抗体噬菌体展示技术

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,*,*,Antibody Phage Display,Meiling Xiong,20180629,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,Introduction of Phage Display Technology,The Ff bacteriophage structure,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,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,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,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),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.,Antibody,Formats,Single domain antibody,V,HH: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,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,.,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 into cDNA,and the variable regions of expressed antibodies are amplified via PCR and cloned into a phage display vector.,Advantages:,Matured in vivo,Immune libraries can be generated from any animal and even humans:mouse,human,chicken,rabbit,camel,Any species that have been immunized,infected,or exposed to an antigen.,Useful in analyzing natural humoral responses,for example,in patients with autoimmune disease,viral infection,neop