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为解决P53蛋白难以进入细胞内部发挥治疗作用的瓶颈难题。将p53基因融合插入带有9个精氨酸作为穿膜肽的表达载体中表达融合蛋白CPPs-P53,并与没有穿膜肽的P53蛋白进行比较,利用Western blotting方法检测蛋白的表达情况,MTT及Annexin V/PI双染法检测细胞生长抑制率及细胞凋亡率。Western blotting检测表明已成功在原核表达系统中表达融合蛋白CPPs-P53和P53蛋白,且蛋白纯度均已达到90%以上;MTT检测表明,P53蛋白对肿瘤细胞的生长虽有一定的抑制作用,但融合蛋白CPPs-P53与之相比,对肿瘤细胞生长的抑制效果显著增强,细胞生长抑制率有明显的提升,并且细胞生长抑制率呈现剂量依赖性;Annexin V/PI双染检测细胞凋亡情况也表明P53虽可以在一定程度上诱导肿瘤细胞的凋亡,但与P53蛋白相比较,融合蛋白CPPs-P53诱导的凋亡细胞明显增加,凋亡率是P53蛋白的2~3倍。由此说明在抑制肿瘤细胞的生长和诱导细胞凋亡方面,CPPs-P53比没有穿膜肽的P53蛋白的效果更显著。
To solve the P53 protein difficult to enter the cell to play a therapeutic role of the bottleneck problem. The p53 gene was fused and inserted into the expression vector containing 9 arginine as a transmembrane peptide to express the fusion protein CPPs-P53, and compared with the P53 protein without penetrating peptide. The protein expression was detected by Western blotting. MTT And Annexin V / PI double staining method to detect the cell growth inhibition rate and apoptosis rate. The results of Western blotting showed that the fusion proteins CPPs-P53 and P53 were successfully expressed in prokaryotic expression system, and the purity of the protein was over 90%. MTT assay showed that P53 protein had some inhibitory effect on the growth of tumor cells, Compared with that, the inhibitory effect of fusion protein CPPs-P53 on tumor cell growth was significantly enhanced, cell growth inhibition rate was significantly increased, and cell growth inhibition rate showed a dose-dependent; Annexin V / PI double staining detection of apoptosis The results also showed that although P53 can induce apoptosis of tumor cells to a certain degree, compared with P53 protein, the apoptotic cells induced by CPPs-P53 of fusion protein increased obviously, and the apoptosis rate was 2 ~ 3 times of P53 protein. Thus, CPPs-P53 was more effective than P53 without membrane-bound peptide in inhibiting the growth of tumor cells and inducing apoptosis.