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目的研究穿膜融合蛋白His-T1-绿色荧光蛋白(GFP)的跨膜效率及其对细胞存活的影响。方法以浓度为500mg·L-1的His-T1-GFP与人鼻咽癌CNE2或大鼠肾小管上皮NRK52E细胞孵育6h,应用荧光显微镜观察His-T1-GFP跨膜进入细胞的情况。应用多功能酶标仪检测细胞荧光强度,研究His-T1-GFP跨膜的动力学因素:以浓度为500mg·L-1的His-T1-GFP与CNE2或NRK52E细胞孵育10min至24h,观察孵育时间对穿膜作用的影响;以浓度为25mg·L-1至1.0g·L-1的His-T1-GFP与两种细胞孵育6h,观察蛋白浓度对跨膜效率的影响;以浓度为500mg·L-1的His-T1-GFP与两种细胞分别在4℃和37℃的条件下孵育6h,观察温度对蛋白跨膜效率的影响。用细胞乳酸脱氢酶(LDH)试剂盒和MTT法来评价5.0g·L-1浓度的His-T1-GFP对细胞存活的影响。结果在一定浓度范围内,His-T1-GFP能够有效穿透CNE2和NRK52E细胞膜,且对NRK52E细胞的跨膜效率明显高于His-TAT-GFP。His-T1-GFP在10min内就能有效跨膜进入细胞,并且在6h内进入细胞的量与时间成正相关。在一定浓度范围(25mg·L-1~1.0g·L-1)内,该蛋白进入细胞的量与自身浓度成正相关,而在4℃时该蛋白仍具有跨膜能力。当其终浓度高达5.0g·L-1时,对CNE2和NRK52E两种细胞几乎无毒性作用。结论His-T1-GFP蛋白是一种跨膜效率高且低毒的穿膜融合蛋白。
Objective To investigate the transmembrane efficiency of transmembrane fusion protein His-T1-green fluorescent protein (GFP) and its effect on cell survival. Methods His-T1-GFP with a concentration of 500 mg · L-1 was incubated with human nasopharyngeal carcinoma CNE2 or rat NRK52E cells for 6 h. The transmembrane entry of His-T1-GFP into cells was observed by fluorescence microscopy. The fluorescence intensity of His-T1-GFP transmembrane was measured by multifunctional microplate reader. His-T1-GFP with concentration of 500mg · L-1 was incubated with CNE2 or NRK52E cells for 10min to 24h. The incubation was observed Time on the transmembrane effect; the concentration of 25mg · L-1 to 1.0g · L-1 of His-T1-GFP and two kinds of cells incubated 6h, observe the effect of protein concentration on transmembrane efficiency; concentration of 500mg · His-T1-GFP with L-1 was incubated with both cells for 6h at 4 ℃ and 37 ℃, respectively. The effect of temperature on the transmembrane efficiency was observed. The effect of 5.0 g · L-1 His-T1-GFP on cell viability was evaluated using the cellular lactate dehydrogenase (LDH) kit and the MTT method. Results His-T1-GFP could effectively penetrate the membrane of CNE2 and NRK52E cells at a certain concentration range, and the transmembrane efficiency of NRK52E cells was significantly higher than that of His-TAT-GFP. His-T1-GFP efficiently transmembrane into the cell within 10 min, and the amount entering the cell within 6 h is positively correlated with time. Within a certain concentration range (25mg · L-1 ~ 1.0g · L-1), the amount of protein entering the cell was positively correlated with its own concentration, while at 4 ℃ the protein still has a transmembrane capacity. When the final concentration of up to 5.0g · L-1, almost no toxic effect on both CNE2 and NRK52E cells. Conclusion His-T1-GFP protein is a transmembrane fusion protein with high transmembrane efficiency and low toxicity.