【摘要】 目的将克隆的抗体轻重链可变区基因拼接成单链抗体基因并将其在大肠杆菌中表达。方法通过RT—PCR从两株抗MagaininⅡ杂交瘤细胞株 (2D1 ,3F8)中克隆出VH 和VL 基因 ,然后利用重组PCR技术 ,将VH 和VL 基因通过柔性肽段(GLY4Ser)3 的Linker拼接成单链抗体基因(ScFv) ,将ScFv克隆到表达载体 pCANTAB5E上并将其分别在大肠杆菌E.coliHB2151及TG1 中进行表达。结果SDS PAGE和Western blot分析表明 ,ScFv在大肠杆菌E.coliHB2151中得到了分泌表达。间接ELISA结果显示可溶性ScFv及其表面展示噬菌体均具有抗原结合活性。结论成功地获得了ScFv 2D1和ScFv 3F8基因并在大肠杆菌中得到了功能性表达 ,为新型抗菌肽的设计打下了基础。更多还原

【Abstract】 Aim To obtain the ScFvs by splicing the VH and VL genes and then express the ScFvs in E.coli. Methods VH and VL genes were cloned from two hybridoma cell lines 2D1 and 3F8 against Magainin II and then spliced together through a flexible linker(Gly4Ser)3 to create ScFv using recombinant PCR. The ScFv genes were then cloned into expression vector pCANTAB5E and expressed in E.coli HB2151 and TG1 respectively. Results SDS PAGE and western blot analyses showed the ScFvs were secretingly expressed in E.coli HB 2151. The result of indirect ELISA showed the soluble and phage displayed ScFv had antigen binding activity. Conclusion The successful cloning and expression of ScFv laid the foundation for the design of new antibacterial peptides.更多还原