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目的制备抗IV型胶原酶单链抗体(scFv),用于构建小型化抗肿瘤抗体靶向药物。方法构建重组表达质粒pET-scFv,并在大肠杆菌进行诱导表达。SDS-PAGE和Western-blot法对表达蛋白进行鉴定,分步透析复性。ELISA法、免疫细胞化学染色法检测scFv对靶抗原和肿瘤细胞的结合活性,明胶酶谱法检测对IV型胶原酶活性的抑制作用。Boyden Chamber法测定对肿瘤细胞侵袭的影响。动物试验肿瘤模型检测在小鼠体内的抗肿瘤活性。结果表达的单链抗体scFv(3G11)以包涵体的形式存在,经变性和复性后,对抗原IV型胶原酶和肿瘤细胞的免疫反应呈阳性,抗体亲和常数为6×107/mol/L。不仅可以抑制HT-29细胞和HT-1080细胞分泌的Ⅳ型胶原酶活性,还可以体外抑制肿瘤细胞的侵袭,抑制程度与浓度呈剂量依赖关系。scFv(3G11)4mg/kg对小鼠移植性肝癌H22的抑瘤率为49.4%(P<0.01)。结论scFv(3G11)保留了完整抗体的抗原结合和抑制活性,能够与肿瘤细胞特异性结合,并在小鼠体内有中度抑瘤效果。scFv(3G11)的相对分子质量远小于完整抗体,可作为肿瘤靶向药物的理想载体。
Objective To prepare anti-type IV collagenase single-chain antibody (scFv) for the construction of miniaturized anti-tumor antibody targeting drugs. Methods The recombinant plasmid pET-scFv was constructed and expressed in E. coli. The expressed proteins were identified by SDS-PAGE and Western-blot, and refolded by stepwise dialysis. ELISA and immunocytochemical staining were used to detect the binding activity of scFv to target antigen and tumor cells. Gelatin zymography was used to detect the inhibitory effect of collagenase type IV. Effect of Boyden Chamber Method on Tumor Cell Invasion. Animal Testing Tumor models were tested for antitumor activity in mice. The results showed that the scFv (3G11) expressed in the form of inclusion bodies. After denaturation and renaturation, the immunoreactivity against type IV collagenase and tumor cells was positive. The antibody affinity constant was 6 × 107 / mol / L. Not only can inhibit HT-29 cells and HT-1080 cells excreted collagenase activity, but also in vitro inhibition of tumor cell invasion, the degree of inhibition and concentration in a dose-dependent manner. The inhibitory rate of scFv (3G11) 4mg / kg on mouse transplanted liver cancer H22 was 49.4% (P <0.01). Conclusion The scFv (3G11) retains the antigen-binding and inhibitory activity of intact antibodies, specifically binds to tumor cells and has a moderate antitumor effect in mice. The relative molecular mass of scFv (3G11) is much smaller than that of intact antibody, which can be used as an ideal carrier for tumor targeting drugs.