中华皮肤科杂志 ›› 2002, Vol. 35 ›› Issue (6): 432-434.

• 论著 • 上一篇    下一篇

淋病奈瑟菌外膜Porin I蛋白基因的构建、表达和鉴定

岑建萍1, 程浩1, 曾凤英1, 方永明3, 周强1, 叶俊1, 高锦程1, 王琦2   

  1. 1. 浙江大学医学院附属邵逸夫医院皮肤科 杭州 310016;
    2. 浙江大学医学院附属肿瘤研究所 杭州 310016;
    3. 浙江大学医学院附属邵逸夫医院中心实验室 杭州 310016
  • 收稿日期:2001-11-16 出版日期:2002-12-15 发布日期:2002-12-15
  • 基金资助:
    浙江省卫生厅科研基金(491030-W10001);浙江省科委科研基金(491030-J30017)

Construction,Expression and Identification of Structural Gene for Porin I,the Major Outer Membrane Protein of Neisseria gonorrhoeae

CEN Jianping1, CHENG Hao1, ZENG Fengying1, FANG Yongming3, ZHOU Qiang1, YE Jun1, GAO Jincheng1, WANG Qi2   

  1. Department of Dermatology, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou 310016, China
  • Received:2001-11-16 Online:2002-12-15 Published:2002-12-15

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摘要： 目的 构建、克隆淋病奈瑟菌外膜PorinI(PI)蛋白基因,并进行表达、纯化和鉴定.方法 用PCR法克隆淋病奈瑟菌外膜PI蛋白基因,再与pGEX-4T-2载体连接成表达重组体pGEX-4T-2/PI;经大肠杆菌P2392表达后,用SDS-PAGE、切胶、电洗脱回收等方法纯化GST-PI融合蛋白;然后用特异性淋球菌外膜PI蛋白的单克隆抗体进行斑点免疫层析试验鉴定该蛋白.结果 成功地获取了pGEX-4T-2/PI表达重组体,经诱导表达后能获得高表达的GST-PI融合蛋白,其相对分子质量为60000;斑点免疫层析试验显示其为淋病奈瑟菌外膜PI蛋白特异性的.结论 本研究将有利于进一步研究淋病奈瑟菌外膜PI蛋白的功能.

关键词: 奈瑟氏球菌,淋病, 细菌外膜蛋白质类, 重组融合蛋白质类

Abstract: Objective To construct,express,purify and identify the gene encoding majorouter membrane protein of Neisseria gonorrhoeae (Porin I, or PI). Methods The gene encoding for PI of N.gonorrhoeae was amplified by PCR and cloned into expression plasmid pGEX-4T-2 to form pGEX-4T-2/PI recombinants. A high level expression of GST-PI fusion protein was obtained in GST gene fusion system (GST:glutathione S transferase). The analysis indicated that the expressed protein was present predominantly in the insoluble form. Therefore, the induced protein was purified by SDS-PAGE, and bands corresponding to polypeptides of GST-PI fusion protein were excised and subjected to electroelution. A dotimmunoch romatographicassay was employed to demonstrate whether the purified protein was gonococcal PI specific. Results The pGEX-4T-2/PI expression recombinants were constructed,expressed,purified and identified successfully. SDS-PAGE analysis and dot immunochromatographic assay suggested that the recombinant GST-PI fusion protein was a 60 000 molecular weight protein andidentical in size to native PI and reacted with anti-PI monoclonal antibody. Conclusion Our results may lead to a potentiality for further study of diagnostic kits and vaccine for Neisseria gonorrhoeae.

Key words: Neisseria gonorrhoeae, Bacterialouter membrane proteins, Recombinant fusion protein