| IP-10 is one kind of chemokine that secreted by antigen presenting cells that stimulated by cytokines, belong to the CXC chemokine family. IP-10 can selectively raise T lymphocytes, natural killer cells and monocytes to injury sites in infectious diseases, autoimmune diseases, allograft rejection, tumor, chronic inflammatory diseases, and play an important roles in host immune protection. Recent research has shown the change of IP-10 expression level related to infectious diseases, the diseases included inflammatory bowel disease, immune dysfunction and the development of tumor. IP-10 is also used as the important biological marker in the diagnosis of bovine tuberculosis and other infectious diseases, and has the similar effect compared with IGRAs. Studies have shown that joint detection of IFN-y and IP-10 could improve the diagnosis sensitivity of bovine tuberculosis (bTB). Therefore, in this study, we cloned and expressed BoIP-10 gene and prepared monoclonal antibodies (mAbs) against BoIP-10 protein, which provide an important biological materials for the diagnosis of bTB.1. Cloning and prokaryotic expression of BoIP-10 geneBoIP-10 gene was amplified by RT-PCR with the template of total RNA isolated from ConA-stimulated peripheral blood lymphocytes of healthy cow. The amplified fragment was inserted into prokaryotic expression vector pGEX-6p-1 and pET-30a(+) respectively. The recombinant plasmids were separately transformed into E.coli BL21. After induced by IPTG, the expression products of positive recombinants were analyzed by SDS-PAGE. Results of SDS-PAGE showed that the fusion were rGST-BoIP-10 and rHis-BoIP-10 were expressed successfully with a relative molecular weight about 36 kDa and 16 kDa, respectively. The expression products were purified by GST and His*Bind affinity chromatography. Results of Western Blot showed that the recombinant proteins had good immunoreactivity. These results confirm that two labels of recombinant proteins rGST-BoIP-10, rHis-BoIP-10 were expressed successfully.2. Preparation and identification of mAbs for BoIP-10The six weeks of BALB/c mice were immunized with the purified protein rHis-BoIP-10. After three times of immunizations, the cell fusion was conducted using the lymphocyte hybridoma technology. Putting the purified protein rGST-BoIP-10 as detecting antigen, indirect ELISA was used to screen positive clones of fusion cells secreting specific antibodies. After two times of subcloning, we obtained 8 hybridoma cell lines secreting mAbs against BoIP-10, theses mAbs were named 3E11,2A5,1C6,3B12,3E12,2D5,2H8 and 3A12, respectively. Their subtypes were identified that 2A5 and 3E11 were IgM,3E11 and 2D5 were IgG2a, 2H8 was IgG3,1C6,3B12 and 3A12 were IgGl. The indirect ELISA results showed that the supernatant titers of 3E11,2A5,3B12,3E12 and 2H8 were not very high, but the remaining 3 mAbs were higher than 1.0 ×104. The ascites titers of 7 mAbs were higher than 1.0×106 besides 2A5. It was also shown that 3E11,2A5,2D5 and 2H8 reacted well with commercialized IP-10 expressed in E.coli system.2A5,2D5 and 2H8 reacted well with commercialized IP-10 expressed in Yeast system. Western blot assay demonstrated that all 8 mAbs reacted with fusion protein rGST-BoIP-10. The FACS analysis documented that 3E11,2D5 and 3A12 reacted with natural BoIP-10. The inhibition ELISA results further confirmed that 2D5 reacted with natural BoIP-10. The results suggested that 2D5 has a good application potential. |
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