| Tropical theileriasis,which transmitted Theileria annulata by infecting cattle,occurs around the North Africa,southern Europe,Asia.In our country,it mainly occurs in arid and semi-arid regions in the north.If it is not treated in time,the mortality rate is as high as 80%,causing serious economic losses to the animal husbandry.In recent research,TaPIN1 is a prolyl isomerase,which maintains host leukocyte transformation secreted by Theileria parasites.TaPIN1 interacts with the host ubiquitin ligase FBW7 resulting in degradation by its ubiquitination.If the function of FBW7 is lost,the expression of negative regulators Jun/Fos family proteins will appear to rise,which promote the ability of infinite proliferation and infection of host cells.According to the pathogenesis of the Tropical theileriasis,it is pointed the importantce of host leukocyte transformation by Theileria annulata.It's possible to provide new ideas and possibilities for prevention and treatment of the disease if we can dock this pathway.Therefore,it is necessary to analyze the structure of TaPIN1 and FBW7 complex to develop specific drugs for the parasite.The TaPIN1 and FBW7 were constructed by a standard protocol using PCR and combined with the expression vector PPGH.To study its structure and function,we tried to express TaPIN1 and FBW7 protein with E.coli expression system,which proved by PPase.We successfully cloned TaPIN1 and FBW7 and then applied affinity chromatography,ion-exchange chromatography and size-exclusion chromatography to purify the protein.We used the most common methods for protein crystallization,hanging drop to set up trays for large screen.The initial screening kit comes from three different companies:Hampton Research,Emerald Bio Systems and Molecular Dimensions,which compensate for the deficiency of crystal growth conditions.Finally,we obtained crystallization for TaPIN1(22-142)protein after a lot of initial screening,which will be an important step for future study. |
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