重组靶向毒素hIL6(T22)-PE38在大肠杆菌中的表达及其细胞毒性

目的 在大肠杆菌中表达、纯化重组靶向毒素hIL6(T22)-PE38,并检测其细胞毒性。方法以PHis-hIL6(T22)-PE38质粒为模板,PCR扩增hIL6(T22)-PE38基因,插入表达载体pET-28a(+)的NcoⅠ和XhoⅠ多克隆位点,构建重组表达质粒,分别转化至大肠杆菌BL21(DE3)、Rosettablue(DE3)和BL21(DE3)pLysS中,IPTG诱导表达,并对表达条件进行优化。表达产物经亲和层析纯化后,检测其细胞毒性。结果重组表达质粒pET-28a-hIL6(T22)-PE38经双酶切和测序证明构建正确。表达的重组毒素相对分子质量约56 000,在大肠杆菌Rosettablue(DE3)中表达量最高。最适诱导表达条件为:1 mmol/L IPTG 28℃诱导4 h。重组毒素能选择性地杀伤人骨髓瘤细胞U266和鼠骨髓瘤细胞SP2/0,体外对U266和SP2/0细胞的IC50分别为0.5～1.0和1.0～1.5μg/ml。结论重组靶向毒素hIL6(T22)-PE38在大肠杆菌中成功获得可溶性表达,纯化的蛋白可明显选择性杀伤U266和SP2/0细胞。

Expression of Recombinant Target Toxin hIL6(T22)-PE38 in E.coli and Cytotoxicity of Expressed Product

Objective To express recombinant target toxin hIL6(T22)-PE38 in E.coli,purify the expressed product and determine its cytotoxicity.Methods The hIL6(T22)-PE38 gene was amplified by PCR using plasmid PHis-hIL6(T22)-PE38 as a template and inserted into the NcoⅠand XhoⅠpolyclonal sites of expression vector pET-28a.The constructed recombinant plasmids were transformed to E.coli BL21(DE3),Rosetta blue(DE3)and BL21(DE3)pLysS respectively for expression under induction of IPTG,and the condition for induction and expression was optimized.The expressed product was purified by affinity chromatography then determined for cytotoxicity.Results Both restriction analysis and sequencing proved that recombinant plasmid pET-28a-hIL6(T22)-PE38 was constructed correctly.The expression level of recombinant toxin hIL6(T22)-PE38,with a relative molecular mass of about 56 000,was the highest in E.coli Rosetta blue(DE3).The condition for expression was optimized as induction with 1 mmol / L IPTG at 28℃ for 4 h.The recombinant toxin selectively killed human myeloma U266 and SP2 / 0 cells,with IC50 in vitro of 0.5 ～ 1.0 and 1.0 ～ 1.5 μg / ml.Conclusions Recombinant target toxin hIL6(T22)-PE38 was successfully expressed in E.coli in a soluble form and,after purification,selectively killed U266 and SP2 / 0 cells significantly.