淋球菌nspA和大肠杆菌ltB融合基因的构建、表达及鉴定

Cloning, expression and identification of the fusion gene between Neisseria gonorrhoeae nspA and Escherichia coli ltB

摘要:

通过基因工程的方法构建奈瑟氏淋球菌表面蛋白A(Neisseria gonorrhoeae surface protein A, nspA)和大肠杆菌不耐热肠毒素B亚单位(B subunit of Escherichia coli heat-labile enterotoxin, ltB)融合基因的原核表达载体, 对其进行表达与鉴定, 为后续融合蛋白LTB-NspA的生物活性分析及其作为淋球菌粘膜免疫疫苗的研究奠定基础。用PCR法从标准菌株分别扩增出nspA、ltB基因, 用重组PCR法通过接头将ltB与nspA融合, 将其插入pET-30a中, 转入BL21中表达。经测序、SDS-PAGE和Western blot分析, 证实成功构建了ltB-nspA融合基因的原核表达载体, 并在BL21中表达。ltB-nspA融合基因的成功表达, 为进一步研究其生物活性及淋球菌粘膜免疫疫苗的研究奠定了一定基础。

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.