Construtcion of Neisseria gonorrhoeae porin B plasmid recombinant and its expression in E. coli.
A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E. coli DE3 was constructed in order to provide a basis of research in detection, prophylactic and therapeutic vaccine against the pathogen infection. The gene encoding PIB was amplified by PCR from Neisseria gonorrhoeae and cloned into prokaryotic expression plasmid pET-28a(+) to construct a pET-PIB recombinant, which was verified by restriction endonuclease and DNA sequencing. Protein PIB was expressed in E. coli DE3 induced with IPTG. The antigenicity of the expressed protein was evaluated by indirect ELISA. Rabbits were immunized with the protein and serum was collected after immunization. To assess the immunogenicity of the protein, the titer of serum to protein PIB was determined by ELISA. DNA sequence analysis showed that the nucleic acid sequence of PIB gene was 99.28% of homology compared with that (NGPIB18) published in GenBank. A 41 kD fused protein was detected by SDS-PAGE and was proven to have reactivity with anti-PIB polyclonal antibody from mouse. A polyclonal antibody to PIB of 1:4000 titer determined by indirect EISA was obtained from rabbit immunized with the purified product. Recombinant plasmid encoding PIB of Neisseria gonorrhoeae was constructed. Protein PIB with antigenicity and immunogenicity was successfully expressed.
Song
Qifa
Q
Department of Biopathogen, School of Basic Medical Sciences, Tong ji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Liao
Fang
F
Ye
Siying
S
Cui
Bing
B
Xiong
Ping
P
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009344:Neisseria gonorrhoeae / Q000235:genetics*; D010957:Plasmids / Q000096:biosynthesis* / Q000235:genetics; D018272:Porins / Q000096:biosynthesis* / Q000235:genetics; D011387:Prokaryotic Cells / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014614:Vaccines, Synthetic / Q000276:immunology
D018272:Porins; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; C057699:porin protein, Neisseria