摘要
目的 了解轮状病毒（RV）的LLR疫苗株全基因组基因和蛋白特征、完善关键基因遗传稳定性研究,为疫苗的质量控制和研发提供依据.方法 将LLR株毒种第38代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方法扩增LLR株（38代）全基因组11个dsRNA片段和传代病毒VP6基因,分别将其克隆到pGEM-T载体中,进行序列测定与分析.结果 LLR株全基因组11条RNA,由18 498个核苷酸组成,共编码5 796个氨基酸;全基因组研究表明,所克隆的LLR株属于G10P[15]/NSP4[A]/SG Ⅰ基因型.LLR株VP6基因全长1 356 bp,含编码397个氨基酸的单一的开放读码框架（ORF）.各代次病毒的VP6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（L11595）同源性分别为99.9％和99.7％.与16株SG Ⅰ亚群RV代表株之间,核苷酸与推导的氨基酸序列同源性分别为84.0％ ～ 99.7％和97.0％ ～ 99.2％;与不同亚群RV代表株之间,VP6基因核苷酸与推导的氨基酸同源性分别为77.7％ ～ 82.2％和92.2％ ～93.5％;LLR株各代病毒VP6基因核苷酸、氨基酸序列高度保守,各关键功能区未发生变异.结论 LLR疫苗株关键基因遗传特性稳定,为在分子水平保证LLR株毒种及其生产疫苗的安全性提供了依据;其全基因组克隆,为进一步研究RV生物学、免疫学和确定该病毒的分类学地位提供了科学依据.
Objective To understandthe molecular characteristics for the genes and proteins of rotavirus （RV ） vaccine strain LLR, to improve study of the key genetic stability, and to provide a basis for the quality control and development of vaccine. Methods Passage 38 （ LLR38 ） of rotavirus strain LLR was subcultured in primary bovine kidney cells to passage 49, and the viral RNAs of passages 38, 43, 44, 49 were extracted for amplification of DNA fragments of VP6 gene by RT-PCR. The amplified gene fragments were cloned into plasmid pGEM-T for sequencing. Results Nucleotide sequences analyses showed that whole full genome of strain LLR contains 18 498 nucleotide,encoding 5 796 amino acids. Genotyping results showed that the strain LLR belongs to genotype G10P [ 15 ]/NSP4 [ A]/ SG I. The VP6 gene of LLR, at a full length of 1 356 bp, contains a single open reading frame （ORF} encoding 397 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 （ L11595 } in Gen- Bank, 84.0% -99.7% and 97.0% -99.2% to other RV strain of subgroup SG I , 77.7% -82.2% and 92.2% -93.5% to the representing RV strain in various subgroups, respectively. LLR of various passages were highly conservedand no significant Both the nucleotide and amino acid sequences of strain change was observed in key domain. Conclusion The key gene of RV strain LLR showeda stable genetic character, which provided a basis at molecular level for ensuring the safety of virus seeds of strain LLR and the produced vaccine. The whole genome cloning is provided a scientific basis for further study the RV biology, immunology and.the taxonomic status of the virus determined too.
出处
《微生物学免疫学进展》
2015年第1期1-7,共7页
Progress In Microbiology and Immunology
基金
"十一五"国家科技支撑计划(2008BA154B03)
作者简介
赵雅静，医学生物学高级工程师，主要从事生物制品质量控制及分子生物学研究。
通讯作者：高雪军，E-mail：xjgao@vacmic．com
