Expression Of VP1 And VP2 Proteins Of Chicken Infectious Anemia Virus And Preparation Of Monoclonal Antibody Against VP2 Protein

Chicken infectious anemia caused by chicken infectious anemia virus(CIAV)is an immunosuppressive disease characterized by aplastic anemia and systemic lymphatic tissue atrophy in chickens.The disease spreads all over the world,which make it difficult to the development of poultry industry.The CIAV genome is about only 2.3 kb,and encodes three proteins.Among them,VP 1,as its only capsid protein,plays a key role in the process of CIAV inducing the host to produce neutralizing antibodies.VP2 is considered to be an accessory protein or conformation protein of VP1,which helps VP1 protein to fold correctly.It has dual-specifcity phosphatase activity,and plays an important role in the infection of CIAV.VP3,as a kind of apoptin,has the function of inducing cell apoptosis and been widely studied in the medical field.This study focused on three parts:expression of VP1 and VP2 proteins of CIAV in baculovirus expression system,prokaryotic expression of CIAV VP2 protein and preparation of its monoclonal antibodies,identification of the antigenic epitope of CIAV VP2 protein.These research results will be benefit for the research and development of the recombinant subunit vaccine of CIAV,establishment of detection methods,prevention and control of CIAV,and study of the related functions of VP2 protein.1.Expression of VP1 and VP2 proteins of chicken infectious anemia virus in Sf9 cellsIn order to explore the expression of VP1 and VP2 proteins in baculovirus system under different expression ways,the VP1 and VP2 genes of CIAV were amplified by PCR.The flexible peptide was added as linker between VP1 and VP2 genes.All the gene was ligated to the pFastBacHTA vector to construct recombinant donor plasmids pFastBacHTA-VP1,pFastBacHTA-VP2 and pFastBacHTA-VP1-linker-VP2 respectively.The recombinant donor plasmids were transformed into DH10Bac competent cells to produce recombinant bacmids,then they were transfected into Sf9 cells to generate three recombinant baculoviruses rBac-VP1,rBac-VP2 and rBac-VP1-linker-VP2.IFA and Western-blot results showed that VP1,VP2 and VP 1-linker-VP2 fusion protein were successfully expressed in Sf9 cells infected with rBac-VP1 and/or rBac-VP2,and rBac-VP1-linker-VP2.These results provided materials basis for the future research and development of the CIAV subunit vaccine and the prevention and control of CIAV.2.Prokaryotic expression of chicken infectious anemia virus VP2 and preparation of its monoclonal antibodiesIn order to prepare monoclonal antibodies against VP2 protein,the CIAV VP2 gene was cloned into prokaryotic expression vector pET-32a in this study to construct recombinant plasmid pET-32a-VP2.BL21(DE3)competent cells transformed with the recombinant plasmid pET-32a-VP2 was induced with 0.1 mM IPTG at 37? for 4 h to express the VP2 protein.The recombinant protein was found in SDS-PAGE and its molecular weight was about 50 kDa.The myeloma cells were fused with mouse spleen cells immunized with the recombinant protein VP2 for four times.The positive hybridomas were screened with rBac-VP2 infected Sf9 cells and pCAGGS-VP2-Flag transfected DF1 cells by IFA.Two hybridoma cell lines that can stably secrete monoclonal antibodies against CIAV VP2 protein were successfully obtained,which were named as CIAV-VP2-4A12 and CIAV-VP2-6B5.Western-blot results showed that the two monoclonal antibodies could react well with the VP2 protein expressing in different systems,which provided materials for the study of epitopes of VP2 protein.3.Identification of the antigenic epitope of chicken infectious anemia virus VP2 proteinIn order to map the epitope recognized by the monoclonal antibody CIAV-VP2-4A12,VP2 protein was gradually expressed in E coli using pET-32a as the expression vector which ligated with the truncated VP2 gene.The reactivity of truncated proteins reacted with CIAV-VP2-4A12 was verified by Western-blot.The results showed that the shortest epitope recognized by the CIAV-VP2-4A12 was 155KTVRW159.The epitope is highly conserved among 23 different strains of domestic and foreign CIAV strains in the GenBank.Our results laid the foundation for the study of the function of VP2 protein and the monitoring of CIAV.