改构人源化免疫促凋亡蛋白的表达及对HER2阳性肿瘤细胞的特异杀伤作用

Expression of modified humanized immunoapoptotic protein and its specific killing effect on HER2-positive tumor cells

目的哺乳动物细胞表达切割位点改构的人源化免疫促凋亡蛋白,验证其对人表皮生长因子受体2(HER2)阳性肿瘤细胞的杀伤作用。方法将Kozak序列、人源化抗HER2单链抗体P1h3、Fc标签、组织蛋白酶D切割位点D1和促凋亡效应分子tBid基因依次连接,克隆入真核表达载体pLVX-EGFP;转染HEK293F细胞,Western blot法鉴定培养上清中目的蛋白的分泌表达;通过显微镜观察计数和萤光素酶活性检测法,验证目的蛋白对HER2阳性PC9肺癌细胞的杀伤作用。结果成功构建真核表达载体pLVX-P1h3-Fc-D1-tBid-EGFP;转染HEK293F细胞后,Western blot结果证明培养上清中有目的蛋白表达;杀伤实验结果显示目的蛋白可以显著诱导HER2阳性PC9细胞死亡。结论哺乳动物细胞表达的改构人源化免疫促凋亡分子可以特异性杀伤HER2阳性肿瘤细胞。

Objective To express the humanized immunoapoptotic protein with a modified cleavage site in mammalian cell expression system and verify its killing effect on human epidermal growth factor receptor 2(HER2)-positve tumor cells. Methods Kozak sequence, the humanized anti-HER2 single chain antibody P1h3, Fc tag, cathepsin D cleavage site D1 and pro-apoptotic effector tBid gene were linked in sequence and cloned into the eukaryotic expression vector pLVX-EGFP. Cell culture supernatant was harvested and subjected to Western blot analysis to detect the secretory expression of the recombinant plasmid after its transfection into HEK293F cells. Killing effect of the recombinant plasmid on HER2-positive PC9 tumor cells was evaluated by cell counting and luciferase activity assay. Results The eukaryotic expression vector pLVX-P1h3-Fc-D1-tBid-EGFP was successfully constructed and its expression was confirmed by Western blot analysis. The humanized immunoapoptotic proteins significantly induced the death of HER2-positive PC9 cells. Conclusion The modified humanized immunoapoptotic proteins obtained by mammalian cell expression system could specifically kill HER2-positive tumor cells.