沙眼衣原体ompA基因真核表达载体的构建及在HeLa细胞中的表达

目的　构建沙眼衣原体 (Ct)ompA基因真核表达重组质粒 ,利用脂质体体外转染HeLa细胞 ,为核酸疫苗的研制作准备。　方法　应用PCR技术从D型Ct基因组中扩增ompA全长基因 ,重组入 pUCm -T克隆载体。将pUCm -T/ompA中的ompA外源基因片段经酶切、连接等反应 ,亚克隆入 pcDNA3 .1真核表达载体的相应位点 ,进行序列分析和酶切鉴定后 ,运用脂质体将重组体 pcDNA3 .1/ompA转染HeLa细胞 ,免疫组化法观察目的基因的表达。　 结果　PCR扩增得到约 1.2kb的特异性ompA基因片段 ;序列测定证实与发布序列一致 ;构建得到真核表达重组体 ;重组质粒pcDNA3 .1/ompA在HeLa表达MOMP。　结论　CtompA基因能够在体外真核细胞中表达 ,为进一步研究CtDNA疫苗以及研究该蛋白的生物学功能提供实验基础。

Construction of Eukaryotic Expression Vector for OmpA Gene From Chlamydia Trachomatis and its Expression in HeLa Cells

Objective To construct the recombinant plasmid containing ompA gene from Chlamydia trachomatis ,and transfect it into HeLa cells to express the major outer membrane protein(MOMP). Methods ompA gene was amplified from the genomic DNA of Chlamydia trachomatis by polymerase chain reaction (PCR), the gene was inserted into cloning vector pUCm-T.The inserted ompA gene was subcloned into appropriate site of pcDNA3.1 vector. After identification by sequencing and restrictive enzymes digestion, The recombinant plasmid was transfected into HeLa cells using Liposome .The expressed protein was identified by immunocytochemistry. Results The specific gene ompA about 1.2kb was obtained,The DNA sequence of ompA gene was consistent with the nucleotide sequence that had been published. Immunocytochemistry analysis showed that ompA gene was expressed in HeLa cells .Conclusion OmpA gene can be expressed in eukaryotic system which lay the foundation for studying the biological activities and the development of the Chlamydia trachomatis vaccine against this pathogen.