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.
出处
《国际检验医学杂志》
CAS
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)
