论文部分内容阅读
目的观察硫糖铝对抗幽门螺杆菌重组细胞空泡毒素抗原(VacA)卵黄抗体(IgY)的保护作用,为制备能耐受胃酸和蛋白酶的IgY制剂奠定基础。方法诱导重组菌DH5α-vacA-pQE30,大量表达并纯化VacA重组蛋白,免疫洛曼母鸡,制备VacAIgY,并进行纯化。在不同pH值的IgY液中加入不同浓度的硫糖铝和不同浓度的胃蛋白酶,将含不同浓度硫糖铝的IgY反复冻融7次,将含30%硫糖铝的IgY室温放置1d～4周,ELISA法测定各实验组IgY的相对抗体活性(AT/AC)。结果在pH1.5、2.0和3.0的条件下,含30%～60%硫糖铝的IgY的相对抗体活性高于不加硫糖铝的IgY;在pH1.5条件下,60%硫糖铝可使IgY的相对抗体活性保持68.7%;在pH2.0和3.0条件下,50%和40%以上的硫糖铝几乎可完全保持IgY的相对抗体活性。在pH值为2.0和3.0时,正常胃蛋白酶浓度(0.02mg/ml)下,30%以上的硫糖铝均可有效保护IgY的相对抗体活性。30%以上的硫糖铝可增强IgY的抗冻融能力。室温放置4周后,含30%硫糖铝的IgY仍能保持80%以上的相对抗体活性。结论30%以上的硫糖铝可增强VacAIgY对低pH和胃蛋白酶的耐受能力及抗冻融能力,是较理想的IgY保护剂。
Objective To observe the protective effect of sucralfate against the vacuolating toxin antigen (VacA) of Helicobacter pylori (IgY) and lay the foundation for the preparation of IgY that can tolerate gastric acid and protease. Methods The recombinant baculovirus DH5α-vacA-pQE30 was induced, VacA recombinant protein was expressed and purified, and Lohmann’s chicken was immunized. VacAIgY was prepared and purified. Different concentrations of sucralfate and different concentrations of pepsin were added into IgY solution with different pH values. The IgY containing sucralfate at different concentrations was repeatedly frozen and thawed seven times, and the IgY containing 30% sucralfate was placed at room temperature for 1 d ~ 4 weeks. The relative antibody activity of IgY in each experimental group was measured by ELISA (AT / AC). Results The relative antibody activity of IgY containing 30-60% sucralfate was higher than that of IgY without sucralfate at pH 1.5, 2.0 and 3.0; at pH 1.5, 60% sucralfate The relative antibody activity of IgY can be kept at 68.7%; at pH 2.0 and 3.0, 50% and 40% of the sucralfate can almost completely retain the relative antibody activity of IgY. At pH 2.0 and 3.0, more than 30% sucralfate effectively protected IgY relative antibody activity at normal pepsin concentration (0.02 mg / ml). More than 30% of sucralfate can enhance the anti-freeze-thaw ability of IgY. After 4 weeks at room temperature, IgY containing 30% sucralfate retains more than 80% relative antibody activity. Conclusions More than 30% of sucralfate can enhance the tolerance of VacAIgY to low pH and pepsin and its anti-freeze-thaw ability. It is an ideal IgY protectant.