HSP65-PEAⅠ融合蛋白表达纯化及免疫效果研究

Study on the expression,purification and immune efficacy of fusion protein HSP65-PEAⅠ

目的通过基因工程手段在大肠杆菌中表达并纯化融合蛋白HSP65-PEAⅠ,建立小鼠实验模型,检测其抗体效价水平,初步评价免疫效果。方法扩增HSP65-PEAⅠ片段插入表达载体pET-28a中,转化E.coli BL21(DE3),IPTG诱导表达,优化纯化条件,以HSP65-PEAⅠ为免疫原对小鼠进行免疫接种。结果双酶切和测序鉴定结果证实HSP65-PEAⅠ成功克隆入pET-28a载体中,表达的重组蛋白相对分子量约为97 000,主要以包涵体形式表达,纯化后的重组蛋白纯度达90%,浓度为0.498mg/ml。免疫小鼠均在首免1周后采集的血清中检测到特异性抗体,抗体效价水平持续增长且在42天均达到了210。结论成功在E.coli中表达并经过纯化得到重组蛋白HSP65-PEAⅠ,确定了动物免疫程序,为制备抗烧烫伤多器官衰竭疫苗奠定了基础。

Objective Express protein HSP65-PEAⅠ in E.coli and obtain purified protein,establish mice model, preliminary evaluate the immune efficacy by detecting antibody titer levels.Methods Amplify HSP65-PEAⅠand then inserted into expression vector pET-28a,transform E.coli BL21（DE3）,express with the induction of IPTG,optimize the purification condition,then the protein was inoculated in mice as immunogen.Results Double digestion and sequencing results proved that the HSP65-PEAⅠ gene was cloned into vector pET-28a,the relative molecular mass of exressed recombinant protein was about 97000,mainly expressed as inclusion body.After purification,purity of recombinant protein was more than 90%,concentration was 0.498 mg/ml.The specific antibody was detected in the serum of mice in the first week after immunization,the titer level continued growing and rise to 2 10 after 42 days.Conclusion Recombinant protein HSP65-PEAⅠwas successfully expressed in E.coli and purified.Animal immunization program was further established which laid a foundation for preparation of vaccine for multiple organ failure by burns.