Hepcidin 20基因克隆及在毕赤酵母中的表达

论文摘要

Hepcidin是一种低分子量的富含半胱氨酸的肝脏多肽激素,最初从人类血浆和尿液中分离得到。Hepcidin对体内铁稳态有重要调节作用,hepcidin的缺乏会导致多种疾病。另外,hepcidin在体外表现出良好的抗细菌及真菌活性。化学合成及从组织中提取hepcidin成本较高且存在许多困难。至今,对于在真核生物中表达像hepcidin这样的小分子多肽的报道还很少。酵母像细菌一样容易进行连续培养,与高等动物表达蛋白的方式相似,因此在本研究中我们希望在酵母中高效表达有活性的重组hepcidin,在本研究中,我们在毕赤酵母中表达了人类hepcidin多肽,并对表达条件进行了优化。根据hepcidin的氨基酸序列以及毕赤酵母中密码子的偏好性,合成了全长的hepcidin基因,克隆到酵母表达载体pPIC9K上并转化毕赤酵母GS115菌株。在涂有合适浓度的遗传霉素抗性平板上筛选转化予,并经PCR验证。在MM和MD平板上鉴定转化子表型,验证后发现均为His+Mut+。重组毕赤酵母菌株在BMGY培养基中培养（28℃,260 rpm）,每24h加入0.5%的甲醇诱导。转化空pPIC9K质粒的菌株作为对照。Tricine-SDS-PAGE显示2.2kD条带,表明异源蛋白在毕赤酵母中成功表达。多肽表达水平和活性分别通过SDS-PAGE和免疫反应检测。对重组酵母的培养及诱导条件进行了优化。结果表明胰蛋白胨,酵母提取物,稳定的PH对重组酵母生长和蛋白表达有显著影响。28℃0.5%的申醇诱导48h,BMMY培养基与其他培养基相比（BMM和MM）最适合hepcidin表达。收集诱导60 h的发酵液,重组多肽通过等电点沉淀和凝胶过滤色谱纯化。含有hepcidin的部分通过反向HPLC进一步纯化。另外,Hepcidin（11min）洗脱时间与化学合成的hepcidin相同。Hepcidin的分子量与计算的分子量（2191.77Da）一致。收集的每一部分通过ELISA验证是否含有重组Hepc,并验证得到的物质确实为hepcidin。另外,LC.ESI-MS图谱显示2192.0 Da的hepcidin分子离子峰。最后重组hepcidin对金黄色葡萄球菌和枯草芽孢杆菌具有明显的抗菌活性。

论文目录

• 摘要
• ABSTRACT
• LIST OF FIGURES
• LIST OF TABLES
• CHAPTER 1:INTRODUCTION AND LITERATURE REVIEW
• 1.1 FUNCTION OF IRON IN THE BODY
• 1.1.2 Harms caused by Iron Overload and Deficiency
• 1.1.3 Mechanism of Iron Metabolism in Body
• 1.1.4 Regulation of Iron Metabolism in Human Body
• 1.2 HEPCIDIN:A NEW MEDIATOR OF INNATE IMMUNITY
• 1.2.1 Structure of Hepcidin
• 1.2.2 Synthesis and Degradation of Hepcidin
• 1.2.3 Hormonal Activity of Hepcidin
• 1.2.4 Regulation of Hepcidin Synthesis
• 1.2.5 Regulation of Cellular Iron Efflux by Hepcidin
• 1.2.6.Biological Functions of Hepcidin
• 1.2.7 Applications in Health
• 1.3.Pichia pastoris;A MODEL HOST SYSTEM FOR RECOMBINANT EXPRESSION
• 1.3.1.Pichia pastoris as a Methylotrophic Expression System
• 1.3.2.Two Alcohol Oxidase Proteins
• 1.3.3.Pichia pastoris Expression Vectors
• 1.3.4.Pichia Host Strains
• 1.3.5.Intracellular and Secretory Expression of Recombinant Protein
• 1.3.6.Optimization of Secretory Protein Expression
• 1.4 PRESENT RESEARCH WORK
• CHAPTER 2:CLONING OF RECOMBINANT EXPRESSION VECTOR
• 2.1 DESIGN AND SYNTHESIS OF HEPCIDIN SEQUENCE
• 2.2 RECOMBINANT EXPRESSION VECTOR PPIC9K-H
• 2.2.1.Different Media and Methods of Preparation
• 2.2.1.1.Growth Media for E.coli
• 2.3 CONSTRUCTION OF RECOMBINANT EXPRESSION PLASMID pPIC9K-H
• 2.3.1.Strains and Plasmids
• 2.3.2.Chemicals and Reagents
• 2.3.3.Equipment
• 2.3.4.Experimental Methods
• 2.3.4.1.PCR Amplification of the Hepcidin Fragment
• 2.3.4.2.Extraction of Plasmid DNA
• 2.3.4.3.DNA Double Digestion
• 2.2.4.4.Gel Electrophoresis for Extractions of Required Fragments
• 2.2.4.5.Phenol-Chloroform Extraction
• 2.2.4.6.Ligation of pPIC9K Vector with the Hepcidin Gene Fragment
• 2.3.4.7.Transformation of pPIC9K-H into E.coli
• 2.4.RESULTS AND ANALYSIS
• 2.4.1.Results of the Hepcidin Sequence
• 2.4.2.The Aonfirmation of the Amplified Fragment
• 2.4.3.DNA Sequencing
• 2.5.SUMMARY OF THE CHAPTER
• CHAPTER 3:PICHIA PASTORIS GS115 AS A HOST FOR HEPCIDIN EXPRESSION
• 3.1.EXPERIMENTAL MATERIALS
• 3.1.1.Pichia Growth Media and Reagents
• 3.1.1.1.10X YNB（Yeast Nitrogen Base）
• 3.1.1.2.500X B（0.02%Biotin）
• 3.1.1.3.100X H（0.4%Histidine）
• 3.1.1.4.10X D（20%Dextrose）
• 3.1.1.5.10X M（5%Methanol）
• 3.1.1.6.10X GY（10%Glycerol）
• 3.1.1.7.100X AA（0.5%of each Amino Acid）
• 3.1.1.8.Potassium Phosphate buffer（1 M） pH 6.0:
• 3.1.1.9.YPD;Yeast Extract Peptone Dextrose Medium
• 3.1.1.10.Preperation of YPD-Geneticin plates
• 3.1.1.11.MGY and MGYH Minimal Glycerol Medium+Histidine
• 3.1.1.12.RD and RDH Liquid Media
• 3.1.1.13.RDB and RDHB Agar Plates
• 3.1.1.14.MD and MDH Minimal Dextrose Medium+Histidine（1 liter）
• 3.1.1.15.MM and MMH Minimal Methanol+Histidine
• 3.1.1.16.Buffered Minimal Glycerol and Buffered Minimal Methanol
• 3.1.1.17.BMGY and BMMY
• 3.1.1.18.DTT（1M）
• 3.2.EXPERIMENTAL METHODS
• 3.2.1.Preparation of Pichia pastoris GS115 Eelectrocompetent Cells
• 3.2.2.Electroporation of Pichia pastoris
• 3.2.2.1.Linearization of the Recombinant Plasmid DNA（pPIC9K-H）
• 3.2.2.2.Electroporation
• 3.2.3.Analysis of the Results
• 3.2.4.Screening and Selection of Recombinant Transformants（His+Mut+）
• 3.2.5.Analysis of Pichia Integrants
• 3.2.6.Isolation of the Genomic DNA from Transformed Pichia Strains
• 3.2.7.PCR analysis of Pichia integrants
• 3.2.7.1.PCR Analysis with Pichia Purified Genomie DNA
• 3.3.SUMMARY OF THE CHAPTER
• CHAPTER 4:EXPRESSION OF PEPTIDE IN THE RECOMBINANT PICHIA PASTORIS
• 4.1.EXPERIMENTAL MATERIALS
• 4.1.1.Strains
• 4.1.2.Media and Reagents
• 4.1.3.Equipment
• 4.2.EXPERIMENTAL METHODS
• 4.2.1 Induced Expression of Recombinat Yeast Plasmid
• 4.2.2.Determination of Protein Concentration
• 4.2.2.1.Coomassie Brilliant blue G250 dye:
• 4.2.3.Tricine-SDS-PAGE Electrophoresis
• 4.2.3.1.Reagents and Equipments
• 4.2.3.2 Liquid and gel electrophoresis buffer
• 4.2.3.3.Preparation of Electrophoresis Sample（Secreted Expression）:
• 4.2.4.Western Blot Analysis of the Protein
• 4.2.5.Optimization of Culture and Expression Conditions
• 4.2.5.1.Comparison of the Effect of Various Media and Temperature
• 4.2.5.2.Effect of the Final Concentration of Methanol during Induction on Protein Expression
• 4.3.RESULTS AND ANALYSIS
• 4.3.1.Trieine-SDS-PAGE and Determination of Protein Concentration
• 4.3.2.Expression of Peptide and Western Blot
• 4.3.3.Optimization of Expression Conditions
• 4.3.3.1.Effect of the Final Concentration of Methanol on Yeast Growth
• 4.4.SUMMARY OF THE CHAPTER
• CHAPTER 5:SEPARATION AND PURIFICATION OF RECOMBINANT HEPCIDIN PROTEIN
• 5.1.EXPERIMENTAL MATERIALS AND METHODS
• 5.1.1.Reagents
• 5.1.2.Instruments
• 5.2.PROTEIN PRECIPITATION
• 5.2.1.Isoelectric Point Precipitation
• 5.2.2.Protein Seperation by Gel Chromatography
• 5.2.2.1.Gel Processing
• 5.2.2.2.Chromatography Column Preparation
• 5.2.3.HPLC and MS analysis of the Hepcidin Protein
• 5.2.3.1.Reagents
• 5.2.3.3.Procedure
• 5.2.4.ELISA Competitive Binding Assay
• 5.2.5.Antibacterial activity of recombinant protein
• 5.3.RESULTS AND ANALYSIS
• 5.3.1.Precipitation and Seperation by Sephadex G-25 gel Chromatography
• 5.3.2.Reverse Phase Liquid Chromatography（RP-HPLC）
• 5.3.3.Mass Spectroscopic（MS） Analysis
• 5.3.4.Detection by ELISA and Characterization of Recombinant Hepcidin
• 5.3.5:Antimicrobial Activity of Recombinant Protein
• 5.4.SUMMARY OF THE CHAPTER
• CHAPTER 6:SUMMARY
• REFERENCES
• ANNEXURE
• CURRICULUM VITAE
• Dedicated To My Parents
• ACKNOWLEDGEMENTS