| 摘要 | 第9-14页 |
| Abstract | 第14-19页 |
| List of Abbreviations | 第20-23页 |
| Chapter 1 Introduction | 第23-51页 |
| 1.1 Progress of molecular pharming | 第23-24页 |
| 1.2 Progress of protein glycosylation | 第24-29页 |
| 1.2.1 The function of N-glycosylation in the quality control and folding of protein | 第27-28页 |
| 1.2.2 The role of N-linked glycosylation in therapeutic protein | 第28-29页 |
| 1.3 Rice endosperm as bioreactor to produce recombinant protein | 第29-31页 |
| 1.4 Progress of study on toxicity and immunogenicity of host cell protein | 第31-33页 |
| 1.5 Development of gene knock out technologies | 第33-51页 |
| 1.5.1 Targeted genome engineering | 第33-34页 |
| 1.5.2 ZFNs | 第34-35页 |
| 1.5.3 TALEN Technology | 第35-42页 |
| 1.5.3.1 TALENs application in Knock-out and Knock-in | 第37-38页 |
| 1.5.3.2 TALENs Applications in different organisms, specifically plants | 第38-41页 |
| 1.5.3.3 Progress of TALENs applications in Rice | 第41-42页 |
| 1.5.4 CRISPR/Cas9 | 第42-51页 |
| 1.5.4.1 Application of CRISPR/Cas9 in plants | 第45-49页 |
| 1.5.4.2 What is the trump card of CRISPR over TALENs and ZFNs? | 第49页 |
| 1.5.4.3 Potential off-target effects of CRISPR/Cas system | 第49-51页 |
| Chapter 2 Evaluation of the immunogenicity of OsrHSA and its residual hostcell proteins | 第51-81页 |
| 2.1 Introduction | 第51-53页 |
| 2.1.1 Importance of HCP research on novel biopharmaceutical development | 第52-53页 |
| 2.2 Research purpose | 第53页 |
| 2.3 Materials and Methods | 第53-58页 |
| 2.3.1 Preparation of HCPs from rice seeds | 第53-54页 |
| 2.3.2 Animal maintenance conditions | 第54页 |
| 2.3.3 Dose design and animal grouping | 第54页 |
| 2.3.4 Hematological analysis | 第54-55页 |
| 2.3.5 Biochemical analysis | 第55页 |
| 2.3.6 T-lymphocyte subset typing | 第55页 |
| 2.3.7 Cytokine assays | 第55-56页 |
| 2.3.8 C-reactive protein (CRP) assay | 第56页 |
| 2.3.9 Plasma circulating immune complex assay | 第56-57页 |
| 2.3.10 Complement measurement | 第57-58页 |
| 2.3.11 ELISA for residual HCP-specific antibody | 第58页 |
| 2.3.12 Statistical analysis | 第58页 |
| 2.4 Results | 第58-78页 |
| 2.4.1 The clinical observations | 第58-59页 |
| 2.4.2 Pathological changes in hematology | 第59-68页 |
| 2.4.3 The OsrHSA did not show immunotoxicity | 第68-71页 |
| 2.4.4 The HCPs showed low immunogenicity | 第71-72页 |
| 2.4.5 The HCPs and OsrHSA did not promote immune responses | 第72-75页 |
| 2.4.6 The pathological changes of OsrHSA could be recovered | 第75-78页 |
| 2.5 Discussion | 第78-81页 |
| Chapter 3 The study on humanization of glycosylation of protein in riceendosperm cell using TALEN technology | 第81-139页 |
| 3.1 Introduction | 第81-89页 |
| 3.1.1 Humanization of N-glycosylation in plant cell | 第83-86页 |
| 3.1.2 The progress of glyco-engineering in higher plants | 第86-89页 |
| 3.2 Research strategy and purpose | 第89页 |
| 3.3 Materials and Methods | 第89-107页 |
| 3.3.1 Plant materials | 第89页 |
| 3.3.2 Assembling TALENs constructs using TALE-NT online software | 第89页 |
| 3.3.3 Preparation of binary vectors for co-transformation with TALENs plasmids | 第89-94页 |
| 3.3.4 Construction of Agrobacterium-based vectors | 第94-97页 |
| 3.3.5 Agrobacterium-mediated Rice transformation | 第97-100页 |
| 3.3.6 PCR reaction | 第100-101页 |
| 3.3.7 Isolation of Protein from rice seed and SDS-PAGE | 第101页 |
| 3.3.7.1 Protein preparation | 第101页 |
| 3.3.7.2 SDS-PAGE | 第101页 |
| 3.3.8 Western Blotting | 第101-102页 |
| 3.3.9 Primers used in this study | 第102-103页 |
| 3.3.10 Isolation and preparation of N-glycans | 第103-105页 |
| 3.3.10.1 Preparation of storage protein from transgenic grain for glycan analysis | 第103-104页 |
| 3.3.10.2 Trypsin digestion and protein purification | 第104页 |
| 3.3.10.3 Preparation of N-glycans | 第104-105页 |
| 3.3.11 N-glycan analysis by MALDI-TOF | 第105-107页 |
| 3.4 Results | 第107-134页 |
| 3.4.1 Designed TALEN pair combinations (2*3)for OsXylT and OsFUT | 第107-110页 |
| 3.4.2 Generation of TALEN cassettes for OsFUT and OsXylT genes | 第110-113页 |
| 3.4.3 Construction of TALEN expression cassettes | 第113-116页 |
| 3.4.4 Generation of transgenic rice expressed TALEN and target genes | 第116-122页 |
| 3.4.4.1 Confirmation of the target genes integration on the DSB created by TALEN | 第120-122页 |
| 3.4.5 Identification of transgenic lines to overexpress two genes | 第122-125页 |
| 3.4.6 N-glycosylation patterns of the transgenic lines | 第125-134页 |
| 3.4.6.1 Plant-specific N-glycans reduced in the overexpression of GalT transgenic lines | 第125-130页 |
| 3.4.6.2 The overexpression of GaIT in rice exhibited galactosylated and sialylated glycans | 第130-134页 |
| 3.5 Discussion | 第134-139页 |
| References | 第139-160页 |
| List of publications | 第160-161页 |
| Supporting Information | 第161-163页 |
| Acknowledgements | 第163页 |
