SA11轮状病毒反向遗传学方法的初步应用

Preliminary Application of Reverse Genetics Methods for Rotavirus SA11

轮状病毒(Rotaviruses,RV)属于呼肠孤病毒科,是分十一节段的dsRNA病毒。直到2017年,轮状病毒才建立起完全基于质粒的反向遗传学系统。本文采用美国John T.Patton教授实验室报道的11+1质粒方法,尝试了基于SA11骨架质粒和我国流行株Hu05(G9P[8])VP4和VP7基因的重配。通过结合致细胞病变效应(Cytopathic effect,CPE)和RT⁃PCR结果验证,证明我们分别成功拯救出rSA11和重配Hu05 VP7的单基因重配毒株(rSA11⁃VP7Hu05)。进一步通过蚀斑试验和qRT⁃PCR方法,我们又比较了rSA11⁃VP7Hu05与亲本株rSA11和Hu05的滴度和基因组复制动力曲线。本文通过对SA11轮状病毒反向遗传方法应用于重配病毒拯救的初步探索,为我国开展下一代轮状病毒疫苗的开发奠定了技术基础。

Rotavirus(RV)is an eleven segmented,double⁃stranded(ds)RNA virus belonging to the family Reoviridae.In 2017,an entire plasmid⁃based reverse genetics system was developed.Here,we adopted the 11+1 methods reported by the laboratory of Dr.John T.Patton in America,the reverse genetics system based on SA11 genetic backbone was recombined with VP4 and VP7 encoding genome segments from Hu05(G9P[8])circulating in China.The results of cytopathic effects(CPE)and RT⁃PCR demonstrated that rSA11 and the VP7 reassortant virus rSA11⁃VP7Hu05 were successfully rescued.Plaque assay and qRT⁃PCR were performed to compare the titers and genome copy equivalents of rSA11⁃VP7Hu05 and the parental rSA11 and Hu05.With the preliminary exploration of the reverse genetic method of rotavirus in reassortant virus,this article laid a foundation for the development of the next generation rotavirus vaccine in China.