Expression Of SPAG Gene Of Theileria Annulata And Establishment Of ELISA Method

Bovine tropical theileriosis is a tick borne hemaprotozoosis caused by the parasite of Theileria annulata.The parasite mainly dwells in lymphocytes and red blood cells.The infected animal shows fever,anemia,jaundice,emaciation and enlargement of the lymph nodes,and even death in serious cases,and it causes important economic loss for the breeding industry.It is a typical insect-borne disease,and transmitted by the genus of Hyalomma ticks,including H.anatolicum,H.detritum and H.scupense.The disease is tightly related to the activity of ticks.Usually,it occurs from May to June,peaks in July,and gradually disappears in August and September.In order to establish a specific diagnostic method for tropical theileriosis,the sporozoite surface antigen（SPAG）was chose as the target gene in this study.A pair of specific primers was designed for clone the gene.Then a real-time PCR detection method was developed and used to analyze the transcriptional level of SPAG gene in three development stages of Theileria annulata.The recombinant plasmid was constructed and the recombinant SPAG protein（rSPAG）was expressed by prokaryotic expression system.The reactionogenicity and specificity of rSPAG was analyzed.An indirect ELISA method was established to detect T.annulata infection using rSPAG as antigen.The method was evaluated and applied with the field serum samples.The major works are as follows:1.According to the sporozoite surface antigen gene sequence obtained from GenBank database,a pair of primers was designed for cloning and sequencing the full length of SPAG gene from three T.annulata strains,Kashi,Yili and Inner Mongolia,respectively.It was found that the gene length of Kashi strain,Yili strain and Inner Mongolia strain was 2706 bp,3051 bp and 2733 bp,respectively,and the homology among them was more than 90%.A real-time PCR detection method was developed targeting on the SPAG gene,and the react conditions of this method were optimized.Confirmed by experiments,the method has a high specifity for DNA T.annulata with the minimum detectable amount of 10 copies/μL of the gene,and no cross-reaction with DNAs of T.sinensis,T.orientalis,Babesia bovis,B.bigemina,B.ovata and B.major.The coefficients of variations were less than 5%for both intra-assay and inter-assay test.The transcriptional level of SPAG gene at different developmental stages such as sporozoite,schizont and merozoite was evaluated by this method,and it was found that the gene was expressed in all three developmental stages,the highest in the sporozoite stage and the lowest in the schizont stage.At the same time,422 samples collected from Hubei,Gansu,Shanxi and Yunnan provinces were detected by this method,and the positive rates were 10.00%（6/60）,3.38%（7/207）,0（0/60）and 4.21%（4/95）,respectively,which were more sensitive than ordinary PCR method.2.The features of SPAG gene sequences of three T.annulata strains were analyzed using bioinformatics techinques.A pair of primers was desinged targeting on the conservative region.The fragment was amplified and inserted into expresion plasmid.The constructed recombinant plasmid of SPAG was used for protein expression and purification.The length of the target gene is 882bp,and the size of the recombinant protein is about 42 ku.Western Blot test shows that the induced recombinant protein can be specifically recongnized by positive sera of calves infected by T.annulata,rather than by those of positive sera of T.sinensis,T.orientalis,B.bovis,and B.bigemina.It indicates that the recombinant protein can be used as a candidate antigen for developing serologically diagnostic method of tropical theileriosis.3.An indirect ELISA method of tropical theileriosis was developed using purified recombinant SPAG protein as an antigen by optimizing the reaction conditions.The positive sera of Theileria annulata can only be detected,and no cross-reaction with positive sera of T.orientalis,T.sinensis,B.bigemina,and B.bovis.The kinetics of anti-T.annulata antibodies in experimentally infected animals was evaluated using the ELISA.The animals had seroconversion on the 7^th day post-infection（dpi）.The highest level of antibodies was detected on the 27^th to 33^rd dpi,and on 145^th dpi,antibody level dropped to that of before infection.A total number of 1,654 of field-collected samples from Xinjiang,Gansu,Qinghai,Shanxi,Hubei,Guangxi,Hunan,Guizhou and Sichuan provinces were tested by this indirect ELISA method.The positive rates of the 9 provinces were 27.15%,22.99%,14.22%,16.67%,50.00%,46.88%,53.57%,35.96%and 59.32%,respectively.