EGFRvⅢ单链抗体治疗小鼠脑胶质瘤的研究

Curative effect of Single chain antibodies of EGFRvⅢ on brain gliomas in mice

目的探讨表皮生长因子受体III型突变体单链抗体(EGFRvIIIscFv)对小鼠脑胶质瘤的治疗效果。方法人工合成EGFRvIIIscFv基因序列并构建到原核表达载体中,诱导表达重组蛋白,经电泳和western blotting鉴定;再通过纯化和复性,得到有功能的EGFRvIIIscFv;用酶联免疫吸附分析(ELISA)测定抗体的亲和常数后,通过立体定向仪将其注射到胶质瘤小鼠的脑内,观察荷瘤鼠的生存期,免疫荧光法检测肿瘤血管内皮生长因子(VEGF)的表达。结果双酶切鉴定和测序结果证实人工合成的EGFRv Ⅲ scFv序列与基因数据库中完全一致;经过诱导表达,电泳显示重组蛋白在大肠杆菌中以包涵体形式表达;western-blotting鉴定蛋白分子量为63kDa,与预期大小基本一致;该单链抗体亲和常数为0.203×10-8mol/L;将蛋白注射到荷瘤鼠的脑内,与对照组相比,实验组肿瘤VEGF表达量明显减少、荷瘤鼠中位生存期明显延长(P<0.01)。结论 EGFRvscFv重组蛋白可与EGFRvⅢ特异性结合;脑内注射EGFRvⅢscFv可明显降低胶质瘤血管形成,延长荷胶质瘤小鼠生存期。

Objective To observe the curative effect of the single chain antibodies of epidermal growth factor receptor variant Ⅲ （EGFRvⅢ） single chain antibody variable fragment （EGFRvⅢ scFv） on cerebral gliomas. Methods The expression of the recombinant protein in Escherichia coli was induced by the constructed plasmid inserted into with EGFRvⅢ scFv gene whose sequence was in accordance with the one published in Gene Bank. However, the single chain functional antibodies of EGFRvⅢ scFv were identified by SDS-PAGE and western blotting, and then obtained by purification and refolding. The affinity constant of the single chain antibody was determined by enzyme linked immunosorbent assay and then the antibodies were stereotaxically injected into the brain in the mice with brain gliomas. The median survival time of the mice with brain gliomas was observed and the expression of vascular endothelial growth factor （VEGF） was determined by immunohistochemical technique. Results The double digestion and gene sequencing proved that the sequence of the synthesized GFRvⅢ scFv was in accord with Gene bank. The recombinant protein in Escherichia coli was expressed in the inclusion body form. The molecular weight of the protein was 63 kDa which was in accordance with the expected value. The affinity constant was 0.203×10^-8 mol/L. The level of VEGF expression in the tumors was significantly lower （P〈0.05） and the median survival time of the mice with tumors was significantly longer （P〈0.01） in the experimental group than those in the control group. Conclusions The expression plasmid of pET30a-EGFRvⅢ scFv is successfully constructed. The recombinant protein of EGFRvⅢ scFv after the purification and refolding is capable of specific binging to EGFRvⅢ. The expression of VEGF in the gliomas may be decreased and the median survival time of the mice with gliomas was prolonged by the intracerebral injection of EGFRvⅢ scFv.