Construtcion ofNeisseria gonorrhoeae porin B plasmid recombinant and its expressionin E. coli

Abstract

A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) ofNeisseria 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 fromNeisseria 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 expressedin 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. A41 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 ELISA was obtained from rabbit immunized with the purified product. Recombinant plasmid encoding PIB ofNeisseria gonorrhoeae was constructed. Protein PIB with antigenicity and immunogenicity was successfully expressed.

Keywords

Neisseria gonorrhoeae / porin B / prokaryotic expression

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