论文目录 | |
| 摘要 | 第1-7页 |
| ABSTRACT | 第7-15页 |
| LIST OF FIGURES | 第15-17页 |
| LIST OF TABLES | 第17-18页 |
| CHAPTER 1:INTRODUCTION AND LITERATURE REVIEW | 第18-46页 |
| · FUNCTION OF IRON IN THE BODY | 第18-26页 |
| · Harms caused by Iron Overload and Deficiency | 第19-20页 |
| · Mechanism of Iron Metabolism in Body | 第20-21页 |
| · Regulation of Iron Metabolism in Human Body | 第21-26页 |
| · HEPCIDIN:A NEW MEDIATOR OF INNATE IMMUNITY | 第26-36页 |
| · Structure of Hepcidin | 第28-31页 |
| · Synthesis and Degradation of Hepcidin | 第31页 |
| · Hormonal Activity of Hepcidin | 第31-32页 |
| · Regulation of Hepcidin Synthesis | 第32页 |
| · Regulation of Cellular Iron Efflux by Hepcidin | 第32-34页 |
| ·Biological Functions of Hepcidin | 第34页 |
| · Applications in Health | 第34-36页 |
| ·Pichia pastoris;A MODEL HOST SYSTEM FOR RECOMBINANT EXPRESSION | 第36-44页 |
| ·Pichia pastoris as a Methylotrophic Expression System | 第37-38页 |
| ·Two Alcohol Oxidase Proteins | 第38-39页 |
| ·Pichia pastoris Expression Vectors | 第39-40页 |
| ·Pichia Host Strains | 第40-43页 |
| ·Intracellular and Secretory Expression of Recombinant Protein | 第43页 |
| ·Optimization of Secretory Protein Expression | 第43-44页 |
| · PRESENT RESEARCH WORK | 第44-46页 |
| CHAPTER 2:CLONING OF RECOMBINANT EXPRESSION VECTOR | 第46-64页 |
| · DESIGN AND SYNTHESIS OF HEPCIDIN SEQUENCE | 第46-47页 |
| · RECOMBINANT EXPRESSION VECTOR PPIC9K-H | 第47-49页 |
| ·Different Media and Methods of Preparation | 第47-49页 |
| ·Growth Media for E.coli | 第47-49页 |
| · CONSTRUCTION OF RECOMBINANT EXPRESSION PLASMID pPIC9K-H | 第49-60页 |
| ·Strains and Plasmids | 第49-50页 |
| ·Chemicals and Reagents | 第50-51页 |
| ·Equipment | 第51页 |
| ·Experimental Methods | 第51-60页 |
| ·PCR Amplification of the Hepcidin Fragment | 第51-53页 |
| ·Extraction of Plasmid DNA | 第53页 |
| ·DNA Double Digestion | 第53-54页 |
| ·Gel Electrophoresis for Extractions of Required Fragments | 第54-55页 |
| ·Phenol-Chloroform Extraction | 第55-56页 |
| ·Ligation of pPIC9K Vector with the Hepcidin Gene Fragment | 第56-57页 |
| ·Transformation of pPIC9K-H into E.coli | 第57-60页 |
| ·RESULTS AND ANALYSIS | 第60-63页 |
| ·Results of the Hepcidin Sequence | 第60页 |
| ·The Aonfirmation of the Amplified Fragment | 第60页 |
| ·DNA Sequencing | 第60-63页 |
| ·SUMMARY OF THE CHAPTER | 第63-64页 |
| CHAPTER 3:PICHIA PASTORIS GS115 AS A HOST FOR HEPCIDIN EXPRESSION | 第64-84页 |
| ·EXPERIMENTAL MATERIALS | 第65-71页 |
| ·Pichia Growth Media and Reagents | 第65-71页 |
| ·X YNB(Yeast Nitrogen Base) | 第65-66页 |
| ·X B(·%Biotin) | 第66页 |
| ·X H(·%Histidine) | 第66页 |
| ·X D(20%Dextrose) | 第66页 |
| ·X M(5%Methanol) | 第66页 |
| ·X GY(10%Glycerol) | 第66页 |
| ·X AA(·%of each Amino Acid) | 第66页 |
| ·Potassium Phosphate buffer(1 M) pH 6.0: | 第66-67页 |
| ·YPD;Yeast Extract Peptone Dextrose Medium | 第67页 |
| ·Preperation of YPD-Geneticin plates | 第67-68页 |
| ·MGY and MGYH Minimal Glycerol Medium+Histidine | 第68页 |
| ·RD and RDH Liquid Media | 第68-69页 |
| ·RDB and RDHB Agar Plates | 第69页 |
| ·MD and MDH Minimal Dextrose Medium+Histidine(1 liter) | 第69页 |
| ·MM and MMH Minimal Methanol+Histidine | 第69-70页 |
| ·Buffered Minimal Glycerol and Buffered Minimal Methanol | 第70页 |
| ·BMGY and BMMY | 第70-71页 |
| ·DTT(1M) | 第71页 |
| ·EXPERIMENTAL METHODS | 第71-81页 |
| ·Preparation of Pichia pastoris GS115 Eelectrocompetent Cells | 第71-72页 |
| ·Electroporation of Pichia pastoris | 第72-74页 |
| ·Linearization of the Recombinant Plasmid DNA(pPIC9K-H) | 第72-73页 |
| ·Electroporation | 第73-74页 |
| ·Analysis of the Results | 第74页 |
| ·Screening and Selection of Recombinant Transformants(His+Mut+) | 第74-75页 |
| ·Analysis of Pichia Integrants | 第75页 |
| ·Isolation of the Genomic DNA from Transformed Pichia Strains | 第75-76页 |
| ·PCR analysis of Pichia integrants | 第76-81页 |
| ·PCR Analysis with Pichia Purified Genomie DNA | 第79-81页 |
| ·SUMMARY OF THE CHAPTER | 第81-84页 |
| CHAPTER 4:EXPRESSION OF PEPTIDE IN THE RECOMBINANT PICHIA PASTORIS | 第84-98页 |
| ·EXPERIMENTAL MATERIALS | 第84-85页 |
| ·Strains | 第84-85页 |
| ·Media and Reagents | 第85页 |
| ·Equipment | 第85页 |
| ·EXPERIMENTAL METHODS | 第85-93页 |
| · Induced Expression of Recombinat Yeast Plasmid | 第85-86页 |
| ·Determination of Protein Concentration | 第86-87页 |
| ·Coomassie Brilliant blue G250 dye: | 第87页 |
| ·Tricine-SDS-PAGE Electrophoresis | 第87-92页 |
| ·Reagents and Equipments | 第88页 |
| · Liquid and gel electrophoresis buffer | 第88-90页 |
| ·Preparation of Electrophoresis Sample(Secreted Expression): | 第90-92页 |
| ·Western Blot Analysis of the Protein | 第92页 |
| ·Optimization of Culture and Expression Conditions | 第92-93页 |
| ·Comparison of the Effect of Various Media and Temperature | 第92页 |
| ·Effect of the Final Concentration of Methanol during Induction on Protein Expression | 第92-93页 |
| ·RESULTS AND ANALYSIS | 第93-97页 |
| ·Trieine-SDS-PAGE and Determination of Protein Concentration | 第93-94页 |
| ·Expression of Peptide and Western Blot | 第94页 |
| ·Optimization of Expression Conditions | 第94-97页 |
| ·Effect of the Final Concentration of Methanol on Yeast Growth | 第96-97页 |
| ·SUMMARY OF THE CHAPTER | 第97-98页 |
| CHAPTER 5:SEPARATION AND PURIFICATION OF RECOMBINANT HEPCIDIN PROTEIN | 第98-114页 |
| ·EXPERIMENTAL MATERIALS AND METHODS | 第100-101页 |
| ·Reagents | 第100-101页 |
| ·Instruments | 第101页 |
| ·PROTEIN PRECIPITATION | 第101-108页 |
| ·Isoelectric Point Precipitation | 第102页 |
| ·Protein Seperation by Gel Chromatography | 第102-104页 |
| ·Gel Processing | 第103页 |
| ·Chromatography Column Preparation | 第103-104页 |
| ·HPLC and MS analysis of the Hepcidin Protein | 第104-106页 |
| ·Reagents | 第105-106页 |
| ·Procedure | 第106页 |
| ·ELISA Competitive Binding Assay | 第106-108页 |
| ·Antibacterial activity of recombinant protein | 第108页 |
| ·RESULTS AND ANALYSIS | 第108-112页 |
| ·Precipitation and Seperation by Sephadex G-25 gel Chromatography | 第108-109页 |
| ·Reverse Phase Liquid Chromatography(RP-HPLC) | 第109页 |
| ·Mass Spectroscopic(MS) Analysis | 第109-111页 |
| ·Detection by ELISA and Characterization of Recombinant Hepcidin | 第111页 |
| ·:Antimicrobial Activity of Recombinant Protein | 第111-112页 |
| ·SUMMARY OF THE CHAPTER | 第112-114页 |
| CHAPTER 6:SUMMARY | 第114-118页 |
| REFERENCES | 第118-130页 |
| ANNEXURE | 第130-132页 |
| CURRICULUM VITAE | 第132-136页 |
| Dedicated To My Parents | 第136-138页 |
| ACKNOWLEDGEMENTS | 第138-139页 |
