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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 an eukaryotic expression vector of HIV polyplex gene with unmethylated CpG ODN sequence and cholera toxin B subunit (CTB) and to express it in vitro. Methods The CTB gene primers containing CpG ODN sequence and linker sequence were designed and synthesized. CpG-CTB gene (CC) was amplified by PCR and ligated into eukaryotic expression vector pVL. Then, Gene MEGNp24, construct recombinant plasmid pVL-CC-MEGNp24, identified by restriction enzyme digestion. The recombinant plasmids were transfected into 293T cells. The transcription and expression of CTB and p24 genes were detected by RT-PCR and cell immunostaining respectively. Plasmid pVL and blank control were also constructed. Results The recombinant plasmid pVL-CC-MEGNp24 was constructed and transfected into 293T cells. The CTB and multi-epitope genes were expressed in 293T cells stably. Conclusions The HIV multi-epitope eukaryotic expression vector containing the immunological adjuvant has been successfully constructed, which lays the foundation for the research of the later vaccine.