Establishment Of An Indirect ELISA For Detecting Antibodies Of Subgroup J ALV And Preparation Of Monoclonal Antibodies Against Gp85Protein

Subgroup J avian leukosis virus (ALV-J) belongs to the group of avian leukosis virus or avian sarcoma virus in a Papovaviridae, Retroviridae. ALV-J was first isolated from broiler chickens by Payne in1991; it induced avian myelcytomatosis (ML) in chickens and caused great economic losses of the chicken industry in many countries. The virus has a capacity of horizontal transmission and vertical transmission. To sweep ALV-J away from the populations is the only effective way to prevent this disease by now, so, establishing an accurate, rapid and sensitive diagnostic method is in urgent need.In this study, the gp85gene of ALV-J was amplified from a field strain by PCR and obtained GenBank accession number:JF911743, and cloned into expression vectors pET-32a(+) and pGEX-6P-1. Constructed recombinant plasmid were named pET-32a(+)-gp85and pGEX-6P-1-gp85and transformed into E.coli BL21(DE3) or BL21respectively. Expression of recombinant fusion proteins were induced by IPTG and identified by SDS-PAGE. Recombinant fusion proteins His-gp85and GST-gp85were purified with High-Affinity Ni-IDA Resin and High-Affinity GST Resin respectively. SPF chickens were intramuscular injected with purified His-gp85protein emulsified with Freund’s complete adjuvant, then used the same way to immune every two weeks for positive serum. The indirect ELISA method which used purified GST-gp85protein to coat the ELISA plate and took the positive serum as first antibody and took the goat anti-chicken IgG labelled by HRP as second antibody was established to detect antibodies against ALV-J gp85.Six-week-old BALB/c mice were subcutaneously injected with purified GST-gp85 protein emulsified with Freund’s complete adjuvant, each one with200μg, then used the same way to immune with purified GST-gp85protein emulsified with Freund’s incomplete adjuvant every two weeks, each one with100μg. An intraperitoneal booster of the same dose of GST-gp85was given4days before splenocytes were collected and fused to SP2/0myeloma cells. The ELISA plates were coated with purified His-gp85protein and hybridoma culture supernatants were screened by indirect ELISA method, then selected the positive to subclone and obtained6hybridoma cell lines secreting monoclonal antibody anti-gp85antigen. Each BALB/c mouse was intraperitoneal injected5×105hybridoma cells to prepare ascites and the biological character of McAbs was identified. Indirect immunofluorescence and Western-blot approved that the McAbs were specific against purified protein.