CN110244039B

CN110244039B - ELISA kit for detecting J subgroup avian leukosis virus antibody and application thereof

Info

Publication number: CN110244039B
Application number: CN201910367890.2A
Authority: CN
Inventors: 高玉龙; 王笑梅; 潘青; 祁小乐; 王永强; 刘长军; 王牟平; 崔红玉; 张艳萍; 李凯; 高立; 高宏雷
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2019-05-05
Filing date: 2019-05-05
Publication date: 2021-12-24
Anticipated expiration: 2039-05-05
Also published as: CN110244039A

Abstract

The invention discloses an ELISA kit for detecting avian leukosis virus subgroup J antibody and application thereof. The ELISA plate comprises an ELISA plate coated with truncated ALV-J gp85 protein, wherein the amino acid sequence of the truncated ALV-J gp85 protein is shown as SEQ ID NO. 2. The invention utilizes the truncated and expressed J subgroup avian leukosis virus (ALV-J) gp85 protein to establish an ALV-J antibody indirect ELISA detection method, tests show that the method has good specificity, is not crossed with the AB subgroup ALV antibody, and has better sensitivity and specificity than similar imported kits.

Description

ELISA kit for detecting J subgroup avian leukosis virus antibody and application thereof

Technical Field

The invention relates to a virus antibody detection kit and application thereof, in particular to an ELISA kit for detecting avian leukosis virus subgroup J antibody and application thereof, and belongs to the technical field of virus detection.

Background

Avian leukemia Virus (Avian Ieukosis Virus, ALV) belongs to the family of retroviruses, and Avian type C retroviruses, and can cause various neoplastic diseases in birds. Avian leukemia has two main hazards, namely, tumor generation and death of chicken; secondly, the disease belongs to egg-borne disease, and the virus can be transmitted to offspring chicks through reproductive tracts, so that the next generation chicks are attacked; moreover, it can also cause immunosuppression of chickens, secondary infection with other bacteria and viruses, and cause serious economic loss. In 1997 and 1998, avian leukemia subgroup J has exploded worldwide, causing enormous economic losses to the poultry industry. Since 2008, the laying hens and local breeders in China outbreak severe avian leukemia, the pathogeny mainly takes subgroup J avian leukosis virus (ALV-J), which causes serious attack to the poultry industry in China and seriously threatens the provenance safety of poultry in China. For this reason, avian leukemia is preferably prevented and purified from epidemic diseases in Long-term programs for prevention and treatment of animal epidemic diseases in China (2012 and 2020), development programs for veterinary health care in China (2016 and 2020), and comments by Ministry of agriculture (2016 and 2016) on promotion of development of modern animal and poultry industries.

At present, no effective vaccine or medicine is available for the disease, and the main method for preventing the disease is to eliminate positive chickens and purify the population. Since 1868, Roloff reported avian leukemias, researchers conducted long-term research on them and achieved considerable success in the isolation, identification, detection, and diagnosis of viruses. A plurality of methods for detecting avian leukosis pathogeny are established, such as virus neutralization test, agar diffusion test, complement fixation test, ELISA, radio immunity, immunofluorescence technology and the like. However, antibody detection methods are less reported. Since 2008, the wide prevalence of leukemia of subgroup J of laying hens and local breeding hens in China causes huge economic loss to the poultry industry in China, so that the purification of avian leukemia is urgently needed to be carried out. Although the antigen detection reagent is mainly used for purifying the avian leukemia, the antibody detection is an important technical means for evaluating the purifying effect and whether the chicken flock is infected with the avian leukemia virus.

Therefore, establishing a method capable of rapidly and sensitively detecting the avian leukosis virus subgroup J antibody is an effective means for controlling the large-scale epidemic of the disease.

Disclosure of Invention

The invention aims to provide an ELISA kit for detecting avian leukosis virus subgroup J antibody and establish a detection method based on the kit.

In order to achieve the purpose, the invention adopts the following technical means:

the envelope protein gp85 of ALV-J contains antigenic determinants, is a protein determining the viral subgroup, and has a plurality of viral antigenic sites. However, the full-length gp85 expression product is taken as a coating antigen, has certain cross reaction with antibodies induced by other subgroup avian leukosis viruses, and the specificity is not very good, in order to improve the specificity, the inventor analyzes and compares gp85 amino terminal sequences of A (ALV-A), B (ALV-B) and J subgroup ALV (ALV-J) which frequently cause chicken flock diseases, selects an amino acid sequence segment with the lowest homology, constructs a stable expression cell line for truncating and expressing ALV-J gp 35 85, and realizes the stable expression of the truncating gp85 protein. Early studies demonstrated that eukaryotic expressed gp85 protein can bind to the cellular receptor of ALV-J, with completely correct conformation and function. In the invention, the inventor establishes an indirect ELISA method for detecting ALV-J antibody by using truncated ALV-J gp85 protein expressed by eukaryotic cells as a coating antigen. The results show that the lowest detected serum dilution for this method is 1: 102400 and ALV-J antibody detection kit of IDEXX company of USA is 1: 25600, sensitivity is 4 times higher than that of the imported kit. In addition, in a specificity experiment, the ALV-J antibody detection kit of IDEXX company not only reacts with the serum of ALV-J, but also has cross reaction with the positive serum of ALV-A and ALV-B, and the ELISA method established by the invention can only react with the positive serum of ALV-J, and the specificity is superior to that of an imported kit.

On the basis of the research, the invention provides an ELISA kit for detecting J subgroup avian leukosis virus antibody, the kit comprises an ELISA plate coated with truncated and expressed ALV-J gp85 protein, wherein the amino acid sequence of the truncated and expressed ALV-J gp85 protein is shown as SEQ ID NO. 2.

Preferably, the kit further comprises rabbit anti-chicken IgG labeled with HRP, a positive control, a negative control, a coating solution, a diluent, a developing solution, a washing solution and a stop solution.

Wherein, preferably, the positive control is avian leukosis virus positive animal serum of subgroup J; the negative control is serum of a healthy animal, preferably SPF chicken serum; the coating liquid is 50g/L of skimmed milk powder; the diluent is a PBST buffer solution containing 1% bovine serum albumin; the color development liquid is color development liquid TMB; the washing solution is PBST buffer solution; the stop solution is 2M H₂SO₄。

Preferably, when the kit is used for detecting avian leukosis virus subgroup J antibody, the kit specifically comprises the following steps:

(1) coating an ELISA plate of truncated and expressed ALV-J gp85 at 4 ℃ overnight, washing with a washing solution, sealing with a confining solution at 37 ℃ for 1h, and washing the plate;

(2) adding control and serum samples to be detected, incubating at 37 ℃, and washing the plate;

(3) adding rabbit anti-chicken IgG labeled by HRP, incubating at 37 ℃, and washing the plate;

(4) adding TMB developing solution, developing at 37 deg.C, adding stop solution to stop reaction, and measuring OD_405nmValue, when OD_450nmWhen the value is not less than 0.20, the test piece is judged to be positive.

Wherein, preferably, the coating amount of the truncated expression ALV-J gp85 is 2 mug/ml, the dilution multiple of the serum sample is 1:500, the dilution degree of the HRP-marked rabbit anti-chicken IgG is 1:3000, the action time of the serum sample, the HRP-marked rabbit anti-chicken IgG and the TMB is respectively 30min, 30min and 15min, and the confining liquid is 50g/L skimmed milk powder.

Preferably, the truncated ALV-J gp85 protein is obtained by expression and purification of a 293F cell line with the preservation number of CGMCC NO.17418 and capable of stably expressing truncated ALV-J gp 85.

Furthermore, the invention also provides application of the kit in preparation of a reagent for detecting avian leukosis virus subgroup J antibody.

Furthermore, the invention also provides a 293F cell line for stably expressing the truncated ALV-J gp85, which is named as 293F-JTgp85, and is classified and named as a 293F stably expressing cell line for expressing the J subgroup avian leukosis virus truncated gp85, wherein the cell line is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the microbial research institute of China academy of sciences, No. 3 of West Lu 1 of North Chen, Naja, Beijing, and the strain preservation number is 17418, and the preservation time is CGMCC NO. 3 and 28 days in 2019.

The amino acid sequence of the ALV-J gp85 protein which is generated by the expression of the 293F cell line for stably expressing the truncated ALV-J gp85 and is truncated and expressed is shown as SEQ ID NO. 2.

Nucleotide sequences encoding the truncated expressed ALV-J gp85 protein are also within the scope of the invention. Wherein, preferably, the nucleotide sequence is shown as SEQ ID NO. 1.

Furthermore, the invention also provides application of the truncated and expressed ALV-J gp85 protein in preparation of a reagent for detecting the J subgroup avian leukosis virus antibody.

Compared with the prior art, the invention has the beneficial effects that:

the invention utilizes the truncated and expressed J subgroup avian leukosis virus (ALV-J) gp85 protein to establish an ALV-J antibody indirect ELISA method and a kit thereof, has good specificity, is not crossed with the AB subgroup ALV antibody, and has better sensitivity and specificity than similar imported kits.

Drawings

FIG. 1 shows the result of fluorescence of 293T cell packaging lentivirus infected by pLVX-J-Tgp85 plasmid;

wherein: a: observing results by a fluorescence microscope; b: bright field cell observation;

FIG. 2 shows the fluorescence detection results of lentivirus-infected 293F cells after sorting;

FIG. 3 shows the result of the expression and identification of truncated ALV-J gp85 protein;

FIG. 4 shows the results of indirect ELISA sensitivity analysis;

FIG. 5 shows the results of indirect ELSIA specificity analysis.

Detailed Description

The invention will be further described with reference to specific embodiments and drawings, the advantages and features of which will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

EXAMPLE 1 construction of a leukocyte line stably expressing truncated ALV-J gp85 protein

1 Material Process

1.1 reagents and instruments

Fetal bovine serum was purchased from EXCELL corporation; DMEM high-glucose medium was purchased from trans gen; 293

CD 293M serum-free medium purchased from Shanghai culture Biotech; DH5 alpha competence colibacillus, eukaryotic expression vector pLVX, 293T cell, 293F cell strain and plasmid containing ALV-J gp85 purpose fragment are preserved by this laboratory; restriction enzymes BamH I and Xho I from Thermo; t4 ligase from NEB.

1.2 construction of eukaryotic expression vector for expressing truncated ALV-J gp85 protein

In order to ensure that the expressed ALV-J gp85 protein only reacts with ALV-J specific serum and does not have cross reaction with ALV (ALV-A or ALV-B) antibody of A or B subgroup, the inventor analyzes and compares ALV-J, ALV-A and ALV-B gp85 gene sequences, selects the gp85 gene of ALV-J domestic isolate JL08CH03-1, 121-777bp for codon optimization and synthesis, the optimized gp85 gene sequence is shown as SEQ ID NO.1, a signal peptide sequence is added upstream of the optimized gp85 gene sequence, and an Fc tag is carried downstream of the optimized gp85 gene sequence. Two ends of the sequence are Xho I and BamH I restriction enzyme sites respectively. After double digestion of the sequence, the gel is recovered, and the digested product is connected to a lentiviral expression vector pLVX digested by Xho I and BamH I by T4 ligase to construct a recombinant plasmid pLVX-J-Tgp 85. Transforming the recombinant plasmid into DH5 alpha competent cells, extracting the plasmid, carrying out double enzyme digestion identification on Xho I and BamH I, selecting positive clones for sequencing, and finally obtaining the recombinant plasmid named as pLVX-J-Tgp 85.

1.3 Lentiviral packaging

293T cells in DMEM Medium containing 10% fetal bovine serum at 37 ℃ and 5% CO₂Culturing in an incubator with saturated humidity. When the cells grew to 80-90% density, 0.1% PEI, plasmid pLVX-J-Tgp85 were expressed as 3: adding the mixture of 1 into a serum-free culture medium, mixing, incubating at room temperature for 15min, transfecting 293T cells, continuously culturing for 48h, observing the expression and distribution of green fluorescence in the cells under a fluorescence microscope, and collecting cell supernatant as packaged virus liquid.

1.4 construction of cell line stably expressing truncated ALV-J gp 85293F, expression, purification and identification of target protein

293F cells in 293M serum-free Medium in serum-free, 2% penicillin mixed solution at 37 ℃ and 5% CO₂Culturing in a shaking incubator with saturated humidity. And (3) carrying out passage on the 293F cell, adding the packaged virus liquid into the 293F cell, continuously culturing, observing the expression and distribution of a green fluorescent signal in the cell under a fluorescent microscope every 12h until the fluorescence rate of the cell is not increased any more, and sorting the cell expressing the green fluorescent protein by using an ultra-high-speed flow sorter. And placing the sorted cells into a culture flask for continuous culture. The supernatant of the sorted 293F cells was collected, protein A resin was added, and protein purification was performed after 12 hours of binding at 4 ℃. The collected eluate was subjected to SDS-PAGE gel electrophoresis, transferred to an NC membrane, and washed 3 times with PBST after transfer, the membrane was blocked in 5% skim milk for 1.5h, and then washed 5 times with PBST. Fc monoclonal antibody 1:3000 PBS was diluted to 30ml, incubated at room temperature for 1.5h, and the membrane was washed 5 times with PBS.After incubation for 1h at room temperature with the corresponding secondary antibody, the membrane was washed 5 times with PBS. The results were scanned using an Odyssey scanner.

2. Results

2.1 eukaryotic expression plasmid pLVX-J-Tgp85 identification

The plasmid pLVX-J-Tgp85 was digested by BamH I and Xho I, and agarose gel electrophoresis showed that about 1.4kb of the desired fragment was obtained, corresponding to the expected size. Sequencing the positive clone, wherein the result is consistent with the original sequence, and the result shows that the plasmid pLVX-J-Tgp85 is successfully constructed.

2.2 packaging and characterization of lentiviruses expressing truncated ALV-J gp85

The plasmid pLVX-J-Tgp85 is transfected into 293T cells, and after 48 hours, the observation under an inverted fluorescence microscope shows that the green fluorescence is obvious, thereby proving that the slow virus packaging is successful (figure 1).

2.3 construction and identification of the leukocyte line stably expressing truncated ALV-J gp85 protein

After the packaged lentivirus is infected into 293F cells, fluorescence appears in 24 hours, the fluorescence rate reaches the highest in 48 hours, the fluorescence rate is about 30%, and the fluorescence is stably expressed. As shown in FIG. 2, the green fluorescence positive rate of the cells after sorting by flow cytometry is more than 85%. Cell culture supernatants were collected and purified on protein A resin, and Western Blot analysis of purified products showed that the cell line could express truncated ALV-J gp85 protein efficiently with an expression level of 8mg/100ml (FIG. 3), and these results showed successful construction of 293F cell line stably expressing truncated ALV-J gp 85.

The 293F cell line stably expressing the truncated ALV-J gp85, which is obtained by the invention, is named as 293F-JTgp85, and is classified and named as 293F stably expressing cell line expressing the J subgroup avian leukosis virus truncated gp85, the cell line is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the address of the cell line is the institute of microbiology, China academy of sciences, No. 3, West Lu No.1 Hopkin, the Shanghai province, Beijing, the strain preservation number is CGMCC NO.17418, and the preservation time is 2019, 3 and 28 days.

Example 2 establishment of ELISA method

1 Material Process

1.1 reagents and instruments

Rabbit anti-chicken IgG and goat anti-mouse FITC secondary antibodies marked by HRP are purchased from Sigma company; protein a resin was purchased from GE: color developing solution TMB, available from tianggen corporation; enzyme-labeled plates were purchased from COSTAR.

1.2 selection of optimal reaction conditions

Under different reaction conditions, the reaction conditions such as the coating concentration (4, 2, 1, 0.5 and 0.25 mu g/ml) of the truncated ALV-J gp85, the working concentration (1: 125, 1: 250, 1:500, 1: 1000, 1: 2000 and 1: 4000) of the serum of a sample, the type (S-Blocking Buffer, 50g/L skimmed milk powder, 10g/L fish gelatin and an enzyme label plate stabilizer) of a Blocking solution, the dilution (1:8000, 1:5000 and 1: 3000) of rabbit-chicken-resistant IgG marked by HRP, the optimal working time (5, 10, 15 and 20min) of TMB work and the like are optimized. The optimum condition was chosen to be an OD450nm value close to 1.0 and a maximum P/N value.

1.3 determination of the decision criterion

184 SPF chickens were collected and sera were collected as negative sera for ELISA detection. And setting a negative and positive control. Calculating the average value OD450nm of the tested negative serum

And the deviation(s), under the condition that the judgment standard is that the negative control and the positive control are satisfied, the OD450 > +3s of the detection sample is judged to be positive, and the negative is not.

1.4 sensitivity and specificity assays

Under optimal reaction conditions, standard positive sera were diluted to 1: 100. 1: 200. 1: 400. 1: 800. 1: 1600. 1: 3200. 1: 6400. 1: 12800. 1: 25600. 1: 51200. 1: 102400, 1: 204800 and respectively detecting to determine the sensitivity of the method. 10 common avian virus antibodies such as ALV-A, ALV-B, avian anemia virus (CAV), avian reticuloendotheliosis virus (REV), avian Infectious Bursal Disease Virus (IBDV), H9 subtype avian influenza virus (AIV-H9), Newcastle Disease Virus (NDV), viral arthritis virus (ARV), infectious laryngotracheitis virus (ILTV) and the like 1: the specificity was evaluated by 500 dilutions.

2 results

2.1 determination of reaction parameters and establishment of Indirect ELISA method

The parameters of the indirect ELISA method for detecting ALV-J antibody are determined through experiments: the optimal coating amount of the truncated and expressed ALV-J gp85 is 2 mug/ml, the optimal dilution multiple of a serum sample is 1:500, the optimal dilution of HRP-labeled rabbit anti-chicken IgG is 1:3000, and the optimal action time of the serum sample, the HRP-labeled rabbit anti-chicken IgG and the TMB is 30min, 30min and 15min respectively. The confining liquid is 50g/L skimmed milk powder.

The indirect ELISA detection method comprises the following specific steps:

(1) coating an ELISA plate (coating amount is 2 mu g/ml) of truncated and expressed ALV-J gp85, washing with a washing solution after overnight at 4 ℃, sealing with 50g/L skimmed milk powder for 1h at 37 ℃, and washing the plate;

(2) adding a control and a serum sample to be detected with the dilution ratio of 1:500, incubating for 30min at 37 ℃, and washing the plate;

(3) adding HRP-labeled rabbit anti-chicken IgG with the dilution of 1:3000, incubating for 30min at 37 ℃, and washing the plate;

(4) adding color developing solution, developing at 37 deg.C for 15min, adding stop solution to stop reaction, and measuring OD_405nmThe value is obtained.

2.2 determination of Positive and negative criteria

Under the conditions of the reaction parameters, 184 parts of ALV-J negative anal serum are used for indirect ELISA, the average value is 0.076, the standard deviation is 0.031, the OD is taken according to the statistical principle and the formula x +3s is 0.167_450nmWhen the value is not less than 0.20, the test piece can be judged to be positive with a reliability of 99.9% and used as a judgment criterion for the detection result.

2.3 sensitivity and specificity assays

By using the indirect ELISA method established by the invention and the ALV-J antibody detection kit of IDEXX company, the determination method of 1: 100. 1: 200. 1: 400. 1: 800. 1: 1600. 1: 3200. 1: 6400. 1: 12800. 1: 25600. 1: 51200. 1: 102400 ALV-J positive sera at different dilutions. The result shows that the minimum detected serum dilution of the indirect ELISA method established by the invention is 1: 102400 (FIG. 4), ALV-J antibody detection kit of IDEXX company 1: 25600. the indirect ELISA method established by the invention is shown to be 4 times more sensitive than ALV-J antibody detection kit of IDEXX company (figure 4).

The detection results of 10 common infectious avian viruses such as ALV-A, ALV-B, CAV, REV, IBDV, AIV-H9, NDV, IBD, ARV, ILTV and the like show that the indirect ELISA method established by the invention is used for detecting OD_450nmAll values were less than 0.1 and all were negative, while ALV-J antibody detection kit from IDEXX corporation reacted with ALV-J positive sera as well as ALV-A and ALV-B positive sera (FIG. 5). The indirect ELISA method established by the invention is proved to have better specificity than ALV-J antibody detection kit of IDEXX company.

Sequence listing

<110> Harbin veterinary institute of Chinese academy of agricultural sciences (Harbin center of Chinese center of animal health and epidemiology)

<120> ELISA kit for detecting J subgroup avian leukosis virus antibody and application thereof

<130> KLPI190320

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 657

<212> DNA

<213> ALV-J gp85

<400> 1

ggcattccac agtaccccct gagcgccttc aagggatatg tcactaatgt tactgcttgc 60

caaaacgata ccgatttagc cagccaaaca gcatgcttga tacagactct aaatacgacc 120

ctcccttggg acccccaaga attggatatt ttagggtccc agatgatcaa gaacggcacc 180

aaccggacct gcgtgacctt cggttcggtg tgctataaga acaatggcag tagagtctgc 240

cacatttttg atgggaattt taatgggact ggtggggcgg aagcagaatt gcgtgacttc 300

ataacaaaat ggaaaggtga tgaccatctt ataaggccct atgtcaacca atcatggacc 360

atggtgtccc ccatcaacac cgagagcttc agcatcagca gccggtactg cggcttcacc 420

agcaacgaga cacggtacta caagggcaac ctttctgatt ggtgtaattc aaaaggggga 480

gaatggtcag cggggtacag caacgggaca caatgttcca gcaacaccac cgactgtgcc 540

ggcaattgca ccgccgagtg gaactactac gcctacggct tcacattcgg caagcagccc 600

gaggtgctgt ggaacaacgg aaccgctaag gcactccccc caggtatttt cttgatt 657

<210> 2

<211> 219

<212> PRT

<213> ALV-J gp85

<400> 2

Gly Ile Pro Gln Tyr Pro Leu Ser Ala Phe Lys Gly Tyr Val Thr 15

Asn Val Thr Ala Cys Gln Asn Asp Thr Asp Leu Ala Ser Gln Thr 30

Ala Cys Leu Ile Gln Thr Leu Asn Thr Thr Leu Pro Trp Asp Pro 45

Gln Glu Leu Asp Ile Leu Gly Ser Gln Met Ile Lys Asn Gly Thr 60

Asn Arg Thr Cys Val Thr Phe Gly Ser Val Cys Tyr Lys Asn Asn 75

Gly Ser Arg Val Cys His Ile Phe Asp Gly Asn Phe Asn Gly Thr 90

Gly Gly Ala Glu Ala Glu Leu Arg Asp Phe Ile Thr Lys Trp Lys 105

Gly Asp Asp His Leu Ile Arg Pro Tyr Val Asn Gln Ser Trp Thr 120

Met Val Ser Pro Ile Asn Thr Glu Ser Phe Ser Ile Ser Ser Arg 135

Tyr Cys Gly Phe Thr Ser Asn Glu Thr Arg Tyr Tyr Lys Gly Asn 150

Leu Ser Asp Trp Cys Asn Ser Lys Gly Gly Glu Trp Ser Ala Gly 165

Tyr Ser Asn Gly Thr Gln Cys Ser Ser Asn Thr Thr Asp Cys Ala 180

Gly Asn Cys Thr Ala Glu Trp Asn Tyr Tyr Ala Tyr Gly Phe Thr 195

Phe Gly Lys Gln Pro Glu Val Leu Trp Asn Asn Gly Thr Ala Lys 210

Ala Leu Pro Pro Gly Ile Phe Leu Ile 219

Claims

1. An ELISA kit for detecting J subgroup avian leukosis virus antibody is characterized by comprising an ELISA plate coated with truncated and expressed ALV-J gp85 protein, wherein the amino acid sequence of the truncated and expressed ALV-J gp85 protein is shown as SEQ ID NO. 2.

2. The kit of claim 1, further comprising an HRP-labeled rabbit anti-chicken IgG, a positive control, a negative control, a blocking solution, a diluent, a developing solution, a washing solution, and a stop solution.

3. The kit of claim 2, wherein the positive control is avian leukosis virus subgroup J positive animal serum; the negative control is serum of a healthy animal; the confining liquid is 50g/L skimmed milk powder; the diluent is PBST buffer solution containing 1% w/v bovine serum albumin; the color development liquid is color development liquid TMB; the washing solution is PBST buffer solution; the stop solution is 2M H₂SO₄。

4. The kit as claimed in claim 1, wherein the kit is used for detecting avian leukosis virus subgroup J antibody, and comprises the following steps:

(4) adding TMB developing solution, developing at 37 deg.C, adding stop solution to stop reaction, and measuring OD_{405 nm}Value, when OD_450nmWhen the value is not less than 0.20, the test piece is judged to be positive.

5. The kit of claim 4, wherein the amount of coating of truncated-expressed ALV-J gp85 is 2 μ g/ml, the dilution factor of the serum sample is 1:500, the dilution factor of HRP-labeled rabbit anti-chicken IgG is 1:3000, the action time of the serum sample, HRP-labeled rabbit anti-chicken IgG and TMB is 30min, 30min and 15min, respectively, and the blocking solution is 50g/L skimmed milk powder.

6. The kit of claim 1, wherein the truncated ALV-J gp85 protein is obtained by expression and purification of a 293F cell line with a preservation number of CGMCC NO.17418 and capable of stably expressing truncated ALV-J gp 85.

7. Use of a kit according to any one of claims 1 to 6 in the preparation of a reagent for detecting avian leukosis virus subgroup J antibodies.

8. The 293F cell line for stably expressing the truncated ALV-J gp85 is characterized by being named as 293F-JTgp85, being preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the culture preservation number of CGMCC NO. 17418.

9. A truncated ALV-J gp85 protein expressed by the cell line 293F of claim 8 stably expressing truncated ALV-J gp85, wherein the amino acid sequence is shown in SEQ ID No. 2.

10. A nucleotide sequence encoding the truncated expressed ALV-J gp85 protein of claim 9.

11. The nucleotide sequence of claim 10, wherein the nucleotide sequence is set forth in SEQ ID No. 1.

12. Use of the truncated-expressed ALV-J gp85 protein of claim 9 in the preparation of a reagent for detecting avian leukosis virus subgroup J antibody.