摘要
背景:幽门螺杆菌(H.pylori)是慢性活动性胃炎和消化性溃疡的重要致病菌,以减毒鼠伤寒沙门菌为载体构建活疫苗已成为探索新型H.pylori疫苗的重要途径。目的:构建携带H.pylori热休克蛋白B亚单位(hspB)基因的重组活减毒鼠伤寒沙门疫苗菌。方法:应用基因工程技术将1640 bp的hspB基因克隆入原核表达质粒pTrc99A。对重组质粒进行序列测定,并将测序结果与基因文库中H.pylori-hspB的基因和蛋白序列进行BLAST分析,再将重组质粒导入活减毒鼠伤寒沙门菌SL3261。结果:重组质粒经聚合酶链反应(PCR)和双酶切,证实构建了携带hspB基因的重组原核表达质粒pTrc99A.hspB,后者成功转化活减毒鼠伤寒沙门菌SL3261。所构建的重组质粒pTrc99A-hspB中所含的H.pylori-spB与基因文库中H.pylori-hspB基因和蛋白的同源性均为97％。结论:成功构建并鉴定了携带H.pylori-hspB基因的重组活减毒鼠伤寒沙门疫苗菌,为研制H.pylori口服疫苗奠定了基础。
Background: Helicobacter pylori (H. pylori) is an important pathogen of chronic active gastritis and peptic ulcer. It has been an important approach to explore the construction of new type H. pylori vaccine by using attenuated Salmonella typhimurium (S. typhimurium) as live vector. Aims: To construct a recombinant live attenuated S. typhimurium vaccine strain carrying H. pylori heat shock protein subunit B (hspB) gene. Methods: By genetic engineering method, 1 640 bp hspB gene was cloned into a prokaryotic expression plasmid pTrc99A. After sequence analysis, the result was aligned with the sequence of H. pvlori-hspB gene and protein provided by genebank using BLAST analysis. The identified recombinant plasmid was then used to transform a live attenuated S. typhimurium strain SL3261. Results: Using polymerase chain reaction (PCR) and restriction enzyme digestion, a recombinant prokaryotic expression plasmid pTrc99A-hspB harbouring hspB gene was constructed, and the recombinant plasmid was then introduced into a live attenuated S. typhimurium strain SL3261 successfully. Most of the H. pylori-hspB in recombinant plasmid pTrc99A-hspB was homologized with the H. pylori-hspB sequence provided by genebank; the homologv of gene and protein were both 97%. Conclusions: The fact that a recombinant live attenuated S. typhimurium vaccine strain harbouring H. pylori-hspB gene has been constructed and identified successfully may help develop an oral vaccine against H. pylori infection.
出处
《胃肠病学》
2003年第1期20-24,共5页
Chinese Journal of Gastroenterology
基金
本课题由广东省科技计划重点项目(No.99MO4802G)
广东省自然科学基金(No.990077)
广东省高教厅"千百十工程"优秀人才基金(No.2000-08)
2000年度教育部骨干教师基金(No.200065)
CMB-SUMSScholarProgram(No.98-677)资助
