title: [Cloning, expression and identification of the fusion gene between Neisseria gonorrhoeae nspA and Escherichia coli ltB].
abstract: Neisserial surface protein A (NspA) of Neisseria gonorrhoeae is a potential anti-gonorrhea vaccine, and the heat-labile enterotoxin subunit B (LTB) of Escherichia coli is a kind of mucosal adjuvant that can assist mucosal immune response. We constructed a prokaryotic expression vector of the fusion gene ltB-nspA, and expressed and identified the fusion protein LTB-NspA. The nspA gene and the ltB gene were amplified from the genomic DNA of standard strains of Neisseria gonorrhoeae and Escherichia coli respectively by polymerase chain reaction(PCR). Two fragments were obtained by agarose gel electrophoresis. One was about 525bp of nspA gene, and the other was about 372bp of ltB gene. Fragment nspA and fragment ltB were fused with a linker encoding 6 amino acids (Asp-Pro-Arg-Val-Pro-Ser) by recombination PCR and a 900bp fragment of the fusion gene ltB-nspA was found on the gel. The fusion gene ItB-nspA was cloned into the prokaryotic expression vector pET-30a after digestion with BamH I and Hind III. Recombinants were selected by enzyme digestion and sequencing. The recombinant plasmid with ltB-nspA gene was then transformed into E. coli BL21 with IPTG to induce and express the fusion protein. SDS-PAGE analysis showed that the relative molecular weight of the expressed recombinant protein was about 39 kDa equivalent to the theoretically predicted value. The specificity of the expressed fusion protein was confirmed by Western-blot. The prokaryotic expression vector was constructed correctly and the fusion protein LTB-NspA was successfully expressed, which provided a basis of investigating its biological functions and developing of a Neisseria gonorrhoeae mucosal vaccine.
authors.affiliation: Institute of Pathogenic Biology, Nanhua University, Hengyang 421001, China. qyyp03@yahoo.com.cn
authors.forename: Yong
authors.initials: Y
authors.lastname: Qin
authors.forename: Sihai
authors.initials: S
authors.lastname: Hu
authors.forename: Yukuai
authors.initials: Y
authors.lastname: Zhang
authors.forename: Minjun
authors.initials: M
authors.lastname: Yu
authors.forename: Ying
authors.initials: Y
authors.lastname: Tang
authors.forename: Gang
authors.initials: G
authors.lastname: Liu
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004768:Enterotoxins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C105506:NspA protein, Neisseria; D011993:Recombinant Fusion Proteins; C027538:heat-labile enterotoxin, E coli