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目标蛋白TmSm是研发出的一种能够较强抑制肿瘤细胞生长和促进其凋亡的基因工程抗肿瘤蛋白,其由介导目的蛋白进入细胞的转导肽HIV-TAT突变体(Tm)和Survivin突变体(Sm)融合而成。原先的TmSm制备工艺以包涵体表达、变性和复性为主,其过程不仅复杂、收率低成本高,而且所得蛋白活性较低。通过将TmSm基因与编码类弹性蛋白(elastin-like polypeptides,ELPs)和内含肽(intein)的标签序列(EI)进行融合,利用ELPs的可逆相变特性和intein的自切割功能,经简单的温度及pH调控,获得了纯度为99.00%的TmSm蛋白。将纯化后的蛋白作用于肺癌细胞A549,24 h后MTT结果显示,63.21μg/ml的目的蛋白对A549的抑制率为57.83%;流式细胞仪检测结果表明,当TmSm浓度为40μg/ml时,24 h后细胞凋亡率为41.72%。因此,这种新型的以类弹性蛋白和内含肽介导的表达纯化技术不仅成本低廉,而且对抗肿瘤蛋白药物的质量提高具有重要应用价值。
The target protein TmSm is a genetically engineered anti-tumor protein developed to inhibit tumor cell growth and promote its apoptosis. It is composed of the transduction peptide HIV-TAT mutant (Tm) and Survivin Mutant (Sm) fusion. The original TmSm preparation process to inclusion body expression, denaturation and renaturation, the process is not only complicated, low yield and high cost, and the resulting protein activity is low. Through the fusion of the TmSm gene with the tag sequences (EI) of elastin-like polypeptides (ELPs) and inteins, the reversible phase transition characteristics of ELPs and the intein self-cleaving function were used. Temperature and pH control, to obtain a purity of 99.00% TmSm protein. The MTT assay showed that the target protein of 63.21μg / ml could inhibit the A549 cell proliferation rate by 57.83% at 24 h. The results of flow cytometry showed that when the concentration of TmSm was 40μg / ml After 24 h, the apoptosis rate was 41.72%. Therefore, this novel expression and purification technology mediated by elastin and intein is not only low in cost but also has important application value for improving the quality of antitumor protein drugs.