Isolation And Expression Of VP2 Gene Of Chicken Anemia Virus And Epidemiology Of Chicken Infectious Anemia

A survey on the prevalence of chicken anemia virus (CAV) infection was performed by using the hematocrit value (HCT) test, polymerase chain reaction (PCR) and indirect immunofluorescent assay (IFA) in Liaoning, Jilin and Heilongjiang provinces in china. CAV was found in 2 of 4 grandparents broiler industries, 8 of 10 parents broiler industries and 89 of 95 commercial broiler industries.The pooled liver homogenates from field chickens was inoculated into specific-pathogen-free (SPF) chickens. After 14 days, we tested the HCTs of SPF chickens and infected MSB1 cells with the liver homogenates from SPF chickens. The results of IFA, PCR and other test confirm we isolated a CAV strain. It was designated J0105.The complete CAV genome of isolate of J0105 strain was amplified in two segments with two sets of primers. The two segments were inserted into PMD18-T vector and being sequenced. Analysis of the complete sequence of isolate of Belgium strain indicates that, the entire genome DNA of J0105 strain is 2298bp long, in which, VP1 gene contains 1350 residues, VP2 gene contains 651 residues, VPS gene contains 366 residues. The entire genome DNA of J0105 strain displayed 97. 8%, 97. 0% and 96. 6% nucleotide identities withCux-1 (clone 33) strain, SJ1 strain, and Netherlands avirulent strain respectively.We designed a set of primers for amplifying VP2 gene of isolate of J0105 strain, and inserted VP2 gene into PMD18-T vector. Then VP2 gene was inserted into Escherichia coli expressing vector PET~30b and baculovirus expressing vector pFASTBACHTa. The recombinant vectors were designated as PET-30bVP2 and pFBHTaVP2, respectively. To get recombinant baculovirus transposon rBacmidVP2, we transformed pFBHTaVP2 into DH10BAC competent cells. Recombinant baculovirus rBacVP2 was obtained by transfected sf9 insect cell with rBacmidVP2.SDS-PAGE analysis showed that VP2 gene was successfully expressed in E.coli BL21 strain and sf9 insect cell. The VP2 proteins which expressed by insect cell reacted with sera from CAV infected chickens in western blot analysis, but the VP2 proteins which expressed by BL21 not.