Construction and identification of specific anti-human β-amyloid peptide ScFv by phage antibody library display system

AIM:To construct and identify the specific anti-human β-amyloid peptide ScFv by phage antibody library display system. METHODS: Total RNA was extracted from spleen B cells of BALB/c mice immunized with β-amyloid peptide, and mRNA was purified from the total RNA, then the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified by primary PCR and the amplified VH and VL PCR fragments were further joined into a ScFv gene with a 15-peptide linker DNA (Gly4Ser)3 with fill-in reactions. Then the assembled ScFv gene fragments were amplified and restriction sites were added by the second PCR. The digested ScFv gene fragments were finally ligated with the phagemids pCANTAB 5E and the phagemids containing ScFv gene were transformed into competent E. coli TG1 cells. The transformed cells were then infected with M13KO7 helper phage to rescue all recombinant phagemids with ScFv gene insert, thus an immune phage ScFv library was constructed. Purity of ScFv was analyzed by SDS-PAGE. RESULTS: Antibody VH and VL gene fragments amplified by primary PCR were about 350 bp and 325 bp after analysis by agarose gel electrophoresis and matched the expectancy of VH and VL gene fragments. The purified VH and VL PCR fragments were further joined into a ScFv gene with fill-in reactions and reached the desired 750 bp expectancy. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 1.1×108 was successfully constructed. After analysis by SDS-PAGE, a purified ScFv gene was got with a relative molecular weight of about 30000 u. CONCLUSION: An immune phage ScFv library is successfully constructed. The successful preparation of anti-Aβ ScFv will serve as a useful tool for studying the etiological and pathological mechanism, diagnosis and treatment of patients with Alzheimer's disease.