Abstract:
Objective To construct a large naive chicken-derived phage antibody library for antibody screening in infectious disease diagnosis.
Methods Five five-week old White Leghorn chickens, including ordinary ones and specific pathogen free ones, Roman chicken, Los Angeles Red chickens, Los Angeles White Chickens and Imperial chickens were selected respectively. Total RNA was extracted from bursa of Fabricius of 30 non-immunized chickens, then reversely transcribed to cDNA, and amplified by polymerase chain reaction to obtain the gene encoding the variable region of antibody. Splicing by overlap extension was used to assemble the gene coding the single-chain variable fragment. By ligation with the phage vector, the recombinant phage was electroporated into competent cells of Escherichia coli TG1 to construct a naive chicken-derived phage antibody library.
Results The naive antibody library with capacity of 1010 was successfully constructed. The titer of phage display library exceeded 1012 CFU/mL. A total of 165 clones were randomly selected, resulting in a positive transformation insertion rate of 96.97% (160/165). Seventy-two positive clones were randomly selected for sequencing, and significant variations in the base sequence and length of the complementarity determining region 3 were found, indicating the good diversity of antibody library.
Conclusion A naive single chicken-derived phage antibody library was successfully constructed in this study, which is fundamental for the valuable single chain antibody screening for specific antigens.
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