【作者】 米荣升；

【导师】 岳城； 陈兆国；

【作者基本信息】 新疆农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 隐孢子虫是一种严重危害人和动物健康的寄生原虫,广泛寄生于人、哺乳动物、鸟类、两栖类和鱼类的消化道和呼吸道中,引起腹泻等症状。对免疫功能正常的动物,腹泻可自限,但免疫功能缺陷时,常表现水泻不止甚至死亡。隐孢子虫不同种性状有别,寄生宿主的对象不一。本研究利用PCR-RFLP技术成功鉴定了本实验室收集的上海地区分离的鼠源、兔源、鸡源、猪源、牛源隐孢子虫虫种和基因型,共有3个虫种和3个基因型,其中鼠源隐孢子虫虫种为微小隐孢子虫鼠基因型(C.parvum Mouse genotype);兔源隐孢子虫虫种为微小隐孢子虫兔基因型(C.parvum Rabbit genotype);猪源隐孢子虫虫种为微小隐孢子虫猪基因型II(C.parvum Pig genotype II);鸡源隐孢子虫虫种为贝氏隐孢子虫(C.baileyi);牛源隐孢子虫虫种为安氏隐孢子虫(C.andersoni)。成功克隆了上海地区分离的微小隐孢子虫鼠基因型、兔基因型、猪基因型II和安氏隐孢子虫的子孢子表面抗原CP15基因,其编码基因为大小为357bp,编码119个氨基酸残基,不同来源的CP15基因序列高度一致。其中安氏隐孢子虫的CP15基因成功克隆为本研究首次报道。成功克隆了上海地区分离的微小隐孢子虫鼠基因型、兔基因型和猪基因型II的子孢子表面抗原P23基因,大小为342bp,编码113个氨基酸,不同来源的P23基因序列高度一致。成功构建了微小隐孢子虫鼠基因型的原核重组表达质粒pET-28a(+)-CP15和pET-28a(+)—P23,实现了CP15和P23基因在大肠杆菌中的表达。ELISA初步分析表明CP15和P23基因具有良好的抗原性。上述工作的进展,为逐步查明上海地区目前流行的隐孢子虫病原种类、掌握隐孢子虫病流行状况和特点提供了基础,具有重要的公共卫生学意义。隐孢子虫子孢子表面抗原CP15和P23基因的成功克隆和表达,为进一步利用基因的表达产物进行免疫预防和诊断研究提供了条件。更多还原

【Abstract】 Cryptosporidium spp. is a protozoan parasite and it is harmful severely to men and animals. It grows in epithelial cells and digestive organs of men, animals, birds, creeperes or fishes and it causes diarrhea. Cryptosporidium spp. is known to be ubiquitous and to have many hosts. The diarrhea can be cure by themselves about the normal immune people, but when people who had immune deficiency, the diarrhea maybe serious even take to death.There were different characteristics and hostes about Cryptosporidium spp. The species and genotypes of Cryptosporidium spp. isolated from mouse, rabbit, chicken, pig and dairy calves in Shanghai district were identified by nested polymerase chain reaction linked restriction fragment length polymorphism (PCR-RFLP). The results showed that there were three species and three genotypes, Cryptosporidium spp. which comes from mouse was Cryptosporidium paravum mouse genotype, rabbit was Cryptosporidium paravum rabbit genotype, chicken was Cryptosporidium baileyi, swine was Cryptosporidium paravum pig genotype II and dairy calves was Cryptospori—dium andersoni.The sporozoite surface antigen CP15 gene which came from C.paravum mouse genotype, C. paravum rabbit genotype, C. paravum pig genotype II and C. andersoni were successful amplified and encoded 119 amino acids. Cryptosporidium spp. came from different sources had a highly homoely and C. andersoni was first report in this expriment.The sporozoite surface antigen P23 gene which came from C. paravum mouse genotype, C. paravum rabbit genotype and C.paravum pig genotype II were successful amplified and encoded 113 amino acids. Cryptosporidium spp. came from different sources had a highly homoely. The prokaryotic expressing vector pET-28a(+)-CP15 and pET-28a(+)-P23 were constructed and expressed in E.coli successfuliy. Both of them had a well antigenicity which analyzed by ELISA.The development of our experiments we had done would much helpful to know the disease sources and epidemic conditions about Cryptosporidium spp. in Shanghai district, and had an important sense about public hygieology. The sporozoite surface antigen CP15 and P23 gene which were successful cloned might be much helpful for the next work of using these genes to develop a immunoprophylaxis or detection technique.更多还原