抗重组VacA IgY体内抗幽门螺杆菌感染的实验研究

目的在构建H.pylori的基因工程菌pQE30-v-DH5a的基础上，诱导表达VacA重组蛋白，以此为抗原，制备抗VacA的蛋黄抗体（VacA IgY）。通过小鼠口服试验，证实VacA IgY治疗H.pylori感染的作用，为进一步制备抗H．pylori感染的IgY制剂提供实验依据。方法用重组H.pylori VacA蛋白免疫母鸡，水稀释结合氯仿有机沉淀法提取IgY，ELISA法测定其针对VacA的效价。建立H.pylori感染的Balb／c小鼠动物模型，治疗组在小鼠灌喂菌液后灌喂不同剂量的VacA IgY。以H.pylori培养和病理切片观察胃黏膜H.pylori定植和炎症反应程度。结果制备了高效价的IgY（1：12800）。动物实验阳性对照组H.pylori的总感染率为70．4％，12周后的感染率为88．9％。治疗组的感染率与同期阳性对照组相似，胃黏膜的炎症反应程度比阳性对照组弱，随IgY剂量的增加，炎症减弱明显，IgY剂量为4mg／ml时，能达到较理想的治疗效果。结论成功制备了高效价的特异性VacA IgV，小鼠体内实验证实了口服VacA IgY具有治疗H.pylori感染的作用,可用于制备口服制剂。

Studies of IgY anti-recombinant VacA to cure the infection of Helicobacter pylori in vivo

Objectives To prepare IgY against VacA of Helicobacter pylori from yolk of hen＇s egg immunized with recombinant protein VacA ,verify its effect in curing the infection of Helicobacter pylori in vivo, and lay a foundation for the development of preparation against H. pylori infection. Methods Based on constructed gene engineering bacteria pQF30- VacA-DH5α of Helicobacter pylori ,recombinant VacA proteins were induced and expressed. VacA IgY was prepared, using VacA as the antigen, and administered orally to the mice. Laying hens were immunized with recombinant H. pylori protein to get IgY. IgY was isolated using precipitation method of water dilution combined with chloroform. The titer against VacA was detected by ELISA. Based on created animal model of H. pylori infection, after infected with H. pylori, different doses of VacA IgY were given to the BALB/C miccs in treatment group using an oral feeding needle. Bacterial culture and pathological section were used to observe the field planting of bacteria and inflammation of mucosa in stomach. Results IgY of high titer （1： 12800） were prepared. The total rate of infection in the positive control was 70.4% ,and after 12 weeks,88.9%. The infection rate of the treatment group was similar to that of the positive control group at the same infected period ,while the in- flammation of mucosa in stomach of the treatment group was thinner than that of the positive control group, and the inflammation of mucosa was weakening along with the increasing of IgY dosage. The satisfactory dosage of VacA IgY that can cure the infection of H. pylori was 4 mg/ml. Conclusion VacA IgY with high titer was successfully prepared and has the ability to cure the infection of H. pylori in vito of mice. It could be used to prepare the oral preparation of anti-infection against He- licobacter pylori.