Construction of Camelid Natural Nanobody Phage Display Library and Screening for Anti-GDH Nanobody

doi:10.13523/j.cb.20181207

China Biotechnology

2018, Vol. 38

Issue (12): 49-56 DOI: 10.13523/j.cb.20181207

Orginal Article

Construction of Camelid Natural Nanobody Phage Display Library and Screening for Anti-GDH Nanobody

FANG Yuan¹,XU Guang-xian^1,^2,^**(

),WANG Xian¹,WANG Hong-xia¹,PAN Jun-fei¹

1 Clinical Medical Colleges,Ningxia Medical University,Yinchuan 750004,China
2 General Hospital of Ningxia Medical University,Yinchuan 750004,China

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Abstract

Objective To construct a natural nanobody phage display library as a nanobody platform for screening different antigens.Using GDH antigen to screen and obtain single domain antibody variable region gene (VHH) of the camel targeting GDH.Methods: Isolate the total RNA from the camel by using Oligo DT and synthesize the cDNA.The genes of variable domain of heavy chain(VHH) were amplified by nested PCR,and then were ligated with the vector pCANTAB5E.Next the recombinant vector cloned into TG1 to construct the phage display library ,and make the analysis and identification for the library.Using GDH to screening anti-GDH nanobodies,and identified the monoclonal by phage-ELISA.Result: The positive rate of the phage display library is 95%,the amino acid homology of nine randomly colonies is 66.17%,and it has a good variety after MEGA analyzing.After helper phage rescue,the titer of the phage display library is 4×10 ¹²CFU/ml.And after three times round of screening,it showed significant enrichment of binding phage.In the end,two nanobodies sequence were obtained by sequencing positive clonoies. Conclusion: Successfully constructed a natural camelid nanobody phage display library,and the good diversity laid a foundation for selecting other nanobodies.Two GDH specific sequences derived from camel are obtained through phage display library,which can be used to prepare the diagnostic antibodies.

Key words： Nanobodies Phage display technology GDH

Received: 30 July 2018 Published: 10 January 2019

ZTFLH:

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Corresponding Authors: Guang-xian XU E-mail: 599040064@qq.com

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	Yuan FANG
	Guang-xian XU
	Xian WANG
	Hong-xia WANG
	Jun-fei PAN

Cite this article:

FANG Yuan,XU Guang-xian,WANG Xian,WANG Hong-xia,PAN Jun-fei. Construction of Camelid Natural Nanobody Phage Display Library and Screening for Anti-GDH Nanobody. China Biotechnology, 2018, 38(12): 49-56.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20181207 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I12/49

Table 1 Primer design of Nested PCR

Fig.1 Agarose gel electrophoresis of the first-round PCR M: DNA marker DL1000;1~3: 700bp PCR products

Fig.2 Agarose gel electrophoresis of the second-round PCR M: DNA marker DL1000;1~5: 400bp PCR products

Fig.3 Detection of the library titer by double dilution method (a) The growth of 10^-2 dilution plating (b) The growth of 10^-4 dilution plating (c) The growth of 10^-6 dilution plating (d) The growth of 10^-8 dilution plating

Fig.4 Colony PCR analyses colonies M: DNA marker DL1000;1~20: Randomly colonies

Fig.5 MEGA analysis Phylogenetic tree

Fig.6 Amino acid sequence analysis of nine randomly colonies

Table 2 Selective enrichment of nanobody during panning

Fig.7 Identification of polyclonal phage by Phage-ELISA

Fig.8 Identification of monoclonal phage by Phage-ELISA (a):Phage-ELISA of monoclonal phage (b):Indirect Phage-ELISA of positive colonies


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