Construction Of Infectious Clone Of A CIA Virus Strain Isolated From Anhui Province And Antibody Preparation Of Apoptin

Chicken infectious anemia virus(Chicken anemia virus comes,CIAV)widely distributed in the world,and is one of the main pathogens in poultry raise industry.Chicken is the only natural host of the disease caused considerable economic losses in poultry industry as a result of a co-infection and a secondary affection.When chickens infected by CIAV tend to appear anemia,lymphoid organ atrophy and immunosuppression.In our study,a strain of CIAV from a farm was identified in Anhui province.Infectious molecular clone of CIAV was constructed based on the whole genome of CIAV.Moreover,Apoptin,its virulence factor,was expressed.The studies were laid foundation for vaccine designing and the virus' s pathogenesis.Firstly,we extracted DNA from the bone marrow of the sick chicken and amplifyed the viral genes using specific primers by PCR methods.The genes were connected with pMD18-T vector.The recombinant pasmids were inditified by sequence analysis.The result showed that the sick chicken infected with CIAV named CIAV-HF211.DNA homology of the key structural protein VP1 was analyzed between CIAV-HF211 and other isolated strains.The strain CIAV-HF211 is highest homologous with a strain AY846844 from Tianjin.Secondly,the genome of the CIAV-HF211 was amplifyed by a pair of specific primers and inserted into the eukaryotic expression vector pcDNA3.1(+),to build pcD-CIAV.On this basis,the second full genome sequence of CIAV was inserted into the pcD-CIAV,to construct the pcD-2CIAV.Then one-day-old SPF chickens were injected with the pcD-2CIAV through leg muscle injection.The control groups were injected with PBS or infected tissue grinding fluid.After 12 days,we observed the pathological lesions and detected the gene of the virus.The results show that the infectious molecular clone can cause similar pathological changes compaired with CIAV infection.The gene amplification of CIAV was positive in chikens injected with the clone pcD-2CIAV and tissue grinding fluid.The results suggested that the infectious clone pcD-2CIAV were successfully constructed.The results laid a foundation for the further research in pathogenesis of CIAV-HF211 strains and vaccine development.Finally,we also cloned apoptin gene of CIAV into prokaryotic expression vector pET-32a(+),and transformed it into E.coli Rosetta(DE3).The positive strains was induced with IPTG and analyzed by SDS-PAGE.His-Apoptin fusion protein were refinedby affinity chromatography.The serum separated after immune was used as antigen,an indirect ELISA was established for detecting the antibody level of fusion protein.The immunogenicity of fusion protein was analyzed by western blot method.The result showed that antibody titer could actually reach 1:1280000.The antiserum could react specifically with fusion protein by western blot analysis.Together,an infectious molecular clone pcD-2CIAV was successfully constructed based on the strain of CIAV identified from Anhui province.The indirect ELISA was established for detecting the antibody level of fusion apoptin.Our results lay the groundwork for further study of CIAV pathogenesis and designing new vaccines.