中华皮肤科杂志 ›› 2004, Vol. 37 ›› Issue (12): 709-711.

• 论著 • 上一篇    下一篇

沙眼衣原体主要外膜蛋白在大肠杆菌中的表达与鉴定

李忠玉, 吴移谋, 陈超群, 余敏君   

  1. 南华大学医学院微生物学教研室 湖南省衡阳市 421001
  • 收稿日期:2003-10-15 出版日期:2004-12-15 发布日期:2004-12-15
  • 基金资助:
    湖南省卫生厅科研基金(B2003-079);湖南省科技厅资助项目(01SSY2008-6)

Expression and Identification of Major Outer Membrane Protein of Chlamydia trachomatis in Escherichia coli

LI Zhong-yu, WU Yi-mou, CHEN Chao-qun, YU Min-jun   

  1. Department of Microbiology, College of Medical Science, Nanhua University, Hengyang 421001, China
  • Received:2003-10-15 Online:2004-12-15 Published:2004-12-15

PDF (PC)

187

赞

摘要： 目的 克隆沙眼衣原体主要外膜蛋白(MOMP)基因,构建原核表达重组载体,并通过E.coli BL21实现MOMP的融合表达.方法 用PCR法扩增MOMP基因,克隆插入到pUCm-T载体中,序列测定正确后,将其亚克隆到原核表达载体pET-22b(+)中,采用Ni-NTA亲和层析法纯化表达产物,对表达产物进行SDS-PAGE和蛋白质印迹鉴定.结果 PCR扩增出约1200bpDNA片段,序列测定证实与基因库登陆的D型沙眼衣原体一致;表达产物的相对分子质量为47000,与预期分子质量相符,蛋白印迹证实表达产物为特异性蛋白.结论 沙眼衣原体MOMP基因可以在大肠杆菌中得到表达,其表达产物能与相应的抗体结合,为沙眼衣原体核酸疫苗的研究奠定了基础.

关键词: 基因表达, 衣原体,沙眼, 细菌外膜蛋白质类

Abstract: Objective To construct the recombinant plasmid containing the major outer membrane protein(MOMP) gene of Chlamydia trachomatis and expres s MOMP protein in E.coli BL21.Methods The MOMP gene was amplified by polymerase chain reaction from the genome of Chlamydia trachomatis serovar D.The amplified fragment was directly inserted into pUCm-T vector and verified by DNA sequencing.MOMP gene was then subcloned into the prokaryotic expression vector pET-22 b(+).The recombinant protein of MOMP was purified by Ni-NTA affinity chromatogr aphy and identified by SDS-PAGE and Western blot.Results The MOMP gene, which is about 1200 bp, was successfully amplified and cloned.The DNA sequence of the cloned MOMP gene was the same as that published by the GenBank.SDS-PAGE analysis showed that the relative molecular weight of this fusion protein was about 47 kDa which was consistent with the theoretically predicted value, and the specificity of this recombinant protein was confirmed by Western blot.Conclusions The MOMP gene of Chlamydia trachomatis was successfully cloned and expressed in the prokaryotic expression system, which may lay the foundation for the development of Chlamydia trachomatis vaccine.

Key words: Chlamydia trachomatis, Bacterial outer membrane proteins, Gene expression