| Babesia caballi is tick-borne hae-moprotozoan parasites in blood cell, which is characterised by fever, anaemia, jaundice and oedema in equids(horses, donkeys, mules, and zebras). This disease is classified as Class B animal diseases and one of the most important diseases of horses in the world. The prevelence and of B.caballi is related to the pathogenicity of local strains and the distribution of vectors, the general was endemic. Due to the special geographical conditions of Xinjiang province, the territory of tick species is widely and the number of horses are ranks first in the whole country. This artical is based on the separation of the Bc-Y strian, amplification of different fragments for the Bc48 gene.The construction of the Recombinant expression plasmid, coating highly soluble recombinant protein as rELISA kit. The present study contains three researches:(1)Epidemiological survey that was used blood smears and PCR methods in Hejing and Zhaosu county, Xinjiang. Then separated local epidemic strains, sequencing and compared with other strains which was published in GenBank, phylogenetic relationship clustering.This experiment was successfully conserve the Bc-Y strain(epidemic strains in Yili area). The results revealed that the positive rate of blood smear and PCR examination was 8% and 21.31%, the highest infection rate was 64.9%. Sequencing compared with standard strains the homology results were 96.7%-99.8% and the divergence results were 0.2%~3.3%.(2)Four pair of primers were designed and synthesized according to the sequence of Bc48 gene of Babesia caballi published in GenBank. Four fragments(the complete gene-QCL, A, B, C)of Bc48 gene were amplified, then construct prokaryotic expression recombinant plasmid by genetic engineering technology. The result indicated that QCL, A, B and C, fragments of Bc48 gene were successfully amplified and four prokaryotic expression recombinant plasmid including pGEX-4T-1-QCL,GA、GB、GC were constructed. Sequencing result showed that the higest identity of cloned Bc48 sequence and deduced amino acid was being 99% with the corresponding sequences published in GenBank. Two kinds of fusion proteins were successfully expressed, GST-GB as high expression protein. Western Blotting showed that the purified fusion protein had good immunogenicity and could be used as diagnostic antigen.(3)An indirect ELISA detection method was successfully established using the purified GST-GB protein as antigen through optimalling reaction condition, and the repeatabilitys, ensitivity and specificity of the method were evaluated through evaluation tests. Finally, 250 samples were detected by using this method. The results showed: the antigen concentration for coating was 0.8 μg/mL, and incubated for overnight at 4℃, The plates were subsequently blocked with 3% skim milk for 1h,and then incubated for 1h with diluted serum samples(1:50). The diluted HRP-secondary antibody(1:10000)was added and incubated for 1 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 0.221. The established indirect ELISA had coefficient of variation was less than 10%. The method was used to detect 250 horse suspected serum samples from Hejing, with the positive rate of serum antibody 28.4% and the coincidence rate was 94.1% in one hundred serum. It could provide a theoretical basis for the prevention and treatment of Babesia caballi in this region. |
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