绿色荧光蛋白-流感病毒血凝素蛋白-宫颈癌细胞特异性穿膜肽融合蛋白的表达及其体外穿膜活性分析

Expression and Functional Study of Green Fluorescent Protein-Hemagglutinin A-Cervical Cancer Fusion Protein in Vitro

目的获得具备从内含体逃逸的宫颈癌特异性穿膜肽GFP-HA-PTP融合蛋白,探讨其在宫颈癌细胞中的靶向穿膜效应。方法构建p ET15b-GFP-HA-PTP融合原核表达质粒,并转化大肠杆菌BL21,经IPTG诱导和NI-NTA树脂纯化得到融合蛋白,经Western blotting分析确认后,于荧光显微镜下观察GFP-HA-PTP的靶向穿膜活性。结果大肠杆菌高效表达了经Western blot鉴定正确的FP-HA-PTP融合蛋白,且此融合蛋白能够靶向宫颈癌细胞株Siha和Caski,并高效地从内含体中逃逸进入胞浆。结论 GFP-HA-PTP融合蛋白能够高效靶向穿膜进入培养宫颈癌细胞。

OBJECTIVE To prepare green fluorescent protein-hemagglutinin A-cervical cancer （GFP-HA-PTP） fusion protein trageting HPV transformed cervical cancer cells with endosome escape capability, and further investigate its penetrating ability for cervical cancer cell lines. METHODS pET15b-GFP-HA-PTP expression plasmid was constructed, and E. coli B121 （DE3） was transformed with the plasmid, then the yielded GFP-HA-PTP fusion protein was purified by Ni-NTA His-Bind resin and conformed by Western blotting assay. Specific penetrating analysis of GFP-HA-PTP was performed under fluorescence microscopy. RESULTS GFP-HA- PTP fusion protein was highly expressed in BL21 cells. Western blotting analysis result showed that the fusion protein was expressed correctly. The fusion protein can selectively penetrate into cervical cancer cell lines Siha and Caski with endosome escape efficiently. CONCLUSION GFP-HA-PTP can specificly penetrate into cervical cancer cell lines after endosome escaping with high efficiency.