多杀性巴氏杆菌Lon蛋白酶的原核表达及ATPase活性的检测

Prokaryotic expression and detection of the ATPase activity of Lon protease from Pasteurella multocida

为表达多杀性巴氏杆菌(P.multocida)Lon蛋白及检测其生物活性,本研究通过PCR扩增P.multocida C51-17株Lon基因,将其克隆到质粒pET-30a(+)中,构建重组表达质粒pET30a-Lon,并将其转化至大肠杆菌BL21(DE3)感受态细胞中,经IPTG诱导表达重组蛋白Lon,采用Ni-NTA His-Bind Resin纯化目的蛋白。SDS-PAGE结果表明,重组蛋白Lon分子量大小约为97 ku,主要以可溶性蛋白形式表达;western blot检测结果表明,重组表达蛋白能够被P.multocida阳性血清识别;ATPase活性检测试验表明,纯化的重组蛋白Lon具有ATPase活性,其将ATP降解为ADP的酶活性可以达到27 708.47μmol/(mg·min)。本研究表达了P.multocida Lon重组蛋白,并且测定其ATPase活性,为进一步研究Lon蛋白在P.multocida致病中的作用奠定了基础。

To express and test the ATPase activities of Lon protease from Pasteurella multocida, the Lon gene was amplified from P.multocida C51-17 strain by PCR and cloned to pET-30a（＋） vector for expression in Escherichia coil BL21 （DE3） under induction with IPTG. Then the recombinant protein was expressed and purified by Ni-NTA His-Bind Resin affinity chromatography. The SDS-PAGE analyses showed the expressed recombinant Lon protein was about 97 ku and mainly existed in a soluble form. Western blot analysis demonstrated that the recombinant Lon protein was recognized by positive sera of P.multocida. While, the purified Lon protein also possessed the ATPase activity, and the enzyme activity was 27,708.47 μmol/（mg·min）. This study provided a basis for the further studies on the role of Lon protein during the P.multocida pathogenesis.