抗幽门螺杆菌HP1188蛋黄抗体的研究  

Preparation of immunoglobulin yolk against recombinant HP1188 protein in Helicobacter pylori

作　　者：韩飞[1] 杨致邦[2] 王长本[1] 李建英[1] 周政[1] 牟君成[1] 

机构地区：[1]重庆三峡中心医院微生物免疫科,重庆404000 [2]重庆医科大学基础医学院病原生物学教研室,重庆400016

出　　处：《国际检验医学杂志》2013年第4期390-392,共3页International Journal of Laboratory Medicine

基　　金：重庆市万州区科委项目(201203006)

摘　　要：目的制备抗幽门螺杆菌(H.pylori)HP1188蛋黄抗体(immunoglobulin yolk,IgY),即HP1188-IgY,了解其理化特性和生物学活性,为研制H.pylori HP1188-IgY型抗体口服制剂提供实验依据。方法用纯化的重组HP1188蛋白免疫产蛋鸡,以水稀释法结合氯仿有机沉淀法提取蛋黄抗体(HP1188-IgY),采用SDS-PAGE电泳检测其纯度,Bradford法测定蛋白含量,Western blot分析抗原特异性。间接ELISA评价HP1188-IgY对热、酸及胃蛋白酶消化作用的耐受情况。分别检测不同浓度HP1188-IgY及不同pH相同浓度HP1188-IgY对H.pylori的生长抑制试验。用MTT法检测不同浓度HP1188-IgY对H.pylori细胞毒活性的中和作用。结果 HP1188-IgY纯度约为68%,蛋白浓度为7.79mg/mL,Western blot结果显示在相对分子质量30000附近出现反应条带。HP1188-IgY具有一定的耐热性、耐酸性和耐胃蛋白酶消化的能力。HP1188-IgY在体外可抑制H.pylori生长,并具有浓度依赖性和pH依赖性。HP1188-IgY能阻断H.pylori菌体蛋白对Hela细胞的毒性作用,且该作用具有浓度依赖性。结论成功制备了HP1188-IgY,其具有良好的理化性质和生物学特性,为进一步制备预防幽门螺杆菌感染的口服制剂奠定了基础。Objective To explore the physicochemical and biological properties of HP1188-IgY extracted from the yolk of hen＇s egg immunized with recombinant HPl188 protein of Helicobacter pylori（H.pylori） and provide basis for preparation of oral admin istration HPl188-IgY. Methods The purified HPl188 protein was used to immunize hens and the HPlI88-IgY was extracted from the yolk by water dilution combined with chloroform methods. The purity of HP1188-IgY was analyzed by SDS-PAGE, bradford method and western blot. The indirect ELISA was used to detect heat-stability,acid-stability and pepsin-stability of HPl188-IgY. The HPl188-IgY in certain pH and concentration was added into liquid medium only containing H. pylori and cultured some time in 37 ℃ in order to observe the ability of different concentrational HPl188-IgY in inhibiting H. pytori growth. MTT was applied to assay the neutralization of HPl1188-IgY to the cytotoxity of H. pylori. Results The purity of HPl188 IgY was about 68% ,and the protein content was 7. 79 mg/mL. Western blot exhibited the protein bands with molecular weight of 30 000. The HPl188-IgY showed a well heat-stability,a favorable acid-stability and a good ability of anti-pepsin digestion. HPl188-IgY inhibited the growth of H. pylori in vitro. The growth of H. pylori could be concentration-dependently and pH-dependently blocked by the HP1188-IgY. The HPl188-IgY neutralized the cytotoxity of H. pylori in a concentration-dependent manner. Conclusion The HPl188-IgY was successfully constructed, with good stabilities and well biologic speciality. It might play an important role in further preparing oral product to prevent the infection of H. pylori.

关 键 词：螺杆菌 幽门 黏附素 HP1188蛋白 蛋黄抗体 体外试验 

分 类 号：R392[医药卫生—免疫学]