幽门螺杆菌重组HpaA蛋白包涵体的切胶纯化分析  

Analysis for purification of recombinant HpaA protein of Helicobacter pylori from inclusion bodies in gel slices

作　　者：范贵荣[1] 杨致邦[1] 栗俊杰[1] 郭丽媛[1] 向瑜[1] 韩飞[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实验室,重庆400016

出　　处：《中国微生态学杂志》2009年第3期193-196,199,共5页Chinese Journal of Microecology

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

摘　　要：目的探讨切胶纯化的最佳条件及纯化重组HpaA蛋白包涵体的可行性。方法克隆并表达pQE30-hpaA-DH5α工程菌,获得重组HpaA蛋白包涵体,使用切胶纯化法进行纯化,得到可溶性的重组HpaA蛋白。SDS-PAGE电泳后使用不同浓度、不同pH的醋酸钠溶液以及不同的染色时间染色电泳蛋白条带,分析切胶纯化的最佳条件。Western blot鉴定重组HpaA蛋白的抗原性。将重组蛋白免疫BALB/c小鼠,双向免疫扩散法检测血清抗HpaA抗体,鉴定重组蛋白的免疫原性。结果4 mol/L醋酸钠溶液、pH13.0、作用1 h为切胶纯化的最佳条件。经切胶纯化法获得的重组HpaA蛋白体积为6 ml,浓度为0.2942 mg/ml,蛋白量为1.7652 mg,得率为77.1%,West-ern blot显示该蛋白具有良好的抗原性。小鼠免疫血清能与重组HpaA蛋白反应,双扩效价大于等于1∶16,具有良好的免疫原性。结论切胶能够纯化重组HpaA蛋白包涵体,获得高纯度的可溶性重组HpaA蛋白,该蛋白具有良好的抗原性和免疫原性,可用于进一步的科学研究。Objective To explore the optimal conditions of purification by gel slices and the probability of purifying recombinant HpaA protein from inclusion bodies. Method Recombinant bacteria pQE30-hpaA-DH5α were cloned and expressed. The soluble recombinant HpaA protein was obtained from inclusion bodies purified by gel slices. Target proteins electrophoresed in SDS-PAGE were dyed with different concentrations,different pH of sodium acetate solution and different dying time to analyze the optimal conditions of purification by gel slices. Its antigenieity was identified by Western blot. Its immunogenicity was identified by double agar diffusion with the serum from BALB/c mice immunized with recombinant HpaA proteins. Result 4 mol/L sodium acetate solution, pH 13.0 and dying for 1 h were the optimal conditions of purification in gel slices. The volume,concentration,weight and yield were 6 ml,0. 2942 mg/ml,1. 7652 mg and 77.1% respectively. Western blotting analysis confirmed that the recombinant HpaA protein could be specifically recognized by the serum of anti-HpaA. Immunized serum of BALB/c mice could bond with the recombinant HpaA protein and the titer of double agar diffusion was above 1：16, which showed that the recombinant HpaA protein possessed satisfactory immunogenicity. Conclusion Recombinant HpaA protein could be purified from inclusion bodies by gel slices. Soluble recombinant HpaA protein was obtained with satisfactory antigenicity and immunogenicity.thus oromising a bright future for further research.

关 键 词：幽门螺旋杆菌 粘附素 切胶纯化 

分 类 号：R378.99[医药卫生—病原生物学]