Tam, Hou Si. / 33 in title is subscript. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 174-187). / Abstracts in English and Chinese. / THESIS COMMITTEE --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT --- p.iii / 摘要 --- p.v / TABLE OF CONTENTS --- p.vii / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xvii / LIST OF ABBREVIATIONS --- p.xviii / CHAPTER / Chapter 1. --- INTRODUCTION / Chapter 1.1 --- Malaria --- p.1 / Chapter 1.2 --- Malaria is a public health problem --- p.1 / Chapter 1.3 --- Malarial parasite --- p.3 / Chapter 1.4 --- Life cycle of P. falciparum --- p.3 / Chapter 1.4.1 --- The pre-erythrocytic stage --- p.3 / Chapter 1.4.2 --- The asexual erythrocytic stage --- p.3 / Chapter 1.4.3 --- The sexual transmission stage --- p.6 / Chapter 1.5 --- Chemoprophylaxis and chemotherapy of malaria --- p.7 / Chapter 1.6 --- Drug resistance of malaria parasite --- p.7 / Chapter 1.7 --- The progress for malaria vaccine --- p.10 / Chapter 1.8 --- Vaccine candidates for asexual erythrocytic stage --- p.11 / Chapter 1.9 --- Merozoite Surface Protein-1 (MSP-1) --- p.13 / Chapter 1.9.1 --- Structure of MSP-1 --- p.13 / Chapter 1.9.2 --- The processing of MSP-1 --- p.17 / Chapter 1.9.3 --- MSP-1 as a blood-stage vaccine --- p.19 / Chapter 1.9.4 --- The vaccine potency of MSP-133 --- p.23 / Chapter 1.10 --- Merits of E. coli expression system --- p.25 / Chapter 1.11 --- Aim of study --- p.26 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.30 / Chapter 2.2 --- Methods --- p.39 / Chapter 3. --- EXPRESSION AND PURIFICATION OF RECOMBINANT MSP-l33kv+19 PROTEIN / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Results / Chapter 3.2.1 --- Construction of pET32a/MSP-l33kv+19 expression vector --- p.64 / Chapter 3.2.2 --- SDS-PAGE analysis of the expressed protein --- p.74 / Chapter 3.2.3 --- Western blot analysis of the expressed protein --- p.78 / Chapter 3.2.4 --- Modification of the expression conditions --- p.78 / Chapter 3.2.5 --- Protein purification by IMAC --- p.82 / Chapter 3.2.6 --- Cleavage of fusion partner from the rMSP-133kv+19 protein --- p.82 / Chapter 3.2.7 --- Verification of non-fused recombinant MSPl33kv+19 protein by N-terminal amino acid sequencing --- p.86 / Chapter 3.2.8 --- Separation of target protein from the fusion mixture by IMAC --- p.86 / Chapter 3.2.9 --- Separation of digestion product by Size Exclusion Chromatography --- p.89 / Chapter 3.2.10 --- Conformational test of the purified protein --- p.89 / Chapter 3.2.11 --- Separation of target protein from contaminants by Anion-Exchange Chromatography --- p.92 / Chapter 3.2.12 --- Separation of target protein from contaminants by Immuno-Affinity Chromatography --- p.95 / Chapter 3.3 --- Conclusion --- p.95 / Chapter 4. --- IMMUNOLOGICAL CHARACTERIZATION OF BACTERIAL EXPRESSED rMSP-l33kv+19 / Chapter 4.1 --- Introduction --- p.97 / Chapter 4.2 --- Results / Chapter 4.2.1 --- Immunogenicity of recombinant NfMSP-133kV+19 protein --- p.98 / Chapter 4.2.2 --- Specificity of anti-NfMSP-133kv+19 sera to MSP-l33kv. MSP-l33 and MSP-l19 --- p.98 / Chapter 4.2.3 --- Cross reactivity of anti-MSP-133kv+19 and anti-BVp42 serum --- p.103 / Chapter 4.2.4 --- Competitive ELISA --- p.103 / Chapter 4.2.5 --- Test for the presence of inhibitory B-cell epitopes on rMSP-l33kv+19 --- p.111 / Chapter 4.2.6 --- In vitro parasitic growth inhibition assay --- p.113 / Chapter 4.3 --- Conclusion --- p.115 / Chapter 5. --- EXPRESSION AND PURIFICATION OF RECOMBINANT MSP-l33kc+19 PROTEIN / Chapter 5.1 --- Introduction --- p.116 / Chapter 5.2 --- Results / Chapter 5.2.1 --- Construction of pET32a/MSP-133kv+19 expression vector --- p.117 / Chapter 5.2.2 --- Expression of recombinant MSP-133kc+19 protien (rMSP-133kc+19) --- p.124 / Chapter 5.2.3 --- Purification of rMSP-l33kc+19 by IMAC --- p.127 / Chapter 5.2.4 --- Cleavage of fusion partner from target protein --- p.127 / Chapter 5.2.5 --- Construction of pRSETA/MSP-l3X33kc+19 expression vector --- p.135 / Chapter 5.2.6 --- SDS-PAGE analysis of the protein expression --- p.146 / Chapter 5.3 --- Conclusion --- p.153 / Chapter 6. --- DISCUSSION / Chapter 6.1 --- Expression of rMSP-l33kv+19 --- p.154 / Chapter 6.2 --- Purification of rMSP-l3.3kv+19 --- p.156 / Chapter 6.3 --- Conformational test of rMSP-133kv+19 --- p.157 / Chapter 6.4 --- Biological and immunological activity of NfMSP-133kv+19 --- p.158 / Chapter 6.5 --- Expression of rMSP-133kc+19 --- p.166 / Chapter 6.6 --- Future prospects --- p.167 / REFERENCES --- p.174 / APPENDICES / Chapter 1. --- HiTrap NHS-activated HP for ligand coupling procedure --- p.188 / Chapter 2. --- Reuse of Ni+-NTA Resin procedure --- p.190 / Chapter 3. --- Sequence alignment of MSP-133 (MAD20 & Welcome/Kl alleles) --- p.191 / Chapter 4. --- Nucleotide sequence and amino acid sequence of P. falciparum MSP-l33kv+19 --- p.192 / Chapter 5. --- Nucleotide sequence and amino acid sequence of P. falciparum MSP-l33kc+19 --- p.193 / Chapter 6. --- "Nucleotide sequence and amino acid sequence of P. falciparum MSP-142 (3D7 isolate, MAD20 allele)" --- p.194 / Chapter 7. --- Amino acid sequence of Plasmodium falciparum MSP-l42 --- p.195