【作者】 赵永军；

【导师】 张西臣；

【作者基本信息】 吉林大学 ， 预防兽医学， 2006， 硕士

【摘要】 本研究建立了犬贾第虫(Giardia canis)细胞系,并利用PCR技术从犬贾第虫滋养中扩增出了2004bp大小的MM/Sac-C/1基因,构建了pMD-MM/Sac-C/1克隆载体,进行了序列测定,利用计算机对该序列进行了核苷酸同源性比较。结果表明,与GenBank登陆的蓝氏贾第虫WB株AF236019序列比较,其同源性为98%,蛋白质同源性比较结果显示,与蓝氏贾第虫WB株表面抗原的同源性为90%,获得了犬贾第虫MM/Sac-C/1基因。根据犬贾第虫MM/Sac-C/1基因的序列,用EcoRI、XhoI双酶切pMD-MM/Sac-C/1及pET-28b质粒,构建了原核表达质粒pET-MM/Sac-C/1,并在DE3中进行了表达,重组蛋白为75kDa,占菌体总蛋白的22.4%,Western-Blotting表明该蛋白具有良好的反应原性。本研究的结果为进一步深入研究贾第虫生物学、免疫学提供了丰富的实验材料,为揭示贾第虫的免疫逃避机制、建立免疫诊断方法,以及制订贾第虫病防治措施均有重要意义。更多还原

【Abstract】 Giardia, a binucleated flagellate protozoa, can infect human,mammal and bird worldwidely. Giardia lamblia, which invade human andmammal, can be classified into G. bovis, G. canis, G. caprae and G.cati according to different hosts. In the study, Giardia canis cell linewas established and its MM/Sac-C/1 gene was also cloned andexpressed in E.coli.Establishment of Giardia canis cell line Giardiasisis is a commondisease which has been increasingly thought highly of by veterinarymedicine and medical science researchers. The pure culture in vitro is theprerequisite of the work. Giardia includes cyst and trophozoite, in vitroculture of trophozoite originate from twenty century. In that time thetrophozoite could grow in vitro for five monthes and later cultural methodand technology has been improved and development of HSP-1, HSP-2,TPS-1 and TYI-S-33. The improved TYI-S-33 medium, which iscommonly used, added to mammal bile and made its nutritional ingredientsfurthermore get close to small intestine and markedly promote the parasitesgrow. In the present study, culture of Giardia is restrict at human Giardiaand Giardia canis was discovered natural cyst and trophozoite. Its in vitroculture has not been reported which inhibit the further research of animalGiardia and Giardiasis.The cysts of Giardia canis was isolated and purified by saccharumdensity gradient centrifugation-G1 acid funnel, and the 10-day-oldsuckling gerbil was infected by the cysts. On the day of 8 after oralinoculation, trophonts isolated aseptically from the superior segmentintestine of the infected animals and were inoculated into modifiedTYI-S-33 medium and cultivated at 37℃.The parasites which graduallyadapted enviroment and formated a cellular monolayer on the inner surfaceof the culture tube was passaged every 48～72h and the passaged numberwas 18.It was confirmated that the cell line of Giardia canis haveestablished by freeze in liguid nitrogon and resuscitation. The suitableformula of Giardia canis culture was obtained by optimizing the principalcomponents and concentration of medium.Cloning and sequencing of MM/Sac-C/1 gene in Giardia canis Thegene was amplified by PCR, the cloning vector pMD-MM/Sac-C/1 wasconstructed and sequenced. The homology of MM/Sac-C/1 gene toAF236019 in GeneBank was 98% in the nucleotide,and 90% in the aminoacid levels. It was demonstrated that Giardia canis trophonts haveMM/Sac-C/1 gene.Expression of MM/Sac-C/1 gene in E.coli According to the sequenceof the MM/Sac-C/1 gene,expression vector pET-MM/Sac-C/1 wasconstructed and was transformed into E.coli strain DE3, then inducedby IPTG, 75kDa expression protein which was found by SDS-PAGE andmade up with 22.4% of total protein possesed immunogenicity byWestern-Blotting. It set up a basis on immunology of Giardia.更多还原