Cloning And Expression Of Caprine IL-1β

One of the most serious disease in caprine is mastitis, it can make serious economic losses by decrease the output and the quality of milk. It's reported that Th17 cells play a important role in the clearance of mucosal infections, so we speculate that Th17 cells may participate in mediate the mastitis. Compare to Th1 cells and Th2 cells, Th17 cells have it's special differentiation way. Recent research indicate that human and mouse Th17 cells have different differentiation way, the CD4~+T cells require IL-6 and TGF-βto differentiate into Th17 cells in mouse, but in human,IL-1βplus IL-6 or IL-1βplus IL-23 is required for Th17 cell generation from CD4~+T cells. But we know nothing about Th17 cells differentiation on other animals. In order to illustrate the role of IL-1βin the development of caprine Th17 cells, we cloned and expressed caprine IL-1β, the detail results are as follows:1. Total RNA was extracted from caprine peripheral blood lymphocyte, after RT-PCR we got 759 bp fragment, connect the fragment with pGEM-Teasy vector , Sequence analysis showed that G mutationed to A, but this is nonsense mutation. the sequence analysis suggested that caprine IL-1βwas cloned successfully.2. The CaIL-1βwas cloned into the pET-32a vector, recombinant plasmid pET-32a- CaIL-1βwas transformed into BL21.Induce the positive clone to express target protein.SDS-PAGE assay showed that the best expression condition was 0.3 mmol / L IPTG, 5 h. Most expressed protein are inclusion bodies, only a little protein are soluble protein. bioassay suggested that the soluble protein expressed by E.coli have bioactivity.3. The fragment of CaIL-1βwas subcloned into baculovirus vector pFastBac-HTb, recombinant transfer vector pFastBac-HTb-CaIL-1βwas transformed into DH10 Bac, by site-specifc transposition, CaIL-1βgene was integrated into Bacmid, then we got recombinant shuttle vector Bacmid-CaIL-1β. Transfect Sf9 cells with shuttle vector got reBac-CaIL-1β. Western blot assay suggested that the recombinant CaIL-1βexsits in the supernatant of reBac-CaIL-1βinfected Sf9 cells .