硫糖铝对抗幽门螺杆菌重组VacA卵黄抗体的保护作用  

Protective Effect of Sucralfate on Activity of IgY against Recombinant VacA of Helicobacter pylori

作　　者：郭丽媛[1] 杨致邦[1] 田一玲[1] 黄伟[2] 韩飞[1] 范贵荣[1] 向瑜[1] 

机构地区：[1]重庆医科大学基础医学院病原生物学教研室神经科学研究中心基础医学实验教学中心病原生物学与免疫学实验室,重庆400016 [2]西南大学荣昌校区动物医学系,重庆402460

出　　处：《中国生物制品学杂志》2009年第5期457-460,467,共5页Chinese Journal of Biologicals

基　　金：重庆市教委(渝教科2003-7-3);重庆市科委(CSTC;2005EA5020)

摘　　要：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 on activity of IgY against recombinant vacuolating cytotoxin antigon （VacA） of Helicobacter pylori. Methods Recombinant VacA was expressed in a large quantity in recombinant E. coli DH5ct-vacA-pQE30 under induction of IPTG, purified by Ni2＋-NTA affinity chromatography and used for the immunization of Lohman hens, based on which VacA IgY was prepared and purified. The sucralfate and pepsin, at various concentrations, were added into IgY at various pH values; the IgY containing various concentrations of sucralfate were subjected to 7 cycles of freeze-thawing; the IgY containing 30% sucralfate was stored at room temperature for 1 d to 4 weeks; the relative activities （AT/Ac） of IgY under the above-mentioned conditions were determined by ELISA. Results At pH 1. 5, 2. 0 and 3. 0, the relative activities of IgY containing 30% - 60% sucralfate were significantly higher than those containing no sucralfate. At pH 1. 5, the relative activity of IgY containing 60% sucralfate was 68.7%. However, both the relative activities of IgY containing 50% sucralfate at pH 2. 0 and that containing 40% su- cralfate at pH 3.0 were nearly 100%. At pH 2.0 and 3.0 and a normal pepsin concentration （0. 02 mg/ml）, all the relative activi- ties of IgY containing more than 30% sucralfate were protected effectively. The sucralfate at concentrations of more than 30% also enhanced the ability of IgY against freeze-thawing. After storage at room temperature for 4 weeks, the relative activity of IgY containing 30% sucralfate was more than 80%. Conclusion The sucralfate at a concentration of more than 30% enhanced the resistance to low pH value and pepsin as well as ability against freeze-thawing of VacA IgY, thus was an ideal protective agent of IgY.

关 键 词：幽门螺杆菌 细胞空泡毒素抗原 卵黄抗体 硫糖铝 抗体活性 

分 类 号：R378[医药卫生—病原生物学] R945[医药卫生—基础医学]