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| Construction and Biopanning of Camelid Naïve Single-domain Antibody Phage Display Library |
| TU Zhui1,2, XU Yang1,2, LIU Xia1,2, HE Qing-hua1,2, TAO Yong2 |
1. State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China;
2. Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, China |
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Abstract The objective is to construct a camelid nave single-domain heavy chain antibody phage display library. Total RNA was purified from 30ml blood of two healthy non-immune alpacas (Lama pacos) and directly used for complementary DNA (cDNA) synthesis. Three sets of primers were designed based on the conserved region of heavy-chain antibody. The repertoire of VHH coding sequence was amplified by nested PCR, and the PCR products were cloned into a phagemid vector pHEN1. By electroporation of E.coli TG1, the primary library (designate NAL) was obtained containing more than 107 independence clones. After helper phage rescue, the phage display library (designate SNA-PDL) was generated with a titre up to 1013 CFU/ml. The library exhibited high diversity as judged by the Hinf I restriction pattern. Solid phage biopanning against artificial antigen DON-MBSA showed significant enrichment of binding phage particles. The positive rate of panning round two was 36.4%. The data indicated that a nave single-domain antibody phage display library was constructed, which has good diversity and would be useful for generating VHHs with specific binding affinity.
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Received: 22 November 2010
Published: 26 April 2011
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