小细胞肺癌2F7单抗ScFv的高效表达及复性研究

High level expression and in vitro refolding of 2F7 Single-Chain Fv antibody fragment against small cell lung carcinoma

目的在大肠杆菌中高效表达小细胞肺癌单抗2F7的单链抗体(ScFv),并获得具有生物学活性的ScFv。方法利用:PCR方法将2F7单抗重链可变区(VH)和轻链可变区(VL)通过一人工设计的柔性连接肽(Linker)连接,再将单链抗体基因重组到原核表达载体pQE31中,构建单链抗体高效表达载体pQE-2F7-ScFv。将pQE-2F7-ScFv质粒转化大肠杆菌M15后诱导表达,并对表达产物进行纯化和稀释复性。结果 获得了2F7单链抗体的高效表达,表达蛋白大小约27.4kD,以包涵体形式存在。包涵体蛋白在经过变性、纯化和稀释复性后,获得了有功能的单链抗体。结论 成功地构建和表达了小细胞肺癌单抗2F7的单链抗体,并对其进行了纯化和复性,将进一步促进2F7单抗小分子抗体的应用。

Objective To express Single-Chain Fv (ScFv) of 2F7 monoclonal antibody (mAb) against small cell lung carcinoma (SCLC) in E. coli and geting active product by renaturation in vitro. Methods The variable regions of heavy chain (VH) and light chain (VL) of 2F7 mAb were connected by a gentle linker using an assembly of polymerase chain reaction (PCR), and the expression vector pQE-2F7-ScFv of 2F7 ScFv was constructed by inserting the ScFv gene(VH-Linker-VL) into prokaryote expression vector pQE31. pQE-2F7-ScFv was transformed into E. coli M15 and expression was induced by IPTG, The expressed 2F7 ScFv was purified by Ni-affinity agarose. The fusion protein was diluted with renaturation solutions so that refolding process was done. Results 2F7 ScFv, with a molecular weight of 27.4 kD was expressed in E. coli as inclusion bodies. After the expressed 2F7 ScFv in inclusion body was purified by Ni-affinity agarose in the presence of 8mol/L urea and refolded in renaturation solutions, the functional ScFv binding to its antigen on H128 cell was obtained. Conclusion 2F7 ScFv against SCLC expression vector was successfully constructed and the functional protein was obtained by in vitro refolding. This study will promote the clinical application of 2F7 ScFv.