Development And Preliminary Application Of Monoclonal Antibodies Against Bovine Tumor Necrosis Factor-?

Tumor necrosis factor(TNF)is an inflammatory cytokine found by Carswell.It can directly kill tumor cells but has no obvious toxicity to normal cells.TNF-? is produced by macrophages,lymphocytes and NK cells.It has many biological functions,such as promoting cell proliferation and differentiation,killing or inhibiting tumor cells,and regulating immunity.It can not only help body resist the infection of bacteria such as E.coli and Mycobacterium tuberculosis,but also participate in the development and occurrence of autoimmune diseases with other cytokines.Bovine TNF-?,as an important index for evaluating bovine tuberculosis,bovine mastitis and other bovine diseases,has important application value and clinical significance.The aim of this study is to prepare monoclonal antibodies based on flow cytometry for detection of TNF-? in bovine-derived cells and provide important biomaterials for diagnosis and treatment of related bovine diseases1.Cloning and prokaryotic expression of BoTNF-? genePeripheral blood mononuclear cells(PBMC)of Holstein cows were isolated and then stimulated by phorbol-12-myristate-13-acetate(PMA)for 6 h.Total RNA was extracted and reversely transcripted into cDNA.The target gene BoTNF-? was amplified using the cDNA as template.The target gene was inserted into prokaryotic expression vector pET-30a(+)and pGEX-6p-1 respectively to construct recombinant expression plasmids.After confirmed by PCR,restriction enzyme digestion and sequencing,these two recombinant plasmids were respectively introduced into host bacteria BL21(DE3)and BL21,and the recombinant bacteria BL21(DE3)(pET-30a(+)-BoTNF-?)and BL21-(pGEX-6p-1-BoTNF-?)were constructed.After induced by IPTG,SDS-PAGE analysis confirmed that the sizes of the target bands of expressed proteins were 23.4 kDa and 43.4 kDa,respectively Western blot results showed that two recombinant proteins had good immunoreactivity.2.Preparation and preliminary application of monoclonal antibodies against bovine TNF-?Female BALB/c mice aged 6-8 weeks were immunized with the fusion protein rHis-BoTNF-?.After three times of immunizations,splenocytes of immunized mice were harvested,and fused with myeloma cells SP2/0.The fusion protein rGST-BoTNF-? was used as detecting antigen to screen positive clones.After several times of screening and subcloning,sixteen cell lines secreting BoTNF-? monoclonal antibody stably were obtained.They were named 1A1,1A2,1A5,1B8,1D3,2B10,2H9,2H11,3C1,3C6,3D6,3E1,3G11,4B12,4G4 and 5F3.By identifying subclasses of monoclonal antibodies,the subclasses of 1A1 and 3E1 were IgA,the subclass of 1A2 was IgG2a,the subclasses of 1A5,1B8,2H9,3C6,3D6,3G11 and 4G4 were IgG2b,and the subclasses of other monoclonal antibodies were IgGl.The titers of monoclonal antibody in cell supermatant and ascites were determined by indirect ELISA.Except the ascites titers of 1A1,3E1,2B10 and 2H11 and the supernatants titers of 1A1 and 3E1 were low,the supernatant titers of the other monoclonal antibodies were all above 1:1.0 × 104 and the ascites titers were above 1:1.0 × 107.ELISA results showed that all the sixteen monoclonal antibodies had good reactivity with the commercialized BoTNF-? protein.Specific identification results showed that sixteen monoclonal antibodies did not react with bovine IFN-? and bovine IL-2.1A1,1B8,1D3,2B10,2H9 and 4G4 also did not cross react with recombinant human TNF-?,mouse TNF-?,porcine TNF-? and sheep TNF-? protein,indicating that these six monoclonal antibodies had good specificity.1A2,1A5,2H11 3C1 and 3E1 had cross reaction with sheep TNF-?.3C6,3D6,3G11 and 4B12 had cross reaction with porcine TNF-?,sheep TNF-? and human TNF-?.5F3 had cross reaction with porcine TNF-?,sheep TNF-?,mouse TNF-? and human TNF-? protein.Western blot results showed that all sixteen monoclonal antibodies had good immunoreactivity with fusion proteins rHis-BoTNF-? and rGST-BoTNF-?.Indirect immunofluorescence results showed that 1A2,1A5,1B8,2H9,3C1,3D6,4B12,4G4 and 5F3 could well recognize the eukaryotic expression of BoTNF-?.Flow cytometry analysis showed that 1A2,1A5,2H9,2H11,3C1 and 4G4 could recognize natural BoTNF-? very well.All these results above indicate that these antibodies could have promising application potentials.Flow cytometry was successfully established by exploring the stimulant,the concentration of stimulant and antibody.The optimum stimulant was 50 ng/ml PM A combined with 1 ?g/ml ionomycin,and the optimum concentration of antibody 3C1-FITC was 1.25 ?g/ml.After infected with bovine PBMCs by Mycobacterium smegmatis,TNF-? in infected group increased significantly compared with that in non-infected group.The PBMCs of bovine tuberculosis positive cattle and bovine tuberculosis negative cattle were isolated and detected by flow cytometry after stimulation with specific antigen CFP-10/ESAT-6 fusion protein.The results showed that flow cytometry based on monoclonal antibody 3C1-FITC could be well applied to the detection of BoTNF-?.