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目的构建融合表达幽门螺杆菌(H.pylori)抗原Ure B与Hsp A的乳酸乳球菌(L.lactis)菌株,为H.pylori感染的疫苗和免疫诊断研究奠定基础。方法从前期构建的hsp A-ure B融合基因克隆菌株E.coli TB1/p MAL-c2x-hsp Aure B中提取质粒,用PCR方法从质粒中扩增hsp A-ure B基因,将扩增产物经限制性酶切后与表达载体p NZ8110连接,用于转化E.coli MC1061菌株,从转化子中提取重组质粒p NZ8110-hsp A-ure B,用电穿孔法将其转入L.lactis NZ3900菌株中,用乳链菌素诱导hsp A-ure B表达,应用PAGE分析表达产物。结果 hsp A-ure B基因的PCR扩增产物长度为2 085 bp,酶切、PCR和测序鉴定结果均显示L.lactis表达系统构建正确,PAGE分析未能确定融合基因表达条带。结论首次以L.lactis为宿主菌成功构建H.pylori 2种抗原的融合表达系统,研究发现对H.pylori口服疫苗和诊断技术发展具有重要意义。
Objective To construct a strain of L. lactis which integrates the expression of H. pylori antigen Ure B and Hsp A, and lay a foundation for the research of the vaccine and immunological diagnosis of H. pylori infection. Methods Plasmid was extracted from E. coli TB1 / p MAL-c2x-hsp Aure B of hsp A-ure B fusion gene and the hsp A-ure B gene was amplified from the plasmid by PCR. The amplified product After restriction enzyme digestion and expression vector p NZ8110 for transformation of E. coli strain MC1061, the recombinant plasmid p NZ8110-hsp A-ure B was extracted from the transformant and electroporated into L. lactis NZ3900 Strain, hsp A-ure B was induced with nisin, and the product was analyzed by PAGE. Results The PCR product of hsp A-ure B gene was 2 085 bp in length. The results of restriction enzyme digestion, PCR and sequencing showed that the expression system of L.lactis was correctly constructed and the fusion gene expression band was not confirmed by PAGE analysis. Conclusion The fusion expression system of two H.pylori antigens was successfully constructed with L.lactis as the host strain for the first time. It is of great significance to study the oral vaccine and diagnostic techniques for H.pylori.