幽门螺杆菌融合蛋白HspA-UreB在pMAL-C2x中的表达与免疫学活性研究

目的:在pMAL-C2x中表达幽门螺杆菌(Helicobacter pylori,Hp)融合蛋白HspA-UreB,探索其免疫学活性,为Hp基因工程疫苗的研究奠定基础。方法:从重组质粒pNHA27和pNUB27中纯化回收hspA和ureB基因片段,并以hspA-ureB的顺序连接后插入原核表达载体pMAL-C2x中,表达麦芽糖结合蛋白(MBP)与HspA-UreB的融合蛋白。采用亲和层析法纯化融合蛋白MBP-HspA-UreB,并辅以弗氏佐剂皮下免疫小鼠,蛋白印迹法对免疫小鼠血清进行检测。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,并以融合蛋白形式MBP-HspA-UreB高效表达,相对分子量为119 000,约占细胞总蛋白的31%。通过大肠杆菌抗原吸收法纯化免疫小鼠血清后,与纯化的融合蛋白进行杂交,结果显示在119 000处出现特异杂交带。结论:融合蛋白HspA-UreB具有良好的免疫原性和免疫反应性。

Study on fusion protein HspA-UreB of Helicobacter pylori expressing in pMAL-C2x and its immunocompetence

Objective: To express fusion protein HspA-UreB encoded by heat-shock protein subunit A gene and urease subunit B gene of H.pylori and study its immunocompetence. Methods: The hspA and ureB gene fragments were purified from recombinant plasmid pNHA27 and pNUB27, and cloned together into the fusion expression vector pMAL-C2x . The recombinant plasmid was then transformed into E.coli TB1. The positive clones were identified by PCR and restriction enzyme digestion. The recombinant fusion protein MBP-HspA-UreB was induced to express by IPTG and was analyzed by SDS-PAGE. The fusion protein was purified by using of affinity chromatography and then was immunized to mice. The mice serum were analyzed by Western blot with fusion protein HspA-UreB. Results: The hspA-ureB fusion gene could be amplified from the recombinant fusion expression plasmid pMHU27(pMAL-HspA-UreB) by PCR, and also the hspA-ureB fusion gene fragments could be produced from these plasmids after restriction enzyme digestion. SDS-PAGE and optical density scanning indicated that the fusion protein was expressed in the recombinant vaccine strain TB1(pMHU27) as a protein with 119 000 of molecular weight and was 31 % of the total bacterial protein. The fusion protein purified by affinity chromatography could be recognized by corresponding antibody of mice serum immunized by this fusion protein. Conclusion: The fusion protein HspA-UreB of H.pylori can be expressed in E.coli successfully, and this fusion protein has strong immunoantigenicity and immunoreactivity.