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目的构建融合白喉毒素转位序列的免疫促凋亡蛋白Immuno-caspase-6的真核表达载体,真核表达纯化该重组蛋白后验证其对人表皮生长因子受体2(HER2)阳性细胞的选择性杀伤活性。方法把Kozak序列与单链抗体e23sfv、白喉毒素的促转位片段白喉毒素弗林蛋白酶识别位点(fdt)、caspase-6以及免疫球蛋白Ig G Fc段基因重组,克隆入真核表达载体pc DNA3.1(+)中,命名为pc DNA3.1(+)-AFC。瞬时转染CHO-S细胞,Western blot法检测上清中目的蛋白的表达;收集培养上清并利用蛋白A纯化柱对目的蛋白进行纯化;流式细胞术验证目的蛋白对HER2阳性SGC-7901细胞的杀伤作用。结果成功构建pc DNA3.1(+)-AFR真核表达载体;瞬时转染悬浮CHO-S细胞后,Western blot法证实目的蛋白可分泌性表达于细胞培养上清中,流式细胞术结果显示纯化后的目的蛋白可以显著促进HER2阳性SGC-7901细胞凋亡。结论真核细胞表达的免疫促凋亡蛋白Immuno-caspase-6可选择性高效杀伤HER2阳性SGC-7901细胞。
Objective To construct an eukaryotic expression vector of Immuno-caspase-6, which binds to the translocation sequence of diphtheria toxin, and then verify the selection of human epidermal growth factor receptor 2 (HER2) positive cells by eukaryotic expression and purification of the recombinant protein Sexual killing activity. Methods The Kozak sequence was cloned into eukaryotic expression vector pc (e23sfv), the diphtheria toxin diphtheria toxin recognition site (fdt), caspase-6 and immunoglobulin Ig G Fc fragment of diphtheria toxin DNA3.1 (+) and was named pcDNA3.1 (+) - AFC. CHO-S cells were transiently transfected, and the expression of the target protein in the supernatant was detected by Western blot. The culture supernatant was collected and purified by protein A purification column. Flow cytometry was used to verify the effect of the target protein on HER2-positive SGC-7901 cells The killing effect. Results The pcDNA3.1 (+) - AFR eukaryotic expression vector was successfully constructed. After transient transfection of CHO-S cells in suspension, the secreted protein was expressed in the supernatant of cells by Western blot and the results of flow cytometry Purified target protein can significantly promote the apoptosis of HER2-positive SGC-7901 cells. Conclusion Immuno-caspase-6 expressed in eukaryotic cells selectively and efficiently kill HER2-positive SGC-7901 cells.