MR-1S融合蛋白的表达、纯化与保存及其抗血清的制备

Expression,purification and preservation of myofibrillogenesis regulator 1S recombinant protein and its antiserum preparation

目的通过人肌纤生成调节因子1(hMR-1S)基因重组表达获得MR-1S-His融合蛋白,纯化后制备抗MR-1S抗血清;同时确定该蛋白保存的稳定体系,为研究MR-1S蛋白理化性质及其功能提供关键的原料。方法利用原核表达载体pET21a(+),构建pET21a-MR-1S重组质粒,转化大肠杆菌BL21(DE3),异丙基硫代半乳糖苷(IPTG)诱导MR-1S-His融合蛋白表达,亲和纯化后Western blot鉴定;进一步用该融合蛋白免疫新西兰兔,制备抗MR-1S的多克隆抗体。同时通过在不同温度条件下,对4种不同的稳定体系中保存后蛋白活性测定进行比较,确定最合适的稳定保存体系及条件。结果构建了重组质粒pET21a-MR-1S,并在BL21中进行可溶性表达,经Western blot鉴定确认;免疫新西兰兔获得了抗MR-1S抗血清;确定了合适的稳定体系及保存条件。结论在非变性条件下实现了可溶性MR-1S融合蛋白的表达和稳定保存,进一步制备的抗MR-1S抗血清为研究MR-1S的性质及功能奠定了基础。

Objective To obtain the myofibrillogenesis regulator 1S（MR-1S）-His recombinant protein through the expression of MR-1S gene and prepare anti-MR-1S antiserum after purification.To identify the stable system to preserve the recombinant protein,and to provid the reference for the investigation of MR-1S protein characteristics and biological function.Methods The recombinant plasmid pET21a-MR-1S was constructed with pronucleus expression vector pET 21a（＋） and transferred into Escherichia coli BL21（DE3）,and the expression of MR-1S recombinant protein was induced by isopropyl-beta-D-thiogalactopyranoside（IPTG）.The MR-1S recombinant protein was purified by affinity chromatography and identified by Western blot.The anti-MR-1S polyclonal antibodies were prepared by the rabbit-immunized technique.With comparison of the protein activities of 4 stable systems under different temperatures,the most suitable stable preservation system and condition were picked out.Results Recombinant plasmid pET21a-MR-1S was established,and the MR-1S-His recombinant protein was expressed solubly in Escherichia coli BL21（DE3） by the identification of Western blot.The anti-MR-1S antisera were obtained by immunization rabbit,and the satabilization system and preservation condition were definited.Conclusions Under undenatured conditions,the soluble MR-1S recombinant protein is expressed and can be preserved stably,and it lays the foundation for MR-1S characteristices and biological function studies.