摘要
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.
出处
《世界华人消化杂志》
CAS
北大核心
2006年第8期789-794,共6页
World Chinese Journal of Digestology