Virus Rescue And Study On Biological Characteristics Of Different Virulence G Protein Rabies Recombinant Virus

In this study,the G gene of CVS-11 strain(standard challenge strain)and SAD strain(vaccine strain)was amplified by RT-PCR using the reverse genetics system of rabies virus vaccine LBNSE strain as the backbone.the G gene of CVS-11 strain(standard challenge strain)and SAD strain(vaccine strain)was replaced with the G gene in the pLBNSE plasmid to construct two recombinant plasmids.The reverse genetics system was used to rescue two recombinant viruses rLBNSE-SfG and rLBNSE-CfG,which recombine different virulence rabies G proteins,and then explore the biological characteristics of the two viruses.Primers were designed for Overlap PCR through Snapgene biological software.The Pst I and EcoR I restriction sites of the pLBNSE plasmid preserved in the laboratory were modified and named r-pLBNSE.According to the complete genome sequence of SAD strain and CVS11 strain published in GenBank database to design the G gene primer(G gene plus signal peptide and flag tag)by Primer 5 biological software,and RT-PCR amplification and sequencing.The amplified G gene sequence was replaced by the restriction enzyme digestion and ligation to the transformed r-pLBNSE plasmid to construct a fall-length plasmid of the recombinant G gene and named r-pLBNSE-SfG(flag-labeled SAD strain G gene)and r-pLBNSE-CfG(flag-tagged CVS 11 strain G gene).Recombinant full-length plasmids were co-transfected into BSR cells with helper plasmids pH-N,pH-P,pH-G and pH-L using reverse genetics system to rescue recombinant viruses rLBNSE-SfG and rLBNSE-CfG.The rescued recombinant virus was identified by direct immunofluorescence assay and RT-PCR.By investigating the biological characteristics of the two recombinant viruses rLBNSE-SfG and rLBNSE-CfG,it was found that the growth characteristics of the recombinant virus were consistent with the parent strain.By comparing the expression of glycoprotein,the expression of recombinant viral glycoprotein was significantly lower than that of the parent,the pathogenicity from strong to weak is rLBNSE-CfG?rLBNSE-SfG?LBNSE.This experiment laid the foundation for the study of the pathogenesis of rabies,the packaging,transportation,release of viruses and the relationship between the neurotropism of the virus and glycoprotein.