铜绿假单胞菌外毒素A的表达、纯化与生物学活性研究

Expression, purification of recombinant Pseudomonas aeruginosa exotoxin A and its cytotoxicity determination

目的利用基因工程技术制备铜绿假单胞菌外毒素A（PE），为深入研究PE的致病机理和免疫防治奠定基础。方法应用PCR技术从铜绿假单胞菌基因组中扩增外毒素A全长结构基因，将其克隆于原核表达载体pQE-31中，所构建的重组质粒经测序鉴定后转化大肠埃希菌JM109，IPTG诱导表达；制备融合蛋白包涵体，并采用Ni—NTA柱亲和层析、葡聚糖凝胶过滤和阴离子交换层析分离和纯化目的蛋白；采用透析法对纯化后的目的蛋白进行复性，MTT法测定复性后的重组PE对L929细胞、B16黑素瘤细胞的细胞毒活性。结果通过对PCR反应体系的优化，扩增到了PE全长结构基因。所构建的pQE-PE重组质粒经酶切及测序鉴定与设计序列一致；转化E．coli JM109后，IFTG诱导目的蛋白表达率约为25％；SDS-PAGE初步测定目的蛋白的相对分子质量（Mr）约为66×10^3，与理论预期值一致；破菌后电泳证实目的蛋白主要以包涵体形式表达。经亲和层析、葡聚糖凝胶过滤和阴离子交换层析后蛋白纯度大于95％。MTT法测得重组PE对L929细胞和B16黑素瘤细胞的半数抑制浓度（IC50值）分别为2．13μg/ml、2．58μg/ml。结论通过对PE的表达纯化，获得了具有细胞毒活性的重组PE，为利用基因工程手段大量制备PE的工作奠定了基础。

Objective To prokaryotically express and purify Pseudomonas aeruginosa exotoxin A（PE）, and to investigate its cytotoxicity. Methods The exotoxin A structural gene was amplified from chromosomal DNA of Pseudomonas aeruginosa by PCR and then cloned into pQE-31 vector. The recombinant plasmid pQE-PE was transformed into E. coli JM109. The inclusion bodies of fusion protein were extracted, and then dissolved in 8 mol/L urea. The fusion proteins were purified by Ni-NTA column, Sephadex G-75 column and ion exchange chromatography. After purification, the fusion proteins were renatured through dialysis, then the cytotoxicity against L929 cells and B16 cells were determined by MTT method. Results The gene of exotoxin A was successfully cloned into pQE-31. The results of SDS-PAGE showed that the molecular mass（Mr） of the expressed protein was 66 × 10^3, and the expression rate was 25%. The purity of recombinant PE was up to 95% after Ni-NTA column, Sephadex G-75 column and ion exchange chromatography, and the purified PE had cytotoxicity to both L929 cells and B16 cells. Conclusion The Pseudomonas aeruginosa exotoxin A was successfully expressed in prokaryotically expression system. After purified, it has shown certain cytotoxicity potential.