Construction Of Mutant Infectious Clones Of Chicken Anemia Virus And Its Rescue

Chicken anemia virus(CAV)is one of the important immunosuppressive pathogens that damage the chicken industry,the gene of CAV encodes VP1,VP2 and VP3 proteins,each protein plays a different role in its infection,and they are interrelated.The study based on the previous research of isolation and identification of CAV clinical isolates and the construction of viral infectious clone pcDNA-2CAV,the recombinant plasmids were identified by site-directed mutations of some amino acids at the important functional regions of the three proteins in vivo and in vitro experiments respectively,and then carried out virus rescue.(1)Construction of double-copy CAV mutant infectious clonesAccording to the sequence of CAV gene isolated from our laboratory,the amino acid(amino acid glutamine)at position 394 of VP1,amino acid of VP2 at position 101(arginine in phosphorylated group)and VP3 85 th amino acid(nuclear localization signal within the serine)were determined by Megaprimer PCR mutation method to site-directed mutations.The whole genome sequence of point mutation was amplified by PCR.The recombinant plasmid was transformed into Escherichia coli DH5?,and the positive clones were screened by suicide gene and bacterial liquid PCR.The positive clones were sent to the biotechnology company The recombinant plasmids containing the correct mutants were digested with Kpn?and Xhol?and ligated with the eukaryotic expression vector pcDNA3.1(+).The whole genome of CAV containing Kpn?cleavage site was obtained by PCR and ligated with Kpn?single-copy single-copy infectious clone mutant,and transformed and identified.The results showed that the mutant clones were identified as pcDNA-2CAV-VP1Q394,pcDNA-2CAV-VP2R101,pcDNA-2CAV?VP3 and pcDNA-2CAV?VP3,respectively,according to the location and function of the mutations.PcDNA-2CAV-VP3S85.(2)The rescue of CAV mutant virusFirst,the SPF chicken embryo was used to detect the infectivity of the constructed recombinant mutant plasmid to the body tissue.The 7-day-old SPF chickens were randomly divided into three groups.The chickens were inoculated with different mutant recombinant plasmids and control samples.Each group had three replicates.The chick embryo was incubated until the 19 th day.The tumor was observed and the tissues of the thymus were collected.Method to analyze virus-specific genes.The results showed that only inoculated pcDNA-2CAV-VP1Q394 experimental group of chicken embryo leg bleeding.Virus-specific gene detection revealed that the mutant CAV gene in the experimental group was present in the chicken embryo tissue of SPF,indicating that these mutant recombinant plasmids could proliferate after infecting the chicken embryo.In order to obtain an infectious mutant having a proliferative ability on the MSB1 cell line,the four plasmids constructed above were transfected into MSB1 cells by liposome 3000 for virus rescue.The apoptosis of the 5th generation cells was observed by fluorescence staining,and the expression of the viral genes and proteins were detected by PCR and Western blotting respectively.The results showed that the mutant virus gene could be detected in the process of cell passage,except for the VP3 deletion group.The apoptosis of the mutant group was different,and the virus-related protein could be detected after VP3 deletion mutation expression.These results demonstrate that CAV mutant infectious clones were successfully constructed,and these mutant clones could produce different infective effects on MSB1 cells.The virus was infected with normal cells,and the cells were passaged,and only the pcDNA-2CAV-VP1Q394 rescue virus infection group was found to have mutated virus,and the virus was named CAVVP1Q394.In this study,four major copies of the double-copy CAV mutant clones were constructed and the mutations of the mutants were analyzed by inoculating cells and chicken embryos.Which resulted in the rescue of CAVVP1Q394 mutant virus and the preliminary observation of its ability to treat the cells.These results laid a foundation for further study of the pathogenesis of CAV and provide a reference for the study of new gene vaccine candidate strains.