幽门螺杆菌热休克蛋白A在乳酸乳球菌中的表达及免疫学活性分析

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。

Expression and antigenicity analysis of hspA gene from Helicobacter pylori in Lactococcus lactis

Objective To construct a recombinant plasmid containing gene encoding heat shock protein A (Hs-pA) from Helicobacter pylori (H, pylori) and express it in Lactococcus lactis, and explore the antigenicity. Method The hspA gene was amplified from genome DNA of H. pylori NCTC 11637 strain by polymerase chain reaction (PCR), and then cloned in the E. coli-L. lactis shuttle vector pMG36e and transformed into E. coli DH5α. The positive recombinant plasmid was named pMG36e/hspA. And electroporation was performed to transform the recombinant plasmid into L. lactis MG1363. The expression of HspA protein was detected by Western blot. Result The pMG36e/hspA was constructed by cloning and recombinant DNA technique. The Western blot analysis showed that the relative molecular mass (Mr) of the expressed product was about 13 kDa, and could be recognized by anti-HspA serum produced by oral administration of the L. lactis transformed into mice, suggesting that the recombinant HspA protein had good antigenicity. Conclusion The recombinant L. lactis MG1363 expressing HspA protein of H. pylori has been constructe successfully. The results lay a foundation for fur-ther research of H. pylori vaccine.