Cloning And Expression Of Gp85Gene Of Avian Leukosis Virus Subgroup J And The Application Of Recombinant Protein

Avian leukosis(AL) is a general designation of avariety of avian neoplastic diseases, which is caused by avian leukosis virus(ALV). Avian Leukosis Virus subgroup J(ALV-J) from broilers was identified as a new subgroup in1991. Its epidemic and pathogenicity are stronger than other subgroups, which can cause myeloid leukosis(ML) and enormous economic losses to the global cultivation. Recent years, ALV-J caused epidemic in several provinces and cities in China. gp85gene is the specific nucleotide sequence of ALV-J. In this study, recombinant gp85protein was prokaryoticly expressed in E.coli and an indirect ELISA for detecting the antibody of ALV-J was established. And the monoclonal antibodies against gp85protein were developped by hybridoma cell fusion technology. The study provided the basis for serum monitoring of ALV-J and development of antigen diagnostic kits. The results were showed as follows:(1) According to the gp85gene of ALV-J strains published by GeneBank, a pair of specific primers was designed. The ALV-J strain XT0908was used as template, and the gp85gene was amplified by RT-PCR. The nucleotide sequence of gp85gene had93.6%-98.2%similarity with other ALV-J strains. The recombinant plasmid pET-28a(+)-gp85was constructed. And the recombinant gp85protein was expressed in E.coli and purified with Ni Sepharose6fastflow.(2) The recombinant protein was used as coating antigen and the indirect ELISA method for the detection of serum antibody was established. The method showed good repeatability and speciicity. The clinic serum samples were detected by this method. The result demonstrated an agreement of96.29%with commercial ELISA Kit(IDEXX).(3) Balb/C mice were immunized with the recombinant gp85protein. After the immune program was finished, the spleen cell was taken and fused with SP2/0myeloma cell. Positive cells were subcloned to get monoclonal cell lines by limit dilution. The hybridoma cell was identified and was used to produce ascites. The ascites was purified by bitterness-ammonium sulfate precipitation. Five cell lines secreting the monoconal antibodied against gp85protein were screened and named1G4,1B9,3A1,3D12and5G5. The antibody titer of hybridoma cells was higer than27, and the ascite was higher than100×27. The Western-boltting showed that all the monoclonal antibody had a good reactogenicity with gp85protein.