PD-L1单链抗体的筛
发布时间：2023-03-09 22:01
程序性细胞死亡配体-1(PD-L1),又称为CD274,B7-H1,是由9号染色体上的cd274基因编码的,并由干扰素调节性因子(IRF1),信号传导转录激活因子1(STAT1)反应原件及其启动子所调控。抑制性受体PD-1(CD279)通过与其配体PD-L1和PD-L2(B7-DC,CD273)的结合,免疫应答的减弱和外周耐受性的维持程度被极大地增强。PD-L1与其受体B7.1(CD80)和PD-1的相互作用可以抑制T细胞增殖,迁移和细胞毒性介质的分泌。活化的T细胞和B细胞能表达PD-1,而PD-L1也可以通过炎性细胞因子在巨噬细胞和树突状细胞上诱导。PD-1/PD-L1(B7-H1)表达在肿瘤细胞上,而在大部分的正常细胞上不表达。免疫因子干扰素γ(IFN-γ)可显著诱导PD-L1的表达,并可用于通过抗PD-L1治疗的免疫疗法。抗PD-L1疗法在肿瘤微环境中是高度有效的。尽管PD-L2与PD-1的结合表现出与PD-L1类似的结合亲和力,但由于PD-L2在肿瘤细胞上的表达有限,并且大置存在于树突细胞上限制了其对肿瘤细胞的作用。PD-L1/PD-1疗法表明了主要途径并为免疫缺陷提供了新的疗...
【文章页数】：144 页
【学位级别】：博士
【文章目录】：
ACKNOWLEDGEMTS
INTRODUCTION
ABSTRACT
中文摘要
ABBREVIATIONS
PARTⅠFunctional expression and purification system of PD-L1 extracellular domain byusing Escherichia coli host cell machinery
1.1. Background
1.2. Materials and Methods
1.2.1 Bacterial strains and vectors
1.2.2 Plasmid extraction and gel electrophoresis
1.2.3 Primer design and amplification of PDL1-ECD
1.2.4 Cloning and expression of PDL1-ECD
1.2.5 Purification of recombinant PDL1-ECD
1.2.6 Refolding of recombinant PDL1-ECD
1.2.7 Western blot analysis
1.2.8 Statistical analysis
1.3. Results
1.3.1. PCR amplification and cloning of PDL1-ECD
1.3.2. Expression of recombinant proteins
1.3.3. Purification, dialysis and antigenic analysis of recombinant proteins
1.4. Discussion
1.5. Conclusions
PARTⅡ Systematic development and bioactive confirmation of single chain fragment(scFv) against PD-L1
2.1 Background
2.2 Methodology
2.2.1 Cell lines and reagents
2.2.2 Helper phage enrichment
2.2.3 Library amplification
2.2.4 Biopanning
2.2.5 Screening of positive clones
2.2.6 Phage ELISA
2.2.7 Positive phage enrichment
2.2.8 Construction of recombinant vector
2.2.9 Expression and purification of recombinant scFv antibodies
2.2.10 Purification and refolding of recombinant scFv antibodies
2.2.11 Western blot analysis
2.2.12 ELISA determination of expressed proteins
2.2.13 Immunofluorescence assay
2.2.14 Statistical analysis
2.3 Results
2.3.1 Phage display biopanning
2.3.2 Affinity determination with ELISA and sequence analysis
2.3.3 Selection of positive clone enrichment
2.3.4 Recombinant formation and expression of scFv antibodies
2.3.5 SDS-PAGE and western blot analysis
2.3.6 Binding affinity determination of expressed proteins
2.3.7 Structural prediction of scFv fragments
2.4 Discussion
2.5 Conclusions
PARTⅢ Development of antibody drug conjugates by using single chain variable fragmentsagainst PD-L1 and intracellular tracking
3.1 Introduction
3.2 Methodology
3.2.1 Reagents and cell lines
3.2.2 Generation of drug conjugate
3.2.3 Spectrophotometry analysis
3.2.4 SDS-PAGE
3.2.5 In vitro activity determination
3.2.6 Immunofluorescence
3.2.7 Intracellular trafficking
3.2.8 Statistical analysis
3.3 Results
3.3.1 Engineering scFv-PD-L1 drug conjugate
3.3.2 Spectrophotometry analysis
3.3.3 SDS-PAGE analysis
3.3.4 Western blotting
3.3.5 In vitro studies
3.3.6 Surface binding specificity and trafficking analysis
3.4 Discussion
3.5 Conclusions
PARTⅣ Generation of full length antibody and bioactive confirmation against PD-L1cancer cells
4.1 Introduction
4.2 Methodology
4.2.1 Reagents, cell lines and plasmids
4.2.2 Construction of pMH3-VH/VL recombinant vector
4.2.3 PCR ligation to develop SP-VH/VL fragment
4.2.4 Double digestion and recombinant pMH3-kH/kL vector construction
4.2.5 Transformation and sequence analysis
4.2.6 Transfection of CHO cells
4.2.7 Purification of full length antibody
4.2.8 ELISA determination
4.2.9 Immunofluorescence analysis
4.2.10 Flow cytometry analysis
4.2.11 Statistical analysis
4.3 Results
4.3.1 Construction of optimized recombinant plasmid
4.3.2 CHO cells transfection optimization
4.3.3 Full length antibody purification
4.3.4 Bioactivity determination
4.4 Discussion
4.5 Conclusions
PARTⅤ Major Findings and Future Perspectives
5.1 Introduction
5.2 Future Perspectives
REFERENCES
LISTS OF PUBLICATIONS
本文编号：3758369
【文章页数】：144 页
【学位级别】：博士
【文章目录】：
ACKNOWLEDGEMTS
INTRODUCTION
ABSTRACT
中文摘要
ABBREVIATIONS
PARTⅠFunctional expression and purification system of PD-L1 extracellular domain byusing Escherichia coli host cell machinery
1.1. Background
1.2. Materials and Methods
1.2.1 Bacterial strains and vectors
1.2.2 Plasmid extraction and gel electrophoresis
1.2.3 Primer design and amplification of PDL1-ECD
1.2.4 Cloning and expression of PDL1-ECD
1.2.5 Purification of recombinant PDL1-ECD
1.2.6 Refolding of recombinant PDL1-ECD
1.2.7 Western blot analysis
1.2.8 Statistical analysis
1.3. Results
1.3.1. PCR amplification and cloning of PDL1-ECD
1.3.2. Expression of recombinant proteins
1.3.3. Purification, dialysis and antigenic analysis of recombinant proteins
1.4. Discussion
1.5. Conclusions
PARTⅡ Systematic development and bioactive confirmation of single chain fragment(scFv) against PD-L1
2.1 Background
2.2 Methodology
2.2.1 Cell lines and reagents
2.2.2 Helper phage enrichment
2.2.3 Library amplification
2.2.4 Biopanning
2.2.5 Screening of positive clones
2.2.6 Phage ELISA
2.2.7 Positive phage enrichment
2.2.8 Construction of recombinant vector
2.2.9 Expression and purification of recombinant scFv antibodies
2.2.10 Purification and refolding of recombinant scFv antibodies
2.2.11 Western blot analysis
2.2.12 ELISA determination of expressed proteins
2.2.13 Immunofluorescence assay
2.2.14 Statistical analysis
2.3 Results
2.3.1 Phage display biopanning
2.3.2 Affinity determination with ELISA and sequence analysis
2.3.3 Selection of positive clone enrichment
2.3.4 Recombinant formation and expression of scFv antibodies
2.3.5 SDS-PAGE and western blot analysis
2.3.6 Binding affinity determination of expressed proteins
2.3.7 Structural prediction of scFv fragments
2.4 Discussion
2.5 Conclusions
PARTⅢ Development of antibody drug conjugates by using single chain variable fragmentsagainst PD-L1 and intracellular tracking
3.1 Introduction
3.2 Methodology
3.2.1 Reagents and cell lines
3.2.2 Generation of drug conjugate
3.2.3 Spectrophotometry analysis
3.2.4 SDS-PAGE
3.2.5 In vitro activity determination
3.2.6 Immunofluorescence
3.2.7 Intracellular trafficking
3.2.8 Statistical analysis
3.3 Results
3.3.1 Engineering scFv-PD-L1 drug conjugate
3.3.2 Spectrophotometry analysis
3.3.3 SDS-PAGE analysis
3.3.4 Western blotting
3.3.5 In vitro studies
3.3.6 Surface binding specificity and trafficking analysis
3.4 Discussion
3.5 Conclusions
PARTⅣ Generation of full length antibody and bioactive confirmation against PD-L1cancer cells
4.1 Introduction
4.2 Methodology
4.2.1 Reagents, cell lines and plasmids
4.2.2 Construction of pMH3-VH/VL recombinant vector
4.2.3 PCR ligation to develop SP-VH/VL fragment
4.2.4 Double digestion and recombinant pMH3-kH/kL vector construction
4.2.5 Transformation and sequence analysis
4.2.6 Transfection of CHO cells
4.2.7 Purification of full length antibody
4.2.8 ELISA determination
4.2.9 Immunofluorescence analysis
4.2.10 Flow cytometry analysis
4.2.11 Statistical analysis
4.3 Results
4.3.1 Construction of optimized recombinant plasmid
4.3.2 CHO cells transfection optimization
4.3.3 Full length antibody purification
4.3.4 Bioactivity determination
4.4 Discussion
4.5 Conclusions
PARTⅤ Major Findings and Future Perspectives
5.1 Introduction
5.2 Future Perspectives
REFERENCES
LISTS OF PUBLICATIONS
本文编号：3758369
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