抗体噬菌体展示技术ppt课件
合集下载
相关主题
- 1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
- 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
- 3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。
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
Antibody Formats
• Fab
✓The light chain (VL-CL) and the Fddomain (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.
▪ Amber codon TAG: supE strains (glutamic acid
codon), non-suppressor strains (stop codon)
▪ Protease cleavage site
▪ Promoter
▪ Signal peptides: phage protein translocation, crucial
• Producing the combinatorial library (ideally with 108 to 109 members) of
Antibody Formats
• The most commonly used format: single-chain variable fragment (scFv)
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)
for display level
▪ Selective marker: for selection of infected host cells
Introduction of Phage Display Technology
▪ Nonlytic filamentous phage is the most
Introduction of Phage Display Technology
TFra Baidu biblioteke Ff bacteriophage structure
Introduction of Phage Display Technology
The scheme of phagemid vector
▪ IG region: intergenic region, usually contains the
polystryrene surfaces, or on columns, or is used in solution as biotinylated antigen and
Advantages of Phage Display for Recombinant Antibody Selection
✓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)
• More efficiently than through conventional hybridoma system.
• Cheaper to produce recombinant antibodies using bacteria, rather than mammalian cell line.
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,
• Phage Ab Selection Methods & Strategies
• Phage Ab Screening Applications
• In vitro Affinity Maturation
• Expression & Purification of Phage Ab Fragments
• 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.
Antibody Phage Display
Meiling Xiong 20180629
Contents
• Introduction of Ab phage Display Technology
• Ab Formats for Phage Display
• Ab Libraries Construction
▪ Molecular tag: to facilitate library screening and for
protein analysis
▪ Restriction enzyme recognition sites: useful for
Antibody Formats
• Fab
✓The light chain (VL-CL) and the Fddomain (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.
▪ Amber codon TAG: supE strains (glutamic acid
codon), non-suppressor strains (stop codon)
▪ Protease cleavage site
▪ Promoter
▪ Signal peptides: phage protein translocation, crucial
• Producing the combinatorial library (ideally with 108 to 109 members) of
Antibody Formats
• The most commonly used format: single-chain variable fragment (scFv)
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)
for display level
▪ Selective marker: for selection of infected host cells
Introduction of Phage Display Technology
▪ Nonlytic filamentous phage is the most
Introduction of Phage Display Technology
TFra Baidu biblioteke Ff bacteriophage structure
Introduction of Phage Display Technology
The scheme of phagemid vector
▪ IG region: intergenic region, usually contains the
polystryrene surfaces, or on columns, or is used in solution as biotinylated antigen and
Advantages of Phage Display for Recombinant Antibody Selection
✓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)
• More efficiently than through conventional hybridoma system.
• Cheaper to produce recombinant antibodies using bacteria, rather than mammalian cell line.
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,
• Phage Ab Selection Methods & Strategies
• Phage Ab Screening Applications
• In vitro Affinity Maturation
• Expression & Purification of Phage Ab Fragments
• 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.
Antibody Phage Display
Meiling Xiong 20180629
Contents
• Introduction of Ab phage Display Technology
• Ab Formats for Phage Display
• Ab Libraries Construction