Drugresistance Proteins Study Of Eineria Tenella

The two drugresistance strains of Eimeria tenella, Diclazuril -resistance strain and Maduramasin-resistance strain were induced by Key Laboratory for Animal Parasitology of Ministry of Agricultry, Shanghai Institute of Animal Parasitology . Two weeks age of Roman chickens,fed in coccidian-free condition were inoculated with sporalated occysts of the drugresistance strain and its homologous sensitive strain .The second generation merozoites ,unsporozoite and sporozoited oocysts of Eimeria tenella were collected with PBS and stored in -70℃ after centrifuging. 1,The coccidia suspension submitted to five cycles of alternate freezeing(-70℃ freeze for 10min)and thawing(35℃ water bath for 5min) ,then sonicated for 30min at 150W on ice bath discontinuously and centrifuged at 12000r/min for 1h at 4℃.Then the soluble protein was applied to SDS-PAGE and stained by Commas brilliant blue.The gels were analized by Imagemaster VDS software.The results showed that there were no difference among the second generation merozoites and unsporozoite of Eimeria tenella; there were three different expressed bands in sporozoited oocysts of Diclazuril resistance strain,the molecular weight and content were displayed respectively, 42.0ku,2.1%； 26.5ku,45.8%；25.5ku,23.8%; there was one band in Maduramasin resistance strain,which molecular weight and content displayed ,26.5ku 23.0%.There were the same brand in drugresistance strain which was 26.5ku .It also showed there was no difference brands among the other stages between the sensitive strain and drugresistance strain.2,The protein of the second generation merozoites of Sensitive strain and drugresistance strain was applied to SDS-PAGE and stained by silver nitrate, it was also shown that there was no difference.3,The second generation merozoites of Eimeria tenella were soluted in 40mmol Tris-base(pH9.5), then sonicated on ice bath discontinuously at 150W until there were no completed cells.The protein was quantitied by Bradford method. 80μg protein sample was applied to rehydrated IPG dry strip with a pH range of 3-10L ,13cm(Amersham Parmacia Biotech).The gel were focused in the first dimension (IPGphor) at a maximum set votage of 65560vh.Equlibrated twice and then transferred to the second dimension .Electrophoresis were performed at 5W/gel for 45min and the rest of the run was completed at 20W/gel until the tracking dye reached to the bottom of the gel.The gel were fixed and stained according to the protocols published by GuoYao-jun. Image analysis was performed using the 2D-Elite software.The experimential results implied, Diclazuril resistance strain had four obvious different expressed spots, which were (Mr/pI)32.7ku/6.3,31ku/7.35,29.2ku/6.85,27.3ku/5.7；Maduramasin resistance strain had four obvious different expressed spots,too,they were(Mr/pI)33.5ku/5.9,29.2ku/6.85,26.7ku/5.6,26.7ku/5.6.All the proteins are low molecular weight ,and these proteins perhaps involved in the development of drugresistance. Although detailed functional tests with these proteins have still to be performed. To analysis the different spots by mass spectroetry associated with analysising the bioinformatics of these proteins of different stages of Eimeria tenella and cytomembrane protein would to be helped to built up a quick diagnosic method of drugresistance and understand the mechanisms of the drugresistance of Eimeria tenella.