| ABSTRACT | 第1-5页 |
| 摘要 | 第5-6页 |
| CONTENTS | 第6-8页 |
| CHAPTER Ⅰ:INTRODUCTION | 第8-20页 |
| 1 Vascular Endothelial Growth Factor(VEGF) | 第8-14页 |
| ·VEGF gene family and isoforms | 第8-9页 |
| ·VEGF receptors | 第9-10页 |
| ·Biological functions of VEGF | 第10-11页 |
| ·Regulation of VEGF gene expression | 第11-12页 |
| ·Clinical significance of serum VEGF | 第12-13页 |
| ·Targeting VEGF as cancer therapy | 第13-14页 |
| 2 Recombinant protein expression | 第14-16页 |
| ·Escherichia coli a typical prokaryotic expression system | 第15页 |
| ·Characteristic of protein aggregates in inclusion bodies | 第15页 |
| ·Recovery of bioactive protein from inclusion bodies | 第15-16页 |
| ·Techniques to limit the in vivo aggregation | 第16页 |
| 3 Polyelonal Antibodies Production | 第16-18页 |
| ·Polyclonal Antibodies | 第16页 |
| ·The antigen | 第16-17页 |
| ·The choice of species | 第17页 |
| ·Adjuvants | 第17-18页 |
| ·Polyclonal antibody advantages in fabrication and usage | 第18页 |
| 4 The purpose and significance of this study | 第18-20页 |
| CHAPTER Ⅱ:MATERIALS AND METHODS | 第20-30页 |
| 1 Materials | 第20-21页 |
| 2 Methods | 第21-30页 |
| ·Construction of the recombinant plasmid | 第21-23页 |
| ·Expression and analysis of recombinant VEGF165 protein | 第23-24页 |
| ·Large-scale expression and protein solubility screening | 第24页 |
| ·Purification of His-tagged VEGF Protein by Ni-NTA affinity Chromatography | 第24-25页 |
| ·Refolding and dialysis | 第25页 |
| ·SDS-PAGE and Western blot analysis | 第25-26页 |
| ·Animal Immunization and Production of polyclonal anti-hVEGF Antibody | 第26页 |
| ·Enzyme-Linked Immunosorbent Assay(ELISA) | 第26-27页 |
| ·Purification of Polyclonal Anti-hVEGF Antibody | 第27页 |
| ·Polyclonal Anti-hVEGF Antibody specificity | 第27-28页 |
| ·Bradford Protein Assay Procedure | 第28页 |
| ·Establishment of ELISA procedure for detection of serum VEGF | 第28-29页 |
| ·Statistical analysis | 第29-30页 |
| CHAPTER Ⅲ:RESULTS AND DISCUSSION | 第30-44页 |
| 1 Results | 第30-42页 |
| ·VEGF gene cloning | 第30-32页 |
| ·Recombinant VEGF Protein expression | 第32-35页 |
| ·Optimization of Protein expression | 第33-35页 |
| ·Inclusion bodies isolation | 第35页 |
| ·Protein purification and refolding | 第35-37页 |
| ·Titer and specificity of polyclonal antibody | 第37-38页 |
| ·Purification of polyclonal anti-VEGF Antibody | 第38-39页 |
| ·Establishment of ELISA procedure for detection of serum VEGF | 第39-42页 |
| 2 DISCUSSION | 第42-44页 |
| ·Recombinant hVEGF protein expression and polyclonal antibody production | 第42-43页 |
| ·Establishment of ELISA procedure for detection of serum VEGF in tumor patients | 第43-44页 |
| CONCLUSIONS | 第44-45页 |
| REFERENCES | 第45-50页 |
| ACKNOWLEDGMENT | 第50-52页 |
| APPENDIX | 第52-53页 |
