NSP4基因突变的重组牛轮状病毒的拯救及其鉴定

 【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。

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

【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 △pT7-NSP4/89/D that contains T7 promoter and T7 terminator at 5′ and 3′ end of the manipulated NSP4 cDNA was constructed. △pT7-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.