| Toxoplasma gondii(T. gondii)is an intracellular protozoan prevalent worldwide,it causes serious human and animal parasitic diseases and globally distribute and popular. Its definitive hosts are cats and other felines, meanwhile, It,s intermediate hosts are many animals including humans, birds and domestic animals, the swine is one of the intermediate hosts,shows high infection and mortality rate in T. gondii.This infection in swine is also wide distribution and the pork consumption ratio was high in our country. So swine is an essential source of people infected with T. gondii and causes serious damage for the animal husbandry and human health. There is no specific drug and effective vaccine in the prevention and treatment of this disease. Hence, it was important to establish a convenience and efficient methods to detect T. Gondii infection in swines.A lot of diagnosis methods were used to detection of Toxoplasmosis. For instance, parasitological diagnosis(indeed and reliable, but the detection rate was low), molecular biology diagnosis(sensitive, requirements for special equipment and personnel)and immunology diagnosis(simple,sensitive and specific, the results of judgments intuitive).This study applies the immunological diagnostic methods of Toxoplasmosis,the main contents and results are as follows:1.Clone, prokaryotic expression and purification of the SAG3 gene of mature peptide T. gondii.According the sequence of SAG3 gene published in Gen Bank, the signal peptide was removed, a pair of specific primers was designed and SAG3 gene was amplified by RT-PCR. According to double enzyme digestion identified then it cloned in prokaryotic expression plasmid p ET-28 a and the recombinant expression plasmid p ET-28a-SAG3 was constructed and transformed into E. coli BL21. The expression was induced by IPTG and analyzed by SDS-PAGE. The result showed that the molecular weight of protein was about 43 k Da, and it was expressed mainly in a form of inclusion body. Western-botting analysis showed that the recombinant protein could specially recognize by T. gondii positive serum in swine. The results show that the recombinant protein has adequate reactiongenicity.2.The establishment and application of indirect ELISA detection method of T gondii-specific Ig G in swine serum.This experiment established indirect ELISA method using SAG3 protein as coating antigen, and optimized the related conditions. The results show that: the optimal conditions were coating concentration of antigen was 31.25ng/hole, the optimal dilutions of the antibody was1:320, the best blocking buffer was 5% skim milk in PBST, the working concentration of HRP Affini Pure Goat Anti-Swine Ig G was 1:3000,the dilution of the positive serum of method was 1:6400,The coefficient of variation of intra- and inter-assay of the ELISA reproducibility were less than3% and 12%.The method showed that high sensitivity and reproducibility. 8 positive serum samples were detected using this method were positive, 94 clinical samples in Guangdong and 54 clinical samples in Jilin were detected by this established indirect ELISA and the positive rate were 8.51%(8/94),46.29%(25/54).3.The establishment and application of a sandwich ELISA for the detection of T. gondii. infection in swine.The establishment of a sandwich ELISA using SAG3 monoclonal antibody whice has been prepared by laboratory.The result showed that: the optimal dilutions of Anti-A-SAG3-7 as capture antibody coating of plate was1:3200, the optimal dilutions of the antibody was1:80, the best blocking buffer was1%BSA in PBST, the working dilutions of HRP- Anti-A-SAG3-23 was 1:3200,when the positive samples in swine was diluted to1:640 and two swine Cysticercosis of positive serum had no cross reaction,the coefficient of variation of intra- and inter-assay of reproducibility was less than 11%, it shows that the established method has good sensitivity, specificity and repeatability. 94 clinical samples in Guangdong and 94 clinical samples in Jilin were detected by this established the ELISA method and the positive rate were20.2%(19/94),11.7%(11/94) respectively.4. The development and clinical application of the immune colloidal gold strip for the detection of T. gondii. infection in swine.Colloidal gold of 40 nm particle diameter was prepared by sodium citrat reduction method.The colloidal gold was conjugates with monoclonal antibody(Anti-A-SAG3-7) against SAG3 protein. Another monoclonal antibody(Anti-A-SAG3-23)against SAG3 protein and goat-anti-mouse Ig G were coated on nitrocellulose membrane as the test and control line. Then we have to assemble the strip and optimize the test conditions. The performance of the strip was evaluated and detected of clinical samples.The results were as follows:The optimum PH labeling of 1m L gold solution was added to 4μl 0.2M/L K2CO3 and the best concentration of monoclonal antibodies was 12μg, the optimum spray concentration of antibodies at T and C wire was 0.25 mg/m L and the best spray volume were 0.8μl.The sensitivity of method was 1:160.No cross reaction with two swine Cysticercosis serum were observed.The stability test of the study demonstrated that the strip were sored1 month at room temperature, and at least 3 months at 4℃.Using the colloidal gold strip compared with double sandwich ELISA method, 94 samples in Guangdong and Jilin were detected separately,the coincidence rates were 89.47% and 90.90%. It was high that the coincidence rates of two methods.The results showed that the colloidal gold strip had the advantages of sensitive, specific and simple operation etc. Therefore, this research is more suitable for the grass-roots unit of amount of samples, tight schedule.For the early diagnosis of T. gondii in swine provides a quick and practical method. |
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