2) is New Zealand white rabbit.

Further, the avian leukaemia is caused by ALV-J infection.

Beneficial effects: compared with the prior art, the chicken TRIM45 has the following outstanding advantages: the chicken TRIM45 constructed by the invention truncates the body weight histone, thus avoiding the defect of full-length expression stability, and simultaneously, the immune effect is more stable and the antibody level is higher. At the same time, the polyclonal antibody prepared by the invention can be applied to the research on the mechanism of chicken

TRIMA4%' s regulation of ALV-J replication, which has far-reaching significance and fills the gap in the preparation and research of chicken TRIM45 antibody; It provides a new idea for the mechanism study of anti-ALV-J infection and immune regulation in chickens..

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 is the analysis result of chicken TRIM45 protein signal peptide.

Fig. 2 shows the predicted results of hydrophobicity of chicken TRIM45 protein.

Fig. 3 shows the predicted results of chicken TRIM45 protein transmembrane region.

Fig. 4 is the prediction result of disordered region in transmembrane region prediction.

Fig. 5 shows the analysis results of TRIM45 phylogenetic tree.

Fig. 6 shows the skeleton of pColdl enzyme digestion.

Fig. 7 shows the expression of chicken TRIM45 recombinant protein.

Fig. 8 is that verification of polyclonal antibody against chicken TRIM45.

Fig. 9 shows the titre of chicken TRIM45 polyclonal antibody detected by ELISA.

Fig. 10 shows the verification of polyclonal antibody against chicken TRIM45.

Fig. 11 shows that chicken TRIM45 protein inhibits ALV-J replication.

DESCRIPTION OF THE INVENTION

Next, the technical scheme of the invention will be further explained with the attached drawings.

Embodiment 1

S1: sequence analysis of chicken TRIM45 protein

The sequence information XM_040707211 of chicken TRIM45 gene is obtained by NCBI database, and the SignalP peptide of TRIM45 protein (SEQ ID No.7) is analysed by http://www.cbs.dtu.dk/services/SignalP-4.1/ (Fig. 1). Expasy online tool (http://expasy.org/cgibin/protscale.pl) was used to analyse the hydrophilicity and hydrophobicity (Fig. 2), and TMHMMServer v.2.0 (hitp:/Avww. cbs. diu dK/services/imh-mm/) was used to predict the transmembrane domain of the receptor (Fig. 3). NOVOPRO online tool (https://www.novopro.cn/tools/disordered.html) was used to predict the inherent disorder region of the protein (Fig. 4), the results show that chicken TRIM45 protein contains 441 amino acids, which is the full-length sequence of intracellular segment, with no signal peptide and transmembrane segment, moderate hydrophilicity, disordered domains at the N-terminal and C- terminal of the protein and 25 cysteines. Mega11.0 software was used to compare the proteins of several species and construct the phylogenetic tree (Fig. 5), the results show that the amino acid sequence homology of TRIM45 between chickens and humans is not conservative.

According to the complex region with high GC in the full length of TRIM45, in order to avoid the expression error or failure when constructing the full-length recombinant protein, the truncated 198-441aa sequence (SEQ ID No.2) is finally selected for the construction of chicken TRIM45 truncated recombinant protein.

S2: construction and identification of pColdi-His-TRIM45 recombinant plasmid pColdl (VLTO 550 , Honor Gene Company) is selected as the connecting vector, and

Ndel(CATATG)-Xhol(CTCGAG) is selected as the site of restriction endonuclease digestion. 1 p g of pColdl plasmid is mixed with restriction endonucleases Ndel and Xhol, and then added with

CutSmart buffer, which is placed at 37°C for 3 h, and the digested product is recovered by gel electrophoresis (Fig. 6).

According to the restriction site, the specific primers of the 198-441aa truncated sequence (SEQ ID No.2) of chicken TRIM45 are designed: the upstream primer is 5'-CATATGGGTAGCGATCTGGGCATTCTGG-3' (SEQ ID No.3), and the downstream primer is 5-GAGCTCAATGTCAAGGTGCCATGCCAT-3' (SEQ ID No.4); the truncated TRIM45 sequence is amplified by PCR after RNA is obtained from chicken fibroblasts and cDNA is obtained by reverse transcription. The procedure of PCR reaction is: pre-denaturation: 94°C, 3min; denaturation: 94°C, 15s; annealing: 58°C for 15s; extension: 72°C, 30 s; there are 35 cycles from degeneration to extension; re-extension: 72°C, 5min; the amplified TRIM45 fragment is ligated with pColdl vector at 16°C for 12h by T4-DNA ligase. According to the Standard Operating

Procedures for Plasmid Transformation, 2 | of ligation product is taken to transform BL21(DE3) competent cells, and then LB-resistant plates are coated, and then inverted cultured at 37°C for 16-18h. Single white colony is selected for sequencing, and the sequencing results are compared with the full-length sequence of TRIM45 gene. The positive monoclonal antibody is Escherichia coli containing pColdl-His-TRIM45 recombinant plasmid.

S3: extraction and preliminary identification of chicken TRIM45 recombinant protein

Selecting positive monoclonal antibodies, putting them into a test tube containing ampicillin in 4ml LB medium, and culturing at 37°C and 220 rpm until the OD600 of the bacteria is 0.6-0.8;

IPTG is added to the test tube in the treatment group, while IPTG is not added to the control group for induction. The test tube is placed in a constant temperature incubator at 37°C to induce expression; centrifuge at 8000 rpm for 5min, discard supernatant and collect bacterial sludge; take all that bacterial mud at the bottom, adding a resuspension buffer to resuspend the bacterial 5 mud until there is no obvious massive precipitation, and crushing the bacterial mud in an ice bath in an ultrasonic crusher; After crushing, it is centrifuged at 10000 rpm for 5 min, and the supernatant is analysed by Western blot. The specific steps are as follows: take 100 pul of the above sample, add 25ul of volume reduction Loading buffer, boil in water bath for 10 min, centrifuge at 5000 rpm for 2 min, take the denatured protein samples of the treatment group and the control group for SDS-PAGE, and then transfer the protein on SDS-PAGE to PVDF membrane by wet rotation after constant voltage of 90 V and 45min; adding sealing liquid for room temperature membrane sealing for 2h; His-tag polyclonal antibody (Bioworld, AP0032) is used as the primary antibody and incubated at 4°C for 12h, and then goat anti-rabbit IgG (H+L)-HRP (Bioworld, BS13278) is used as the secondary antibody for 2h at room temperature. Using ECL luminescent kit (Thermo Fisher Company, A34580 ) to develop, the results showed that TRIM45 truncated histone positive band appeared at 28kDa (Fig. 7), with strong specificity and high expression content.

S4: expanded expression of TRIM45 recombinant protein and purification by Ni column

The monoclonal antibody positive in S2 is selected and cultured in a 250 ml shake flask of

LB medium containing ampicillin at 37°C and 220 rpm for 16-18 h as seed solution. Transfer the seed solution to a shake flask containing 500 ml ampicillin-resistant LB medium according to the ratio of 1:100 , culture at 37°C and 220 rpm until the OD800 nm of the bacteria is 0.6-0.8, and add 1mmol/lol IPTG for induced expression; centrifuge at 8000 rpm for 5min, discard supernatant and collect bacterial sludge; take the above-mentioned bacteria mud, add 25ml buffer solution to every 500 ml bacteria liquid, resuspend the bacteria mud until there is no obvious massive precipitation, and crush it in an ice bath in an ultrasonic crusher for 30 min; take 50 ul of the broken sample, centrifuge at 10000 rpm for 35min, discard the supernatant, and use inclusion body purification buffer 1(100 mmol/l Tris, 100 mmol/l NaH2PO,, 8M Urea, 10 mmol/l Imidazole, pH8.3) for ultrasonic dissolution of the precipitate; centrifuge at 10000 rpm for 35min, and take the supernatant, after taking out the chromatographic column, use ultrapure water to clean 5-10 column volumes; washing the column for 5-10 times with purified buffer 1; the filtered sample is purified by chromatographic column, and the sampling flow rate is 2 ml/min. After loading the sample, wash 5-10 column volumes with purification buffer 1; Use 2/3 of purification buffer (purification buffer 2: 100 mmol/l Tris, 100 mmol/l NaH2PQO., 8M Urea, 20 mmol/l imidazole, pH6.3; purification buffer 3: 100 mmol/l Tris, 100 mmol/l NaH:POs, 8M Urea, 250 mmol/l Imidazole, pH8.5) for impurity washing and protein elution; take that eluted sample for gel run analysis, after renaturation, the eluted proteins are dialyzed to renaturation buffers 1, 2 and 3 (renaturation buffer 1: 20 mmol/l tris, 500 mmol/l NaCl, 10 mmol/l arginine, 4M Urea, pH8.0; renaturation buffer 2: 20 mmol/l Tris, 500 mmol/l NaCl, 10 mmol/l arginine, 2M Urea, pH8.0; renaturation buffer 3: 20 mmol/l Tris, 1mmol/l DTT, 1mmol/l EDTA, 1M Urea, pH9.0 }, dialysis ratio > 100 , dialysis time 4- 6 h; dialyze the final sample into storage buffer (20 mmol/l Tris, 1mmol/l DTT, 1mmol/l EDTA, pH9.0), filter with a 0.22um filter, and subpackage the protein.

S5: preparing polyclonal antibody against chicken TRIM45.

The serum of female New Zealand white rabbits (2.5-3 kg) purchased from Qingdao Konde

Biological Company is collected and analysed as a negative control. The protein concentration is determined by BCA method and adjusted to 1mg/ml. The purified and renatured recombinant protein is mixed with Freund's complete adjuvant (Sigma Company, F5881) according to the ratio of 1:1, emulsified on ice for 40 min, and injected subcutaneously on the back of New Zealand white rabbits at 5 points. The immunization is strengthened once every two weeks, emulsified with Freund's incomplete adjuvant (Sigma, F550 6) with the same volume as the protein, and each New Zealand white rabbit is injected with 250 ug of renatured recombinant protein, and the immunization is strengthened for 5 times. Ten days after the last immunization, the whole blood of four different New Zealand white rabbits is collected to prepare TRIM45 antiserum. The results of antibody verification are shown in Fig. 8: The antibody titre of rabbits No.1 and No.2 is higher than that of rabbits No.3 and No.4.

Embodiment 2 Analysis of specificity and effectiveness of polyclonal antibody

Detection of chicken TRIM45 polyclonal anti-epidemic titre by ELISA: the protein concentration of chicken TRIM45 purified in S4 step of Embodiment 1 is determined by BCA method, and the protonucleoprotein is diluted to 3 ug/ml by antigen coating method, and added to a 96-well plate with 100 pl in each well, with a total of 3 repeated wells, and placed at 4°C overnight. After removing the coating solution, it is washed with PBS-T for three times. Add 200 ul of 0.1% skimmed milk powder and seal it at room temperature for 1 hour. After removing the sealing liquid, use PBS-T for washing for three times. Adding non-immunized negative control serum diluted by gradient (dilution ratio: 1:1000 , 1:10000 , 1:20000 , 1:40000 , 1:80000 |, 1:160000 , 1:320000 ) and serum of four immunized rabbits in step S5 of Embodiment 1, 200 pl/well. Wash with 200 pl PBS-T for 3 times, 5min each time, and add 1:1000 diluted secondary antibody Goat anti-Rabbit IgG (H+L) at 37°C for incubation for 1h. PBS-T solution is used for washing for three times, and finally TMB chromogenic solution (A:B=1:1) is added, which is kept out of light for 30 min at 37°C, and 50 pl of stopping solution (1mol/l hydrochloric acid) is added to stop the reaction, after 1-2 min, the absorbance at 450 nm is detected by ELISA. The results show that compared with the control group, the rabbit serum in the immune group still has a higher titre when diluted at the ratio of 1:320000.

Primers are designed by homologous recombination method (SEQ ID No.5: chTRIM45-F: aaactcatctcagaagaggatctgcttgcggccgegATGGCCGCGTCACGCTGC; SEQ ID No.6: chTRIM45-

R: gtgagccagggcattggTCAGAGTTCCACTGTCCTGAGCAAACT, and the underline indicates the homologous arm consistent with the sequence of pCAGGS-Myc vector), and the full-length sequence of chicken TRIM45 is amplified by PCR and connected to the backbone of pCAGGS-

Myc (Miaoling Biological Company, P1372) vector. The constructed eukaryotic expression plasmid pCAGGS-Myc -TRIM45 is transfected into chicken fibroblast cell line, and the cells are placed in a constant temperature incubator of 37°C and 4% CO: for 24h. After adding 100 pl of

RIPA (Beyotime, POO 13B) to lyse the cells for 15min, the supernatant is centrifuged at 12000 rpm at 4°C for 10 min, and the loading buffer with 1/5 of the protein-like volume is added. Protein is separated by 10 %SDS-PAGE electrophoresis, and then the protein is transferred to 0.45 um

NC film by wet rotation. 5% skimmed milk powder is sealed at room temperature for 2 h; According to the predicted molecular weight of the protein, a suitable band is obtained by shearing, and the

TRIM45 polyclonal antibody (1:500 ) prepared in step S5 of Embodiment 1 is used to incubate overnight at 4°C, and B actin is used as internal reference according to the ratio (1:3000 (Abcam

Company, ab200 658). After washing the strips with TBST buffer for three times, they are incubated with goat anti-rabbit IgG (1:10000 ) labelled by HRP (ABCAM Company, ab20 5718) at room temperature for 1 hour. The ultra-sensitive ECL detection kit is used for detection. The results show that there s a strong positive band in the cell protein sample transfected with pCAGGS-Myc-TRIM45 at the size of 55kDa, which indicates that the prepared polyclonal antibody of TRIM45 is specific and efficient (Fig. 10).

Embodiment 3

Verification of TRIM45 inhibiting ALV-J replication by TRIM45 polyclonal antibody pCAGGS-Myc-TRIM45 eukaryotic expression plasmid constructed in Embodiment 2 is transfected into DF-1 chicken fibroblast cell line, and the cells are placed in a constant temperature incubator with 37°C and 5% CO: for 24 h, then ALV-J(MOI 1.0) is added to infect them for 24 h. After harvesting the total protein, add 1/5 times of protein-like volume of loading buffer, and boil at 100 °C for 10 min; Protein is separated by 10 % SDS-PAGE electrophoresis, and then transferred to 0.45um NC membrane by wet rotation. 5% skimmed milk powder is sealed at room temperature for 2 h; according to the predicted protein molecular weight, the appropriate bands are obtained by shearing, and incubated overnight at 4°C with TRIM45 antibody (1:500 ) or ALV-J Env antibody (1:1000 ), and B actin is used as internal reference in proportion (1:3000 }. After washing the strip with TBS-T buffer solution for three times, HRP-labelled goat anti-rabbit

IgG(1:10000 ) and goat anti-mouse 1gG(1:10000 (Abcam, ab6728) are incubated at room temperature for one hour. After washing the strip with TBS-T buffer for 3 times; the ultra-sensitive

ECL detection kit is used for detection. The results showed (Fig. 11) that the expression of ALV-

J Env protein is significantly inhibited after transfection of the eukaryotic expression plasmid of

TRIM45, which indicates that the chicken TRIM45 protein had the effect of anti-ALV-J infection.

Therefore, the preparation of polyclonal antibody of chicken TRIM45 can lay a foundation for the follow-up antiviral research of TRIM45.

NL TRIM45 NL CN 202311563075.6 2023-11-22 Yangzhou University Chen Shihao

Application of Chicken TRIM45 Truncated Recombinant Protein or Polyclonal

Antibody Thereof 7 244 AA PAT source 1..244 mol_type protein organism synthetic construct

GSDLGILVTKGVVASRLAKLNSAAYSTHPSVDDSIQFSPQERAGQCHGYEVFGAVICKAVDPA

KCTLQGEGLRSARQNELTGFTLLCSDTTGERMGRGGEAVVVTITHKDKKDCAVKPTVCDNGD

GTYHISYSPEEPGLYAVCVYVKGQHVQGSPFIVMVKSQFRKHQGMFHCCTFCSSGGQKAARC

ACGGTMPGGYQGCGHGHKGHPGCPHWSCCGQVKMSSECLSGLPSDRSQRSLLRTVEL 747

DNA PAT source 1..747 mol_type other DNA organism synthetic construct catatgggtagcgatctgggcattctggttaccaaaggtgttgttgccagccgcctggcaaaactgaatagtgcagcct atagcacccatccgagtgttgatgatagtattcagtttagcccgcaggaacgtgccggccagtgccacggttatgaagt gtttggtgcagttatttgtaaagcagtggaccctgccaaatgcaccctgcagggtgaaggtctgcgcagcgcacgcca gaatgaactgaccggctttaccctgctgtgtagcgataccaccggtgaacgcatgggccgtggtggcgaagccgtgg tggtgaccattacccataaagataaaaaagattgcgccgttaaaccgaccgtgtgtgataatggcgatggtacctatc atattagttatagcccggaagaaccgggtctgtatgccgtgtgtgtgtatgttaaaggccagcatgtgcagggcagtcc gtttattgttatggttaaaagtcagtttcgcaaacatcagggtatgtttcattgctgcaccttttgcagtagtggcggcca gaaagcagcccgctgtgcctgcggcggtaccatgcctggcggttatcagggttgtggccacggtcataaaggtcatcc gggctgcccgcattggagctgttgcggccaggtgaaaatgagtagtgaatgtctgagcggcctgccgagcgatcgca gccagcgtagtctgctgcgtaccgtggaactgtaactcgag 28 DNA PAT source 1..28 mol_type other DNA organism synthetic construct catatgggtagcgatctgggcattctgg 27 DNA PAT source 1..27 mol_type other DNA organism synthetic construct gagctcaatgtcaaggtgccatgccat 54 DNA PAT source 1..54 mol_type other DNA organism synthetic construct aaactcatctcagaagaggatctgcttgcggccgcgatggccgcgtcacgctgc 44 DNA PAT source 1..44 mol_type other DNA organism synthetic construct gtgagccagggcattggtcagagttccactgtcctgagcaaact 70 AA PAT source 1..70 mol_type protein organism Gallus gallus

MLLTCMHRTFSRKSEWAASPRQRRQKKTASHVVMELESTKDCSQAGKPFFCPSHPSEELGLF

CEQCDQPV

Claims

CONCLUSIONS

1. An application of the chicken gene TRIM45, a recombinant vector, a truncated recombinant protein or polyclonal antibody thereof in the manufacture of medicinal products for avian leukemia, wherein the accession number of the chicken gene TRIM45 is Genbank XM_040707211.

The use according to claim 1, wherein the recombinant vector is obtained by inserting into the pCold1 vector the gene sequence encoding truncated chicken TRIM45, wherein the gene sequence encoding truncated chicken TRIM45 is shown in SEQ ID No. 2 and the amino acid sequence is shown in SEQ ID No. 1.

The use according to claim 2, wherein the truncated recombinant protein is obtained by transformation and expression of the chicken TRIM45 recombinant vector.

The use according to claim 1, wherein the antibody is prepared by immunizing animals with the truncated recombinant chicken TRIM45 protein.

5. The use according to claim 1, wherein the preparation method of the truncated recombinant chicken TRIM45 protein comprises the steps of: (1) performing protein sequence analysis on chicken TRIM45; (2) designing a specific primer pair for the gene sequence encoding the truncated chicken TRIM45, and annealing the primer pair with the plasmid pCold1 after PCR amplification to obtain a recombinant pCold1-His-TRIM45 plasmid, and transforming the recombinant pColdI-His-TRIM45 plasmid into competent cells, wherein the gene sequence of the truncated chicken TRIM45 is shown in SEQ ID No. 2; (3) selecting a positive clone obtained in step (2) and culturing it in LB medium with ampicillin, and then inducing the expression of TRIM45 protein by IPTG or isopropyl-β-D-thiogalactoside, and extracting the protein to obtain the recombinant truncated chicken TRIM45 protein.

6. The use according to claim 5, wherein the nucleotide sequence of the primer pair mentioned in step (2) is shown in SEQ ID Nos.3 - 4.

The use according to claim 5, wherein in step (2) the truncated sequence of the chicken TRIM45 and the insertion sites in the pCold! plasmid are NdeI and XhoI.

8. The use according to claim 5, wherein the competent cell in step (2) is BL21(DES3).

9. The use according to claim 4, wherein the method of preparing the polyclonal antibody comprises the steps of: (1) purifying recombinant truncated chicken TRIM45 protein by Ni column chromatography, and then renaturing the protein; (2) immunizing animals with purified protein and renatured protein to obtain serum containing polyclonal antibody of TRIM45.

10. The use according to claim 1, wherein the avian leukemia is caused by ALV-J infection.