生物技术通报 ›› 2023, Vol. 39 ›› Issue (10): 323-331.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0291
收稿日期:
2023-03-30
出版日期:
2023-10-26
发布日期:
2023-11-28
通讯作者:
晁跃辉,男,博士,副教授,研究方向:草地植物生物技术;E-mail: chaoyuehui@bjfu.edu.cn作者简介:
王欣怡,女,硕士研究生,研究方向:草地植物生物技术;E-mail: nqzxwxy@163.com;基金资助:
WANG Xin-yi1(), WANG Xiao-qian2, WANG Hong-jun2, CHAO Yue-hui1()
Received:
2023-03-30
Published:
2023-10-26
Online:
2023-11-28
摘要:
纳米抗体是一种新型的蛋白质工程抗体,其体积小、稳定性强、亲和力高等特性为科学研究提供了新的可能性。FLAG标签是一种广泛应用于生物学研究中的短肽标签,在生物学研究中具有重要的作用。为了制备FLAG标签的纳米抗体,利用酵母双杂交技术筛选出具有高亲和力的纳米抗体,并对制备的FLAG纳米抗体进行性能检验。通过DNA重组技术,构建包含FLAG的诱饵载体,利用酵母双杂交技术,在驼源纳米抗体酵母文库中,筛选针对FLAG标签纳米抗体。在酵母文库中筛选出5个单一的候选抗体DNA序列,为了排除载体本身表达蛋白序列对抗体筛选带来的干扰,通过“点对点”验证方法排除非特异性杂交的可能,通过该操作确认所筛选的5株纳米抗体均能与FLAG标签发生特异性亲和反应。为了制备纳米抗体,构建了5个纳米抗体原核表达载体,并利用大肠杆菌体系进行表达。通过SDS-PAGE和Western杂交(WB)分析,结果显示,成功获得2株可溶性表达的抗FLAG标签蛋白纳米抗体。对这2株纳米抗体与商品化的常规FLAG标签抗体进行效果比对,结果显示制备的2株纳米抗体与商品化抗体均能够识别FLAG多肽及含有FLAG标签的融合蛋白,且在特异性上没有明显区别,表明制备的FLAG纳米抗体具有较好的应用前景。基于酵母双杂交技术,成功筛选并制备了FLAG标签纳米抗体,这一成果不仅丰富了纳米抗体的类型,也为抗体开发及应用提供了新的途径。该研究为进一步研究FLAG标签在生物学研究中的应用,以及纳米抗体在生物工程中的应用提供有力支持。
王欣怡, 王晓倩, 王红军, 晁跃辉. FLAG标签纳米抗体的筛选、表达及验证[J]. 生物技术通报, 2023, 39(10): 323-331.
WANG Xin-yi, WANG Xiao-qian, WANG Hong-jun, CHAO Yue-hui. Screening, Expression, and Validation of Nanobodies with FLAG Tag[J]. Biotechnology Bulletin, 2023, 39(10): 323-331.
引物名称 Primer name | 序列 Sequence(5'-3') |
---|---|
pGBKT7-FLAG-F | GGAGGCCGAATTCCCGGACTATAAAGACGACGACGATAAAGGGATCCGTCGACCT |
pGBKT7-FLAG-R | AGGTCGACGGATCCCTTTATCGTCGTCGTCTTTATAGTCCGGGAATTCGGCCTCC |
T7 promoter | AATACGACTCACTATAGGGC |
3'BD | TTTTCGTTTTAAAACCTAAGAGTC |
3'AD | AGATGGTGCACGATGCACAG |
pCold-VHH-F | GATTACGCTCATATGGCCATGGAGGCCAGTCAGGTTCAGCTGCAGGAGTCTGGRGGAGG |
pCold-VHH-R | GTTTTTCAGTATCTACGATTCATCTGCAGCTTACGCAGAAGAGACGGTGACCWGGGT |
pCold-F | AAACCACTCCACTGCGTCGTCTG |
pCold-R | CAGGGATCTTAGATTCTGTG |
表1 本试验所用引物序列
Table 1 Primer sequences used in the experiment
引物名称 Primer name | 序列 Sequence(5'-3') |
---|---|
pGBKT7-FLAG-F | GGAGGCCGAATTCCCGGACTATAAAGACGACGACGATAAAGGGATCCGTCGACCT |
pGBKT7-FLAG-R | AGGTCGACGGATCCCTTTATCGTCGTCGTCTTTATAGTCCGGGAATTCGGCCTCC |
T7 promoter | AATACGACTCACTATAGGGC |
3'BD | TTTTCGTTTTAAAACCTAAGAGTC |
3'AD | AGATGGTGCACGATGCACAG |
pCold-VHH-F | GATTACGCTCATATGGCCATGGAGGCCAGTCAGGTTCAGCTGCAGGAGTCTGGRGGAGG |
pCold-VHH-R | GTTTTTCAGTATCTACGATTCATCTGCAGCTTACGCAGAAGAGACGGTGACCWGGGT |
pCold-F | AAACCACTCCACTGCGTCGTCTG |
pCold-R | CAGGGATCTTAGATTCTGTG |
图2 FLAG标签纳米抗体序列及结构 FR1-4:4个骨架区;CDR1-3:3个抗原互补决定区
Fig. 2 Sequences and structures of FLAG-tag nanobody FR1-4: Four fragment regions. CDR1-3: Three complementarity determining regions
图4 纳米抗体原核表达载体PCR检测 M:MB2000 DNA marker;1-5:5种纳米抗体原核表达载体
Fig. 4 PCR detection of prokaryotic expression vector for nanobodies M: MB2000 DNA marker. 1-5: Prokaryotic expression vectors of 5 nanobodies
图5 原核表达 M:蛋白质marker;1: Nano-FLAG6;2:Nano-FLAG7;CK-:阴性对照;红色箭头:表达的纳米抗体
Fig. 5 Prokaryotic expression M: Protein marker. 1: Nano-FLAG6. 2: Nano-FLAG7. CK-: Negative controls. Red arrows: Expressed nanobodies
图6 Western blot检测 M:蛋白质marker;1:Nano-FLAG6;2:Nano-FLAG7;CK-:阴性对照
Fig. 6 Western blot detection M: Protein marker. 1: Nano-FLAG6. 2: Nano-FLAG7. CK-: Negative controls
图7 纳米抗体Western blot验证 1:Nano-FLAG6;2:Nano-FLAG7;C:商品化FLAG单抗;D:示意图
Fig. 7 Western blot validation of nanobodies 1: Nano-FLAG6. 2: Nano-FLAG7. C: Commercialized FLAG monoclonal antibody. D: Schematic diagram
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