抗幽门螺杆菌黏附素A卵黄抗体的制备及评估

Preparation and characterization of specific IgY against Helicobacter pylori adhesin A

目的制备及评估一种以幽门螺杆菌黏附素A(HpaA)为靶点的特异性卵黄抗体。方法脲酶阳性胃黏膜样本采自富顺县人民医院,微需氧条件下分离培养幽门螺杆菌;PCR扩增HpaA片段,原核表达形式获得HpaA重组蛋白并以Western blot检测其抗原性;与弗氏佐剂充分乳化后免疫产蛋鸡,水稀释-盐析法纯化卵黄抗体;SDS-PAGE、ELISA及Dot blot分析其生物学特性并评估对分离株体外生长的抑制效果。结果分离株脲酶、氧化酶、触酶反应阳性;HpaA片段长702 bp,重组HpaA蛋白相对分子质量约为45 000,可溶形式表达且具有良好的抗原性;以其制备的IgY-HpaA滴度高达1∶150 000,1∶1 000稀释后仍能良好地识别分离株,5 mg/ml剂量可显著抑制分离株的生长。结论以HpaA为靶点制备的卵黄抗体在体外可特异性识别并抑制幽门螺杆菌的生长。

Helicobacter pylori（Hp） is known as the main cause of gastritis disorder,but traditional antibiotic therapy against Hp infection is challenged by the increasing tolerance.A high-targeted egg yolk antibody（IgY） has expected to be a mean of passive immunization therapy for Hp infections.Here,a specific IgY targeting to Hp adhesion-A（IgY-HpaA） was prepared and evaluated.In brief,a urease-,oxidase-,catalase-positive strain was isolated from a suspected Hp infected patient,and hpa A partial gene（702 bp） was amplified from the isolated strain using PCR reaction with a specific primer.Recombinant HpaA protein,molecular weight is about 45 KDa,was expressed as soluble protein in the E.coli prokaryotic expression system and then purified from supernatant through Ni^（2＋）-NTA affinity chromatography,and its antigenicity was identified by Western blotting with anti-Hp IgY.Anti-HpaA vaccine was prepared from the emulsion of r HpaA protein with the same volume of Freund＇s adjuvant.7 days after immunizing chickens,IgY-HpaA was extracted from yolk using water dilution and sodium chloride precipitation.The purity and titer of IgY-HpaA,evaluated by SDS-PAGE and ELISA,reached to 95% and 1∶150 000 respectively.A specific recognition of IgY-HpaA to Hp still been detected although diluted by 1 000 times.Remarkably,the growth of Hp can be suppressed significantly by IgY-HpaA at 5 mg/ml in vitro.In summary,the prepared IgY-HpaA possesses a high specificrecognition and growth inhibition to Hp in vitro.