重组细粒棘球蚴(中国大陆株)线粒体苹果酸脱氢酶的表达、复性及鉴定

Expression,Renaturation and Identification of Recombinant mMDH of Echinococcus Granulosus( Chinese mainland strain)

目的通过原核表达细粒棘球蚴(中国大陆株)线粒体苹果酸脱氢酶(EgmMDH),获取高纯度的可溶性的重组细粒棘球蚴线粒体苹果酸脱氢酶(rEgmMDH)。方法从细粒棘球蚴(中国大陆株)中获得基因mdh并克隆到原核表达载体pET28a;通过IPTG诱导表达线粒体苹果酸脱氢酶(mMDH),进而采用亲和层析的方法纯化该包涵体目的蛋白并复性,获得可溶性目的蛋白rEgmMDH;利用细粒棘球蚴(中国大陆株)攻击感染家兔后获得血清;分别采用抗原头蚴兔血清和抗His-Tag鼠单克隆抗体对可溶性rEgmMDH进行Western blot鉴定。结果①成功构建原核表达载体pET28a-mMDH/BL21;②通过亲和层析及复性,获得高纯度可溶性目的蛋白rEgmMDH;③Western blot结果表明,抗原头蚴兔血清和抗His-Tag鼠单克隆抗体可分别特异性鉴定该复性可溶性rEgmMDH。结论通过本研究获得高纯度可溶性rEgmMDH,为进一步研究其在细粒棘球蚴感染宿主过程中的作用奠定基础。

Objective In order to obtain the recombinant proein EgmMDH （E.granulosus mitochondrial malate dehydrogenase）,the recombinant His6-tagged EgmMDH from the extract of transformed E.coli BL21 was expressed and refolded.Method Firstly,the mdh gene was isolated from Echinococcus granulosus and cloned as described by our lab before.Plasmid pET28a-mMDH was transformed into E.coli BL21.rEgmM-DH was expressed in plasmids of pET28a-mMDH/BL21.Secondly,the recombinant His6-tagged EgmMDH was purified by nickel chelate affinity chromatography （Novagen）.The purified His6-tagged protein was refolded by dialysis method.Then,The rabbits were challenged infection with Echinococcus granulosus （Chinese mainland strain） to obtain anti-rabbit sera.At last,the renaturalized rEgmMDH was detected by western blot using anti-rabbit sera and anti-his-tag monoclonal antibody.Results ① pET28a-mMDH/BL21 plasmid was constructed successfully.② The high purified soluble protein was harvested after the His6-tagged rEgmMDH protein was purified and refolded.③ The renaturalized rEgmMDH could be recognized with specific antibodies in western blotting.Conclusion In this study,a purified soluble rEgmMDH was expressed and identified successfully,which will be as a basis for further study on hosts and E.granulosus interplay.