沙眼衣原体60 kDa外膜蛋白基因的克隆与表达

Cloning and Expression of 60 kDa Outer Membrane protein Gene from Chlamydia Trachomatis

目的 克隆和表达沙眼衣原体 60kDa外膜蛋白 (Omp2 )基因。 方法 D型沙眼衣原体感染McCoy细胞后 ,抽提基因组DNA ,经PCR扩增出omp2基因片段 ,与 pUCm -T克隆载体连接 ,酶切纯化后克隆到原核表达载体pQE3 0 ,构建重组表达载体 pQE3 0 omp2 ,PCR、酶切及测序鉴定。IPTG诱导表达 ,SDS -PAGE检测有无蛋白的表达。 结果 (1)获得长约 165 0bp的PCR产物 ,酶切结果显示所构建的重组质粒已成功地克隆了omp2基因 ,序列分析结果与已知omp2序列相同 ;(2 )SDS -PAGE检测表达产物 ,在相对分子量 60kDa处有表达条带 ;(3 )诱导表达之菌体超声破碎后 ,目的蛋白主要以包涵体形式存在。 结论 获得了Ctomp2基因片段 ,并在E .coliM

Objective To clone and express the 60kDa outer membrane protein gene (omp2) from Chlamydia trachomatis(Ct). Methods Ct serovar D was cultured through infected the McCoy monolayer cell and then the Ct template DNA was extracted from the cultured cells. PCR was used to amplify the omp2 gene fragment from Ct chromosomal DNA. The PCR products were directly ligated into pUCm-T vector. After being digested with BamHⅠ+ HindⅢ and purified, the omp2 gene fragment was inserted into the compatible site of prokaryotic expression vector pQE30, then the constructed recombinant plasmid was introduced into competent E. coli XL1-Blue. After restriction enzymes cleavage analysis and sequencing, the recombinant plasmid pQE30/omp2 was transferred into an expression stain E. coli M15. The host bacteria harboring the expression plasmid were induced by IPTG and the product was identified by SDS-PAGE. Result (1)The size of amplified omp2 gene was about 1650bp. The correct recombinant plasmid pQE30/omp2 was isolated and confirmed by restriction enzymes cleavage analysis. DNA sequencing showed the DNA sequence of the cloned gene was the same as the published sequence. (2)The fusion protein from the transformants was approximate size of 60 kDa in SDS-PAGE analysis. (3)Most of the expressed recombinant proteins were in inclusion bodies form. Conclusion We obtained omp2 gene of Ct and expressed it successfully in E.coli M15.