Construction,Expression and Identification of Structural Gene for Porin I,the Major Outer Membrane Protein of Neisseria gonorrhoeae
- VernacularTitle:淋病奈瑟菌外膜Porin I蛋白基因的构建、表达和鉴定
- Author:
Jianping CEN
;
Hao CHENG
;
Fengying ZENG
;
Yongming FANG
;
Qiang ZHOU
;
Jun YE
;
Jincheng GAO
;
Qi WANG
- Publication Type:Journal Article
- Keywords:
Neisseria gonorrhoeae;
Bacterial outer membrane proteins;
Recombi nant fusion protein
- From:
Chinese Journal of Dermatology
1994;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct,express,purify and identify the gene encodi ng major outer membrane protein of Neisseria gonorrhoeae (Porin I, or PI). Metho ds The gene encoding for PI of N.gonorrhoeae was amplified by PCR and cloned int o expression plasmid pGEX-4T-2 to form pGEX-4T-2/PI recombinants. A high lev el expression of GST-PI fusion protein was obtained in GST gene fusion system (GST:glutathione S transferase). The analysis indicated that the expressed pr otein was present predominantly in the insoluble form. Therefore, the induced pr otein was purified by SDS-PAGE, and bands corresponding to polypeptides of GST-PI fusion protein were excised and subjected to electroelution. A dot immunoch romatographic assay 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 fusio n 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 diagnosti c kits and vaccine for Neisseria gonorrhoeae.
