论文部分内容阅读
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。
Objective To prepare and evaluate a specific yolk antibody targeting Helicobacter pylori adhesin A (HpaA). Methods Urease-positive gastric mucosa samples were collected from Fushun County People’s Hospital. Helicobacter pylori was isolated and cultured under micro-aerobic conditions. HpaA fragment was amplified by PCR, and HpaA recombinant protein was obtained by prokaryotic expression. The antigenicity was determined by Western blot. After being fully emulsified, the immunized laying hens were immunized. The yolk antibodies were purified by dilution with water and salting out. The biological characteristics were analyzed by SDS-PAGE, ELISA and Dot blot, and the inhibitory effect on the growth of isolates was evaluated. Results The isolates were positive for urease, oxidase and catalase. The length of HpaA fragment was 702 bp. The relative molecular mass of recombinant HpaA protein was about 45 000, which was expressed in soluble form and had good antigenicity. The IgY-HpaA titer Up to 1: 150 000, 1: 1 000 dilution can still well identify isolates, 5 mg / ml dose can significantly inhibit the growth of isolates. Conclusion The yolk antibody with HpaA as the target can specifically recognize and inhibit the growth of Helicobacter pylori in vitro.