One step germline immunoglobulin genes retrieval and diversity enhancement forscFv library construction.

摘要

Nowadays, tailor-made antigen-specific single-chain variable fragment (scFv) can be obtained by isolating immunoglobulin (Ig) mRNA from peripheral immune system and the subsequent construction of scFv phage-displayed library. In general, the quality and quantity of scFvs isolated from a phage library are affected by the characteristics (e.g. size and diversity) and the origin (e.g. naive or immune) of the scFv repertoire library.; However, diversity of Ig gene mRNA repertoire is hampered by the inability of retrieving Ig genes that against poor immunogenic targets (e.g. haptens) and Ig genes that are masked by class-selection, clonal-deletion, self-intolerance and non-productive exon joining. By contrast, germline-templates (genomic source) that bypass all the limitations and show higher diversity and stability during library construction are believed to be a better alternative for retrieving antibody genes.; This thesis is a proof-of-concept demonstration of the proposed use of germline Ig genes for scFv repertoire library construction. Besides, a novel somatic recombination mimicking-polymerase chain reaction (frame-shifting PCR) is introduced to enhance the CDR3 diversity of the retrieved Ig genes, as well as to recover defective Ig genes resulting from non-productive exon joining. The possibility of retrieving somatic rearranged Ig V-region genes, and somatic recombination mimicking were examined by the sequence analysis of retrieved DNA fragments from splenocytic genome, and the generation of heterogeneous populations of Ig gene from a single template, respectively. The feasibility of applying germline-derived and frame-shifted Ig V-region genes for scFv phage-displayed library construction was tested in two models by identifying scFvs against (1) phenyloxazolone (phOx) and (2) SARS coronavirus nucleocapsid protein (SCoV N).; In conclusion, these results not only suggested the possibility of using germline Ig genes for antibody repertoire library construction, but also clearly demonstrated the feasibility of recombination-like diversity-enhancement process. The advantage of using such a library was clearly shown by the easy and rapid retrieval of different quality and quantity of scFvs, even with a small initial library size. (Abstract shortened by UMI.)