程序性細(xì)胞死亡配體-1(PD-L1),又稱(chēng)為CD274,B7-H1,是由9號(hào)染色體上的cd274基因編碼的,并由干擾素調(diào)節(jié)性因子(IRF1),信號(hào)傳導(dǎo)轉(zhuǎn)錄激活因子1(STAT1)反應(yīng)原件及其啟動(dòng)子所調(diào)控。抑制性受體PD-1(CD279)通過(guò)與其配體PD-L1和PD-L2(B7-DC,CD273)的結(jié)合,免疫應(yīng)答的減弱和外周耐受性的維持程度被極大地增強(qiáng)。PD-L1與其受體B7.1(CD80)和PD-1的相互作用可以抑制T細(xì)胞增殖,遷移和細(xì)胞毒性介質(zhì)的分泌�；罨腡細(xì)胞和B細(xì)胞能表達(dá)PD-1,而PD-L1也可以通過(guò)炎性細(xì)胞因子在巨噬細(xì)胞和樹(shù)突狀細(xì)胞上誘導(dǎo)。PD-1/PD-L1(B7-H1)表達(dá)在腫瘤細(xì)胞上,而在大部分的正常細(xì)胞上不表達(dá)。免疫因子干擾素γ(IFN-γ)可顯著誘導(dǎo)PD-L1的表達(dá),并可用于通過(guò)抗PD-L1治療的免疫療法�？筆D-L1療法在腫瘤微環(huán)境中是高度有效的。盡管PD-L2與PD-1的結(jié)合表現(xiàn)出與PD-L1類(lèi)似的結(jié)合親和力,但由于PD-L2在腫瘤細(xì)胞上的表達(dá)有限,并且大置存在于樹(shù)突細(xì)胞上限制了其對(duì)腫瘤細(xì)胞的作用。PD-L1/PD-1療法表明了主要途徑并為免疫缺陷提供了新的療...

【文章頁(yè)數(shù)】：144 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
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



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