| Ovine and caprine theileriosis is a tick-borne disease,caused by genus Theileria that parasitized in macrophages,lymphocytes and erythrocytes of sheep,goats and other ruminants.It's a severe disease characterized by swollen superficial lymph nodes,high raise of fever,anemia and jaundice.Up to date,prevention and control of theileriosis mainly depends on chemical drugs for medication,but it was found that Theileria species had created resistant against some drugs(i.e.Berenil)in the recent years.Moreover,the chemical drug causes environmental pollution and drug residues in animal products.Therefore,vaccination becomes one of the most promising measures for the prevention and control of theileriosis.Theileria luwenshuni surface protein(TlSP),which is a transmembrane protein located in the parasitic membrane,is expressed during the sporozoite and schizont life stage of the parasite,and it become a candidate antigen for the development of anti-Theileria vaccine.In this study,the immune-dominant region of TlSP was expressed in Pichia pastoris.Then,rTlSP was prepared in the form of a subunit vaccine to immunize sheep to evaluate the immune protective effect of subunit vaccine.The content of the research is summarized as follows:1.TISP gene cDNA was amplified by RT-PCR and the sequence was analyzed by bioinformatics.The result showed that the cDNA sequence of TlSP gene was 888 bp,with a 94% identity at nucleotide acids level of other Theileria.TlSP has two transmembrane regions near the C-terminal,and the N-terminal region ranges 1~184 aa is located on the surface of the parasitic membrane and owns B cell epitopes,which was identified as an immune-dominant region.2.The immune-dominant region of TlSP gene was cloned into the pPIC9 K vector to form the recombinant plasmid pPIC9K-TlSP.The recombinant vector was transformed into Pichia pastoris cells(GS115 and KM71),and the multiple copies of transformants with hyper-resistance to Geneticin(G418)were obtained by screening.The result showed that 5 colonies of GS115 and 7 colonies of KM71 resistant to 4.0 mg/mL Geneticin were obtained and the expression level of rTlSP was higher in GS115 than in KM71.This recombinant TlSP reacted with the positive sera of T.luwenshuni,T.uilenbergi,T.ovis and T.annulata.3.Both recombinant TlSP protein and adjuvant(MONTANIDE ISA 61 VG)were mixed and fully emulsified.The antigen dose was 600 ?g and 300 ?g per sheep in the immune group A and B,respectively,and the control group was the equal volume of PBS.The primary immunization,second immunization and the challenge of ticks were carried out on day 0,21,and 35,respectively.The results showed that there were obvious clinical symptoms of high fever and swollen lymph nodes in the control group,while slight anemia in the immune group A and B.In the control group,immune group A and B,the piroplasms in RBC were initially found on day 20,36,and 29,respectively;the highest percentage of parasitemia was 8.6%,1.12% and 3%,respectively.The detection of specific antibody showed that the highest titer of control group,immune group A and group B was 1?8 192,1?65 536 and 1?32 768,respectively.Recombinant TlSP subunit vaccine could stimulate the proliferation of specific T lymphocytes and significantly increase the secretion of cytokines(IL-2,IL-4 and IFN-?). |
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