In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion prot...In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E.coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E.coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.展开更多
A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E.coli DE_3 was constructed in order to provide a basis of research in detection, prophylactic and therapeutic ...A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E.coli DE_3 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 DE_3 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 ELISA 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.展开更多
文摘In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E.coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E.coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.
文摘A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E.coli DE_3 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 DE_3 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 ELISA 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.
