Design and Selection of a Camelid Single-Chain Antibody Yeast Two-Hybrid Library Produced De Novo for the Cap Protein of Porcine Circovirus Type 2 PCV2Report as inadecuate




Design and Selection of a Camelid Single-Chain Antibody Yeast Two-Hybrid Library Produced De Novo for the Cap Protein of Porcine Circovirus Type 2 PCV2 - Download this document for free, or read online. Document in PDF available to download.

Nanobodies or variable domain of the heavy chain of the heavy-chain antibodies, VHHs are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized Camelus Bactrianus VHH yeast two-hybrid Y2H library according to the Clontech Mate and Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×106 cfu-3 µg and 2×109 cfu-ml, which met the demand for Y2H library screening. Using as an example the porcine circovirus type 2 PCV2 Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. Among these sequences, 7 of 9 randomly selected clones were strongly positive as indicated by enzyme-linked immunosorbent assay, either using PCV2 viral lysis or purified Cap protein as coated antigen. Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells. All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy.



Author: Xiangjing Fu, Xiaolong Gao, Shengfang He, Di Huang, Peng Zhang, Xinglong Wang, Shuxia Zhang, Ruyi Dang, Shuanghui Yin, Enqi Du ,

Source: http://plos.srce.hr/



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