抗胶质瘤重组免疫毒素SZ39-PE40的原核表达、纯化及复性研究

目的 构建抗胶质瘤重组免疫毒素SZ39-PE40基因并在大肠杆菌中表达。对主要以包涵体形式表达的产物进行变性和复性研究。方法 从质粒pVC85中克隆PE40基因，与抗胶质瘤的单链抗体（ScFv)-SZ39基因进行拼接，构建重组免疫毒素SZ39-PE40基因，并在大肠杆菌中表达，对表达产物（包涵体）进行分离，纯化，变性及复性处理后，以ELISA及免疫荧光技术，检查其对胶质瘤细胞的结合活性。结果 SDS-PAGE及Western blot分析显示，表达产物的相对分子质量（Mr)为68000左右，与SZ39-PE40融合蛋白的理论推算值相符，凝胶灰度扫描显示，其表达量占菌体总蛋白的20%。ELISA及免疫荧光技术均证实，经复性的SZ39-PE40融合蛋白具有结合胶质瘤细胞SHG-4的活性。结论 成功地构建并在原核细胞中表达SZ39-PE40融合蛋白基因，复性的表达产物具有特异性识别胶质瘤细胞的活性。为进一步应用于胶质瘤的导向治疗奠定了基础。

Study on prokaryotic expression,purification and renturation of a recombinant immunotoxin SZ39-PE40

Aim To construct the recombinant immunotoxin SZ39 PE40 gene and express it in E coli. To observe the binding activity of immunotoxin expressed mainly in the form of inclusion body to glioma cells after denaturation and renaturation. Methods Using the plasmid pVC85 as template, the PE40 cDNA was amplified by PCR, and then the PE40 gene was linked up with ScFv SZ39 gene to construct the recombinant gene SZ39 PE40, and it was expressed in E.coli . After preparation, purification, denaturation and renaturation of the immunotoxin SZ39 PE40, the binding activity of SZ39 PE40 to glioma cells was examined by ELISA and immunofluorescent assay.Results SDS PAGE and Western blot analyses showed that a protein band with M r of 68 000 appeared on SDS PAGE gel and nitrocelulose membrane. Scanning of SDS PAGE gel indicated that the expressed protein accounted for 20% of the total bacterial protein, and existed mainly in the form of inclusion body. After renaturation, the expressed immunotoxin SZ39 PE40 still retained the activity of specifically binding to SHG 4 glioma cells. Conclusion The recombinant immunotoxin against glioma has been constructed and expressed successfully, which provides warrant for further study on application of the recombinant immunotoxin to treatment of glioma.