小细胞肺癌抗独特型单链抗体的表达及活性研究

目的:在大肠杆菌中高效表达小细胞肺癌(SCLC)抗独特型抗体3F6单链抗体(ScFv),并获得具有生物学活性的ScFv。方法:从SCLC抗独特型抗体3F6小鼠杂交瘤细胞中提取总RNA,反转录为cDNA。利用小鼠抗体骨架区共用引物,PCR扩增单抗重链可变区(VH)和轻链可变区(VL)。通过人工设计的柔性连接肽(Gly4Ser)3连接构建3F6ScFv。再将其重组到原核表达载体pQE31中,构建3F6ScFv表达载体。转化大肠杆菌M15,IPTG诱导表达,用NiNTA树脂对表达产物进行变性纯化。通过凝胶(SephacrylS200)柱上在位复性后,用竞争ELISA检测复性的3F6ScFv活性。结果:获得了SCLC抗独特型抗体3F6的VH和VL基因,构建了3F6ScFv表达质粒。在大肠杆菌中高效表达3F6ScFv,表达蛋白的相对分子质量为32×103,以包涵体形式存在。纯化后获得较纯的3F6ScFv蛋白,经复性后可竞争2F7抗体与SCLCNIHH128细胞结合。结论:成功构建、表达、纯化和复性了SCLC抗独特型抗体3F6ScFv,获得有活性的3F6ScFv,为SCLC抗独特型抗体的进一步研究奠定了基础。

Expression and activity measurements of single chain Fv of anti-idiotypic antibody 3F6 against small cell lung cancer

Objective:To express single chain Fv (ScFv) of anti-idiotypic monoclonal antibody (mAb) 3F6 against small cell lung cancer (SCLC) in E coli to get functional product. Methods:The total RNA was extracted using Trizol from the hybridoma cells secreting anti-idiotypic mAb 3F6 and reversely transcribed to cDNA. The variable regions of heavy chain (VH) and light chain (VL) of anti-idiotypic mAb 3F6 were amplified by polymerase chain reaction (PCR) using universal primers at the framework region of mouse antibody. The ScFv of mAb 3F6 was constructed by connecting VH and VL by a linker peptide (Gly4Ser) 3 using an assembly PCR and subcloned into the expression vector pQE31. The pQE-3F6ScFv was transformed into E coli M15 and expression was induced by IPTG. The expressed 3F6 ScFv was purified and refolded by affinity chromatography using Ni-NTA resin. The immunoactivity of 3F6 ScFv was detected by competitive ELISA. Results: The VH and VL genes of mAb 3F6 against SCLC were obtained. The ScFv of mAb 3F6 was constructed and highly expressed in E coli. The relative molecular weight of fusion protein is 32 kDa in the form of inclusion bodies. The purified 3F6 ScFv showed a single band on SDS-PAGE. After renaturation, the ScFv of mAb 3F6 could compete for 2F7 antibody binding to SCLC cell line NIH-H128. Conclusion:We successfully construct,express,purify,and re-nature the anti-idiotypic mAb 3F6 against SCLC and obtain functional 3F6 ScFv. This study will establish a basis for the clinic application of the ScFv of mAb 3F6.