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目的 构建抗胶质瘤重组免疫毒素SZ39 PE4 0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PE4 0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 PE4 0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Westernblot分析显示 ,表达产物的相对分子质量 (Mr)为 6 80 0 0左右 ,与SZ39 PE4 0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 2 0 %。ELISA及免疫荧光技术均证实 ,经复性的SZ39 PE4 0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 PE4 0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
Objective To construct an anti-glioma recombinant immunotoxin SZ39 PE40 gene and express it in E.coli. Products denatured and refolded primarily expressed as inclusion bodies. Methods PE40 gene was cloned from plasmid pVC85 and spliced with scFv SZ39 gene. The recombinant immunotoxin SZ39 PE40 gene was constructed and expressed in E. coli. The expressed products (inclusion bodies) were isolated, purified, denatured and refolded, and the binding activity to glioma cells was examined by ELISA and immunofluorescence. Results SDS PAGE and Western blot analysis showed that the relative molecular mass (Mr) of the expressed product was about 6 80 0, which was consistent with the theoretical value of SZ39 PE40 fusion protein. Gray scale scanning showed that the expression level accounted for 20% of total bacterial proteins. ELISA and immunofluorescence confirmed that the refolded SZ39 PE40 fusion protein has the activity of binding SHG 4 to glioma cells. Conclusion The SZ39 PE40 fusion protein gene was successfully constructed and expressed in prokaryotic cells. The renaturation of the recombinant SZ39 PE40 fusion protein has the specific activity of recognizing glioma cells, which lays a foundation for the further application in glioma targeting therapy