Construction and identification of nanobody T7 phage display library

doi:10.3969/j.issn.1004-1524.2021.01.04

Acta Agriculturae Zhejiangensis ›› 2021, Vol. 33 ›› Issue (1): 27-33.DOI: 10.3969/j.issn.1004-1524.2021.01.04

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

Q939.48

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.

Figures/Tables 8

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

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

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.

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

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.

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.

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

Fig.6 Nanobody amino acid sequence analysis

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Construction and identification of nanobody T7 phage display library

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