MG_7胞内抗体的表达及活性鉴定

Prokaryotic expression and bioactivity identification of MG_7 intrabody

目的制备 MG7胞内抗体并鉴定其分子量和抗原结合活性。方法以含 MG7sc Fv基因的质粒 p CANTAB5 E为模板 ,PCR扩增 MG7sc Fv基因 ,并在其 3’端引入一段内质网滞留序列 (SEKDEL ) ,即形成 MG7胞内抗体基因。 DNA测序后 ,将 MG7胞内抗体基因克隆到表达载体 p GEX- 3X中 ,转化宿主菌 HB2 15 1,IPTG诱导 ,SDS- PAGE分析产物分子量 ,EL ISA检测产物的 MG7抗原结合活性。结果电泳分析发现融合基因片段长约 780 bp。DNA序列分析显示 ,SEKDEL与 MG7sc Fv基因正确融合 ;SDS- PAGE分析发现 MG7胞内抗体的分子量为 32 0 0 0 u;EL ISA检测发现 MG7胞内抗体具有明显的抗原结合活性。结论我们成功地制备并表达了 MG7胞内抗体 ,为利用其探讨

ObjectiveTo prepare MG 7 intrabody and identify its molecular mass and bioactivity Methods Using the MG 7 scFv harbored phagemid pCANTAB5E as template, PCR was conducted to amplify MG 7 scFv and simultaneously introduce the SEKDEL to its 3' end to form the MG 7 intrabody gene After DNA sequencing, MG 7 intrabody gene was subcloned into plasmid pGEX 3X The recombinant was then transformed into E.coli HB2151 and expressed by IPTG induction The molecular mass of product was measured by SDS PAGE, and finally its bioactivity was identified by ELISA Results MG 7 intrabody gene was found to be approximately 780 bp in length DNA sequencing revealed that MG 7 scFv gene and SEKDEL were correctly spliced SDS PAGE found that the molecular mass of MG 7 intrabody was 32 000 u ELISA suggested that MG 7 intrabody had apparent antigen binding affinity Conclusions We have successfully develop MG 7 intrabody and lay a experimental foundation for its application in elucidation of the biological function of MG 7 antigen and the gene therapy of gastric cancer