铜绿假单胞菌外毒素A表达载体的构建及可溶性表达

目的 实现铜绿假单胞菌外毒素A(PEA)全基因的可溶性表达。方法 用基因重组技术构建pMAL-PEA原核分泌型表达载体，并在大肠杆菌BL21中进行表达，然后利用直链淀粉树脂对表达蛋白进行亲和层析纯化。结果 酶切鉴定及测序结果表明pMAL-P2X载体上成功地插入了PEA全基因。用IPTG诱导目的基因表达，表达的融合蛋白量约占细菌总蛋白的20％。表达产物经超声处理后，80％的融合蛋白存在于上清液中，说明实现了PEA的可溶性表达。表达的融合蛋白经亲和层析纯化后进行SDS-PAGE电泳，显示为一条蛋白带，分子量约为112kD。结论成功地对PEA全基因进行了可溶性表达和纯化，获得了稳定表达的工程菌，为深入研究：PEA的致病机制和制备用于免疫导向治疗的重组毒素奠定了基础。

Construction of recombinant plasmid of Pseudomonas aeruginosa exotoxin A and its secreting expression in E.coli

Objective To clone and to express the full-length Pseudomonas aeruginosa exotoxin A gene and to purify the expressed protein using affinity chromatography. Methods Exotoxin A gene was amplified from the recombinant plasmid pSK/PEA-T vector and subcloned into the pMAL-P2X vector. Then the recombinant plasmid pMAL- PEA was transformed to E.coli BL21. After induction with IPTG, the expressed protein was analyzed by SDS-PAGE and purified with affinity chromatography. Results The recombinants expressed the fusion protein at a level of about 20% of total cell proteins, and 80% of the fusion protein was secreted into the supernatant. Conclusion Successful expression and purification of PEA are of significance for in-depth study of the pathogenic mechanism of Pseudomonas aeruginosa and preparing immunotoxin.