抗磺胺二甲嘧啶单链抗体基因的克隆与序列分析

Sequence Analysis and Construction of Anti-sulfadimidine Molecule ScFv Gene

采用RT-PCR方法成功克隆出抗磺胺二甲嘧啶(SM2)单抗的杂交瘤细胞中396 bp的VH和339 bp的VL可变区基因,再通过重叠延伸PCR方法,由引入的(Gly4Ser)3柔性肽体外组装抗717bp的SM2-ScFv基因。测序经NCBI Blast比较分析和Expasy预测分析。结果表明:所得ScFv具有重组功能性鼠抗体可变区基因的特征,单链抗体的结构符合抗体可变区的功能区的框架区和可变区的结构特点,是抗磺胺二甲嘧啶的可变区基因,且经lingker连接后为影响轻重链的结构。

To construct a gene of ScFv against-SM2 molecule. The VH and VL genes cloned from McAb 3D7 hybridoma cell were ligated with a flexible linker （Gly4Ser）3 to construct ScFv gene, then corresponding restriction endonuclease digestion sites were introduced into 5＇ and 3＇ end of ScFv gene. Restriction endonuclease digestion and DNA sequencing proved that ScFv gene was correctly constructed. NCBI comparison certificated that VL gene was mouse g chain and had the characteristics of the mouse variable region gene. The ScFv-pMD18-T vector was constructed successfully, and the normal exposure of the epitope made the solid foundation for the further application of the drug residues.