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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。
The purpose of this study was to understand the genetic stability of the non-replicating recombinant vaccinia virus (rNTV-C) expressing the six genes of HIV-1, including the viral vector and six foreign genes: gp160, gag, polr, evt, at and nef ). We continuously passaged rNTV-C in primary chicken embryo fibroblasts (CEFs) to passage 25 for the stability of the 9th, 12th, 15th and 25th generation viral vector genes, the stability of the six exogenous genes, the exogenous The stability of gene expression and the rate of exogenous gene loss were investigated. The results showed that all the viruses maintained the characteristics of vaccinia virus virus of non-replicating type of Tiantan strain and the passage was stable. The sequence of HIV-1 gene was consistent with the original designed sequence. The recombination site was correct and genetically stable. The 25-nucleotide mutation The target protein was expressed efficiently in each generation of rNTV-C, and there was no significant difference between the expression levels and molecular weights of all the virus generations. The expression of Gag and Nef protein was used as a marker and rNTV-C The gene loss rate of the virus is below 5%. The results of this study provide key information for vaccine production to ensure stable species.