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为研究细胞穿透肽TAT融合小鼠存活素T34A(survivinT34A)重组蛋白(TAT-msvT34A)转导细胞的效能,该实验将表达TAT-msvT34A的原核表达载体pTAT-msvT34A转化大肠杆菌表达株E.coli BL21(DE3),异丙基硫代半乳糖苷(IPTG)诱导,表达的融合蛋白主要以包涵体形式存在。通过亲和层析、离子交换柱层析、分子筛等步骤纯化,得到TAT-msvT34A融合蛋白的纯度可达98%。纯化的融合蛋白用异硫氰酸荧光素(FITC)标记(FITC-TAT-msvT34A)后,分别转导HepG2、TC-1、B16及HEK293细胞株,流式细胞仪检测显示,较低浓度的重组蛋白即对HepG2、TC-1、B16及HEK293等细胞株具有较高的转导效能,在100 nmol/L时的转导效率均可达到60%以上,作为对照,FITC标记的牛血清白蛋白(FITC-BSA)在100 nmol/L时对上述细胞株的转导效率极低,结果具有显著性差异(P<0.01)。荧光显微镜观察可见,50 nmol/L和100 nmol/L的TAT-msvT34A融合蛋白对HepG2细胞的转导效率均可达50%,而对照FITC-BSA对HepG2细胞几乎无转导。表达纯化的TAT-msvT34A融合蛋白对HepG2、TC-1、B16及HEK293等细胞株均有较高的转导效能,可以用于后续抗肿瘤研究。
In order to investigate the effect of cell penetrating peptide TAT fusion mouse TAT-msvT34A transduced cells, the prokaryotic expression vector pTAT-msvT34A expressing TAT-msvT34A was transformed into E.coli E.coli E. coli. The fusion protein induced by E. coli BL21 (DE3) and isopropylthiogalactoside (IPTG) mainly existed in the form of inclusion bodies. The purity of TAT-msvT34A fusion protein was 98% by affinity chromatography, ion-exchange column chromatography and molecular sieve. The purified fusion protein was transfected into HepG2, TC-1, B16 and HEK293 cell lines respectively by FITC-labeled FITC-TAT-msvT34A. Flow cytometry analysis showed that the lower concentration of The recombinant protein had high transduction efficiency on HepG2, TC-1, B16 and HEK293 cell lines, and the transduction efficiency was above 60% at 100 nmol / L. As a control, FITC-labeled bovine serum albumin The transduction efficiency of FITC-BSA at 100 nmol / L was extremely low, with significant difference (P <0.01). Fluorescence microscopy showed that the transduction efficiency of HepG2 cells with 50 nmol / L and 100 nmol / L TAT-msvT34A fusion protein was up to 50%, while the control FITC-BSA had almost no transduction of HepG2 cells. The expressed TAT-msvT34A fusion protein has high transduction efficiency on HepG2, TC-1, B16 and HEK293 cell lines, which can be used in subsequent antitumor research.