新穿膜融合蛋白His-T1-绿色荧光蛋白的跨膜效率及其对细胞存活的影响

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。

Transmembrane efficiency of His-T1-green fluorescence protein and its effect on cell survival

AIM To study the transmembrane efficiency of a novel cell penetrating peptide(CPP) fusion protein, His-T1-green fluorescence protein (GFP), and its effect on cell survival. METHODS His-T1-GFP 500 mg·L^-1 was incubated with CNE2 and NRK52E cells separately for 6 h. And its cell penetrating efficiency to CNE2 and NRK52E cells was observed by fluorescence microscopy. For its transmembrane dynamics analysis, His-T1-GFP 500 mg·L^-1 was incubated with CNE2 and NRK52E cells separately from 10 min to 24 h to investigate the influence of incubation time; His-T1-GFP 25 mg·L^-1-1.0 g·L^-1 was incubated with CNE2 and NRK52E cells for 6 h to detect the effect of incubation protein concentration; His-T1-GFP 500 mg·L^-1 was incubated with CNE2 and NRK52E cells at 4℃ and 37℃ for 6 h to analyze the effect of incubation temperature. All the dynamic data were calculated with fluorescence intensity immunoanalyzer. Finally, the toxicity of His-T1-GFP 5.0 g·L^-1 on CNE2 and NRK52E cells was evaluated with lactate dehydrogenase (LDH) kit and MTT assay. RESULTS His-T1-GFP could effectively penetrate through CNE2 and NRK52E cell membranes, with a higher efficacy compared with its positive control His-TAT-GFP when penetrating NRK52E cell. His-T1-GFP could penetrate into target cells in 10 min, and could increase its concentration in cells within 6 h; also, the concentration of the protein in cells was relevant to its protein concentration: in the concentration of 25 mg·L^-1 to 1.0 g·L^-1, the concentration of the protein in cells could be increased with higher incubation concentration; and the permeation capability of His-T1-GFP could be maintained even at 4℃. When its final concentration was up to 5.0 g·L^-1, there was no obvious toxic effect to either cell membrane integrity or cell proliferation. CONCLUSION His-T1-GFP is a novel CPP fusion protein with high permeating efficiency and low cytotoxicity.