沙眼衣原体真核表达质粒pcDNA4/CT703的构建及其表达

Construction and Expression of Eukaryotic Expression Plasmid pcDNA4/CT703 of Chlamydia trachomatis

目的:构建含沙眼衣原体(Chlamydia trachomatis,Ct)基因CT703的真核重组表达质粒pcDNA4/CT703,并检测其在HeLa细胞中的表达。方法:利用RT-PCR扩增CT703基因,然后将其亚克隆到真核表达载体pcDNA4,PCR、双酶切和测序检测重组质粒。将正确的重组质粒瞬时转染HeLa细胞,免疫荧光和WesternBlot实验检测重组质粒目的蛋白表达。结果:经PCR、双酶切和测序鉴定后,成功构建了真核重组表达质粒pcDNA4/CT703,将其转染HeLa细胞后,免疫荧光和Western Blot实验能检测到目的蛋白的表达。结论:成功构建了重组质粒pcDNA4/CT703,并能在HeLa细胞中表达,为进一步研究CT703的功能奠定了基础。

Objective：To construct a recombinant eukaryotic expression plasmid of pcDNA4/CT703 containing CT703 gene of Chlamydia trachomatis and study its expression in HeLa cells. Methods：CTT03 gene was obtained by RT-PCR and subcloned into the eukaryotic expression vectors pcDNA4, the recombinant plasmid was detected by PCR,restriction enzyme digestion and sequence analysis, pcDNA4/CTT03 was transiently transfected into HeLa cells, Immunofluorescence and Western Blot assay was used to detect the expression of recombinant proteins. Results ： Enkaryotic expression plasmid pcDNA4/CTT03 had been constnicted successfully, which was demonstrated by PCR, restriction enzyme digestion and sequence analysis; after transfected into HeLa cells, the expression of CT703 protein was demonstrated by Immunofluorescence and Western Blot assay. Conclusion：The cukaryotic expression plasmid pcDNA4/CTT03 had been constructed successfully and could express CT703 protein in HeLa cells, which laid the foundation for further study.