Cloning And Expression Of The EMA1 Gene Of Xinjiang Strain Of Theileria Equi And Establishment Of RELISA For Its Application

Theileria equi is a tick-borne disease of equids(including horses,donkeys,mules and zebras)caused by intra erythrocyte/lymphocyte protozoa.T.equi belongs to the reporting disease from The World Organization for Animal Health(Office International des Epizooties,OIE),and it is the Ⅱ kinds of animal diseases in China.These signs range from mild to moderate and may go unnoticed,but they generally include low physical performance,anemia,hemoglobinuria,jaundice,et al.It is manifested in acute,subacute,or chronic disease phases,with variable and nonspecific clinical signs.It is an economically important protozoan disease of horses in tropical and subtropical regions,including America,Africa,Asia and Southern Europe.The infected horse may die depending on the pathogen strain and infection intensity.overall health status,and treatment administered.The control measures of equine piroplasmosis require effective diagnostic approaches that can detect chronically infected horses.And the key question of EP is to control infection source(positive horse),although there are foreign merchandise kit is not bad,but its not only expensive price and low specificity but also not appropriate for the local serum samples,therefore,grassroots need effective diagnostic methods to detect chronic infection of horses.The erythrocytic-stage surface protein,Equi Merozoite Antigen 1(EMA-1),is a major candidate for the development of a diagnostic antigen for equine piroplasmosis.So the study of EMA1 gene and establish an rEMA1-ELISA is important for practical use.(1)According to the specific sequence of EMA1 of Theileria equi genes,the primers were designed and synthesized.The EMA1 gene of Xinjiang strain was inserted into the prokaryotic vector pGEX-4T-1,the recombinant plasmid(pGEX-4T-1/EMA1 & PET-28a/EMA1)were transformed into Escherichia coli BL12(DE3)and the combination protein(GST-EMA1 & His-EMA1)was obtained with the induction of IPTG.The fusion protein was purified by extracting the inclusion bodies with gel slices,and the purified protein was used as coated antigen for the establishment of rELISA method to detect the antibody of Theileria equi.The results indicated the expressed EMA-1 was largely consistent with its theoretical value,and expression of protein was identified by Western blot and IFAT,which confirmed that the protein had highly specificity,antigenicity and immunogenicity.(2)The recombinant purified GST-EMA1 protein from(1)was tested in an rEMA1-ELISA for the detection of antibodies anti-T.equi in horses,the rELISA clearly differentiated T.equi-infected from Babesia caballi-infected,equiperdum-infected horse sera and normal horse sera;The intra-and inter-assay demonstrated that the coefficient of maximum variation was 14.79% and 11.06% respectively;The antigen concentration for coating was 4.0 μg/mL,and incubated for overnight at 4℃,The plates were subsequently blocked with 0.05% skim milk for 1 h,and then incubated for 1h with diluted serum samples(1:400).The diluted HRP-secondary antibody(1:20000)was added and incubated for 0.5 h at 37℃,and then substrate of TMB solution was added for incubation 15 min at 37℃ to develop color.The thresholds for indirect ELISA was 0.353.96 serum samples collected from horses in the state of Yili,Xinjiang,the comparison between rELISA and cELISA commercial kit showed that the positive of T.equi infection were27.08%(26/96)and 25.0%(24/96)respectively,and the total coincidence rate was 95.8%.In conclusion,the EMA1 protein expressed in E.coli could be a reliable immunodiagnostic antigen for rELISA test and that had highly specificity,sensitivity and stability,so that the rELISA can provided the means of effective detecting and monitoring for T.equi(especially in recessive infection)in the State of Xinjiang.