中华皮肤科杂志 ›› 2009, Vol. 42 ›› Issue (5): 359-360.

• 技术与方法 • 上一篇    下一篇

沙眼衣原体E型MOMP基因原核表达载体的构建和纯化

李艳飞1,刘原君2,王艳2,齐蔓莉3,刘全忠3   

  1. 1. 天津医科大学总医院皮肤性病科
    2. 天津市医科大学总医院皮肤性病科
    3. 天津医科大学总医院皮肤科
  • 收稿日期:2008-04-11 修回日期:2008-05-17 出版日期:2009-05-15 发布日期:2009-05-13
  • 通讯作者: 李艳飞
  • 基金资助:

    国家自然科学基金项目(30671879);国家自然科学基金资助项目（编号）

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

  • Received:2008-04-11 Revised:2008-05-17 Online:2009-05-15 Published:2009-05-13

PDF (PC)

331

赞

摘要：

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。

关键词: 沙眼衣原体；主要外膜蛋白；克隆；蛋白疫苗

Abstract:

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.