Analysis Of The Proteome Of The Difference Stages Of Eimeria Tenella Maduramycin Resistant Strian And That Of Maduramycin Sensitive Strain

Eimeria tenella is a parasite of great importance as a disease causing agent in the poultry industry.The disease has largely been controlled through the use of anticoccidial drugs.But the development of drug resistance results from the overuse of anticoccidial drugs especially the longtenn use of anticoccidial drug as feed additive in livestock and poultry.So study on the drug resistant mechanism in chicken coccidia is key to prevention and new anticoccidial drug development.Eimeria tenella life cycle is complex that compose of oocysts stage,sporozoites stage and agamont stage.It is differential protein,enzyme,morphology and biochemical characteristics along E.tenella life cycle.Protein expression studies have been proposed as a powerful method to study drug resistant mechanism.Study on protein expression in E.tenella is still at the starting stage. Few reports in this field has been found yet..In this research,previous studies have found differentially expressed protein spots in different stages of E.tenella Maduramicin resistant strains and sensitive strains by two dimensional electrophoresis(2-DE).We studied the function of the differentail expressing proteins by PMF and bioinformation.The works which we did are showed as follows:1 Comparison of different stages of E.tenella Maduramicin resistant strains with that of sensi -tive strains by protein electrophoresisThe Maduranlicin resistant strains and sensitive strains of E.tenella oocysts were obtained by passage through coccidia-free-2-week-old broiler chickens.The second generation merozoites, unsporozoited and sporozoited oocysts of Eimeria tenella were collected with PBS and stored in 4℃. In this experiment,Eimeria tenella purified by EDAE gel.After the comparison of test TCA/acetone precipitation method,the direct grinding of high-speed centrifugation and effect of Liu Wenjiang protein extraction,solubilised protein opts for direct grinding of high-speed centrifugation and put onto HiTrap Desalting,then put it into freeze-drying system to be concentrated.This program solved the troubled of high concentration of salt ions and low concentration of protein for two dimensional electrophoresis study.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 shows that there was one different expressed bands in sporozoited oocysts of Maduramicin resistance strain,the molecular weight and content were displayed respectively,21.5kD,23.0%;there was no difference brands among the the second generation merozoites and unsporozoite between the sensitive strain and drugresistance strain. In further study,150μg protein sample was applied to rehydrated IPG dry strip with a pH range of 3-10,17cm(BIO-RAD).The gel were focused in the first dimension(PROTEAN IEF Cell) at a maximum set votage of 60,000vh.Equlibrated twice and then transferred to the second dimension electrophoresis.The second dimension was performed on a 0.5ram 12%SDS-PAGE in PROTEANⅡxi Cell.Gels were stained with sliver.Image analysis was performed using the 2D-Elite software. The experimential results implied:the protein spots of 2-DE maps of E.tenella Maduramicin resistant strains and the homologous sensitive strains second-generation merozoites,unsporozoited, sporozoited are respectively identified on 900±73,900±25,200±14,200±11,200±51,200±43 and the average matching rate about 88%～92%.Most of protein spots are molecular weight of 15kD～100kD,and isoelectric point between 3.5 to 9.5.Comparison of these protein spots,it is found that Maduramicin sensitive strains and resistant strains have difference in expression points at different stages.These spots mostly distributing in 21～28KD,35～45KD,66～90KD with pH3.5～8.0.2.Analysis of the different protein of E.tenella Maduramiein resistant strains and Maduramicin sensitive strains by MALDI-TOF MSFifteen uniquely obvious expressed spots from 2-DE map of E.tenella Maduramicin resistant strains and homologous sensitive strains(nine spots of Maduramicin strains,six spots of sensitive strains) for MAIDI-TOF-MS peptide fingerprint analysis by Mascot software online(http://www. Matrixscience.com)in the NCBInr database.By the database search,we identified twelve proteins and three hypothetical proteins.The results showed that the majority of fifteen differentially expressed proteins were proteinase.Those differentially expressed proteins are related to cell Glucose Metabolism,Polyamine Metabolism and Energy Metabolism such as endonucleaseⅤ, carboxy-lyase,porphobilinogen deaminase,beta-N-acetylglucosaminid-ase isoform;Some proteins related to cell-mediated immunity such as 1C2(immune protein),A6(immune protein),LT2(ATP enzyme molecular chaperone),ATCC49188(secretion protein) and ANA-3(chromosome segregation protein):Parts of the proteins related to cell signal transduction such as HTCC2150(ABC transporter protein),RAB36(RAS family transporter protein)and cellulosity such as Strongylocentrotus purpuratus(action protein)There were proteins expressed specifically including DSM3645,SD-21,ATCC 49188, Strongylocentrotus purpuratus and highly expression proteins including endonucleaseⅤ, HTCC2150,RAB36,ANA-3,LT2 in second generation merozoites stage and sporozoites stage of E.tenella Maduramicin resistant strains.It was conclude that resistant strains coccidian may adapt to environment with drug by the improvement of metabolism ability.Therefore,study on the mechanism of coccidian immune tolerance should based on expressed protein.