细胞穿透肽YARA介导增强型绿色荧光蛋白转导入人结直肠癌细胞的实验研究  

Transduction of enhanced green fluorescent protein mediated by a cell penetrating peptide-YARA into human colorectal cancer SW480 cells

作　　者：陈思思 王家宁 黄永章 郭凌郧 孔霞 郑飞 Chen Sisi Wang Jianing Huang Yongzhang Guo Lingyun Kong Xia Zheng Fei(Department of Endocrinology,Affiliated the People＇s Hospital of Hubei Medical College, Shiyan 442000, Hubei, China In- stitute of Clinical Medicine. Affiliated the People＇s Hospital of Hubei Medical College, Shiyan 442000, Hubei, China.)

机构地区：[1]湖北医药学院附属人民医院内分泌科,湖北十堰442000 [2]湖北医药学院附属人民医院临床医学研究所,湖北十堰442000

出　　处：《贵州医药》2017年第8期787-791,共5页Guizhou Medical Journal

基　　金：十堰市重大科技项目(NO:2006030Z);湖北省高等学校优秀中青年科技创新团队计划(NO:T200811)

摘　　要：目的构建原核表达载体pET15b-YARA-EGFP,表达并纯化出融合蛋白YARA-EGFP,观察其穿透人结直肠癌细胞的情况。方法用分子克隆技术构建出表达型载体pET15b-YARA-EGFP,在E.coli BL21(DE3)中表达融合蛋白YARA-EGFP,将鉴定和纯化后的融合蛋白加入体外培养的人结直肠癌细胞。结果得到纯化的融合蛋白YARA-EGFP,能高效地转导入体外培养的人结直肠癌细胞,并表现出剂量和时间依赖性,MTT法检测在其高浓度时仍对细胞无毒性。结论 YARA-EGFP融合蛋白能高效地转导入体外培养的人结直肠癌细胞。Objective To construct the expression vector pET15b-YARA-EGFP and purify the fusion protein YARA EGFP. And evaluate the penetrating ability of YARA in the transduction of enhanced green fluorescent pro- tein（EGFP） into human colorectal cancer line SW480. Methods The recombinant plasmid pET15b YARA-EGFP was constructed by inserting YARA-encoding DNA into pET15b-EGFP. The fusion protein YARA-EGFP expressed in E. coli BL21 （DE3）, which were purified and added in cultured human colorectal cancer line SW480. Results The high- ly purified YARA-EGFP fusion protein was obtained,and it could be transduced into SW480 cells in a time-and dose- dependent manner when added exogenously in a culture media. The cytotoxieity of YARA-EGFP detected by MTT method demonstrated there were no statistical significantly differences between different concentrations. Conclusion The fusion protein YARA EGFP could efficiently transduce into human colorectal cancer line SW480 which provides a basis for YARA-mediated biomacromolecule transduction in protein therapy.

关 键 词：细胞穿透肽 蛋白转导结构域 增强型绿色荧光蛋白 人结直肠癌细胞 

分 类 号：R33[医药卫生—人体生理学] R735.3[医药卫生—基础医学]