沙眼衣原体ompA基因克隆及其在真核细胞中的表达

摘要

目的克隆D型沙眼衣原体(Ct)ompA基因,构建真核表达重组质粒,转染真核细胞,为核酸疫苗的研制作准备.方法用PCR技术从D型Ct基因组DNA中扩增ompA基因片段,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,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的基因的表达.结果从D型Ct基因组DNA中扩增出特异的ompA基因片段;序列测定证实与GenBank登陆的D型Ct一致;重组质粒pcDNA3.1/ompA在HeLa中获得表达.结论Ct ompA基因能够在体外真核细胞表达,为进一步研究Ct致病机制及DNA疫苗的研究提供理论依据.%Objective: To clone and construct the recombinant plasmid containing ompA gene from Chlamydia trachomatis, and transfect it into mammalian cells to express the major outer membrane protein (MOMP). Methods: Amplifed from the genomic DNA of Chlamydia trachomatis using polymerase chain reaction (PCR), ompA gene was inserted into cloning vector pUCm-T. The inserted ompA gene was subcloned to pcDNA3.1 eukaryotic expression vector by linking reactions. After identifying by sequencing and restrictive enzymes digestion. The recombinant plasmid was transfected into HeLa cells using Liposome. Results: The specific gene fragment about 1.2 kb was successfully amplified. The DNA sequence of ompA gene was found to be the same as the nucleotide sequence published by GenBank. Immunocytochemistry analysis showed that om pA gene was expressed in HeLa cells and located in cytoplasm. Conclusion: OmpA gene can be expressed in eukaryotic system which lay the foundation for studing the pathogenic mechanism and the development of the Chlamydia trachomatis vaccine against this pathogen.