| 摘要 | 第1-11页 |
| ABSTRACT | 第11-13页 |
| TABLE OF CONTENTS | 第13-15页 |
| LIST OF ABBREVIATIONS | 第15-16页 |
| CHAPTER 1.Introduction | 第16-25页 |
| ·Background | 第16-24页 |
| ·Aim and Purpose | 第24-25页 |
| CHAPTER 2.Materials and Methods | 第25-41页 |
| ·Materials | 第25-29页 |
| ·Nucleic acids,vector,bacterial strains,enzymes and enzymes buffers | 第25页 |
| ·Media,buffers,solutions,and reagents | 第25-29页 |
| ·Methods | 第29-41页 |
| ·WT rat liver mitochondrial VLCAD protein subcloning | 第29-35页 |
| ·PCR amplication | 第29-30页 |
| ·Agarose gel electrophoresis | 第30-31页 |
| ·DNA extraction from agarose gel | 第31-32页 |
| ·PCR products purification | 第32页 |
| ·Double restriction digestion of plasmid and PCR products | 第32-33页 |
| ·Ligation into the PLM1 plasmid expression vector | 第33页 |
| ·Transformation of recombinant pLM1::VLCAD plasmid int #0 TOP10 competent cells for screening purposes | 第33-34页 |
| ·Single and double restriction digestion of positive recombinant pLM1::VLCAD colony | 第34-35页 |
| ·Transformation of recombinant pLM1::VLCAD plasmid int #0 E.coli strain BL21(DE3)for expression purposes | 第35页 |
| ·Construction of mutant-types VLCAD expression plasmids | 第35-37页 |
| ·Expression of the wild- and mutant-types VLCAD proteins | 第37-38页 |
| ·Purification of the wild- and mutant-typesVLCAD proteins | 第38页 |
| ·SDS-PAGE gel electrophoresis | 第38-39页 |
| ·Determine protein concentration | 第39-40页 |
| ·Enzyme activity assays | 第40-41页 |
| CHAPTER 3 RESULT | 第41-56页 |
| ·Recombinant WT pLM1::VLCAD plasmid subcloning | 第41-43页 |
| ·Expression and purification of WT protein | 第43-44页 |
| ·Multiple sequence alignments of various sources VLCAD and ACADs | 第44-48页 |
| ·Three dimensional structure of VLCAD protein | 第48-50页 |
| ·Construction of G441 mutant-types protein | 第50-51页 |
| ·Spectral analyses of wild- and mutant-types protein | 第51-52页 |
| ·Quantization of wild- and mutant-types protein expression | 第52-53页 |
| ·Enzymatic activity assay of wild- and mutant-types protein | 第53-54页 |
| ·Kinetic studies of wild- and mutant-types protein | 第54-56页 |
| CHAPTER 4 DISCUSSION | 第56-67页 |
| ·Sequence-based analysis | 第58-59页 |
| ·Structural-based analysis | 第59-63页 |
| ·Functional-based analysis | 第63-67页 |
| CHAPTER 5.Conclusion | 第67-68页 |
| References | 第68-75页 |
| Review | 第75-95页 |
| REFERENCES | 第86-95页 |
| Acknowledgments | 第95页 |
