Establishment Of Cell Line, Cloning And Prokaryotic Expression Of MM/Sac-C/1 Gene Of Giardia Canis

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.