幽门螺杆菌HpaA重组蛋白的表达及免疫效果检测  

Expression and Immune Effect of Recombinant HpaA Protein of Helicobacter pylori

作　　者：栗俊杰[1] 杨致邦[1] 张任飞[1] 周侠[1] 夏丽君[1] 

机构地区：[1]重庆医科大学基础医学院病原生物学教研室,重庆400016

出　　处：《中国生物制品学杂志》2008年第6期493-496,共4页Chinese Journal of Biologicals

摘　　要：目的表达幽门螺杆菌hpaA基因的重组蛋白,并检测其抗原性和免疫原性。方法用PCR方法从H.pylori DNA中扩增hpaA基因片段,插入原核表达载体pQE-30,转化大肠杆菌DH5α,进行诱导表达,SDS-PAGE分析表达蛋白的相对分子质量和表达形式,Ni2+-NTA树脂纯化后,用Westernblot鉴定其反应原性。用重组蛋白免疫家兔,检测其免疫原性。用重组蛋白免疫小鼠,分别检测胃黏膜、PP细胞悬液及小肠黏液中的IgG和sIgA的含量。结果重组表达质粒pQE30-hpaA构建正确,所得序列完整,插入的基因片段全长783bp,与基因文库中的hpaA基因同源性达97.3%。表达产物相对分子质量为30000,目的蛋白表达量占全菌总量的31.67%,主要存在于碎菌后上清中,纯度在90%以上。Westernblot显示该蛋白可与病人血清特异性结合,免疫血清能与重组蛋白反应,双扩散效价为1:16。小鼠体内IgG、IgA、sIgA含量明显高于PBS对照组。结论已获得高纯度的HpaA重组蛋白,该蛋白具有良好的抗原性和免疫原性,可用于研制幽门螺杆菌疫苗。Objective To express recombinant HapA protein of Helicobacter pylori and study its antigenicity and immunogencity. Methods Amplify hpaA gene fragment from H. pylori DNA by PCR and insert into prokaryotic expression vector pQE-30. Transform the constructed recombinant plasmid pQE30-hpaA to E. coli DH5α for expression under induction of IPTG. The expressed product was determined for form and relative molecular mass by SDS-PAGE, purified by Ni^2＋-NTA chromatography then identified for reactogenicity by Western blot and for immunogenicity by immunizing rabbits. Immunize mice with the expressed recombinant HpaA （rHpaA） protein and determine the IgG and sIgA contents in gastric mucosa, Peyer patches （PP） and intestinal mucus. Results Both PCR and restriction analysis showed that recombinant plasmid pQE30-hpaA was correctly constructed. The inserted gene fragment, at a full-length of 783 bp, was 97. 3% homologous to hapA gene reported in GenBank. The expressed product, with a relative molecular mass of 30 000, contained about 31.67% of total somatic protein, mainly existed in the ultrasonic supernatant of recombinant strain and reached a purity of more than 90% after purification. Western blot showed specific reaction of rHpaA protein with the sera of H. pylori-positive patients. The titer of rHpaA protein determined by double immunodiffusion test with immune sera of rabbits was 1 ： 16. The IgG, IgA and sIgA levels of mice immunized with rHpaA were significantly higher than those of control（ PBS ）. Conclusion The highly purified rHapA protein was obtained. The protein showed good antigenicity and immunogenicity, and might be used for the development of H. pylori vaccine.

关 键 词：幽门螺杆菌 HpaA蛋白 表达 免疫效果 

分 类 号：Q782[生物学—分子生物学] R392-33[医药卫生—免疫学]