沙眼衣原体pORF5蛋白真核表达系统的构建与鉴定

目的构建沙眼衣原体(Ct)pORF5基因重组质粒pDSRed-C1/pORF5,建立pORF5稳定转染的表达系统并对其进行鉴定。方法PCR法扩增pORF5基因,定向插入pDSRed-C1真核表达载体构建pDSRed-C1/pORF5重组体,重组体经酶切、PCR及测序鉴定后,用脂质体介导的基因转染法,分别将pDSRed-C1/pORF5和pDSRed-C1导入HeLa-229细胞,通过G418筛选获得转入目的基因的阳性细胞克隆;荧光显微镜和Western blot检测pORF5蛋白在细胞中的表达。结果所克隆的pORF5基因片段经测序完全正确;转染pDSRed-C1/pORF5的HeLa-229细胞大部分有pORF5蛋白的表达,而转染空载体pDSRed-C1或未转染的HeLa-229细胞,均未见pORF5蛋白表达。结论成功构建了稳定表达外源新基因pORF5的细胞系,为进一步研究该蛋白的结构功能、阐明Ct的致病机制、研制基因工程疫苗提供了有利的条件。

Construction and Identification of Eukaryotic Expression System for pORF5 Gene of Chlamydia Trachomatis

Objective To construct eukaryotic expression plasmid pDSRed-C1/pORF5 based on gene pORF5 of Chlamydia trachomatis,and establish a stable expression system. Methods pORF5 gene was amplified by PCR and cloned into appropriate site of pDSRed-C1 vector to construct eukaryotic expression plasmid pDSRed-C1/pORF5.After identification by PCR,restrictive enzymes digestion and sequencing,the recombinant plasmid was transfected into HeLa-229 cells by using Liposome.Finally,the pORF5 gene expression of protein was dete...