摘要
TAT蛋白转导肽是HIV-1病毒编码的一段富含碱性氨基酸序列的多肽,能够高效介导多种外源生物大分子通过多种膜性结构,如细胞质膜和血脑屏障等。为探索TAT蛋白转导肽介导的秀丽线虫体内外源蛋白跨膜转导作用,以EGFP为报告基因结合常规分子克隆技术构建了原核表达载体pET28b-EGFP和pET28-TAT-EGFP,继而利用诱导剂IPTG(终浓度1mmol/L)诱导表达了靶蛋白并结合荧光显微观察、SDS-PAGE和Western blot等鉴定技术获得表达靶蛋白的大肠杆菌BL21(DE3)细胞,最后将其涂布到含有Kana+的LB固体培养基上直接饲喂野生型N2株系线虫,利用荧光显微镜观察绿色荧光信号在线虫体内的分布。结果证明,TAT-EGFP融合蛋白较之于EGFP可高效、可溶性表达,而且通过直接饲喂秀丽线虫表达靶蛋白的大肠杆菌48小时后,TAT-EGFP荧光信号明显分布于线虫肠壁细胞,而EGFP荧光信号则分布在秀丽线虫肠腔,空载体对照组未见任何荧光信号,说明TAT蛋白转导肽能够高效介导外源蛋白在秀丽线虫体内跨膜转导。同时,通过比较空载体对照组与实验组线虫微分干涉图像,未见线虫出现明显的细胞形态变化,说明TAT蛋白转导肽介导的外源蛋白跨膜转导作用是安全的,为在秀丽线虫体内直接研究外源蛋白的功能以及进行蛋白药物的研发提供了重要参考。
The TAT protein transduction peptide was enriched in basic amino acids and encoded by the HIV-1 virus.Previous studies have revealed that it could safely and efficiently mediate various heterologous biological macromolecules across a variety of biomembranes,such as the plasmid membrane and the blood-brain barrier et al.To further study its function in mediating heterologous proteins transduction in nematode in vivo,the prokaryotic expression vector pET28b-EGFP and pET28b-TAT-EGFP were constructed and induced by IPTG（final concentration 1 mmol/L）,followed with the analysis on the expressed protein by fluorescence microscopy,SDS-PAGE and Western blot.Subsequently,the bacterial cells were coated to the LB medium and directly fed to the nematodes followed with capturing the image at 48 h.Results showed when fed to the nematodes for 48 h,the TAT-EGFP fluorescence signals were clearly distributed in the intestinal cells of the worm,while the EGFP fluorescence signals were mainly distributed in the intestinal cavity of the animal.Furthermore,the cellular morphology of TAT-EGFP had no distinct change compared with the EGFP group and controls.Taken together,the data suggested the TAT protein transduction peptide could mediate heterologous protein expression in C.elegans and provided an alternative approach for development of new drug transporter.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2011年第3期39-45,共7页
China Biotechnology
基金
国家自然科学基金(30973107
30772293)
国家"973"计划(2006CB504100)
国家"重大新药创制"科技重大专项课题(2009ZX09503-002
2009ZX09301-002
2009ZX09103-616)资助项目
关键词
TAT蛋白转导肽
原核表达
线虫
跨膜转导
TAT protein transduction peptide Prokaryotic expression C.elegans Transmembrane transduction
