HPV阳性宫颈癌细胞特异性穿膜肽PTP的穿膜特性研究  

Expression of GFP-PTP fusion protein and functional analysis of the HPV transformed cervical cancer cell membrane penetrating activity

作　　者：钟春燕[1,2] 王琥[1] 柳长柏[1] 黄宇彬[3] 

机构地区：[1]三峡大学分子生物学研究所,宜昌443002 [2]杭州市第三人民医院检验科,杭州310009 [3]中科院长春应用化学研究所高分子物理与化学国家重点实验室,长春130022

出　　处：《中国新药杂志》2010年第13期1164-1170,共7页Chinese Journal of New Drugs

基　　金：国家自然科学基金项目(20774094/B040203)

摘　　要：目的:证实宫颈癌细胞特异性细胞膜穿透肽特异性穿膜并优化穿膜条件。方法:将编码人乳头瘤病毒(HPV)阳性宫颈癌细胞特异性穿膜肽(PTP)的cDNA片断与编码绿色荧光蛋白(GFP)的基因片断融合,克隆至原核表达质粒pET15b克隆位点,构建pET15b-GFP-PTP重组原核表达载体。转化大肠杆菌BL21(DE3)并表达GFP-PTP融合蛋白,纯化后的GFP-PTP融合蛋白作用于体外培养的HPV阳性宫颈癌细胞株HeLa,Siha,肺癌细胞株A549及正常脐静脉内皮细胞ECV304。利用合成的标记短肽探讨优化穿膜条件。结果:荧光显微镜观察发现,与融合蛋白GFP-PTP共孵育后的Hela和Siha细胞胞浆及胞核内有绿色荧光均匀分布,而A549及ECV304胞内无绿色荧光。与GFP共孵育的任何细胞内均无绿色荧光。流式细胞术分析结果显示PTP对HeLa,Siha细胞的穿膜效率分别为33.2%和28.9%。10%DMSO处理细胞后可增强PTP特异性穿透HPV阳性宫颈癌细胞株的胞膜并改善胞浆内分布。结论:本研究结果为进一步探讨PTP作为宫颈癌防治的靶向导入载体奠定了基础。Objective：To confirm the specific membrane penetrating activity of PTP（HPV transformed cervical cancer cell targeting penetrating peptide） and to further investigate PTP as a potential targeting delivery tool for the prevention and therapy of cervical cancer.Methods：The cDNA encoding PTP was fused to the sequence encoding GFP,and then cloned into plasmid pET15b,generating a construct of pET15b-GFP-PTP which expressed GFP-PTP fusion protein.The target fusion protein was yielded in E.coli BL21（DE3） and purified by Ni-NTA His-Bind Resin.Purified GFP-PTP was then added to cultured HPV transformed cervical cancer cell lines HeLa and Siha,lung cancer cell lines A549 and normal cell line umbilical vein endothelial cell（ECV304）.The membrane penetrating experiments conditions were optimized using synthetic peptides.Results：Fluorescent microscope analysis showed that GFP-PTP only entered HeLa and Siha cells,but not A549 or ECV304 cells.FCM assay revealed that the trans-membrane efficiency of PTP to HeLa and Siha was 33.2% and 28.9% respectively.The membrane penetrating activity was enhanced by treating cells with 10% DMSO.Conclusion：The results confirmed that PTP was a HPV transformed cervical cancer cell targeting penetrating peptide,which laid a foundation for further development of PTP as potential targeting delivery tool for prevention and therapy of cervical cancer.

关 键 词：细胞膜穿透肽 宫颈癌 靶向导入 

分 类 号：R730[医药卫生—肿瘤]