狂犬病病毒重组免疫毒素的表达及其生物活性鉴定

Expression of a Recombinant Immunotoxin against Rabies Virus-infected Cells and Its Biological Activity

将狂犬病病毒中和性单链抗体基因克隆入原核表达载体pET-PE40,经酶切鉴定及序列测定,成功构建了重组免疫毒素原核表达载体。IPTG诱导后目的蛋白获得高效表达,SDS-PAGE分析目的蛋白主要以不溶性包涵体的形式存在于菌体中,表达量占菌体总蛋白的32.29%。包涵体蛋白经体外复性及离子交换色谱柱、疏水作用色谱柱、Sephadex G200凝胶过滤层析柱三步纯化后获得纯度大于96%的目的蛋白,间接免疫荧光染色检测表明重组免疫毒素与狂犬病病毒感染细胞具有抗原结合活性,MTT试验显示,重组免疫毒素对狂犬病病毒感染细胞具有明显的杀伤作用,而对正常细胞无杀伤作用。

The single-chain Fv cDNA of a monoclonal antibody specific for glycoprotein of rabies virus,was cloned into the PE40 （a truncated form of pseudomonas exotoxin）gene inserted fusion protein expression plasmid pET-PE40 to form the expression vector pET-ScFv-PE40 ,The recombinant plasmid was identified by restriction enzyme digestion and sequencing. After transfection of E. coli BL21 and induction by IPTG, the recombinant immunotoxin fusion protein was highly expressed in E. coli BL-21 in the form of inclusion bodies（ up to 32.29% of total cellular proteins）. The fusion protein was refolded by constant gradient dialysis and purified with anion exchange chromatography, hydrophobic interaction and gel filtration chromatography, the interest protein was about 96% pure. Indirect immunofluorescent staining showed that recombinant immunotoxin could specifically bind to rabies virus-infected cells. Cytotoxicity analysis by MTT showed that the fusion proteins could kill rabies virus- infected cells specifically ,while having no toxicity to normal cells.