Fusion Expression And Evaluation Of Two T Cell Epitopes Of Mpsp Gene Of Theileria Orientalis

Theileria orientalis is a tick-borne protozoan which parasitizes in bovine erythrocytes and lymphocytes, causing high fever, anemia, weight loss and lymphadenectasis. Cattle infected with the disease often become carriers and the growth of cattle was continuously impacted.MPSP (Major piroplasm surface protein) is the immunodominant antigen of T. orientalis and it can be recognized by host immune system. However, the protein has antigenic diversity in order to evade host immune system. Therefore, vaccine prepared by single MPSP can not have the completely immune protection for host. In this study, two dominant T cell epitopes (E1, E2) of MPSP were screened. They were linked by overlap extension PCR (SOE-PCR). The fusion epitopes were cloned into the pMD-18T simple vector. Then, the prokaryotic expression vector pGEX-4T-E1-E2and the eukaryotic expression vector pVAX-E’1-E’2were constructed, respectively. E.coli BL21with the correct pGEX-4T-E1-E2was inducted by IPTG in the condition of37℃for4h. The size and reactogenicity of the expressed protein were analyzed by SDS-PAGE electrophoresis and Western blot. Coated with cationic liposome, the correct pVAX-E’1-E’2was transfected into Vero cells. The expression of two T epitopes fusion gene was detected by RT-PCR and IFA. The results of SOE-PCR showed that the size of two T cell epitopes fusion gene was approximately361bp. SDS-PAGE electrophoresis showed that the size of the expressed protein was about39ku. Western blot analysis showed that the two T epitope fusion protein has reactogenicity. RT-PCR and IFA test showed that the the two T epitope fusion gene was transiently expressed in Vero cells.48female BALB/c mice were randomly divided into6groups (n=8) used for the animal experiment: BALB/c mice in group A, B, C, D, E, F were immunized by R-fusion epitopes, R-MPSP protein, pVAX-E’1-E’2, pVAX-MPSP, pVAXl vector and PBS, respectively. The level of humoral immunity in different groups were measured by ELISA. The levels of IFN-y and IL-4were detected using mouse IFN-y and IL-4ELISA kits. The results showed that group A and group B were significantly higher than group F, but there was no significant difference between group A and group B; group C and group D were significantly higher in group E, but there was no significant difference between group C and group D in all the detection.The results indicated that cocktail vaccine can be used for the prevention of theileriosis. This research settles a theoretical foundation for the development of cocktail vaccine against T. orientalis.