幽门螺杆菌融合蛋白HspA-UreB在大肠杆菌中的表达与鉴定

Expression and identification of fusion protein HspA-UreB from Helicobacter pylori in E.coli

目的在大肠杆菌中表达幽门螺杆菌(简称Hp)HspA-UreB融合蛋白,并探索其免疫反应性,为Hp基因工程疫苗的研制奠定基础。方法用PCR方法扩增郑州分离Hp菌株MEL-HP27的hspA和ureB基因,分别克隆入pNEB193中。测序后,回收两种基因片段,并以hspA-ureB的顺序连接插入原核表达载体pMAL-C2x进行融合表达。采用蛋白印迹法对表达产物进行鉴定。结果特异PCR法和酶切鉴定证实融合基因hspA-ureB克隆入表达载体中;重组质粒转化大肠杆菌TB1后,经IPTG诱导3h,SDS-PAGE电泳显示在119kDa处出现一条特异蛋白带,即麦芽糖结合蛋白(MBP)与HspA-UreB的融合表达形式,约占细菌总体蛋白含量的31%;该融合蛋白与Hp免疫小鼠血清和Hp阳性病人血清的Westernblot分析结果显示,在119kDa处出现特异杂交带。结论成功地在大肠杆菌中实现了Hp融合蛋白HspA-UreB的高效表达,并证实其具有良好的免疫反应性。

To express the fusion protein of the heat shock protein subunit A gene(hspA) and urease subunit B gene(ureB) from Helicobacter pylori in E.coli,and to determine the immunoreactivity of its encoded proteins with specific antibodies.These two genes were amplified by PCR from clinical isolate strain of H.pylori MEL HP27 and cloned into vector pNEB193.After sequencing, they were digested with two restriction enzymes separately and then cloned into the fusion expression vector pMAL C2x. The recombinant plasmid was then transformed into E.coli TB1. And the positive clones were identified by PCR and restriction fragment electrophoresis. For the induction of expression on the recombinant fusion protein MBP HspA UreB, IPTG was applied for 3 hours, and the fusion protein was identified by SDS PAGE and Western blot analysis. It was found that the hspA weB fusion gene could be amplified from the recombinant fusion expression plasmid pMHU27 (pMAL hspA ureB) by PCR,and the hspA ureB fusion gene fragment could be produced from these plasmids after restriction enzyme digestion.Results in SDS PAGE and optical density scanning demonstrated that this fusion protein was expressed in the recombinant strain of E.coli TB1 (pMHU27) as a protein with molecular weight of 119 kDa and was equivalent to 31% of the total bacterial proteins.As demonstrated on the Western blot analysis, a specific band of hybridization was demonstrated between the fusion protein and the mouse immune sera immunized with H.pylori or the sera of patients with H.pylori infection. It concludes that highly efficient expression of the fusion protein HspA UreB from H.pylori in E.coli is successfully obtained in the present study, and this protein is proved to possess good immunoreactivities.