轮状病毒LLR疫苗株VP7基因的遗传变异研究

Heredity and Variation of VP7 Gene of Rotavirus Vaccine Strain LLR

目的研究轮状病毒(RV)LLR疫苗株VP7基因的遗传特征,为疫苗的质量控制和发展提供依据。方法将LLR株轮状病毒毒种LLR38在原代牛肾细胞上连续传至49代。提取第38、43、44、49代病毒RNA。通过RT-PCR扩增VP7基因片段,将其克隆入质粒pGEM-T中,进行序列测定与分析。结果LLR株VP7基因全长1062bp,含编码326个氨基酸的单一的开放读码框架(ORF)。各代次病毒的VP7基因核苷酸与推导的氨基酸变化完全一致,与GenBanK中LLR参考株(L11602)同源性分别为99.9%和99.7%。与14株G10血清型RV毒株之间,核苷酸与推导的氨基酸序列同源性分别为84.3%～87.4%和92.7%～94.9%;与不同血清型RV代表株间,VP7基因核苷酸与推导的氨基酸同源性分别为62.2%～76.8%和72.9%～83.1%;在A、B、C3个重要抗原表位,LLR株与同一血清型RV代表株B223氨基酸同源性高达97.5%,而与不同血清型仅为42.5%～62.5%。结论轮状病毒LLR疫苗株VP7基因具有良好的遗传稳定性,与来自不同国家同一型别的RV株VP7蛋白氨基酸序列之间无明显差异,在遗传上密切相关,从VP7基因分子水平证明LLR株毒种及其制备的疫苗是安全可靠的。

Objective To study the heredity and variation of VP7 gene of rotavirus （RV） vaccine strain LLR and provide a basis for the quality control and development of vaccine. Methods Rotavirus strain LLR of passage 38 （LLR38）was subeuhured in primary bovine kidney cells to passage 49, and the viral RNAs of passages 38, 43, 44 and 49 were extracted for amplification of DNA fragments of VP7 gene by RT-PCR. The amplified gene fragments were cloned into plasmid pGEM-T for sequencing. Results The VP7 gene of LLR strain, at a full-length of 1 062 bp, Contained a single open reading frame （ORF） encoding 326 amino acids. The homologies of nucleotide and deduced amino acid sequences of strain LLR were 99. 9% and 99. 7% to the reference strain LLR（L11602） in GenBank, 84. 3% - 87.4% and 92. 7%-94. 9% to other RV strains of serotype G10, and 62. 2% - 76. 8% and 72. 9% ～83.1% to the representing RV strains of various serotypes, respectively. The changes of nucleotides and deduced amino acids of strain LLR of various passages were completely in agreement. The homologies of amino acids at three key antigenic epitopes A, B and C of strain LLR were 97. 5% to those of RV strain of the same serotype, while only 42. 5% ～ 62. 5% to those of different serotype. Conclusion The VP7 gene of rotavirus vaccine strain LLR showed good genetic stability. LLR strain showed no significant difference with the RV strains of the same serotype from various countries in amino acid sequence of VP7 protein, indicating that the former was closely related to the latter in genetics. It proved good safety of LLR strain and prepared vaccine at molecular level.