人胃蛋白酶原原核表达、纯化及性能研究

Prokaryotic Expression,Purification and Properties of Human Pepsinogen

构建人胃蛋白酶原原核表达载体,建立胃蛋白酶原大肠杆菌表达条件和纯化工艺,通过测试该重组蛋白的反应性及稳定性,评价重组胃蛋白酶原在制备免疫学检测标准物质中的应用前景。采用同源重组的方法构建表达载体,在N端加入6×His和SUMO标签帮助提高胃蛋白酶原表达量和纯化;用镍亲和柱和阴离子交换层析方法纯化蛋白,用蛋白酶切除SUMO标签后再通过镍亲和柱层析方法获得高纯度胃蛋白酶原重组蛋白;按照试剂说明书测定重组胃蛋白酶原与自产胶乳的反应性,将纯化的重组胃蛋白酶原蛋白用储存液稀释至100 mg/L后等量分装,置于4℃保存,每隔5 d取出一份,通过SDS-PAGE分析粗略判断蛋白的稳定性。结果表明人胃蛋白酶原在大肠杆菌中大部分为包涵体形式且表达量高,产物纯度可达90%以上,稳定性较好,重组抗原与自产胶乳有良好的反应性。利用SUMO标签的促表达作用,成功在大肠杆菌中表达了胃蛋白酶原重组蛋白。该方法制备的蛋白适合作为制备胃蛋白酶原免疫检测的校准品的原材料。

This study aims to obtain pepsinogen recombinant protein by constructing pepsinogen prokaryotic expression vector,expressing pepsinogen recombinant protein in E.coli and establishing its purification procedure,the reactivity and stability of the recombinant protein were tested to evaluate the application prospect of recombinant pepsinogen in the preparation of standard materials for immunological detection.Pepsinogen expression vector was constructed by homologous recombination,with 6×His and SUMO tag at the N-terminal for improving expression and purification.Highly purified recombinant protein pepsinogen was obtained by Ni2+affinity column,anion-exchange chromatography and Ni2+affinity column for removing tags after cleavage with SUMO protease.The reaction between recombinant pepsinogen and self-produced latex was measured according to the reagent specifications.The purified recombinant pepsinogen protein was diluted with storage solution to 100 mg/L and then stored at 4℃.One portion was taken out every 5 days and the stability of the recombinant protein was roughly determined by SDS-PAGE analysis.Homo pepsinogen was highly expressed in E.coli in inclusion body form.Purified pepsinogen protein had high purity(>90%)and good stability.In addition,recombinant antigen had good reactivity with self-produced latex.Recombinant pepsinogen was succesfully expressed in E.coli with the assisstance of SUMO tag,which played an important role in improving expression.The purified pepsinogen recombinant protein produced by this way is suitable as the key material of calibrator for pepsinogen immunoassay.