Expression Of Gp85Gene Of Subgroup A、B Aavian Leukosis Viruse And Comparison Of Antigenic Relatedness Of The Two Anti-gp85Serum

In order to get a rapid specific diagnostic reagent for subgroup A/B Avian Leukosis Virus detection and compare the antigenic relatedness of anti-gp85serum of Chinese strain SDAU09C1of subgroup A avian leukosis viruses and SDAU09C2of subgroup B avian leukosis viruses. The recombinant gp85protein had been expressed and anti-serum was made by immunizing rabbits with purified gp85protein.Env-gp85gene was amplified by polymerase chain reaction (PCR) with a pair of primers designed according to the published gp85gene sequence from plasmids respectively containing the env gene of Chinese strain SDAU09C1of subgroup A avian leukosis viruses and SDAU09C2of subgroup B avian leukosis viruses. The amplified products of1017bp and1038bp in size were cloned into pMD18-T and sequenced. The correct DNA fragment was then subcloned into prokaryotic expression vector pET-32a(+) at the location between restriction endonucleases BamH I and Hind Ⅲsites. The recombinant plasmids pET-SDAU09C1-gp85and pET-SDAU09C2-gp85were transformed into E coli. BL21for gp85gene expression and induced. The expression products with molecular weight of55.7kDa and56.5kDa were identified by SDS-PAGE as expected. The best inductive conditions were established:30℃,1mM IPTG concentration, induced expression5h. Because N-terminal of fusion protein contained six histidine, the target protein can be purifieated from the bacterial protein by nickel metal affinity chromatography. The His-tagged recombinant protein was purified using a His-tag affinity chromatography column on Ni2+-nitrilotriacetate(NTA)resin. The target protein was refolded in the method of dialysis, its concentration was relatively high, it is suitable to animal immunity. The recombinant proteins were proved to be specific with western blotting.Two rabbits were immunized by the recombinant fusion protein respectively and the serum were collected, identified by indirect immunofluorescence(IFA). The results showed that the titer of obtained gp85anti-serum was500,the optimal concentration for SDAU09C1anti-serum was1:100, while1:50for SDAU09C2anti-serum. Anti-gp85serum produced was reactive with ALV-A and ALV-B with different titers but not ALV-J. It demonstrated that the recombinant ALV-A and ALV-B gp85fusion protein demonstrated good antigenecity.The serological relatedness of SDAU09C1strain and SDAU09C2strain was determined using a virus-neutralization(VN) test. One serum was reacted against the other in a beta VN test in DF-1. The anti-serum produced by SDAU09C1strain had higher antibody titer, which having a high neutralizing titer for both the two isolates. Also its neutralizing titer value for SDAU09C2strain was even higher than that of the SDAU09C2anti-serum for its own virus. But the value for one’s own virus was significantly higher than the neutralization value for the heterologous virus. The results, determined by cross VN, revealed that the Relatedness(R) value between the two serums was36.6%.