关键词: 轮状病毒, 反向遗传技术, NSP4基因

Abstract: 【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.

Key words: rotavirus, reverse genetic, NSP4 gene.