The construction and bio-panning of mouse ScFv phage-displayed antibody library against pemphigus vulgaris antigen / 中国免疫学杂志

ABSTRACT

Objective:To construct mouse ScFv against pemphigus vulgaris antigen (PVA) through phage displayed antibody technology Methods:Total RNA was isolated from the spleen cells of mice immunized with recombinant pemphigus vulgaris antigen (rPVA) after six weeks, and then reverse transcripted into cDNA Primers specific for mouse V H and V L were synthesized to amply the V H and V L by ploymerase chain reaction (PCR) V L gene was modified and amplified with linker primer (which contain 3′ region of V H, linker, and 5′ region of V L), the resultant gene called V L′ Purified V L′ and V H were subjected to SOE (splicing by overlap extension) ligation, and resultant ScFv gene was digested with SfiI and NotI, cloned to the identically digested pCantab 5E Library of TG1 transfected with recombinant phagemid were derived with diversity of 1 5?10 6 Results:The pemphigus vulgaris phage antibody library was constructed, and one positive TG1 clone was obtained, which can highly expresses phage displayed antibody Conclusion:Specific genetic antibody against PVA can be made from phage displayed antibody library