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目的分段表达幽门螺杆菌(HP)尿素酶B亚单位(UreB),并对其进行免疫学性质分析,为精确定位其保护性表位奠定基础。方法用生物信息学软件分析UreB全长基因,选择分段点,PCR分别扩增其5个片段,构建于pET-11c(+)原核表达载体,并转化大肠杆菌BL21(DE3),用IPTG诱导表达,SDS-PAGE分析蛋白表达情况,ELISA及Western blot分别鉴定其免疫原性及抗原性。结果成功克隆了UreB的5个基因片段,基因测序结果与Genbank公布的序列一致.经SDS-PAGE分析,5个蛋白表达相对分子质量大小都与预期相符,在大肠杆菌中实现了表达。ELISA和Western blot分析显示,表达的5个蛋白表现出良好的抗原性和免疫原性。结论 UreB的分段表达为进一步研究UreB保护性表位及HP疫苗鉴定奠定了新的基础。
Objective To segregate Helicobacter pylori (UreB) subunit (UreB) in segments and analyze its immunological properties, which will lay the foundation for the precise mapping of its protective epitopes. Methods The full-length UreB gene was analyzed by bioinformatics software. Five segments of the UreB gene were amplified by PCR and sequenced. The prokaryotic expression vector pET-11c (+) was constructed and transformed into E.coli BL21 (DE3) The protein expression was analyzed by SDS-PAGE and the immunogenicity and antigenicity were identified by ELISA and Western blot respectively. Results Five of the UreB gene fragments were successfully cloned, and the results of sequencing were consistent with the published sequences of GenBank. SDS-PAGE analysis showed that the relative molecular mass of the five proteins were in line with expectation and expressed in E. coli. ELISA and Western blot analysis showed that the expressed five proteins showed good antigenicity and immunogenicity. Conclusion The segmented expression of UreB lays a new foundation for the further study of UreB protective epitope and HP vaccine identification.