两种纯化幽门螺旋杆菌VacA-HpaA融合蛋白包涵体方法的比较

目的比较Ni^2＋-NTA树脂和切胶纯化重组VacA-HpaA融合蛋白的纯化效果,探讨纯化蛋白包涵体的简易方法。方法克隆并表达重组pQE30-vacA-hpaA-DH5α工程菌,获得重组VacA-HpaA融合蛋白的包涵体,分别进行Ni^2＋-NTA树脂和切胶纯化,获得可溶性重组蛋白,Western blot鉴定其抗原性;用纯化蛋白免疫BALB/c小鼠,ELISA法检测小鼠血清抗VacA、HpaA和VacA-HpaA的IgG水平,鉴定其免疫原性。结果两种方法均能纯化重组VacA-HpaA融合蛋白包涵体,得到可溶性的重组蛋白,Ni^2＋-NTA树脂纯化法获得的可溶性重组蛋白的纯度为95.8%,得率为63.5%;切胶纯化法获得可溶性重组蛋白的纯度为93.3%,得率为75.2%。Western blot鉴定纯化蛋白均具有良好的抗原性。ELISA法检测显示小鼠血清均能与重组蛋白VacA、HpaA和VacA-HpaA发生反应,两组IgG水平比较,差异无统计学意义（P〉0.05）,均具有良好的免疫原性。结论切胶纯化重组VacA-HpaA融合蛋白包涵体是一种简单、经济的可行方法,有利于重组蛋白包涵体的纯化。

Comparison of two methods of purifying recombinant VacA-HpaA fusion protein inclusion bodies of Helicobacter pylori

Objective To compare the effect of purification of recombinant VacA-HpaA fusion protein of Helicobacter pylori from inclusion bodies by gel slices and Ni^2＋-NAT chromatography, and to explore a simple method for purifying recombinant protein from inclusion bodies. Methods The recombinant bacteria pQE30-vacA-hpaA-DH5α were cloned and expressed. Inclusion bodies of recombinant VacA-HpaA fusion protein obtained were purified by gel slices and Ni^2＋- NAT chromatography, respectively. Antigenicity of soluble recombinant protein obtained was identified by Western blot, and immunogenicity of soluble recombinant protein was identified by testing the anti-VacA, anti-HpaA and anti-VacAHpaA IgG of BALB/c mice immuned with soluble recombinant protein by ELISA. Results Two methods both can purify recombinant VacA-HpaA fusion protein from inclusion bodies. The soluble recombinant VacA-HpaA fusion protein was obtained; purity and yield were 95.8% and 63.5% by Ni2＋ NAT chromatography; 93.3% and 75.2% by gel slices, respectively. Western blot confirmed that both soluble recombinant VacA-HpaA fusion proteins had satisfactory antigenicity. Immune serum of BALB/c mice all could be bond with the recombinant protein VacA, HpaA and VacA-HpaA. The difference of IgG level hadn＇t statistical significance（P〉0.05）, which showed that the recombinant VacA-HpaA fusion protein both had the satisfactory immunogenicity. Conclusion Purification of the recombinant VacA-HpaA fusion protein from inclusion bodies in gel slices is a relatively simple, effective, economic, useful and practical method, which could be put into purification for protein from inclusion bodies.