抗体噬菌体展示技术(课堂PPT)
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)。
▪ Producing the combinatorial library (ideally with 108 to 109
members) of functional antibodies to generate a larger repertoire of antibodies than those available through conventional hybridoma technology.
▪ 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.
1
Antibody Phage Display
Meiling Xiong 20180629
Contents
2
▪ 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
for display level
▪ Selective marker: for selection of infected host cells
5
Introduction of Phage Display Technology
▪ Nonlytic filamentous phage is the most
▪ 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.
6
▪ 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.
3
Introduction of Phage Display Technology
The Ff bacteriophage structure
Introduction of Phage Display Technology
The scheme of phagemid vector
4
▪ 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
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.
polystryrene surfaces, or on columns, or is used in solution as biotinylated antigen and
Advantages of Phage Display for
Recombinant Antibody Selection
▪ Amber codon TAG: supE strains (glutamic acid
codon), non-suppressor strains (stop codon)
▪ Protease cleavage site
▪ Promoter
▪ Signal peptides: phage protein translocation, crucial
▪ pIII protein is essential for infection of
bacteria
▪ Helper phage: wild-type pIII helper phage
and special helper phage
▪ Antigen immobilized on magnetic beads,
recombination and gene manipulation; multiple cloning sites (MCS)
▪ Coat protein: PIII (larger protein, less than 5 copies,)
PVIII (mo源自文库e than 5 copies, decreased length)
members) of functional antibodies to generate a larger repertoire of antibodies than those available through conventional hybridoma technology.
▪ 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.
1
Antibody Phage Display
Meiling Xiong 20180629
Contents
2
▪ 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
for display level
▪ Selective marker: for selection of infected host cells
5
Introduction of Phage Display Technology
▪ Nonlytic filamentous phage is the most
▪ 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.
6
▪ 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.
3
Introduction of Phage Display Technology
The Ff bacteriophage structure
Introduction of Phage Display Technology
The scheme of phagemid vector
4
▪ 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
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.
polystryrene surfaces, or on columns, or is used in solution as biotinylated antigen and
Advantages of Phage Display for
Recombinant Antibody Selection
▪ Amber codon TAG: supE strains (glutamic acid
codon), non-suppressor strains (stop codon)
▪ Protease cleavage site
▪ Promoter
▪ Signal peptides: phage protein translocation, crucial
▪ pIII protein is essential for infection of
bacteria
▪ Helper phage: wild-type pIII helper phage
and special helper phage
▪ Antigen immobilized on magnetic beads,
recombination and gene manipulation; multiple cloning sites (MCS)
▪ Coat protein: PIII (larger protein, less than 5 copies,)
PVIII (mo源自文库e than 5 copies, decreased length)