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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。
Objective To clone the cloned variable light chain variable region genes into single chain antibody genes and express them in E. coli. Methods VH and VL genes were cloned by RT-PCR from two anti-MagaininⅡ hybridoma cell lines (2D1, 3F8). Then the VH and VL genes were spliced by Linker of flexible peptide (GLY4Ser) 3 Single chain antibody gene (ScFv). The ScFv was cloned into the expression vector pCANTAB5E and expressed in Escherichia coli E. coli HB2151 and TG1, respectively. Results SDS PAGE and Western blot analysis showed that ScFv was secreted in E.coli HB2151. Indirect ELISA showed that both soluble ScFv and its surface-displayed phage had antigen-binding activity. Conclusion The ScFv 2D1 and ScFv 3F8 genes were successfully obtained and expressed in Escherichia coli, which laid the foundation for the design of novel antimicrobial peptides.