生物技术通报 ›› 2021, Vol. 37 ›› Issue (12): 198-204.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0317

• 研究报告 • 上一篇    下一篇

细胞穿膜肽M918偶联抗体的表达纯化及其内吞效率研究

李雪(), 李俊敏, 张雷, 李杉()   

  1. 华南理工大学生物科学与工程学院,广州 510006
  • 收稿日期:2021-03-17 出版日期:2021-12-26 发布日期:2022-01-19
  • 作者简介:李雪,女,硕士,研究方向:医药生物学;E-mail: 865747654@qq.com
  • 基金资助:
    国家“新药创制”重大专项(2018ZX09201002)

Expression and Purification of Cell-penetrating Peptide M918 Conjugate Antibody and Study on Its Uptake Efficiency

LI Xue(), LI Jun-min, ZHANG Lei, LI Shan()   

  1. School of Biology and Biological Engineering,South China University of Technology,Guangzhou 510006
  • Received:2021-03-17 Published:2021-12-26 Online:2022-01-19

摘要:

细胞穿膜肽由于存在靶细胞摄取效率低等问题,使其在药物递送载体领域的应用受到限制。本研究用基因工程手段将穿膜肽M918与靶向HER2的单链抗体偶联,通过大肠杆菌表达系统和镍亲和层析表达并纯化了重组蛋白M918-scFv。利用微量热泳动技术检测重组蛋白M918-scFv与HER2抗原的亲和能力;利用激光共聚焦显微镜和流式细胞仪检测重组蛋白的内吞效率。实验结果表明亲和层析获得的重组蛋白M918-scFv纯度较高,偶联M918,不会改变scFv与抗原的亲和能力。在HER2阳性细胞中,重组蛋白M918-scFv的内吞效率是scFv的1.8倍,说明重组蛋白M918-scFv发挥了穿膜肽M918的穿膜特性,表现出更高的内吞效率。穿膜肽M918偶联scFv既表现出穿膜肽的穿膜特性又兼具了scFv的靶向性,提供了一种新的靶向递送的策略。

关键词: 细胞穿膜肽, M918, 单链抗体, 靶向递送

Abstract:

Application of cell-penetrating peptides in the field of drug delivery are limited in their due to the low uptake efficiency of target cells. In this study,genetic engineering methods were used to conjugate the cell penetrating peptide M918 with single-chain antibody targeting HER2,and the recombinant protein M918-scFv was expressed by Escherichia coli expression system and purified by nickel affinity chromatography. The microscale thermophoresis was applied to detect the affinity of the recombinant protein M918-scFv with the HER2 antigen. Laser confocal microscopy and flow cytometry were employed to detect the uptake efficiency of the recombinant protein. The results showed that the recombinant protein M918-scFv obtained by affinity chromatography was of high purity. Conjugating M918 did not affect the affinity of scFv and antigen. In HER2 positive cells,the uptake efficiency of the recombinant protein M918-scFv was 1.8 times that of scFv,indicating that M918-scFv exerted the penetration properties of cell-penetrating peptide M918 and presented higher uptake efficiency. Cell-penetrating peptide M918 conjugating with scFv not only demonstrated the cell-penetrating feature but also owned the targeting of scFv,thus it may provide a new targeted delivery strategy.

Key words: cell-penetrating peptide, M918, single chain antibody, targeted delivery