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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 investigate the optimal conditions of gel purification and the feasibility of purification of recombinant HpaA protein inclusion bodies. Methods The recombinant plasmid pQE30-hpaA-DH5α was cloned and expressed. The inclusion body of recombinant HpaA protein was purified and purified by gel excision. The soluble recombinant HpaA protein was obtained. SDS-PAGE electrophoresis using different concentrations, different pH of sodium acetate solution and different staining time electrophoresis protein bands, gel electrophoresis analysis of the best conditions. Western blot identification of recombinant HpaA protein antigenicity. BALB / c mice were immunized with the recombinant protein, and anti-HpaA antibody was detected by two-dimensional immunodiffusion to identify the immunogenicity of the recombinant protein. Results 4 mol / L sodium acetate solution, pH 13.0, the role of 1 h gel cut the best conditions. The size of recombinant HpaA protein obtained by gel-cutting purification was 6 ml with a concentration of 0.2942 mg / ml and a protein yield of 1.7652 mg, with a yield of 77.1%. West-ern blot showed that the protein had good antigenicity. Mouse serum can react with recombinant HpaA protein, double expansion titer greater than or equal to 1:16, has good immunogenicity. Conclusion Ablation of recombinant HpaA protein inclusion bodies can be purified by gel cutting to obtain highly purified soluble recombinant HpaA protein. The protein has good antigenicity and immunogenicity and can be used for further scientific research.