伴放线放线杆菌细胞膨胀致死毒素重组蛋白的表达

Expression of recombinant cytolethal distending toxin of ActinobacUlus actinomycetemcomitans

目的通过分子克隆技术构建细胞膨胀致死毒素（cytolethaldistendingtoxin，CDT）的重组表达载体，并诱导重组CDT蛋白的表达，以期为重组CDT蛋白的生物学功能研究奠定基础。方法采用聚合酶链反应（PCR）扩增获得CDT的编码基因cdtABC，通过TA克隆和限制性酶切将cdtABC与目的载体pQE60连接，转化感受态大肠杆菌后诱导重组CDT蛋白的表达，收集细菌总蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳和蛋白质印迹法鉴定。结果pQE60-cdtABC转染的感受态大肠杆菌中均携带cdtABC基因，该片段与GenBank中的DNA一致性高达99％。细菌总蛋白中21000、25000、32000左右的蛋白增多，蛋白质印迹法检测发现带有6-His标记的目的蛋白。结论本研究成功构建了CDT的重组表达载体，并诱导重组CDT蛋白的表达。

Objective To examine the expression of recombinant cytolethal distending toxin （CDT） produced by Actinobacillus actinomycetemcomitans （Aa）. Methods CDT encoding gene cdtABC was amplified by PCR. Through TA clone and restriction endonuclease digestion, gene cdtABC and vector pQE60 were ligated to form pQE60-cdtABC expression system which transformed into competent cells. Protein expression was induced by IPTG and examined by SDS-PAGE and Western-blotting. Results Random colony PCR of pQE60-cdtABC transformed cells demonstrated that all strains contained cdtABC gene. The DNA sequence was blast with cdtABC gene from GenBank and 99% homology was obtained. SDS-PAGE and Western-blotting confirmed that recombinant CDT was obtained. Conclusions CDT protein expression system was reconstructed and recombinant protein was obtained.