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

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%.