重组人可溶性DR5蛋白的分离纯化及其生物活性检测

Isolation and purify of recombinant human soluble DR5 and detection of its biological activity

目的:提纯毕赤酵母GS115菌株表达的重组人可溶性死亡受体5(sDR5)蛋白,并研究其对肿瘤坏死因子相关凋亡诱导配体(TRAIL)所诱导细胞的阻断效应。方法:大量扩增可溶性DR5酵母表达载体转化阳性的毕赤酵母GS115菌株,收集上清,利用Ni-NTA Agarose亲和层析柱纯化获得重组可溶性DR5,用SDS-PAGE检测纯化产物的纯度。用流式细胞仪检测纯化的可溶性DR5对TRAIL诱导Jurkat细胞凋亡作用的阻断效应。结果:转化阳性的毕赤酵母GS115菌株可以成功表达分子量为23kD的可溶性DR5蛋白,经过Ni-NTAAgarose亲和层析柱纯化,SDS-PAGE鉴定显示获得了高纯度的重组人可溶性DR5蛋白,培养上清产量达28.69mg/L;体外活性检测结果显示,纯化的可溶性DR5蛋白几乎完全可以阻断TRAIL诱导的Jurkat细胞凋亡作用。结论:经纯化后的重组人可溶性DR5蛋白可以有效阻断TRAIL诱导的细胞凋亡作用,显示DR5在TRAIL诱导Jurkat细胞凋亡作用中起着十分关键的作用。

Objective： To isolation and purify of recombinant human soluble death receptor 5 （sDR5） expressed in Pichia Pastoris（Pp） GS115 strain and study its reversion effects on the apoptosis induced by TNF-related apoptosis-inducing ligand. Methods： Pichia Pastoris（Pp） GS115 strain, transformed with yeast expression vector containing soluble DR5, were produced in abundance, and the culture supernatant was collected. Ni - NTA Agarose affinity chromatography column was used to isolation and purify recombinant human soluble DR5. The sublime product was detected by SDS- PAGE. The blocking effects of purified sDR5 on TRAIL-induced apoptosis of Jurkat cell line were observed by fly cytometry assay. Results： Pichia Pastoris（Pp） GS115 strain, transformed with yeast expression vector containing soluble DR5, could successfully express soluble DR5（23kD）. The results of SDS-PAGE showed that recombinant human soluble DR5 highly purified was obtained. The yield was up to 28.69mg/L of yeast culture. Purified soluble DR5 could very effectively block the apoptosis of Jurkat cells induced by TRAIL in vitro by fly cytometry assay. Conclusion： The purified sDR5 could effectively block the apoptosis induced by TRAIL and show that DR5 plays a very key role in TRAIl. induced apoptosis in Jurkat cells.