Isolation And Identification Of Chicken Infectious Anemia Virus And Preparation Of Monoclonal Antibody Against VP3

Chicken infectious anemia virus(CIAV)is an important pathogen associated with immunosuppressive disease in poultry.The disease caused by CIAV has been reported in the major poultry countries and has been spread globally,resulting in huge economic losses to the poultry industry.In this study,the isolation and identification of chicken infectious anemia virus and the whole genome sequencing of CIAV-CZC1602,the construction of prokaryotic CIAV-VP3 expression vector and the development of monoclonal antibody against CIAV-VP3 protein were performed which will be of great significance for further studying on CIAV epidemiology,genetic variation and development of CIAV detection methods..1.Isolation and identification of chicken infectious anemia virus and the genetic evolution analysis of CIAV-CZC1602 isolateThe chickens suspected with the infection of chicken infectious anemia virus(CIAV)in farmns from Jiangsu and the vaccines suspected with the contamination of CIAV were first detected and diagnosed for CIAV through PCR amplification for CIAV specific DNA fragment.The PCR positive tissue samples and vaccine samples were then homogenated and inoculated into MDCC-MSB1 cells for virus isolation.Four CIAV viruses were successfully isolated and named CIAV-CZC1602,CIAV-LHC1710,CIAV-HJC1709 and CIAV-YC1711 respectively.To understand the variation of the whole genome of CIAV isolates,the whole genome of CIAV-CZC1602 was amplified and analyzed.The results showed the full-length genome of CIAV-CZC1602 strain was 2298bp,and the nucleotide homology with other 21 CIAV strains from GenBank was between 96.1%-99%.Phylogenetic tree revealed that the CIAV-CZC1602 showed the closest relationship with the Beijing strain based on its genome.2.Construction of prokaryotic expression vector of VP3 protein of chicken infectious anemia virusIn this study,a pair of specific primers for CIAV-VP3 based on the gene sequence of CIAVcux-1 strain published by Genbank(accession number:M55918)were designed and the CIAV-VP3 gene from CIAV-CZC 1602 was successfully amplified by PCR.At the same time,a pair of linearized primers against the pGEX-6p-1 vector were designed,and the linear pGEX-6p-1 vector was amplified by PCR.The CIAV-VP3 gene fragment was then ligated with the linear pGEX-6p-1 vector according to one-step cloning method,and the recombinant plasmid pGEX-6p-1-VP3 was successfully generated and confirmed by sequencing.After induction by IPTG,the fusion protein GST-VP3 in the BL-21 cells transformed with pGEX-6p-1-VP3 was expressed with the size of about 35kDa and purified.It has good antigenicity.The purified GST-VP3 fusion protein here could provide a good antigen for the preparation of monoclonal antibody against CIAV-VP3.3.Preparation of monoclonal antibody against VP3 protein of chicken infectious anemia virusIn this study,the purified GST-VP3 fusion protein was used as an immunogen to immunize Ba1B/c mice,and the spleen cells from the vaccinated mice were harvested and fused with SP2/0 cells to prepare monoclonal antibodies against CIAV-VP3 protein.Through two screening methods including indirect ELISA and indirect immunofluorescence,two hybridoma cell lines secreting mAbs specific to different epitopes of VP3 protein were successfully identified,named CIAV-VP3-4D7 and CIAV-VP3-4G8,The subtype of CIAV-VP3-4D7 and CIAV-VP3-4G8 both were IgG1 and Kappa chains.Western blot assay confirmed that mAbs CIAV-VP3-4D7 and CIAV-VP3-4G8 could react with the GST-VP3 recombinant protein,but not with GST.Immunofluorescent assay also showed that mAbs CIAV-VP3-4D7 and CIAV-VP3-4G8 could efficiently recognize the VP3 protein expressed in DF-1 cells transfected with pCDNA3.1-VP3.The generation of mAbs CIAV-VP3-4D7 and CIAV-VP3-4G8 specific to VP3 laid the foundation for the subsequent study on the molecular characteristics of CIAV-VP3 and the development of VP3-based diagnostics of CIAV.