表达狂犬病毒糖蛋白的非复制型重组痘苗病毒的构建与鉴定  

Construction and characterization of nonreplicating recombinant vaccinia virus expressing rabies glycoprotein.

作　　者：李萍[1,2] 胡巧玲[1,2] 孙朝晖 王继麟[1,2] 朱家鸿[1,2] 阮力 

机构地区：[1]卫生部武汉生物制品研究所基因工程室 [2]中国预防医学科学院病毒学研究所

出　　处：《中华实验和临床病毒学杂志》1999年第2期170-174,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：湖北省科委重点项目;国家863高科技生物领域基金;国家自然科学基金

摘　　要：目的提高表达狂犬病毒糖蛋白（ＲＧ）的重组痘苗病毒的安全性。方法将编码中国狂犬病毒５ａＧ株糖蛋白的基因，插入痘苗病毒天坛株的ＴＫ区，获得重组病毒ＶＴＫＲＧ，通过两步同源重组，删除ＣＫ片段间与痘苗病毒毒力及宿主范围相关的基因，得到非复制型重组痘苗病毒ＶＴＫＲＧ△ＣＫ。结果经ＰＣＲ鉴定，ＣＫ间的核酸片段被成功删除，其缺失性状能稳定地遗传。ＷｅｓｔｅｒｎＢｌｏｔ及间接免疫荧光结果表明，ＶＴＫＲＧ△ＣＫ能有效地表达ＲＧ。该病毒在鸡胚成纤维细胞中繁殖，在人源细胞如ＴＫ１４３中几乎不增殖。动物实验证实，ＶＴＫＲＧ△ＣＫ免疫小鼠后能诱生中和抗体并能保护小鼠抵抗致死剂量狂犬病毒（ＲＶ）的攻击。结论ＶＴＫＲＧ△ＣＫ具有良好的免疫原性和更高的安全性。Objective To improve the safety of recombinant vaccinia rabies virus, the nonreplicating recombinant vaccinia rabies virus VTKRGCK was constructed. Methods The rabies glycoprotein(RG) gene was introduced into the TK locus of Chinese Tiantan strain of vaccinia virus(VTKRG), and a fragment between fragment C and fragment K involving vaccinia virus virulence and hostrange related genes were deleted by homologous recombinant. Results The ability to replicate in primary chick embryo fibroblast(CEF) and dramatically reduced replicating capability in mammalian cells of the virus were observed, whereas VTKRGCK could express high level of RG efficiently. The expression of RG could be detected in CEF by indirect immunofluorescence assay and Western blot analysis. The VTKRGCK could elicit neutralizing antibody against CVS rabies virus and protect the animals from lethal rabies virus challenge in inoculated mice. Conclusion VTKRGCK possesses immunogenicity and higher safety.

关 键 词：VTKRGΔCK 狂犬疫苗 减毒 瞬时显性选择 

分 类 号：R373.9[医药卫生—病原生物学]