单域抗体T7噬菌体展示文库构建与鉴定

doi:10.3969/j.issn.1004-1524.2021.01.04

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (1): 27-33.DOI: 10.3969/j.issn.1004-1524.2021.01.04

单域抗体T7噬菌体展示文库构建与鉴定

徐海¹(), 王健¹, 郭长明¹, 董洪燕¹, 邓碧华², 侯继波²^,^*()

1.江苏农牧科技职业学院江苏省兽用生物制药高技术研究重点实验室,江苏泰州 225300
2.江苏省农业科学院动物免疫工程研究所,江苏南京 210014

收稿日期:2020-05-14 出版日期:2021-01-25 发布日期:2021-01-25
通讯作者: 侯继波
作者简介:*侯继波,E-mail:houjibo@jaas.ac.cn
徐海(1982—),男,江苏扬州人,硕士,副研究员,主要从事噬菌体展示技术研究与应用。E-mail:hai_x@126.com
基金资助:
江苏农牧科技职业学院院级课题(NSF201902)

Construction and identification of nanobody T7 phage display library

XU Hai¹(), WANG Jian¹, GUO Changming¹, DONG Hongyan¹, DENG Bihua², HOU Jibo²^,^*()

1. Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Jiangsu Agri-animal Husbandry Vocational College, Taizhou 225300, China
2. Institute of Veterinary Immunology & Engineering, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China

Received:2020-05-14 Online:2021-01-25 Published:2021-01-25
Contact: HOU Jibo

摘要/Abstract

摘要：

优化噬菌体展示文库构建策略,获得高品质羊驼源天然纳米抗体T7噬菌体展示文库,并对文库质量进行鉴定。分离羊驼外周血淋巴细胞,提取总RNA,RT-PCR扩增羊驼重链抗体可变区(VHH)基因,插入T7 select 415-1b噬菌体载体构建文库,计算文库的库容量,测定文库传代稳定性。测序分析VHH基因的多样性和纳米抗体氨基酸序列特征。Western-blot检测纳米抗体在噬菌体表面的表达情况,电镜观察文库中重组噬菌体颗粒形态。结果表明,构建羊驼源纳米抗体T7噬菌体展示文库,有效库容达到2.73×10⁹ PFU;纳米抗体与羊驼源抗体同源性高,氨基酸序列显示纳米抗体特征区域,并其在噬菌体表面高拷贝表达;重组噬菌体颗粒结构完整,但随着传代出现插入基因的丢失。

关键词: 羊驼, 纳米抗体, T7噬菌体, 表面展示

Abstract:

To construct a high-quality natural alpaca nanobody phage display library by strategy optimization, and systematically identified the quality of the library, the total RNA was isolated form peripheral blood mononuclear cell of 6 alpacas and then reverse transcripted into cDNA. The VHH gene was amplified and insert into plasmid and T7 select 415-1b vector, respectively, to construct plasmid and phage display library. The phage library was identified by volume calculating, propagating stability testing and sequencing. The expression of nanobody was tested by Western-blot, and the structure of phage particles were detected by electron microscopy. Results showed that the effective volume of phage library was 2.73×10⁹ PFU, and VHH nanobody was successfully displayed on T7 phage surface in high copy number. Sequence analysis showed that VHH nanobody had high homology with alpaca antibody. Recombinant phage presented intact particle structure the same as the donate T7 select 415-1b phage. However, during continuous passage a part of recombinant phages would lost the insert VHH gene, which indicated that only lower-generation phage library could be used for affinity screening.

Key words: alpaca, nanobody, T7 phage, surface display

中图分类号:

Q939.48

徐海, 王健, 郭长明, 董洪燕, 邓碧华, 侯继波. 单域抗体T7噬菌体展示文库构建与鉴定[J]. 浙江农业学报, 2021, 33(1): 27-33.

XU Hai, WANG Jian, GUO Changming, DONG Hongyan, DENG Bihua, HOU Jibo. Construction and identification of nanobody T7 phage display library[J]. Acta Agriculturae Zhejiangensis, 2021, 33(1): 27-33.

图/表 8

图1 VHH基因扩增与噬菌体表面展示示意图

Fig.1 Schematic of VHH gene PCR amplification and phage surface display

表1 文库构建与鉴定引物

Table 1 Primers used in library construction and identification

引物名称 Primer	引物序列 Primer sequence(5'-3')
F1	CTGGGTGGTCCTGGCTGCTCTT
R1	GCGGTACGTGTGTTGAACTGTT
F2	GAATTCGGGTGGTCAGKTGCAGCYCGTGGAGNC
R2-1	AAGCTTTTATTGTGGTTTTGGTGTCTTGGG
R2-2	AAGCTTTTAGGGGTCTTCGCTGTGGTGCG
T7 select up	GGAGCTGTCGTATTCCAGTC
T7 select down	AACCCCTCAAGACCCGTTTA

图2 VHH基因扩增与质粒文库鉴定 A、B,套式PCR扩增羊驼重链抗体可变区;M,DL2000 marker;1-6,6份羊驼样品。C,酶切鉴定VHH质粒文库;M,DL5000 marker;1,VHH质粒文库双酶切鉴定。

Fig.2 PCR amplification of VHH gene and enzyme digestion of plasmid library A, B, Heavy chain antibody variable region amplification by nested PCR; M, DL2000 marker; 1-6, Six alpaca samples. C: Identification of VHH plasmid library; M, DL5000 marker; 1, Enzyme digestion of VHH plasmid library.

表2 质粒文库(A)与噬菌体文库(B)库容

Table 2 The volume of plasmid library (A) and phage library (B)

文库 Library	构建批次Batches										累计 Total
文库 Library	1	2	3	4	5	6	7	8	9	10	累计 Total
A/CFU	2.9×10⁷	3.4×10⁷	2.8×10⁷	4.3×10⁷	5.4×10⁷	3.8×10⁷	4.1×10⁷	5.1×10⁷	4.5×10⁷	4.2×10⁷	4.05×10⁸
B/PFU	5.4×10⁸	6.2×10⁸	4.7×10⁸	7.3×10⁸	3.7×10⁸	—	—	—	—	—	2.73×10⁹

图3 噬斑PCR检测噬菌体展示文库重组率 A,第1代文库重组率测定;M,DL2000 marker;1-20,20个噬菌体噬斑PCR检测;B,第5代文库重组率测定;M,DL2000 marker;1-20,20个噬菌体噬斑PCR检测。

Fig.3 Identification of recombinant phage by plaque PCR testing A, The recombination rate of the first generation of library; M, DL2000 marker; 1-20, PCR test of 20 unique phage clones; B, The recombination rate of the fifth generation of library; M, DL2000 marker; 1-20, PCR test of 20 unique phage clones.

图4 T7噬菌体衣壳蛋白Western-blot鉴定 M,预染蛋白Marker;1,T7 select 415-1b 噬菌体;2,第1代T7噬菌体展示文库;3,第5代T7噬菌体展示文库。

Fig.4 Western-blot of T7 phage capsid protein M, Protein marker; 1, T7 select 415-1b phage; 2, The first generation of T7 phage display library; 3, The fifth generation of T7 phage display library.

图5 电镜检测噬菌体形态 A,T7 select 415-1b噬菌体;B,T7噬菌体纳米抗体展示文库。

Fig.5 Electron microscopy observation of T7 phages A, T7 select 415-1b phage; B, T7 phage displayed nano-antibody library.

图6 VHH纳米抗体随机克隆氨基酸多序列分析

Fig.6 Nanobody amino acid sequence analysis

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单域抗体T7噬菌体展示文库构建与鉴定

Construction and identification of nanobody T7 phage display library

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