抗体噬菌体体展示技术.pptVIP

Phage VOLUME 178 Display Antibody Meiling Xiong Phage disMethods and protocols a M O Br Robert Aite a Introduction of Ab phage Display technology Ab Formats for Phage Display a Ab Libraries Construction Contents,Phage A electon Mthd stages a Phage Ab Screening Applications a In vitro Affinity Maturation a Expression Purification of Phage Ab fragment Introduction of Phage Display Technology The Ff bacteriophage structure ,2r Introduction of Phage Display Technology a IG region: intergenic region, usually contains the packing sequence and replication origin of minus ne scheme of phagemid vector Molecular tag: to facilitate library screening and for Replication origin of phasmid Promote a Restriction enzyme recognitio / Restriction enaymes recognition sites DNA recombination and gene multiple cloning sites (MCS) Coat protein: Pll (larger protein, less than 5 copies, I PVIl(more than 5 copies, decreased length) a Amber codon TAG: SupE strains glutamic aci codon), non-suppressor strains(stop codon) a Protease cleavage site Signal peptides: phage protein translocation, crucial a Selective marker: for selection of infected host cells Introduction of Phage Display Technology a Nonlytic filamentous phage is the most ofter used for phage display, primarily the M13 and Fd strain s Proteins to be selected are infused to all five at proteins, with pill and pVll most commonly used 3 Remove unspec pIll protein is essential for infection of bacteria selection rounds Helper phage: wild-type pill helper phage and Amplification ecial helper phage E utio Antigen immobilized on magnetic beads olystryrene surfaces, or on columns, or is used in solution as biotinylated antigen and later captured by imme obilized strept Colony picking E coli infection a More efficiently than through conventional hybridoma system Easier to maintain and grow bacterial cultures for recombinant Bypass immunization in antibody selection Advantages of a Producing the cor with 108 to 10 Phage Disp