声明
List of Abbreviations
Chapter One: Introduction
1.1 Introduction to Diabetes and Insulin
1.2 Single-chain Antibody
1.2.1 Introduction to Single-chain Antibodies
1.2.2 Single-chain Antibodies Structure
1.2.3 Applications of Single-chain Antibodies
1.3Fusion Proteins
1.3.1Introduction to Fusion Protein
1.3.2 Progress in Fusion Protein Research
1.4Green Fluorescent Protein
1.4.1Introduction to Green Fluorescent Protein
1.4.2 Green Fluorescent Protein Structure
1.4.3 Applications of Green Fluorescent Protein
1.5Protein Expression System
1.5.1 Introduction to Protein Expression System
1.5.2 Yeastas an Expression System for Foreign Proteins
1.5.3 Promoter of Foreign Protein-glyceraldehyde-3-phosphate dehydrogenase
1.5.4 Exogenous Protein Expression Vector pGAPZαA
1.6Research Purposes, Significance and Prospects of the Thesis
1.7The Previous Related Work of the Construction of Anti-human Proinsulin Single-chain Antibody
1.8Technical routes of the research
Chapter Two: Expression of Human Proinsulin and Preparation
2.1 Materials
2.1.1 Strains and Plasmid Materials
2.1.2 Main Instruments
2.1.3 Main Reagents
2.1.4 Configuration of Medium, Antibiotics and Common Solutions
2.2 Methods
2.2.1 Preparation of E. coli BL21 (DE3) Competent Cells by CaCl2 Method
2.2.2 Transformation
2.2.3Expression of Human Proinsulin Protein
2.2.4 Extraction and Dissolving of Inclusion Bodies with Triton X-100
2.2.5 Dissolving the inclusion bodies
2.2.6 Extracting and Dissolving Inclusion Bodies with Inclusion Body Washing Solution
2.2.7 Coomassie Blue Method for Measuring Protein Content
2.3 Results
2.3.1 Transformant Cells
2.3.2Expression of Human Proinsulin
2.3.3 Extraction and Dissolution of Inclusion Bodies with Uera
2.3.4 Extracting and Dissolving Inclusion Bodieswith Triton X-100 Results
2.5 Discussion
2.4 Summary
Chapter Three:Expression of scFv-EGFP Fusion Protein and Preparation
3.1 Materials
3.1.1 Strainsand Plasmid Materials
3.1.2 Main Instruments
3.1.3 Main Reagents
3.1.4 Preparation of Main Reagents
3.2 Methods
3.2.1Screening the Positive Transformants and PCR Identification
3.2.2 Detection of Positive Transgenic P.pastoris by Super-resolution Fluorescence Microscopy
3.2.3 Expression of Recombinant scFv-EGFP Fusion Protein
3.2.4. Precipitation of the Protein by Ammonium Sulfate
3.2.5Purification of Expression Proteins by His-Taq Purification Method
3.2.6Purification of the Expression Protein by Cellulose DE-52 Ion-exchange Chromatography
3.2.7 Examination of Nucleotides Sequence of the pGAPZαA-scFv-EGFP Plasmid
3.2.8Precipitation of Medium Protein by Acetone
3.2.9Identification of Histidine-Taqs of the Fusion Proteins by Western Blot
3.3 Results
3.3.1 Screening of the Positive Transformants and PCR Results
3.3.2Expression of Recombinant scFv-EGFP Fusion Protein
3.3.3Purification of scFv-EGFP Fusion Proteinby His60-Ni Column Affinity Chromatography
3.3.4Purification of scFv-EGFP Fusion Proteinby Cellulose DE-52 Ion-exchange Chromatography
3.3.5Intracellular Localization of Expressed scFv-EGFPFusion Protein
3.3.6Identification of the scFv-EGFP Fusion Proteins by Western Blot
3.4 Discussion
3.5 Summary
Chapter Four: Sensitivity and Specificity of Human Proinsulin to scFv-EGFP Fusion Protein
4.1 Materials
4.1.3 Preparation of Main Reagents
4.2 Methods
4.2.1Sensitivity and Specificity Detection of scFv-EGFP Fusion Proteins to Human Proinsulin by Dot Blot
4.3 Results
4.3.1 Sensitivity and Specificity of scFv-EGFP Fusion Protein to Human Proinsulin
4.4 Discussion
4.5 Summary
参考文献
致谢
兰州理工大学;
