华支睾吸虫线粒体苹果酸脱氢酶基因的原核表达及重组蛋白的功能鉴定

目的克隆肝吸虫线粒体苹果酸脱氢酶基因并在大肠杆菌内表达，同时对表达的重组蛋白进行初步的功能分析，为进一步的研究和药物筛选打下基础。方法通过大规模测序从肝吸虫cDNA文库中确定肝吸虫线粒体苹果酸脱氢酶基因．采用PCR方法扩增出目的片段，与表达质粒pGEX-4T-1连接构建重组质粒，IPTG诱导表达；研究重组蛋白的理化性质及酶动力学特征，并寻找可能的抑制剂。结果成功表达出约64kDa的重组蛋白，凝血酶切后为36kDa蛋白，其酶活性为63．6U／mg。1．14mM4，4’-二甲氨基联苯甲醇处理30min酶活性下降了60％；吡喹酮、灭滴灵和阿苯哒唑对酶活性无抑制作用；而5’-单磷酸腺苷对重组酶有竞争性抑制作用，其Ki为0．49。结论成功地表达了目的蛋白，为药物筛选提供了一个易于获得的候选蛋白分子。

Prokaryotic expression of gene encoding the mitochondrial malate dehydrogenase of Clonorchis senensis and the functional identification of the recombinant protein

In order to provide the basis for further studies and for the screening of drugs for treatment,the gene encoding the mitrochondial malate dehydrogenase of Clonorchis senensis(Cs-mMDH) was cloned by high throughput sequencing from the cDNA library constructed from adult C.senesis.The recombinant protein was expressed in E.coli as a molecule with molecular weight of 64 kDa.When it was cleaved by thrombin as a 36 kDa protein,its enzyme activity was 63.6 U/mg,and it reduced up to 60% when it was treated with 1.14 mM of 4,4'-bisdimethylamino-diphenylcarbinol.Paraziquantel,metronidazol and albendazol showed no inhibitory effect on the activity of Cs-mMDH,but adenosine 5' monophosphate demonstrated competitive inhibition to Cs-mMDH with the Ki of 0.49 mM.The results of this study provide the foundation to supply sufficient amount of protein as target for drug screening.