CAJ | 학술논문

目的研究人类细胞色素C中Cyt c77-101对HeLa细胞的穿膜活性.方法人工合成编码Cyt c77-101的寡核苷酸链,构建重组质粒pET28a-EGFP-Cyt c77-101,并转化大肠杆菌BL21进行原核表达,进一步通过Ni2+-NTA亲和层析纯化带有绿色荧光蛋白EGFP的Cyt c77-101融合蛋白,并经荧光显微镜和激光共聚焦检测Cyt c77-101的穿膜活性.结果测序分析表明重组质粒pET28a-EGFP-Cyt c77-101构建成功,且在大肠杆菌BL21中成功表达.融合蛋白EGFP-Cyt c77-101与HeLa细胞保温10 h后,可在细胞内观察到强绿色荧光.结论人源性穿膜肽Cyt c77-101具有较强的穿膜活性.
목적연구인류세포색소C중Cyt c77-101대HeLa세포적천막활성.방법인공합성편마Cyt c77-101적과핵감산련,구건중조질립pET28a-EGFP-Cyt c77-101,병전화대장간균BL21진행원핵표체,진일보통과Ni2+-NTA친화층석순화대유록색형광단백EGFP적Cyt c77-101융합단백,병경형광현미경화격광공취초검측Cyt c77-101적천막활성.결과측서분석표명중조질립pET28a-EGFP-Cyt c77-101구건성공,차재대장간균BL21중성공표체.융합단백EGFP-Cyt c77-101여HeLa세포보온10 h후,가재세포내관찰도강록색형광.결론인원성천막태Cyt c77-101구유교강적천막활성.
@@@@Objective To study the transduction activity of Cyt c77-101, a human-derived cell-penetrating peptide(CPP), in HeLa cells. Methods The oligonucleotide chains encoding Cyt c 77-101were synthesized artificially, and a recombinant vector of pET28a-EGFP-Cyt c77-101 was constructed. The fusion protein was expressed in E.coli BL21 and purified by Ni2+-NTA affinity chromatography. The penetration activity of the CPP in HeLa cells was tested by fluorescence microscope and laser scanning confocal microscope(LSCM). Results Sequencing analysis proved that the recombinant plasmid pET28a-EGFP-Cyt c77-101 was constructed successfully. And the fusion protein was expressed in E.coli BL21. After about 10 h incubation of HeLa cell with EGFP-Cyt c77-101, an intensive green fluorescence was observed. Conclusion The CPP of Cyt c 77-101shows a strong transduction activity.