单击此处编辑母版标题样式,Edit Master text styles,Second level,Third level,Fourth level,Fifth level,6,7,8,9,*,完整版课件,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,二级,三级,四级,五级,完整版课件,*,Antibody Phage Display,Meiling Xiong,20180629,1,完整版课件,Antibody Phage DisplayMeiling,Contents,Introduction of Ab phage Display Technology,Ab Formats for Phage Display,Ab Libraries Construction,Phage Ab Selection Methods&Strategies,Phage Ab Screening Applications,In vitro Affinity Maturation,Expression&Purification of Phage Ab Fragments,2,完整版课件,ContentsIntroduction of Ab pha,Introduction of Phage Display Technology,The Ff bacteriophage structure,3,完整版课件,Introduction of Phage Display,Introduction of Phage Display Technology,The scheme of phagemid vector,IG region,:intergenic region,usually contains the packing sequence and replication origin of minus and plus strands,Molecular tag,:to facilitate library screening and for protein analysis,Restriction enzyme recognition sites,:useful for DNA recombination and gene manipulation;multiple cloning sites(MCS),Coat protein,:PIII(larger protein,less than 5 copies,)PVIII(more than 5 copies,decreased length),Amber codon TAG,:supE strains(glutamic acid codon),non-suppressor strains(stop codon),Protease cleavage site,Promoter,Signal peptides,:phage protein translocation,crucial for display level,Selective marker,:for selection of infected host cells,4,完整版课件,Introduction of Phage Display,Introduction of Phage Display Technology,Nonlytic filamentous phage is the most often used for phage display,primarily the M13 and Fd strains.,Proteins to be selected are infused to all five coat proteins,with pIII and pVIII most commonly used.,pIII protein is essential for infection of bacteria,Helper phage:wild-type pIII helper phage and special helper phage,Antigen immobilized on magnetic beads,polystryrene surfaces,or on columns,or is used in solution as biotinylated antigen and later captured by immobilized streptavidin,5,完整版课件,Introduction of Phage Display,Advantages of Phage Display for Recombinant Antibody Selection,More efficiently,than through conventional hybridoma system.,Cheaper,to produce recombinant antibodies using bacteria,rather than mammalian cell line.,Easier,to maintain and grow bacterial cultures for recombinant antibody production.,Bypass immunization,in antibody selection.,Bypass the use of animal cells,for production of antibodies.,Producing the combinatorial library,(ideally with 10,8,to 10,9,members)of functional antibodies,to generate a larger repertoire of antibodies,than those available through conventional hybridoma technology.,Easy isolation and expression,of the cloned gene in a bacterial host.,Excellent potential,to further improve binding properties of the selected antibody by protein engineering techniques.,Capable of generating antibodies,against almost any desired antigen,including highly conserved or self-antigens,conformational variants,low immunogenic antigens,and also toxic components,which is not possible by in vivo immunization of animals.,A number of starting material:proteins,peptides,haptens,cel,l,lines,tissue slides,or virus particles,6,完整版课件,Advantages of Phage Display fo,Antibody,Formats,The most commonly used format:,single-chain variable fragment(scFv),Simplicity of cloning process,Fast and easy library generation,A high display rate(small protein size 25 kDa),Less stable than Fab fragments,Tend to form dimers(can be reduced with linker more than 20 amino acids),7,完整版课件,Antibody FormatsThe most commo,Antibody,Formats,Fab,The light chain(VL-CL)and the Fd-domain(VH-CH1)of the heavy chain of an antibody.,During bacterial expression,these two chains are,synthesized separately,and secreted into the,periplasm,where they fold to form heterodimers.,Fab exhibit higher stability than scFvs,Possess better PK and PD qualities than scFvs,Easier to convert into full-length antibodies,Clinical applications:abciximab,lucentis,cimzia.,8,完整版课件,Antibody Formats Fab8完整版课件,Antibody,Formats,Single domain antibody,VHH:VH domain of camelid antibody,heavy chains only,IgNAR(new antigen receptor):shark antibody,heavy chains only,Unique CDRs,Affibodies,Anticalins,DARPins,Avimers,Affimers,Monobodies,vNAR,9,完整版课件,Antibody Formats Single domain,Antibody,Formats,Multivalent fragments,Miniantibodies,are scFvs or Fabs connected via a flexible linker to self-associating structures such as helix bundles or leucine zippers.,Diabodies,are noncovalent dimers of scFvs,which spontaneously form depending on the linker length between VH and VL.Another form of diabodies is two scFvs connected with a short linker.,Fab-A,is created by genetic fusion of the Fab Fd gene with the alkaline phosphatase(PhoA)gene and coexpressing the light chain gene.,scFv-Fc,are scFvs dimerized by the Fc domain.,10,完整版课件,Antibody Formats Multivalent f,Immune libraries,:first,immunize an animal with an antigen and isolate the mRNA from B lymphocytes(for immunized animals)or peripheral blood B cells(for immunized donors).The mRNA is then reverse transcribed in