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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。
TAT protein transduction peptide is a polypeptide rich in basic amino acid sequence encoded by HIV-1 virus, which can effectively lead a variety of exogenous biological macromolecules through a variety of membranous structures such as the plasma membrane and the blood-brain barrier. In order to explore the transmembrane transduction of foreign proteins mediated by TAT protein transduction peptide, the prokaryotic expression vectors pET28b-EGFP and pET28-TAT-EGFP were constructed by EGFP reporter gene and conventional molecular cloning techniques, The target protein was induced by IPTG (final concentration of 1mmol / L) and combined with fluorescence microscopy. SDS-PAGE and Western blot were used to obtain E. coli BL21 (DE3) cells expressing the target protein, Wild type N2 strain nematodes were fed on Kana + LB solid medium, and the distribution of green fluorescence signal in nematodes was observed by fluorescence microscopy. The results showed that the TAT-EGFP fusion protein was highly efficient and soluble in comparison with EGFP, and the fluorescence signal of TAT-EGFP was obviously distributed in the nematode intestinal wall cells by direct feeding E. coli expressing the target protein of C. elegans at 48 hours. EGFP Fluorescence signals were distributed in the intestinal cavity of C. elegans without any fluorescence signal in empty vector control group, indicating that TAT protein transduction peptide can efficiently transduce the foreign protein transmembrane transduction in C. elegans. At the same time, no significant change of cell morphology was observed in the nematodes of the control group and the experimental group, indicating that transmembrane transduction mediated by the TAT protein transduction peptide is safe, Nematode direct study of the function of foreign proteins and the development of protein drugs provide an important reference.