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目的构建含幽门螺杆菌(H.pylori)热休克蛋白A编码基因的重组载体,并电转入乳酸乳球菌MG1363,表达目的蛋白并分析其免疫原性,为H.pylori基因工程口服疫苗的研究和开发奠定基础。方法以H.py-loriNCTC 11637株基因组DNA为模板,PCR扩增hspA基因,并克隆至乳酸乳球菌表达载体pMG36e中。将重组质粒转化E.coliDH5α,经鉴定的阳性重组质粒命名为pMG36e/hspA。以电穿孔法将pMG36e/hspA转化乳酸乳球菌MG1363并用Western blot检测HspA蛋白的表达。结果克隆重组后得到pMG36e/hspA。将pMG36e/hspA电转化MG1363后,收集菌体蛋白进行Western blot分析,在HspA的相对分子质量(Mr≈13 kDa)处出现特异性条带。结论首次成功构建了表达H.pyloriHspA的重组乳酸乳球菌MG1363,为进一步口服疫苗的相关研究奠定了基础。
OBJECTIVE: To construct a recombinant vector containing the gene coding for HSP70 of H.pylori and electroporation into L. lactis MG1363, express the target protein and analyze its immunogenicity, and to study the oral vaccine against H.pylori gene engineering And lay the foundation for development. Methods The genomic DNA of H.py-loriNCTC 11637 was used as a template to amplify the hspA gene by PCR and cloned into the Lactococcus lactis expression vector pMG36e. The recombinant plasmid was transformed into E. coli DH5α, the positive recombinant plasmid was identified as pMG36e / hspA. PMG36e / hspA was transformed into L. lactis MG1363 by electroporation and the expression of HspA protein was detected by Western blot. Results After cloning and recombination, pMG36e / hspA was obtained. After electrotransformation of pMG36e / hspA into MG1363, the bacterial proteins were harvested and analyzed by Western blot. A specific band appeared at the relative molecular mass of HspA (Mr≈13 kDa). Conclusion The recombinant Lactococcus lactis MG1363 expressing H.pylori HspA was successfully constructed for the first time, which laid the foundation for the further study of oral vaccine.