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

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

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。

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 TB 1 （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.