幽门螺杆菌重组VacA蛋黄抗体的体外活性

In vitro activity of IgY against recombinant VacA protein of Helicobacter pylori

目的:制备幽门螺杆菌(Helicobacter pylon)重组VacA的鸡蛋黄抗体IgY(VacA-IgY),了解其理化特性和生物学活性,评价其抗H pylori感染的作用．方法:以纯化的重组VacA免疫产蛋鸡,水稀释法提取蛋黄抗体(VacA-IgY),硫酸铵沉淀法纯化IgY．将一定浓度的VacA-IgY在不同温度的水浴中维持一定时间,评价其耐热性;在不同 pH的Tris-HCI中孵育一定时间,评价其耐酸性;加入胃蛋白酶并作用一定时间后,评价其对酶消化的耐受作用．以上评价均采用ELISA 法测定抗体效价变化．采用Hela细胞体外培养 MTT法分析VacA-IgY对H pylori细胞毒活性的中和作用．结果:本实验制备的VacA-IgY在70℃水浴 15 min后活性保持约50％;在pH≥5时,抗体活性几乎无改变,pH<5时,抗体活性下降较快,pH2．0左右,抗体活性几乎丧失;pH4．0时, 60 kU／L胃蛋白酶作用1 h,抗体活性几乎无变化,2 h后活性仍保持50％以上．VacA-IgY能浓度依赖性地中和H pylori菌体蛋白的Hela细胞毒活性．20 mg／L超声提取物即可降低1／2的 Hela细胞增殖能力,80-320 mg／L的VacA-IgY 能完全中和H pylori菌体蛋白的Hela细胞毒活性(P<0．01)．结论:成功制备了重组VacA的IgY,且具有较好的耐热性、耐胃酶消化和一定的耐酸性; 在其体外具有中和H pylori细胞毒活性的作用．

AIM： To explore the physicochemical and biological properties of IgY extracted from the yolk of hen＇s egg immunized with recombinant VacA protein of Helicobacter pylori. METHODS： The purified antigen of recombinant VacA was used to immunize the hens and the VacA-IgY antibody was extracted from the yolk of hen＇s egg by water dilution methods and then purified by deposition technique with ammonium sulfate. In order to evaluate VacA- IgY heat stability, the titer of VacA-IgY was detected by enzyme-linked immunosorbent assay （ELISA） after being heated for 15 min under different temperatures. VacA-IgY was added into Tris-HC1 of different pH values and incubated under a temperature of 37℃ to access its acid stability using ELISA. The competence of anti-pepsin digestion was also measured by ELISA, The VacA^＋ H pylori were co-cultured with Hela cells and VacA-IgY antibody was added to observe its neutralization on the cytotoxity of H pylori by MTr assay in vitro. RESULTS： The activity of VacA-IgY was maintained about 50% after water baths （70℃, 15 min）. The activity of VacA-IgY seldom changed at pH values 5.0-7.0, but declined rapidly below the pH value 5.0, even absolutely disappeared at pH value 2.0. After incubation with pepsin （pH4.0, 60 kU/L） for 1 h, the activity of VacA-IgY had no changes, and 2 h after incubation, it still maintained over 50%. The VacA-IgY antibody neutralized the cytotoxity of VacA^＋ H pylori in a concentrationdependent manner. VacA^＋ H pylori ultrasonic extracts inhibited 50% proliferation of Hela cells at a concentration of 20 mg/L, while VacA-IgY completely neutralized the cytotoxity of H pylori at concentrations of 80-320 mg/L （P 〈 0.01）. CONCLUSION： The VacA-IgY antibody shows a good heat stability, certain acid stability, and competence of anti-pepsin digestion, and can neutralize the cytotoxity of VacA protein of H pylori effectively in vitro.