细胞穿膜肽M918偶联抗体的表达纯化及其内吞效率研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0317

摘要/Abstract

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

李雪, 李俊敏, 张雷, 李杉. 细胞穿膜肽M918偶联抗体的表达纯化及其内吞效率研究[J]. 生物技术通报, 2021, 37(12): 198-204.

LI Xue, LI Jun-min, ZHANG Lei, LI Shan. Expression and Purification of Cell-penetrating Peptide M918 Conjugate Antibody and Study on Its Uptake Efficiency[J]. Biotechnology Bulletin, 2021, 37(12): 198-204.

图/表 7

表1 扩增M918-scFv基因片段所需引物

Table 1 Primers used to amplify M918-scFv

Primer	Sequence（5'-3'）
F1	TGTCATATGATGGTGACCGTGCTGTTC
F2	CGGCGGGGGAGGATCTGAGCTCGCATGCGCTTGTG- GAGGAGGAGGATCTG
R1	CTGATCCTCCTCCTCCACAAGCGCATGCGAGCTCA- GATCCTCCCCCGCCG
R2	TGCAAGCTTTTTGTAGAGCTCATCCATGCCATG

图1 重组质粒PET-28b-M918-scFv-EGFP的构建 A:Overlap PCR扩增目的基因片段,1:目的基因M918-scFv-EGFP;M:DNA marker;B:菌落PCR筛选阳性克隆,1-6:转化目的基因的大肠杆菌DH5α菌落;M:DNA marker;C:PET-28b-M918-scFv-EGFP的质粒图谱

Fig.1 Construction of recombinant plasmid PET-28b-M918-scFv-EGFP A:Overlap PCR amplified target gene fragment. 1:Target gene M918-scFv-EGFP. M:DNA marker. B:Screening positive clone via colony PCR. 1-6:E. coli DH5α colonies transformed with target gene. M:DNA marker. C:Plasmid profile of PET-28b-M918-scFv

图2 重组蛋白M918-scFv-EGFP的纯化 A:不同浓度IPTG诱导重组蛋白M918-scFv表达的Western Blot分析,1:未诱导全液;M:蛋白marker;B:SDS-PAGE检测纯化的蛋白,1:纯化前样品;2:穿过液;3:10% Buffer B洗脱;4:20% Buffer B洗脱;5:40% Buffer B洗脱;6:60% Buffer B洗脱;7-8:100% Buffer B洗脱;M:蛋白marker

Fig.2 Purification of recombinant protein M918-scFv A:Western Blot analysis of recombinant protein M918-scFv induced by different IPTG concentrations. 1:Un-induced M918-scFv sample. M:Protein marker. B:Detected and purified protein via SDS-PAGE. 1:Sample before purification. 2:Sample passing through the column. 3:10% Buffer B elution. 4:20% Buffer B elution. 5:40% Buffer B elution. 6:60% Buffer B elution. 7-8:100% Buffer B elution. M:Protein marker

图3 Western Blot鉴定纯化后的重组蛋白M918-scF 1:纯化后的重组蛋白M918-scFv;M:蛋白marker

Fig.3 Identification of the purified recombinant protein M918-scFv by Western Blot 1:Purified recombinant protein M918-scFv. M:Protein marker

图4 scFv（A）和M918-scFv（B）与HER2抗原的结合曲线

Fig. 4 Binding curve of scFv（A）and M918-scFv（B）to HER2 antigen

图5 流式细胞仪检测scFv、M918-scFv在不同细胞系中的内吞效率 A:scFv、M918-scFv与SK-BR-3细胞结合时的水平;B:scFv、M918-scFv与BT474细胞结合时的水平;C:scFv、M918-scFv与MCF-7细胞结合时的水平,红色:空白对照;橘色:4℃处理时,细胞表面的MFI;蓝色:37℃处理时,细胞表面的MFI;D:定量分析scFv、M918-scFv在SK-BR-3（a）和BT474（b）细胞系中的内吞效率,***P < 0.001,**P < 0.01

Fig.5 Uptake studies of scFv and M918-scFv into different cell lines detected by flow cytometry A:Level of scFv,M918-scFv binding to SK-BR-3. B:Level of scFv,M918-scFv binding to BT474. C:Level of scFv,M918-scFv binding to MCF-7. Red:Control. Orange:MFI at 4℃. Blue:MFI at 37℃. D:Quantification of the uptake ratios of scFv and M918-scFv in SK-BR-3（a）and BT474（b）,***P < 0.001,**P < 0.01

图6 激光共聚焦显微镜观察scFv、M918-scFv在SK-BR-3（A）、BT474（B）、MCF-7（C）中的内吞蓝色:经Hoechst 33342染色的细胞核;绿色:重组蛋白;比例尺:20 µm

Fig.6 Uptake studies of scFv,M918-scFv into SK-BR-3（A）,BT474（B） and MCF-7（C）cells observed by confocal laser scanning microscopy Blue:Nucleus stained with Hoechst 33342. Green:Recombinant protein. Scale bars:20 µm

参考文献 16

[1]	Derakhshankhah H, Jafari S. Cell penetrating peptides:a concise review with emphasis on biomedical applications[J]. Biomed Pharmacother, 2018, 108:1090-1096. doi: S0753-3322(18)34018-6 pmid: 30372809
[2]	Milletti F. Cell-penetrating peptides:classes, origin, and current landscape[J]. Drug Discov Today, 2012, 17(15/16):850-860. doi: 10.1016/j.drudis.2012.03.002 URL
[3]	Lin Y, Chen Z, Hu C, et al. Recent progress in antitumor functions of the intracellular antibodies[J]. Drug Discov Today, 2020, 25(6):1109-1120. doi: 10.1016/j.drudis.2020.02.009 URL
[4]	谢洋洋, 王邵娟, 袁权, 等. 细胞穿膜肽研究应用的新进展[J]. 生物工程学报, 2019, 35(7):1162-1173.
	Xie YY, Wang SJ, Yuan Q, et al. Advances in the research and application of cell penetrating peptides[J]. Chin J Biotechnol, 2019, 35(7):1162-1173.
[5]	Rostami B, Irani S, Bolhassani A, et al. M918:a novel cell penetrating peptide for effective delivery of HIV-1 nef and Hsp20-nef proteins into eukaryotic cell lines[J]. Curr HIV Res, 2018, 16(4):280-287. doi: 10.2174/1570162X17666181206111859 URL
[6]	El-Andaloussi S, Johansson HJ, Holm T, et al. A novel cell-penetrating peptide, M918, for efficient delivery of proteins and peptide nucleic acids[J]. Mol Ther, 2007, 15(10):1820-1826. pmid: 17622242
[7]	Nguyen PV, Hervé-Aubert K, David S, et al. Targeted nanomedicine with anti-EGFR scFv for siRNA delivery into triple negative breast cancer cells[J]. Eur J Pharm Biopharm, 2020, 157:74-84. doi: 10.1016/j.ejpb.2020.10.004 pmid: 33059006
[8]	Niesner U, Halin C, Lozzi L, et al. Quantitation of the tumor-targeting properties of antibody fragments conjugated to cell-permeating HIV-1 TAT peptides[J]. Bioconjug Chem, 2002, 13(4):729-736. doi: 10.1021/bc025517+ URL
[9]	Li ZH, Zhang Q, Wang HB, et al. Preclinical studies of targeted therapies for CD20-positive B lymphoid malignancies by Ofatumumab conjugated with auristatin[J]. Invest New Drugs, 2014, 32(1):75-86. doi: 10.1007/s10637-013-9995-y URL
[10]	唐煜, 王相阳, 马晓慧, 等. 抗体偶联药物内吞作用机制的研究进展[J]. 中国新药杂志, 2017, 26(10):1130-1136.
	Tang Y, Wang XY, Ma XH, et al. Progress on internalization study of antibody-drug conjugates[J]. Chin J New Drugs, 2017, 26(10):1130-1136.
[11]	周华华, 董坚. 细胞穿膜肽的研究进展与前景展望[J]. 西部医学, 2012, 24(7):1410-1411, 1414.
	Zhou HH, Dong J. Research progress and prospect of cell-penetrating peptides[J]. Med J West China, 2012, 24(7):1410-1411, 1414.
[12]	张裕丰, 谢梦佳, 周舒蕾, 等. 细胞穿膜肽在肿瘤靶向治疗及疾病诊断中的应用[J]. 中国生物工程杂志, 2019, 39(6):48-54.
	Zhang YF, Xie MJ, Zhou SL, et al. Application of cell-penetrating peptides in tumor targeted therapy and disease diagnosis[J]. China Biotechnol, 2019, 39(6):48-54.
[13]	Packer D. The history of the antibody as a tool[J]. Acta Histochem, 2021, 123(4):151710. doi: 10.1016/j.acthis.2021.151710 pmid: 33721607
[14]	Crivianu-Gaita V, Thompson M. Aptamers, antibody scFv, and antibody Fab’ fragments:an overview and comparison of three of the most versatile biosensor biorecognition elements[J]. Biosens Bioelectron, 2016, 85:32-45. doi: S0956-5663(16)30369-4 pmid: 27155114
[15]	Malpiedi LP, Díaz CA, Nerli BB, et al. Single-chain antibody fragments:Purification methodologies[J]. Process Biochem, 2013, 48(8):1242-1251. doi: 10.1016/j.procbio.2013.06.008 URL
[16]	Bolhassani A, Jafarzade BS, Mardani G. In vitro and in vivo delivery of therapeutic proteins using cell penetrating peptides[J]. Peptides:N Y, 2017, 87:50-63.

编辑推荐 0

Metrics

阅读次数

全文

212

HTML			PDF

最新录用	在线预览	正式出版	最新录用	在线预览	正式出版
0	0	10	0	0	202

来源	本网站	其他网站

次数	204	8
比例	96%	4%

摘要

448

最新录用	在线预览	正式出版

0	0	448

	来源	本网站

	次数	448
	比例	100%