【作者】 周怀瑜；

【导师】 何深一；

【作者基本信息】 山东大学 ， 病原生物学， 2005， 硕士

【摘要】 弓形虫是一种专性细胞内寄生的原虫,能感染包括人类在内的所有哺乳动物,引起人兽共患弓形虫病。弓形虫呈世界性分布,人群感染率高达25%～50%。孕妇孕期首次感染,弓形虫可穿越胎盘屏障感染胎儿,影响其发育,重者导致畸胎、死胎,存活者也常有畸形及智力发育不全等严重后遗症,同时可使孕妇出现流产、早产。健康成人感染弓形虫多为隐性感染,但当机体免疫功能受累时,如患恶性肿瘤、艾滋病或器官移植等,弓形虫的增殖力和致病力明显增强,是其致死的重要原因。弓形虫还可引起禽畜疾病,给畜牧业生产造成严重的经济损失。迄今为止,无任何有效的防治方法。疫苗的研究成为预防与治疗弓形虫病的发展方向。 P30抗原作为弓形虫速殖子表面的主要抗原之一,具有较强的免疫原性,能够诱导机体产生IgG、IgM、IgA抗体和CD8+T细胞杀伤弓形虫,是最有希望的疫苗候选抗原。另外,弓形虫病主要通过粘膜途径感染,应用无毒性的霍乱毒素A2/B(CTA2/B)亚基作为免疫佐剂,能有效增强P30蛋白的免疫原性,可以增强免疫作用,打破免疫耐受,是研制高效的弓形虫疫苗的理想佐剂。 国内外学者曾用多种表达系统来表达P30与CTA2/B蛋白,动物和人体实验结果显示疫苗的效果不理想。原核表达系统操作简单、产量高,但缺乏真核生物的基因表达调控机制和蛋白质的加工修饰能力,表达产物往往没有生物活性,质量不高。哺乳动物细胞表达系统虽能正确表达蛋白,但产量低,无法进行P30蛋白下游研究和大规模动物实验。本研究选用既具有原核生物的优点,又具有真核生物功能的毕赤酵母表达系统。迄今为止,毕赤酵母表达系统已成功地表达了400多种蛋白。酵母是单细胞真核生物,兼有细菌易培养、遗传操作简单、高效表达外源蛋白等优点,又具有真核更多还原

【Abstract】 Toxoplasma gondii (T. gondii) is a harmful intracellar protozoa, which can cause a kind of worldwide , severe parasitic diseases named Toxoplasmosis. T. gondii occurs in almost all mammals, including human beings. The prevalence rate among the population is high range from 25% to 50%. Congenital toxoplasmosis is that toxoplasma crosses the placental barrier from the mother’s blood, which can cause abortions, teratosis and stillbirths. Toxoplasma is an opportunistic protozoan. In immunosuppressed and immunodeficient individuals, including patients with organ transplantation, malignant tumor, acquired immune deficiency syndrome (AIDS), toxoplasma can cause them to death. Toxoplasma is also capable of causing severe disease in animals so it could destroy farming badly. So far we do not have any methods effectively to cure the disease, the prevention of toxoplasmosis is very important. The manufacture of Toxoplasma gondii’svaccine becomes urgent and a common direction among the researches.P30 is the main surface antigen of the tachyzoite. It is highly immunogenic andcould induce IgG、 IgM、 IgA、 antibodies and CD8+T cell. It is regarded as one of the diagnostic antigens and candidated molecular vaccine to preventing Toxoplasma gondii infection. In addition to Toxoplasma gondii invades into the body though the mucous membrane, nontoxic CTX A2/B was used as an adjuvant in order to enhance the immunogenicity of p30, which play a important role in Toxoplasma gondii’s vaccine. Though both home and aboard have expressed p30 and CTX in various system, the effect of the protein in animals and human beings can not cater to the requirement.It is well known that genes of eukaryotic cells can be transcripted and translated in prokaryotic cells in a simple and high productive way. But the prokaryotic cells cannot produce proteins with natural configuration and activity, due to its incompetence of accurate post-translation processings. Although the cell of mammals can express the protein with activity, the product is low in does, and it can not suit to get a great deal of protein required.Yeast pichia pastores expression system was used in the experiment. It was applied popular for the system both have the characters of prokaryotic cells and eukaryotic cells> simplicity in operation n fast in growth and has competence of accurate post-translation processings. Furthermore it also has the character of productivity. The expressing vector used in experiment is pGAPZ a A has a a signal peptide, which can make the product secrete from the cells simpJicizing the purification.The compound gene P30-CTXA2/B, cloning plasmid pMD]8-P30-CTXA2/B and pichia pastores expression piasmid pGAP-P30-CTXA2/B were successfully constructed by means of molecular biological clone-oriented method and gene combination technique. Then the recombinant pichia pastores expression plasmid pGAP-P30-CTXA2/B transfected Yeast pichia pastores cells GS115. Meanwhile, fusion protein of compound gene P30-CTXA2/B was successfully expressed in Yeast pichia pastores cells, which was detected by running SDS-PAGE, showing a specific protein band and was further confirmed by Western Blotting, showing that the specific fusion protein containing P30 immunological activity. And the protein expressed has the characters of high productiveity and purification.The successful expression of P30-CTX A2/B fusion protein in Yeast pichia pastores expression system plays a basic role in the aspect of diagnosis and the research in vaccine at the level of gene and protein. And it also paves way for further study on animals.更多还原