狂犬病病毒CTN株反向遗传系统的改造及拯救  

Reconstruction and rescue of reverse genetic system of rabies virus CTN strain

作　　者：解庭波[1] 明平刚[1] 唐芳[1] 黄思佳[1] 沈智俊[1] 王月[1] 徐葛林[1] 严家新[1] 

机构地区：[1]武汉生物制品研究所有限责任公司,湖北武汉430207

出　　处：《中国生物制品学杂志》2015年第11期1121-1126,共6页Chinese Journal of Biologicals

摘　　要：目的对狂犬病病毒CTN株反向遗传系统进行改造,并拯救改造后的狂犬病病毒。方法应用基因定点突变技术分别对狂犬病病毒CTN株G蛋白的第194位和333位氨基酸进行定点突变,将突变后的G蛋白基因替换原有反向遗传系统中的G蛋白基因,同时对突变后的G蛋白基因进行拷贝,分明构建含有1、2和3个改造后G蛋白基因全长序列的感染性克隆,并将其分别与辅助质粒共转染BSR细胞,采用直接免疫荧光实验(DFA)和RT-PCR法鉴定重组病毒。结果 G蛋白的第194位天冬酰胺突变为丝氨酸,第333位谷氨酰胺突变为谷氨酸,获得含有1、2和3个突变后G蛋白基因的狂犬病病毒CTN株反向遗传系统,并拯救出重组狂犬病病毒。结论成功对狂犬病病毒CTN株反向遗传系统进行了改造,并拯救出了重组病毒,为研制安全、稳定、高效的新型狂犬病病毒疫苗奠定了基础。Objective To reconstruct the reverse genetic system of rabies virus CTN strain and rescue the modified recombinant rabies virus.Methods The amino acids at sites 194 and 333 of G protein of CTN strain were mutated by site-directed mutagenesis technology.The mutated G gene was used as a substitute for the former G gene,based on which a novel system carrying a single,double and triple mutated G gene was constructed.The reconstructed full-length c DNA and auxiliary plasmids were co-transfected to BSR cells,and the rescued virus was identified by direct immunofluorescence assay（DFA）and RT-PCR.Results The mutation of G gene（194 Asn→Ser,333 Gln→Glu）was observed.The reverse genetic systems carrying a single,double and triple mutated G gene were constructed respectively,and the recombinant virus was rescued.Conclusion The reverse genetic system of rabies virus CTN strain was successfully modified,and the recombinant virus was rescued,which laid a foundation of developing a novel,safe,stable and effective rabies vaccine.

关 键 词：狂犬病病毒 CTN株 反向遗传学 定点突变 G基因 

分 类 号：R373.16[医药卫生—病原生物学] Q78[医药卫生—基础医学]