PTD-Apoptin原核表达载体构建及可溶性表达  

Establishment of the PTD-Apoptin Prokaryotic Expression Vector and Its Soluble Expression

作　　者：贲松彬[1] 潘子瑶[1] 刘雪梅[1] 崔剑[1] 谷海峰[1] 李其久[1] 陈长兰[2] 

机构地区：[1]辽宁大学生命科学院,辽宁沈阳110036 [2]辽宁大学药学院,辽宁沈阳110036

出　　处：《生物技术》2013年第4期20-24,共5页Biotechnology

基　　金：辽宁省教育厅的项目("凋亡素抗肿瘤药物膜转运载体构建及药物分子制备";2009A310);辽宁省科学技术计划项目("常见病发病机理及防治新技术";2010225036);沈阳市科技计划项目"新型抗肿瘤药物肿瘤特异性凋亡基因微球制备及在基因治疗中应用研究";2009-15);辽宁大学国家自然基金预资助;博士启动项目共同资助

摘　　要：目的:可溶性表达PTD-Apoptin融合蛋白。方法:亚克隆Apoptin基因并人工合成PTD序列,构建原核表达载体pGEX-6p-1-PTD-Apoptin,使载体在E.coli BL21(DE3)中表达并优化。GST亲和纯化天然状态的融合蛋白,并通过MTT实验验证活性。结果:最佳表达条件是25℃、0.1mmol/mL IPTG过夜诱导表达,经GST亲和纯化得到天然状态的单一组分融合蛋白,与胃癌823细胞共孵育后细胞存活率为10.86%,与对照组相比具显著差异。结论:成功获得高生物活性的原核可溶性表达融合蛋白PTD-Apoptin,使胃癌823细胞存活率降至10.86%。Objective ：To express the PTD - Apoptin fusion protein solubly. Method： Subcloning of Apoptin gene and using a synthetic method to obtain the PTD sequence to construct the pGEX -6p - 1 - PTD - Apoptin prokaryotic expression vector, the expression vector was expressed and optimized in E. coli BL21 （DE3）. The natural fusion protein was purified by GST affinity - purification and using MTY reduction assay to verify its activity. Result：The best expression condition was 25℃, the natural and single component fusion protein was obtained by using overnight inducible expression of 0. 1 mmol/mL IPTG and GST affinity purification, incubated the purified fusion protein together with BGC- 823cells, it was determined that the cells viability was 10. 86% ,and it was significant different from the control group. Conclusion： The prokaryotic soluble fusion protein PTD - Apoptin was successfully constructed, which had a high biological activity through GST affinity purification and made BGC -823cells viability decreased to 10. 86%.

关 键 词：APOPTIN PTD 可溶性表达 BL21(DE3) MTT 

分 类 号：Q786[生物学—分子生物学]