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.
Highlights
Phage-displayed antibody library has been widely used to derive high-affinity target-specific antibodies, such as antibodies that were specific for angiogenesis marker fibronectin [1], melanoma-specific B3 and B4 antigens [2], epidermal growth factor receptor [3], HIV Vpr protein [4], and spike protein of SARS-associated coronavirus (SCoV) [5,6]
To retrieve the variable regions of rearranged Ig genes from genomic DNA, degenerate primer pair that is complementary to the antibody framework segment 1 (FR1) and framework 4 (FR4) of immunoglobulin chain was used for semi-nested PCR
There was on distinct difference between clones derived from naıve or immunized libraries, except more positive clones were isolated from the immunized library. In this proof-of-concept study, we have demonstrated the possibility of using rearranged germline immunoglobulin variable region genes as the source of antibody repertoires for construction of germline scFv library
Summary
Phage-displayed antibody library has been widely used to derive high-affinity target-specific antibodies, such as antibodies that were specific for angiogenesis marker fibronectin [1], melanoma-specific B3 and B4 antigens [2], epidermal growth factor receptor [3], HIV Vpr protein [4], and spike protein of SCoV [5,6]. Antibody repertoires of phage-displayed library are conventionally created by harvesting mRNAs from peripheral blood lymphocytes, spleen, bone marrow, tonsil or similar sources using RT-PCR and familybased oligonucleotides [7,8,9,10]. Potential Ig genes may not be recovered from antibody cDNA library due to reading frame shifted or the presence of stop codon(s) which are generated by imprecise somatic recombination and P- and N- additions [15,16]; or the immunoglobulins are self-reactive and eliminated by the host immune system [17,18]. On the other hand, activated B cells undergo clonal expansion and the antibody repertoires of a cDNA-derived scFv library would be dominated by antigenstimulated humoral response. Recombinant antibody repertoires of a cDNA-derived antibody library are limited
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