FLAG标签纳米抗体的筛选、表达及验证

doi:10.13560/j.cnki.biotech.bull.1985.2023-0291

摘要/Abstract

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。

关键词: FLAG标签, 纳米抗体, 酵母双杂, 筛选, 表达, 特异性

Abstract:

Nanobodies are a novel type of engineered antibodies with small size, high stability, and strong affinity, offering new possibilities for scientific research. The FLAG tag is a short peptide tag widely used in biological research, playing a significant role in various studies. In order to prepare nanobodies against the FLAG tag, high-affinity nanobodies were screened using yeast two-hybrid technology and the prepared FLAG nanobodies were subsequently tested for performance. Through DNA recombination technology, a bait vector containing the FLAG tag was constructed. Using yeast two-hybrid technology, nanobodies against the FLAG tag were screened from a camel-derived nanobody yeast library. Five single candidate antibody DNA sequences were screened from the yeast library. To exclude interference from the expression of protein sequences in the carrier itself on antibody screening, the possibility of non-specific hybridization was ruled out through a ‘point-to-point’ verification method. This operation confirmed that all five screened nanobodies specifically reacted with the FLAG tag. For the production of nanobodies, five nanobody prokaryotic expression vectors were constructed and expressed using the Escherichia coli system. SDS-PAGE and Western blot（WB）analysis showed that two soluble anti-FLAG tagged protein nanobodies were successfully obtained. The effects of these two nanobodies were compared with commercially available conventional FLAG tag antibodies. The results showed that both the prepared nanobodies and the commercial antibodies recognized FLAG peptides and fusion proteins with FLAG tags, and there were no significant differences in specificity, indicating that the prepared FLAG nanobodies had good application prospects. Based on yeast two-hybrid technology, FLAG tag nanobodies were successfully screened and prepared. This achievement not only enriches the types of nanobodies but also provides a new approach for the development and application of antibodies. This study provides strong support for further research on the application of FLAG tags in biological research, as well as the application of nanobodies in bioengineering.

Key words: FLAG tag, nanobody, yeast two-hybrid, screening, expression, specificity

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.

图/表 8

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1 FLAG标签酵母双杂交筛选

Fig. 1 Yeast two-hybrid screening of FLAG tag

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3 互作蛋白酵母双杂交验证

Fig. 3 Yeast two-hybrid assay for protein-protein interaction *: pGADT7-Nanobody+pGBD T7-FLAG. **: pGADT7-Nanobody+pGBDT7 empty vector

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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