沙眼衣原体E型MOMP基因原核表达载体的构建和纯化

目的:构建沙眼衣原体E型主要外膜蛋白(MOMP)基因的原核表达载体，并在大肠杆菌(BL-21)中融合表达，为沙眼衣原体疫苗的研究提供材料。方法:用PCR技术扩增E型沙眼衣原体MOMP基因片段,再将其定位插入到原核表达载体pGEX中,构建重组表达质粒。然后将重组表达质粒转化入大肠杆菌(BL-21)中，并用酶切分析、PCR扩增及部分序列测定等方法对重组质粒进行了鉴定。然后诱导表达，用SDS-PAGE及蛋白印迹进行鉴定，然后进行蛋白纯化。结果:PCR扩增出约1202bp DNA片段,序列测定证实与GenBank登陆的E型沙眼衣原体一致；表达产物的相对分子量为66KD,与预期分子量相符，蛋白印迹证实表达产物为特异性蛋白，并纯化获得大量蛋白。结论:成功的构建了原核表达载体pGEX/MOMP,在大肠杆菌中得到了表达，并得到了纯化后的蛋白。

Construction and purification of a prokaryotic expression vector carrying major outer membrane protein gene of Chlamydia trachomatis serovar E

Objective:To clone MOMP gene from genomic DNA of Chlamydia trachomatis serotype E and to construct prokaryotic expression plasmid of pGEX/MOMP,and achieve the fussion expression in the Bacterium coli(BL-21).This has lay the foundation for future Ct vaccine research.Methods:MOMP gene of Ct serotype E was amplified by polymerase chain reaction(PCR) and the fragment was cloned into the vector pGEX.The positive recombinant was transformed into Bacterium coli(BL-21),and it was identified by enzyme digestion,PCR amplification and sequencing.Then it was induced to expression and identified by SDS-PAGE and Western-blotting,and it was purified. Result:About 1.2kb MOMP gene was successfully isolated.The DNA sequence of MOMP was found to be the same as the nucleotide sequence published by GenBank; The molecular weight of expression product was 66kd ,which conformity to expectancy molecular weight.And I got a quantity of purified protein.Conclusion:The prokaryotic expression vector pGEX/MOMP was constructed successfully,and it was expressed in Bacterium coli(BL-21),and the protein was purified successfully.