含CpG和CTB的HIV多表位基因真核表达载体的构建及表达

Construction and expression of eukaryotic expression vector of HIV multi-epitope gene containing CpG sequence and CTB gene

目的构建含非甲基化的CpG ODN序列及霍乱毒素B亚单位(CTB)的HIV复合多表位基因的真核表达载体,并在体外进行表达。方法设计并合成含CpG ODN序列和linker序列的CTB基因引物,用PCR方法扩增CpG-CTB基因(CC),定向连接到真核表达载体pVL上,然后在CC基因下游插入HIV复合多表位基因MEGNp24,构建重组质粒pVL-CC-MEGNp24,酶切鉴定。纯化重组质粒,转染293T细胞,RT-PCR和细胞免疫染色方法分别检测CTB和p24基因的转录及表达,同时设质粒pVL及空白对照。结果构建了重组质粒pVL-CC-MEGNp24,该质粒转染293T细胞后,CTB和复合多表位基因在293T细胞中得到稳定表达。结论成功构建了含免疫佐剂的HIV复合多表位真核表达载体,为后期疫苗的研究奠定了基础。

Objective To construct a eukaryotic expression vector of HIV multi-epitope gene containing unmethylated CpG ODN sequence and cholera toxin B subunit（CTB） and expression in vitro.Methods The primers of CTB gene containing CpG ODN and linker sequence were designed and synthesized.CpG-CTB gene sequence（CC） was obtained by PCR,and connected to pVL.HIV multi-epitope gene was inserted into the downstream of CC gene to construct pVL-CC-MEGNp24.The recombination plasmid was confirmed by enzyme digestion.293T Cells were transfected with the purified recombination plasmid pVL-CC-MEGNp24.The transcription of CTB and p24 gene in the transfected 293T cells was detected by RT-PCR,and the expression of CTB and p24 protein was confirmed by cellular immunostaining.The plasmid pVL and blank controls were established simultaneously.Results The results showed that the recombination plasmid of pVL-CC-MEGNp24 was constructed,and CTB and multi-epitope gene were stably expressed in the 293T cells transfected with the plasmid pVL-CC-MEGNp24.Conclusion The eukaryotic expression vector of HIV multi-epitope gene containing immunological adjuvants is successfully constructed,facilitating studies of HIV vaccine.