Screening, Expression and Specificity Detection of Anti-TNF-α Nanobody

doi:10.13523/j.cb.2002034

China Biotechnology

2020, Vol. 40

Issue (7): 15-21 DOI: 10.13523/j.cb.2002034

Screening, Expression and Specificity Detection of Anti-TNF-α Nanobody

LIN Shi-xin¹,LIU Dong-chen²,LEI Yun²,XIONG Sheng²,XIE Qiu-ling^1,^**(

)

1 College of life science and technology, Jinan university, Guangzhou 510632, China
2 National Engineering Research Center of Genetic Medicine, Jinan university, Guangzhou 510632, China

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Abstract

Objective: To construct the phage library of anti-TNF-α (vascular endothelial growth factor) andto screen and express the nanobodies which have specificity and affinity with TNF-α. Methods: (1) The llama was immunized with TNF-α, and the total RNA of peripheral blood lymphocytes was extracted to construct a phage library, the clones having affinity with TNF-α were screened by multiple panning. (2) Then their molecular weight, pI and hydrophilicity were analyzed by ExPASy. And the VHH genes were cloned into the expression vector pNCS to construct the recombinant plasmids (pNCS-Nb_TNF-α) and to express these recombinant nanobodies (Nb_TNF-α) in E.coli DH5α. (3) The recombinant nanobodies were purified by Ni metal chelate affinity chromatography, followed by detection the specificity by enzyme linked immunosorbent assay (ELISA).Results: (1) Ten VHH gene fragments having affinity with TNF-α were obtained after phage library construction and panning. (2) Based on the bioinformatics analysis, it was found that eight nanobodies were hydrophilic proteins with molecular weights of 19.6-20.1kDa. All of Nb_TNF-α were expressed in E.coli DH5α in soluble form. (3) It was showed that five recombinant nanobodies, Nb_TNF-α-1, Nb_TNF-α-2, Nb_TNF-α-3, Nb_TNF-α-4 and Nb_TNF-α-5 could specifically bind to TNF-α. Conclusion: Eight nanobodies with specificity to TNF-α were screened and expressed in E.coli successfully, and five Nb_TNF-α showed good affinity with TNF-α, which could be possible candidates for anti-TNF-α drug.

Key words： Nanobody Tumor necrosis factor Phage library Screening

Received: 22 February 2020 Published: 13 August 2020

ZTFLH:

Q816

Corresponding Authors: Qiu-ling XIE E-mail: txql@jnu.edu.cn

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	Shi-xin LIN
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Cite this article:

LIN Shi-xin,LIU Dong-chen,LEI Yun,XIONG Sheng,XIE Qiu-ling. Screening, Expression and Specificity Detection of Anti-TNF-α Nanobody. China Biotechnology, 2020, 40(7): 15-21.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2002034 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I7/15

Tab.1 The list of primers

Fig.1 Titer of antibody in llama immunized with TNF-α

Fig.2 The pComb-3X-VHH digested by SfiⅠ M:DNA Marker;1-8:The band of pComb-3X-VHH with SfiⅠ digestion

Fig.3 Panning of Nb_TNF-α

Fig.4 Enrichment of Nb_TNF-α

Fig.5 Screening of Nb_TNF-α

Table 2 Physicochemical properties of Nb_TNF-α protein

Fig.6 SDS-PAGE electrophoresis and Western blot analysis of Nb_TNF-α M: Marker; 1-8: The band of Nb_TNF-α-1 -Nb_TNF-α-8

Fig.7 The purified Nb_TNF-α proteins 1-8: The band of Nb_TNF-α-1 -Nb_TNF-α-8

Fig.8 Specificity of Nb_TNF-α proteins


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