Construction And Identification Of The Attenuated Vaccinia Tiantan Strain With Multiple Genes Deleted

Vaccinia virus has many characteristics that make it an excellent research system andexperimental tool, including:(1) its wide mammalian host range,(2) its ability to elicit stronghumoral and cell-mediated immune responses,(3) its large dsDNA genome while retaininginfectivity and immunogenicity,(4) its exceptional thermal stability,(5) the fact that it is easy topropagate and (6) as a vaccine during the smallpox eradication campaign. According tohomologous recombination, using vaccinia virus as a vector to express foreign proteins, requiresinserting exogenous genes into the viral genome region in which genes were non-essential forviral replication. With the function of VACV promoter，the recombinant vaccinia virus couldexpress foreign proteins within cell. In order to conduct a attenuated strain carrier, deletingmany host range genes, virulence genes and other non-essential genes for viral replication fromthe vaccinia virus genome, which also could weaken the diffusion level of virus.Vaccinia Virus TianTan strain was isolated in China, the virulence of the virus is relativelyweak. By analysis of the viral genome, selected seven genes associated with virulence, hostrange and other non-essential gene functions for viral replication to knockout in order to achievereduced viral virluence and narrowed host range of. With homologous recombination, wecombined EGFP gene as color screening with TK gene as tk-negative selection with Brdu,aswell as Cre/Loxp system to konckout EGFP. Vaccinia virus attenuated strain were acquired.Using PCR method for the identification and analysis of genetic stability of the recombinantvirus. The difference between recombinant virus and parent strain in their virulence and hostrange were intected by cell level testing and animal test rating.Results showed that multi-gene deletion recombinant attenuated strain of vaccinia virusTTVAC7has a good genetic stability, and genes knockout did not affect the replication andmorphogenesis of virus. Crystal violet assay indicated the genes deleted reduced the diffusionlevel of vaccinia virus. MTT testing proved that the missing genes diminished cytotoxicity ofthe virus. Intracranial inoculation and nasal inoculation of mice and rabbits pox plaqueformation test had shown that multi-gene deletion made the parent strain virulence significantlyweakened. The attenuated vaccinia virus as a vector of exogenous gene expression system andviral vector has scientific value and potential application prospects.