Preparation And Preliminary Application Of Monoclonal Antibodies Specific For Bovine MCP-1

Monocyte chemoattractant protein-1(MCP-1)which mainly chemotaxes mononuclear cells and T lymphocytes,inducing monocytes and endothelial cells express adhesion molecules,making a variety of inflammatory cells especially monocytes aggregate to the lesion site,thus play an important role in the body defense,chronic inflammation and Anti-tumor is a member of the chemokine c-c subfamily.MCP-1 Monocytes in blood migrate and aggregate in kinds of diseased tissue to play an significant biological effects under the effect of specific chemokines,such as phagocytosing and killing pathogenic microorganisms,presenting antigen and secreting of many bioactive substances et..This study cloned the expression of bovine MCP-1 gene and prepared monoclonal antibody of bovine MCP-1,which provided important biomaterial for MCP-1 in the treatment of inflammatory diseases and immune diseases.1.Clone and prokaryotic expression of BoMCP-1 geneStimulating the peripheral blood lymphocytes of healthy Holstein dairy cows with Con A,extracted the total RNA of cells.RT-PCR was used to reverse transcribe cDNA,and then cDNA was used as template to amplify the not with signal peptide(BoMCP-1)and with signal peptide(BoPreMCP-1),and also amplified the GFP-BoPreMCP-1 gene.Constructed the recombinant plasmid by ligating the BoMCP-1 gene with the expression vector pET-30a(+)and pGEX-6P-1 respectively.The recombinant plasmid of ligating the BoPCPM-1 gene with the vector pVAX 1 performed double enzyme digestion and sequencing appraisal.The appraisal results analyzing by DNA star software shown correct and without any mutations.Transfered the identifiedly correct recombinant plasmid and named pET-30a-BoMCP-1,pGEX-6p-1-BoMCP-1,pVAXl-BoPreMCP-1 and pVAXl-GFP-BoPreMCP-1 respectively after successfully constructed.Then putted the prokaryotic expression vector into a host bacterium,induced expression with 0.5 mM/L IPTG and conducted analysis with SDS-PAGE.The results shown expected bands of 17 kDa and 36 kDa respectively.Through the immune affinity chromatography method for protein purification,Western blot results show that the purified protein has good immune reactivity,thus recombinant protein rHis-and rGST-BoMCP BoMCP-1-1 expressed successful.2.Preparation and preliminary application of bovine MCP-1 monoclonal antibodyThe purified recombinant protein rHis-BoMCP-1 was used to immunized 6-week-old BALB/c mice.After three immunizations,the cells were fused with lymphocyte hybridoma to purify the recombinant protein rGST-BoMCP-1 being as the detecting original.The positive clones were screened by indirect ELISA method.After three subclones,obtained three hybridoma cell lines of stably secreting specific BoMCP-1 monoclonal antibody and named as 3D10,4G9 and 6F5 respectively.Subtype identification shown that 3D10 and 4G9 monoclonal antibody were IgA subtype and 6F5 was IgG1 subtype.The determination of titer by indirect ELISA assay shown that the supernatant titer of 6F5 monoclonal antibody was 3200,and the supernatant titer of 3D 10 and 4G9 were 12800.The preparation of the above three single ascites,determination of its titer by indirect ELISA,the results shown that ascites titer of 3D 10 and 4G9 were 256000 and 512000 respectively,besides 6F5 of 32000 was lower.Indirect ELISA assay also showed that the reactivity of the 3D 10 and 4G9 monoclonal antibodies with the commercialized bovine MCP-1 expressed by Pichia pastoris was good whreas 6F5 monoclonal antibody was not good like 3D 10 and 4G9.Western blot results showed that the three monoclonal antibodies reacted with the fusion proteins rHis-BoMCP-1 and rGST-BoMCP-1.Successful construction of pVAXl-GFP-BoPreMCP-1 transfected 293T cells,analyzed reactivity of monoclonal antibody with MCP-1 protein by indirect immunofluorescence method.The results showed that reactivity of three monoclonal antibodies with GFP-BoPreMCP-1 expressed by eukaryotic was good.The reactivity of monoclonal antibody with natural cow MCP-1 protein was analyzed by flow cytometry method conbaining with intracytoplasmic staining method.First,screened and optimized flow cytometry method of three antibody:① selected two different cell stimulants ConA and LPS;② setted different antibody concentration thus obtained the a more stable results of flow cytometry method.The results showed that only 4G9 has a good reactivity with natural BoMCP-1.4G9 antibody was used to diagnose bovine tuberculosis,and a determination method of flow cytometry based on specific antigen was established.Collected bovine peripheral blood samples that have validated positive and negtive of bovine tuberculosis by TST and bovine tuberculosis y-interferon ELISA detection kit,used specific antigen ESAT-6/CFP-10 recombinant protein to stimulate bovine peripheral blood mononuclear cells and used 4G9 monoclonal antibody conducting detection of flow cytometry method.The results showed that flow cytometry method based on 4G9 has a good potential for application.