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 ob-tained 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 ltB-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 mo-lecular 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 investi-gating its biological functions and developing of a Neisseria gonorrhoeae mucosal vaccine.