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Construction and Characterization of Single-chain Variable Fragment Antibody Library Derived from Germline Rearranged Immunoglobulin Variable Genes
Man Cheng1; Shirley Y. W. Chan1; Qi Zhao1; Elaine Y. M. Chan1; Shannon W. N. Au2; Susanna S. T. Lee2; Wing-Tai Cheung1
2011
Source PublicationPLOS ONE
ISSN1932-6203
Volume6Issue:11
Abstract

Antibody repertoires for library construction are conventionally harvested from mRNAs of immune cells. To examine whether germline rearranged immunoglobulin (Ig) variable region genes could be used as source of antibody repertoire, an immunized phage-displayed scFv library was prepared using splenocytic genomic DNA as template. In addition, a novel frame-shifting PCR (fsPCR) step was introduced to rescue stop codon and to enhance diversity of the complementarity-determining region 3 (CDR3). The germline scFv library was initially characterized against the hapten antigen phenyloxazolone (phOx). Sequence analysis of the phOx-selective scFvs indicated that the CDRs consisted of novel as well as conserved motifs. In order to illustrate that the diversity of CDR3 was increased by the fsPCR step, a second scFv library was constructed using a single scFv clone L3G7C as a template. Despite showing similar binding characteristics towards phOx, the scFv clones that were obtained from the L3G7C-derived antibody library gave a lower non-specific binding than that of the parental L3G7C clone. To determine whether germline library represented the endogenous immune status, specific scFv clones for nucleocapsid (N) protein of SARS-associated coronavirus (SCoV) were obtained both from naïve and immunized germline scFv libraries. Both libraries yielded specific anti-N scFvs that exhibited similar binding characteristics towards recombinant N protein, except the immunized library gave a larger number of specific anti-N scFv, and clones with identical nucleotide sequences were found. In conclusion, highly diversified antibody library can be efficiently constructed using germline rearranged immunoglobulin variable genes as source of antibody repertoires and fsPCR to diversify the CDR3.

DOIhttps://doi.org/10.1371/journal.pone.0027406
Indexed BySCI
Language英语
WOS Research AreaScience & Technology - Other Topics
WOS SubjectMultidisciplinary Sciences
WOS IDWOS:000297553900041
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Cited Times [WOS]:3   [WOS Record]     [Related Records in WOS]
Document TypeJournal article
CollectionFaculty of Health Sciences
Corresponding AuthorWing-Tai Cheung
Affiliation1.School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, China
2.School of Life Sciences, Chinese University of Hong Kong, Hong Kong, China, University of Rome, Italy
Recommended Citation
GB/T 7714
Man Cheng,Shirley Y. W. Chan,Qi Zhao,et al. Construction and Characterization of Single-chain Variable Fragment Antibody Library Derived from Germline Rearranged Immunoglobulin Variable Genes[J]. PLOS ONE,2011,6(11).
APA Man Cheng.,Shirley Y. W. Chan.,Qi Zhao.,Elaine Y. M. Chan.,Shannon W. N. Au.,...&Wing-Tai Cheung.(2011).Construction and Characterization of Single-chain Variable Fragment Antibody Library Derived from Germline Rearranged Immunoglobulin Variable Genes.PLOS ONE,6(11).
MLA Man Cheng,et al."Construction and Characterization of Single-chain Variable Fragment Antibody Library Derived from Germline Rearranged Immunoglobulin Variable Genes".PLOS ONE 6.11(2011).
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