狂犬病毒HEP-dG株的拯救

目的筛选免疫原性高、致病性低的狂犬病疫苗候选株。方法应用反向遗传技术,在狂犬病毒弱毒株HEP-Flury株全基因组3'-N-P-M-G-L-5'的假基因区域(G与L之间),插入一个G基因,构建携带双G基因的全长感染性克隆,其与辅助质粒共转染BHK-21细胞,盲传十代,用荧光抗体和RT-PCR鉴定病毒。结果成功获得携带双G基因的狂犬病毒HEP-dG株,该病毒在BHK-21细胞中稳定生长,其病毒滴度达到1010FFU/mL。结论HEP-dG株会为研制高效的狂犬病病毒基因工程口服疫苗提供了良好的疫苗候选株。

Rescue of the strain HEP-dG of rabies virus used in vaccine

To find out the candidate strain of rabies virus （RV） with high immunogenicity and low pathogenicity used in vaccine preparation, a recombinant RV carrying additional glycoprotein G gene in the HEP-Flury strain （ a rabies vaccine strain of RV widely used in vaccine） was inserted to the pseudogene area situated between G and L genes of the entire genome 3＇-N-P-M-G-L-5＇, and the full-length of cDNA and the auxiliary L, P and L genes were transfected to BHK-21 cells. The rescued virus and the corrected nucleotide sequences of the inserted genes were confirmed by immunostaining with fluorescein isothiocynate- labeled antibody against RV-N protein, RT-PCR and DNA sequencing respectively. On the basis the reverse genetic manipula- tion, chimeric RV HEP-dG which could grow steadily in the GHK-21 cells with a viral titer of 10^10 FFU/mL was successively obtained. This recombinant virus providing strong immunogenicity coupled with loss of pathogenicity should be considered as the good candidate strain of RV used in live engineering oral rabies vaccine.