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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 MG7抗原的功能及胃癌的基因治疗奠定了基础
Objective To prepare MG7 intracellular antibody and identify its molecular weight and antigen binding activity. Methods The MG7sc Fv gene was amplified by PCR using the plasmid pCANTAB5 E containing the MG7sc Fv gene as a template, and an endoplasmic reticulum retention sequence (SEKDEL) was introduced at its 3 ’end to form the MG7 intracellular antibody gene. After DNA sequencing, the MG7 intracellular antibody gene was cloned into the expression vector pGEX-3X and transformed into host strain HB2151. The product was induced by IPTG and the product was analyzed by SDS-PAGE, and the product was detected by ELISA. Results The electrophoresis analysis showed that the fusion gene was about 780 bp in length. DNA sequence analysis showed that the fusion protein of SEKDEL and MG7sc Fv was correctly fused. The molecular weight of MG7 intracellular antibody was 3200uU by SDS-PAGE analysis. The result of ELISA showed that the intracellular antibody of MG7 had obvious antigen-binding activity. Conclusions We successfully prepared and expressed MG7 intracellular antibodies, which laid the foundation for their use in exploring the function of MG7 antigen and gene therapy of gastric cancer