目的 了解兰州羔羊轮状病毒(LLR)株VP4基因遗传变异特点及与人源性轮状病毒基因结构上的相关性.为疫苗的质量控制和对人体的免疫原性提供理论依据.方法 严格按疫苗生产过程将LLR疫苗株毒种LLR38代在新生儿肾原代细胞传至第49代,取LLR38、LLR43、LLR44、LLR49代进行研究.通过逆转录-聚合酶链式反应获得了LLR株传代病毒VP4全基因的cDNA片段,构建成重组质粒,经酶切筛选,对克隆的VP4基因进行序列测定.结果 LLR株VP4基因全长2362 bp,编码776个氨基酸.各代次病毒的核苷酸和氨基酸遗传稳定,核苷酸同源性在99.7%～100.0%,氨基酸同源性在99.0%～100.0%.不同P基因型来源轮状病毒在亲缘关系上没有明显的区分.氨基酸序列比对分析显示,LLR株与13株人源病毒在基因结构上密切相关.结论 LLR疫苗株VP4基因具有很好的遗传稳定性,LLR株毒种及生产的疫苗是安全有效的,能够预防轮状病毒感染.%Objective To study the characteristics of genetic variation of VP4 gene of Lanzhou lamb rotavirus (LLR) strain of rotavirus and its association with the genetic structure of human rotavirus in order to provide theoretic evidence for the vaccine quality control and development. Methods LLR38 strain was amplified in primary fetal calf kidney cells for 38～49 generations according to the procedure of vaccine production,and LLR38,LLR43,LLR44 and LLR49 were analyzed. The cDNA of VP4 gene was obtained by reverse transcription polymerase chain reaction (RT-PCR),the recombinant plasmid was constructed and the cloned VP4 gene was sequenced. Results The whole length of VP4 gene of LLR strain was 2 362 bp encoded 776 amino acids. Sequence analysis indicated that there was high genetic stability among the sequences of every passage. The homology of nucleotide or amino acid was 99.7%～100.0% or 99.0%～100.0%,respec- tively. The analyzing result also indicated that there were no evident changes in their phylogenetic relatives between different kinds of P types and the original rotavirus. Conclusion VP4 gene of the LLR strain has good genetic stability,its vaccine is safe and may be used for the prevention of retrovirus infection.
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