表达HIV-1六种基因的非复制型重组痘苗病毒在CEF细胞中遗传稳定

The Non-replicating Recombinant Vaccinia Virus Expressing Six Genes of HIV-1 Can be Passaged Stably in CEF

本研究目的是了解表达HIV-1六种基因的非复制型重组痘苗病毒(rNTV-C)的遗传稳定性(包括病毒载体和六种外源基因:gp160、gag、polr、evt、at和nef)。我们将rNTV-C在原代鸡胚成纤维细胞(CEF)中连续传代至25代,对第9、12、15以及25代病毒载体基因的稳定性、六种外源基因的稳定性、外源基因表达的稳定性以及外源基因的丢失率进行研究。结果显示:各代病毒均保持了非复制型天坛株痘苗病毒载体特点且传代稳定;HIV-1目的基因序列与原设计序列相符、重组位点正确且遗传稳定,连续传25代核苷酸突变率低于万分之一;目的蛋白在各代rNTV-C中均能有效表达,而且各代病毒之间表达量和分子量无明显差别;以Gag和Nef蛋白表达为标记,rNTV-C各代病毒的基因丢失率均在5%以下。本研究结果为疫苗生产提供了确保毒种稳定的关键资料。

To investigate the genetic stability（including the vector of vaccinia virus and six foreign genes：gp160,gag,pol,rev,tat and nef） of the HIV-1 non-replicating recombinant vaccinia virus（rNTV-C）.rNTV-C was serially passaged to passage 25（P25） in primary chicken embryo fibroblast（CEF）.P9,P12,P15 and P25 were selected to study the genetic stability in four aspects,including the genetic stability of viral vector,the genetic stability of six foreign genes,the expressing stability of foreign genes and the genetic loss of foreign genes.The results showed that the viral vector was non-replicated vaccinia virus of Tiantan strain and was passaged stably;foreign gene sequences matched with designed sequences,the insert sites were right,and the nucleotide mutation rate was less than one over ten thousands within different passages of rNTV-C;the target proteins could be expressed effectively,and the expression level was stable within different passages of rNTV-C;the genetic loss of gag and nef was less than 5% within different passages of rNTV-C.The above results provided important data for the vaccine production.