重组人可溶性DR5蛋白的表达、制备及其生物活性检测

Expression and preparation of recombinant human soluble DR5 and detection of its biological activity

目的:纯化毕赤酵母GS115菌株表达的重组人可溶性死亡受体5(DR5)蛋白,并研究其对肿瘤坏死因子相关凋亡诱导配体(TRAIL)所诱导细胞凋亡的阻断效应?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ProBondTM亲和层析柱纯化获得重组可溶性DR5蛋白,用SDS-PAGE?Western Blot检验纯化产物的特异性和纯度?用MTT法检测纯化的可溶性DR5蛋白对TRAIL诱导人宫颈癌细胞系Hela细胞凋亡作用的阻断效应?结果:转化阳性的毕赤酵母GS115菌株可成功表达分子量为26 kD的可溶性DR5蛋白,经ProBondTM亲和层析柱纯化,SDS-PAGE?Western Blot鉴定,结果显示获得了高纯度的重组人可溶性DR5蛋白,蛋白产量达20 μg/ml;体外活性检测结果显示,纯化的可溶性DR5蛋白可部分阻断TRAIL诱导的Hela细胞的凋亡,阻断率最高达53.6%?结论:经纯化后的重组人可溶性DR5蛋白可有效阻断TRAIL诱导的细胞凋亡反应?

Objective: To purify recombinant human soluble death receptor 5 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. ProBondTM affinity chromatography column was used to purify recombinant soluble DR5.The purity was detected by Western Blot and SDS-PAGE. The blocking effects of purified sDR5 on TRAIL-induced apoptosis of Hela cell line were observed. Results: Pichia Pastoris(Pp)GS115 strain,transformed with yeast expression vector containing soluble DR5, could successfully express soluble DR5 (26kD). The results of SDS-PAGE, Western Blot showed that recombinant human soluble DR5 highly purified was obtained. The yield was up to 20 μg/ml of yeast culture. Purified soluble DR5 could effectively block the apoptosis of Hela cells induced by TRAIL. The highest blocking rate was 53.6%. Conclusion: Purified,soluble DR5 could effectively block the apoptosis induced by TRAIL.