NSP4基因突变的重组牛轮状病毒的拯救及其鉴定

Rescue and Identification of the Recombinant Bovine Rotavirus with Mutational NSP4 Gene

【目的】通过反向遗传技术结合RNAi筛选和蚀斑克隆方法,拯救出毒力减弱的轮状病毒(rotavirus,RV)。【方法】以野生型轮状病毒CHLY基因组RNA为模板,通过重叠PCR方法在NSP4基因93—104 nt引入5个沉默突变核苷酸,并将毒力位点区135aa、136aa和138aa对应的核苷酸进行定向突变,构建了含有突变位点的转录质粒△pT7-NSP4/89/M。将该质粒与携带RNA聚合酶基因的重组质粒pcDNA3.1/T7-RNAP共转染已接种野生型RV病毒的MA104细胞单层,继续培养24 h,获得了携带NSP4变异基因的RV病毒粒子和野生型RV病毒粒子的混合病毒。将获得的混合病毒接种MA104细胞,通过RNA干扰和蚀斑克隆方法逐步筛选纯化以获得拯救RV。【结果】成功拯救出了NSP4基因变异的RV,该病毒在MA104细胞上的病变和致乳鼠腹泻效果较野生型RV明显减弱。【结论】通过反向遗传技术结合RNAi筛选和蚀斑克隆方法成功拯救出毒力减弱的RV;NSP4毒力位点135aa、136aa和138aa的变异对RV毒力改变和腹泻严重程度有影响。

[ Objective ] The objective of the study is to rescue the attenuated rotavirus with mutational NSP4 gene by reverse genetic method and to purify further by RNAi technique and plaque clone method. [ Method ] NSP4 gene of a wild type RV strain CHLY was cloned and five silent mutation nucleotides were introduced at 93 nt-109 nt and five missense mutations at 444 nt-453 nt, which resulted in M135L, I136T, A138P amino acid mutation. A recombinant plasmid ApT7-NSP4/89/D that contains T7 promoter and T7 terminator at 5＇ and 3＇ end of the manipulated NSP4 eDNA was constructed. ApT7-NSP4/89/D plasmid and expression vector pcDNA3.1/T7-RNAP carrying RNA polymerase in vivo was co-transfected MA104 cell layer that had been infected 24 h earlier with wild type RV as helper virus, and the transfected cell was cultured for further 24 h until harvest. The culture fluid colleted was subjected to passage on MA104 cell in the presence of Lentivirus-RNAi-H1-89, which could inhibit the amplification of helper virus specifically. The rescued virus was biologically cloned by five successive plaque purifications in MA104 cells. [Result] A RV strain with mutational NSP4 gene was successfully rescued. Compared to wild type RV, the rescued virus showed attenuated virulence to MA104 cell and mouse pups. [Conclusion] A attenuated virulence RV was rescued by reverse genetic method and RNAi technique. The mutations of 135aa, 136aa and 138aa of NSP4 have effect on virus virulence.