| Tumor necrosis factor(TNF)is an important inflammatory cytokine and mainly secreted by macrophages and monocytes.It is the only cytokine known to exert direct biological activity on tumor cells,not only promoting cell proliferation and differentiation but also killing and inhibiting tumor cells.The functions and detection methods of mammalian TNF-α under physiologically pathological conditions were extensively studied.However,there are few studies on avian TNF-α.Until recently,the authentic chicken TNF-α(chTNF-α)gene was cloned and confirmed.but the function of chTNF-α and its detection methods are still unknown and missing.In this study,the chTNF-α extracellular region was codon-optimized,synthesized and cloned into the expression vector pET-45b(+)and pcDNA3.1(+)and expressed recombinant His-chTNF-α protein.Using this protein as immunogen,mouse and rabbit anti-chTNF-α monoclonal(mAb)and polyclonal(pAb)antibodies were generated,pcDNA3.1(+)-His-chTNF-α was transfected into COS7 cells,screening of mAb that can identify eukaryotic expression of chTNF-α protein by indirect immunofluorescence(IFA).Lastly an antigen-capture ELISA for chTNF-α was developed.Our study provides important immunological tools for future research.The main research and results are as follows:1.Vector construction and recombinant expression of chTNF-αAccording to the published chTNF-α gene sequence(GenBank#:MF000729),the extracellular region was codon-optimized,synthesized and cloned into the expression vector pET-45b(+)and pcDNA3.1(+).The recombinant plasmids were confirmed by sequencing.The pET-45b(+)-His-chTNF-α vector was transformed into E.coli Rosetta strain.After induction with IPTG,SDS-PAGE and western-blot(WB)analysis showed that the recombinant chTNF-α was expressed successfully as a soluble form.The expression conditions were optimized to be induction with a final concentration of 0.1 mM IPTG for 12 h at 18℃.The recombinant protein with a mass of 26 kD were purified by Ni-NTA affinity chromatography,with a yield of 8.3 mg/L.The eukaryotic expression vector pcDNA3.1(+)-His-chTNF-α was transfected into COS7 cell and the expression of chTNF-α protein was confirmed by indirect immunofluorescence(IFA)and WB.2.Development of monoclonal and polyclonal antibodies against chTNF-αThe recombinant His-chTNF-α protein was used to immunize rabbits to produce pAb against chTNF-α protein.After three immunizations,the titer of rabbit anti-chTNF-α pAb reaches 1:51200.Six-week-old BALB/c mice were immunized with the same strategy to generate mouse anti-chTNF-α mAb.Hybridoma cells were screened by IFA and indirect ELISA.Ten hybridoma cell lines that are capable of secreting mAbs against chTNF-α were obtained,named as 1A11,1C8,1G1,1C11,1C12,1E5,2B5,3E9,3D4,5D10.The analysis of immunoglobulin subclasses and isotype of mAbs showed that mAb 1C11 1C8 and 3D4 were IgG2a,1G1 was IgM and the others were IgG1.The ascites titers of these mAbs deter mined by indirect ELISA range from 1.6×10-4 to 1.02×10-6.WB and IFA results showed that all the mAbs had good reactivity with the prokaryotic and eukaryotic chTNF-α protein.Constructed chTNF-α-100-179aa,chTNF-α-159-233aa,chTNF-α-195-285aa to pcDNA3.1(+)and transfected into Cos7 cells in order to identify the epitopes recognized by these mAbs with IFA,the results showed that all the mAbs recognized the region of 195-285aa of chTNNF-α.A peptide of 167-196aa chTNF-α was synthesized,and tested for its reactivity with each mAb by Dot-ELISA,the results showed that none of the mAbs was reactive with this peptide.3.Establishment of an antigen-capture ELISA for the detection of chTNF-αUsing immunoglobulin affinity chromatography,rabbit anti-chTNF-α pAb and mouse anti-chTNF-α mAb were purified.An antigen-capture ELISA for meature chTNF-α was developed using the purified pAb as a capture antibody and the mAb 3E9 as a detection antibody.The optimal concentration of capture antibody and detection antibody was deter mined to be 10 ug/mL and 1:400 by chessboard method.According to the standard curve established by gradient dilution of recombinant chTNF-α,the detection limit of this antigen-capture ELISA was lng/mL.This method can detect a small amount of natural chTNF-α protein expression in the spleen of chicken stimulated by PMA/Ionomycin. |
