表达HIV-1多价合成基因的重组痘苗病毒疫苗株的构建

Construction of Recombinant Vaccinia Virus Expressing HIV-1 Poly-Valent Synthetic Gene for Vaccine

为研制适合我国使用的HIV疫苗,选择具有代表意义的中国流行株HIV CN54(B′/C)病毒gag、pol、nef等基因的合成基因syngpnef,插入到自行构建的能在病毒筛选过程中将标记基因去除掉的双标记基因痘苗病毒载体pVI75的KpnI酶切位点,构建成转移质粒pVI75-syngpnef,与我国的天坛株痘苗病毒共转染鸡胚成纤维细胞,通过蓝白斑筛选得到的重组病毒疫苗株DNA。经PCR鉴定标记基因已被删除并有目的基因的整合,Western blot可检测到目的基因的融合表达。此重组病毒疫苗株有望成为HIV/AIDS候选疫苗。

In order to develop HIV vaccine against Chinese HIV-infectors, HIV-1 poly-valent gene syngpnef was synthesized and inserted into Kpn I restriction site of plasmid pV175 to construct transfer plasmid pV 175-syngpnef, By co-transfecting CEFs with vaccinia virus Tiantan strain, syngpnef gene was recombined into a genomic DNA of the vaccinia virus and the recombinant vaccinia virus was acquired through homologous recombination and screening by color difference in virus dots, Plasmid has double selection markers, but during the process of screening, the selection markers can be deleted, The deletion of selectable markers and the insertion of gene HIV-1 syngpnef have also been confirmed by PCR. Western blot ascertained the expression of target protein in CEFs. The constructed recombinant vaccinia virus would be hopefully used as a candidate HIV/AIDS vaccine with Chinese independent intellectual property.