Specific antigen epitope for avian leukosis virus subgroup J, fusion protein, specific antibody and application thereof

An avian leukemia virus, antigen epitope technology, applied in the direction of virus/bacteriophage, virus, virus peptide, etc., can solve the problems of in vitro diagnosis of avian leukemia antibody subtypes, weak specificity, and inability to exclude antigen recognition sites.

Active Publication Date: 2017-06-20
CHINA AGRI UNIV
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Problems solved by technology

[0005] The current research methods for subgroup-specific antigenic epitopes are limited to the analysis of the specific subgroup gp85 protein under study. For example, to locate the J subgroup epitope is only analyzed for the J subgroup gp85 protein. In the detection method, the samples to be tested The mouse monoclonal antibody prepared for J subgroup-specific serum or ALV-J protein, this method can find the J subgroup epitope but the specificity is not strong, and it cannot be ruled out that the found antigen epitope region contains other avian leukosis subgroups group antigen recognition site
When using recombinant proteins to distinguish different subtypes of avian leukosis virus infection sera, due to the certain homology of the gp85 proteins of each subgroup, it is impossible to diagnose the subtypes of avian leukosis antibodies in vitro

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  • Specific antigen epitope for avian leukosis virus subgroup J, fusion protein, specific antibody and application thereof
  • Specific antigen epitope for avian leukosis virus subgroup J, fusion protein, specific antibody and application thereof
  • Specific antigen epitope for avian leukosis virus subgroup J, fusion protein, specific antibody and application thereof

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Experimental program
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Embodiment 1

[0053] Embodiment 1, polypeptide synthesis:

[0054] Obtain the amino acid sequences of gp85 proteins of ALV-A, ALV-B, and ALV-J viruses representing epidemic strains on NCBI, and the protein sequence numbers are: gi / 559807359, gi / 382933114, and gi / 350606570, respectively. Using ClustalX2 combined with MEGA5.1 software to compare the gp85 proteins of these three subgroups, the results are as follows figure 1 . The rule for selecting polypeptides from the gp85 protein sequences of ALV-A, ALV-B, and ALV-J viruses is the region with a large difference between the AB subgroup and the J subgroup in the comparison results. 15 peptides were selected, including 7 peptides from subtype J and 8 peptides from subtype AB. The synthesized peptide sequence is shown in Table 1 below, synthesized by a peptide solid-phase synthesizer, and desalted for preliminary purification.

[0055] Table 1 Synthetic sequences of ALV-A, ALV-B, and ALV-J subgroup-specific polypeptides

[0056] ...

Embodiment 2

[0057] Embodiment 2, screening epitope recognition region:

[0058] The 15 peptides were screened with AB subgroup and J subgroup specific sera, and the effects of the peptides were detected by indirect ELISA method. The specific detection method is: take the 15 synthesized peptides and resuspend them in DMSO to 20mg / ml, dilute them with pH9.6 carbonate buffer (coating solution) to 20μg / ml respectively and coat them on CORNING microtiter plate, overnight at 4°C , the uncoated group was used as the control for the judgment standard of negative and positive; add 2% skimmed milk to block at 37°C for 2 hours; add 300 μl PBST to each well to wash, shake off the residual water in the microplate each time the liquid is poured out and put it on a clean absorbent paper Dry the upper control, wash 5 times; add AB subgroup and J subgroup specific serum 100 μl / well, serum dilution factor is 50 times, 250 times, 1000 times, incubate at 37°C for 1 hour, wash 5 times; add 1:5000 Double-dilu...

Embodiment 3

[0061] Embodiment 3, detect the specificity and sensitivity of antigenic epitope:

[0062] The polypeptides with specificity are obtained through the above ELISA screening, satisfying that the peptides from the J subgroup are only responsive to the J subgroup specific serum, and the AB subgroup polypeptides are only responsive to the AB subgroup specific serum. Preliminary simulation of the detection effect of the ELISA kit by reacting with field chicken serum, a total of 156 samples: 6 samples of standard negative serum; 82 sera; 77 J-specific sera.

[0063] Use the indirect ELISA method to detect the specificity and sensitivity of the antigenic epitope: the polypeptide is diluted to 20 μg / ml with the carbonate buffer (coating solution) of pH 9.6 to coat the CORNING microtiter plate, overnight at 4°C; add 2% degreasing Seal the milk at 37°C for 2 hours; add 300 μl PBST to each well to wash, shake off the residual water in the microplate plate each time the liquid is poured o...

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Abstract

The invention belongs to the technical field of animal disease detection, and discloses a specific antigen epitope for avian leukosis virus subgroup J, a fusion protein, a specific antibody and an application thereof. The amino acid sequence of the specific antigen epitope for avian leukosis virus subgroup J is shown in SEQ ID NO: 1. Experiments show that the antigen epitope responses to the serum of the subgroup J and does not responses to the serum of the subgroup AB, and can specifically recognize the avian leukosis virus subgroup J. The fusion protein can be prepared into a vaccine to stimulate a receptor to generate an antibody and protect the receptor against the infection of the avian leukosis virus subgroup J. The fusion protein can immunize animal to prepare the specific antibody of the avian leukosis virus subgroup J. The specific antibody of the avian leukosis virus subgroup J can be specifically recognized with the specific antigen epitope for avian leukosis virus subgroup J, and a detection kit is developed to distinguish the avian leukosis subgroup AB and the avian leukosis subgroup J.

Description

technical field [0001] The invention belongs to the technical field of animal disease detection, in particular to J subgroup avian leukosis virus specific antigenic epitope, fusion protein, specific antibody and application thereof, especially J subgroup avian leukemia virus GP85 specific antigenic epitope, fusion Protein, specific antibody and J subgroup avian leukemia detection kit. Background technique [0002] Avian leukosis is a poultry neoplastic disease caused by avian leukosis virus (ALV) and avian sarcoma virus (ASV), which is a serious hazard to the poultry industry in my country. ALV / ASV can be divided into 10 subgroups A-J, among which A, B and J subgroups are exogenous viruses that occurred in field chickens. At present, there is no effective treatment for the disease, and the spread of the disease is mainly controlled by purifying the population of positive chickens. J subtype ALV can be transmitted horizontally and vertically, and vertical transmission can l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/15A61K39/21A61P31/14G01N33/569
CPCA61K39/00C07K14/005C12N2740/11022C12N2740/11034G01N33/56983G01N2469/20
Inventor 王永强曹红李晓齐郑世军陈福勇
Owner CHINA AGRI UNIV
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