麦胚无细胞蛋白合成系统表达恶性疟原虫环子孢子蛋白的研究-病原生物学专业毕业论文.docxVIP

PAGE 4 PAGE 4 华 中 科 技 大 学 硕 士 学 位 论 文 Expression of recombinant plasmodium falciparum circumsporozoite protein by using wheat germ cell-free protein synthesis system Abstract Circumsporozoite protein distributed in surface of plasmodium sporozoite ring is one of the most important protective antigens for plasmodium falciparum in sporozoite period. Therefore it is also one of the important malaria vaccine candidates. However so far there is no any protein expression system that is feasible for expression of protein and preparation of CSP as malaria vaccine for use. Wheat germ cell-free protein synthesis system is a eukaryotic system in the cell free systems of protein synthesis that are different from the systems based on cells. The system may overcome disadvantages of other gene engineering systems for CS protein synthesis. Advantages have been confirmed by some foreign published papers on research of wheat germ cell-free protein synthesis system expressing other proteins of plasmodium. So we try to express of recombinant plasmodium falciparum circumsporozoite protein as basis of a new vaccine candidate by using wheat germ cell-free protein synthesis system. Objective: To express recombinant CSP by using wheat germ cell-free protein synthesis system. Methods: According to the CSP gene sequence in data provided by PlasmoDB, we designed two primers and amplified the CSP gene sequence first by PCR and then extracted and identified PCR product was cloned into vector pIVEX 1.3 WG, producing a recombinant plasmid pIVEX 1.3 WG-CSP. Both PCR product and recombinant plasmid 华 中 科 技 大 学 硕 士 学 位 论 文 were identified by the NcoI and SmaI, which was followed by transformation of pIVEX 1.3 WG-CSP into E.coil DH5α. The harvested recombinant plasmid was identified by conventional methods and DNA sequencing. Then the recombinant plasmid pIVEX 1.3 WG-CSP was expressed in the wheat germ cell-free protein synthesis system. The product expressed from wheat germ cell-free protein synthe