construction and characterization of single-chain variable fragment antibody library derived from germline rearranged immunoglobulin variable genes建设和表征单链可变片段抗体库来自生殖系基因重组免疫球蛋白变量.pdf

Construction and Characterization of Single-Chain Variable Fragment Antibody Library Derived from Germline Rearranged Immunoglobulin Variable Genes 1 1 1 1 2 2 Man Cheng , Shirley Y. W. Chan , Qi Zhao , Elaine Y. M. Chan , Shannon W. N. Au , Susanna S. T. Lee , Wing-Tai Cheung1* 1 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 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 cl