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目的将肺炎克雷伯菌所产CTX-M-3型超广谱β-内酰胺酶(extended-spectrumβ-lactamase,ESBLs)进行表达、纯化及其多克隆抗体的制备。方法将保存的重组质粒pET-28a(+)/CTX-M-3在大肠埃希菌BL21(DE3)中原核表达。IPTG诱导CTX-M-3融合蛋白表达,利用镍琼脂糖凝胶亲和柱层析纯化蛋白,用纯化的CTX-M-3型ESBLs酶蛋白免疫小鼠制备多克隆抗体。结果可溶性检测表明表达产物以可溶性形式(上清中)和包涵体形式(沉淀中)两种形式存在。通过蛋白表达条件的优化实验,SDS-PAGE电泳结果显示18℃,0.8 mmol/L IPTG诱导24 h的CTX-M-3重组蛋白的可溶性表达最佳。SDS-PAGE电泳和Western-blot检测表明获得纯化的重组32 kD蛋白。免疫获得的CTX-M-3型ESBLs酶蛋白抗血清的效价为1∶32。结论 pET-28a(+)/CTX-M-3表达载体在大肠埃希菌BL21(DE3)中表达,用His亲和层析柱纯化可获得高纯度可溶性的重组蛋白,成功制备了效价较高的抗CTX-M-3型ESBLs酶蛋白多克隆抗体。
Objective To express and purify CTX-M-3 extended-spectrum β-lactamase (ESBLs) produced by Klebsiella pneumoniae and to prepare its polyclonal antibody. Methods The recombinant plasmid pET-28a (+) / CTX-M-3 was prokaryoticly expressed in Escherichia coli BL21 (DE3). The CTX-M-3 fusion protein was induced by IPTG. The purified protein was purified by Ni-Sepharose affinity chromatography and the polyclonal antibody was prepared by immunizing mice with the purified CTX-M-3 ESBLs protein. Results Solubility test showed that the expression product existed in both soluble forms (in supernatant) and inclusion bodies (in sediment). The results of SDS-PAGE showed that the optimal expression of CTX-M-3 recombinant protein was induced by 0.8 mmol / L IPTG for 24 h at 18 ℃. SDS-PAGE electrophoresis and Western-blot detection showed that the purified recombinant 32 kD protein was obtained. The CTX-M-3 type ESBLs enzyme protein antiserum obtained by immunization had a titer of 1:32. Conclusion The recombinant plasmid pET-28a (+) / CTX-M-3 was expressed in Escherichia coli BL21 (DE3). The purified recombinant protein was purified by His affinity chromatography. High anti-CTX-M-3 type ESBLs protein polyclonal antibody.