Chicken Anemia Virus Vp1 Gene Cloning, Prokaryotic Expression, Purification And Preparation Of Polyclonal Antibody

Chiken Anemia Viru(sCAV)is the pathogen of Chiken Infectious Anemia(CIA). CAV belongs to Circoviride. CAV genome contains three open reading- frames(ORFs),which encodes three kinds of proteins: VP1(51.6KD), VP2(24.0KD) and VP3(13.6KD). VP1 is the only caspid protein of CAV, VP2 might act as a helper protein to help VP1 form correct conformation ; VP1 can induce chicken to produce immune protection with the help of VP2 protein. VP3 , also named apoptin , is related to the pathogenicity of CAV, which can induce cell apoptosis。Two pairs of primers were designed and synthesized according to the sequence of VP1 gene on the basis of the sequence of CAV genome from Genbank. And the whole of VP1 gene and 608bp VP1 gene was amplified by PCR technique from the cloning plasmid pUC-CAV. Then the recombinant expression plasmids pET 28a(+)-VP1,pET41a(+)-VP1'(608bp) and pET32a(+)-VP1'constructed after double restriction endonucleases digestion and the host strain DH5αwas transformed. The selected clones were verified by double restriction endonucleases and DNA sequencing. DNA sequencing results showed that the sequence of DNA fragments inserted into the vector was identical to the one that had been reported before from Genebank. The plasmid was transformed into E.coli BL-21 (DE3) to express VP1 protein with 28℃-37℃and 1mM IPTG induced, But the fused protein only expressed in the induced DE3 which contains pET32a(+)-VP1',others were not expressed. The expressed protein with molecular weight of Mr 42 000 was detected by SDS-PAGE electrophoresis and Western blotting. The protein VP1 fused with Trx-His tag. And inclusion bodies would form under the kind of condition and most of the fused protein was contained within them.The expressed fuse protein was purified by the method of affinity chromatography according to the manual of the producer. Then the purified protein VP1 fused with His tag was detected by Western blotting. The purified product showed good reactivity to anti-His tag antibody .The purified protein was dialyzed with PBS .The furified protein in the gel of SDS-PAGE electrophoresis was cutted and triturated with PBS.Emulsified it and aduvant to immunize BALB/C Mouse. Antiserum was gained after Mouse immunization and Western blotting were conducted afterwards.In brief, the fragment of 608bp VP1 gene was successfully expressed in BL-21 (DE3) contained the recombined plasmid pET32a(+)-VP1'. The molecular weight of the expressed protein was identical to the anticipant one. Then the expressed VP1 protein was purified by affinity chromatography and prepared as antigen for animal immunization. By Western blotting, the expressed protein with molecular weight of Mr 42 000 was detected by antiserum diluted.This research could provide base for preparation of monoclonal VP1 antibody, and lay foundatioan of strudy the relation of three proteins encoded by CAV.Otherwise, CAV isolates show extremely limited genetic variability worldwide. Given homologous recombination is an important viral evolutionary strategy, it is clearly important to determine the extent to which recombination plays a role in CAV evolution. In order to gain insight into these matters, we have performed a phylogenetic analysis of 31 full-length CAV strains isolated all over the world in order to detect possible recombination events. Putative recombinant sequences were identified with the use of SimPlot program. Recombination events were confirmed by bootscaning, using putative recombinant sequence as a query. A new genotype CAV resulted from recombination was found. And two putative parental-like strains of AF999018, DQ124936 and DQ141672 were identified. It suggests that homologous recombinant plays an extensive role for generating genetic diversity in natural populations of CAV.