Prokaryotic Expression Of Recombinant ALV-A Gp85 Gene And Its Application

Avian leukosis of subgroup A is a contagious disease caused by a retrovirus. The chicken infected by subgroup A always produce lymphoid neoplastic diseases, immunosuppression and other production problems. Avian leukosis of subgroup A become a serious disease,which does severely harm to local strains of chickens and reduce the economic loss in our country. So far, there has been no effective diagnostic methods and vaccine for ALV-A. In this study, by using the env-gp85 gene of ALV-A, the immunoprotection of ALV-A subunit vaccines were evaluated, meanwhile the development of monoclonal antibodies(Mabs) to ALV–A were developed which aims to provide scientific evidence for developing measures to diagnose and control ALV-A.Cloning and expression of gp85 gene of ALV-A. a pair of specific primers was designed based on the published ALV-A gp85 gene sequence..909 bp genes of gp85 was expressed by prokaryotic expression.The expressed product was confirmed using SDS-PAGE and western blot that it is about 46 KD of recombinant protein.Immunoprotection of recombinant ALV-A gp85 protein. The purified recombinant proteins combining with CpG-ODN adjuvant or Freund’s adjuvant were inoculated into the breeder hens, the ALV-A antibodies in serum and in egg-yolk were detected; the fertilized eggs from the vaccinated hens with different titers of egg-yolk antibody were hatched and then challenged with ALV-A-SDAU09C1 strain, all the hatched chickens were weekly detected for the viremias and the cloacal swab p27 antigen and pathological lesions; the neutralizing test of antisera in vitro was conducted. The results are as follows:(1) the recombinant gp85 proteins combining with CpG-ODN adjuvant could induce the breeder hens to produce better antibody responses than gp85 protein with Freund’s adjuvant or without adjuvant.(2) the MAbs with higher titers induced by CpG-ODN+gp85 proteins could obviously decrease the ratios of viremias, cloacal detoxification and death caused by ALV-A infection than control group.(3) the results of the neutralizing test indicated that the antisera from the hatched chickens could neutralize the ALV-A in vitro, but which depends on theantibody titers. The results of IFA confirmed that the serum antibody could combine with the ALV in DF-1 cells.The study of monoclonal antibodies to subgroup A avian leukosis virus. The purified recombinant proteins combining with Freund’s adjuvant were inoculated into female BALB/c mice, 6 weeks old. One strain(A14GZ) was selected after tested by ELISA and IFA.The antibody was determined by ELISA, IFA and immunohistochemistry. The results are as follows:(1) The positive hybridoma cells stain A14 GZ were determined by ELISA and IFA,which ELISA titers of cell supernatants and ascites were 1:26and1:213.(2) the McAb were identified by the indirect immunofluorescence(IFA) method and using ALV-J, ALV-B,ALV-A which were inoculated to CEF cells. The results indicated that the McAb could be used as specific reagent to detect ALV-A.(3) IFA using McAb against ALV-A was established and used to detect ALV-A antigen in paraffin-embedded tissues of death chickens which experimentally infected with ALV-A. The green fluorescent signals were obviously observed in the cytoplasm of virus-infected cells.These results indicate that ALV-A gp85 recombinant protein was successfully prepared using E.Coli expressed system, and combining with adjuvant, could induce the vaccinated breeder hens to produce higher titers of serum antibodies; the maternal antibodies from the hens vaccinated with gp85 protein plus CpG-ODN adjuvant could protect 80% of their offspring chickens against early ALV-A infection. In addition, the positive hybridoma cells stain A14 GZ which could stably secrete monoclonal antibodies(Mabs) to ALV–A were obtained by using recombinant ALV-A gp85 protein, the secreted antibodies could be used as specific reagent to identify and detect ALV-A.