title: 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).
abstract: 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 & Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×10(6) cfu/3 µg and 2×10(9) 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.
authors.affiliation: College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi, China.
authors.forename: Xiangjing
authors.initials: X
authors.lastname: Fu
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Gao
authors.forename: Shengfang
authors.initials: S
authors.lastname: He
authors.forename: Di
authors.initials: D
authors.lastname: Huang
authors.forename: Peng
authors.initials: P
authors.lastname: Zhang
authors.forename: Xinglong
authors.initials: X
authors.lastname: Wang
authors.forename: Shuxia
authors.initials: S
authors.lastname: Zhang
authors.forename: Ruyi
authors.initials: R
authors.lastname: Dang
authors.forename: Shuanghui
authors.initials: S
authors.lastname: Yin
authors.forename: Enqi
authors.initials: E
authors.lastname: Du
authors.forename: Zengqi
authors.initials: Z
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000956:Antigens, Viral / Q000032:analysis* / Q000276:immunology; D002162:Camelus; D036022:Capsid Proteins / Q000032:analysis* / Q000276:immunology; D017925:Circovirus / Q000737:chemistry*; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D015723:Gene Library; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D008297:Male; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D020798:Two-Hybrid System Techniques
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; D000939:Epitopes; D007143:Immunoglobulin Heavy Chains; D057127:Single-Chain Antibodies