牛肿瘤坏死因子α小鼠单克隆抗体的制备
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摘要
目的制备抗牛肿瘤坏死因子α(BoTNF-α)的单克隆抗体(mAb)。方法以原核系统表达的带有组蛋白(His)标签的重组牛TNF-α蛋白(rHis-BoTNF-α)为免疫原,带有谷胱甘肽硫转移酶(GST)标签的重组牛TNF-α蛋白(rGST-BoTNF-α)为检测原,采用杂交瘤细胞技术制备mAb。Western blot法检测mAb与rHis-BoTNF-α和rGST-BoTNF-α的反应性,间接ELISA检测mAb效价、特异性以及mAb与商品化重组BoTNF-α的反应性,流式细胞术分析mAb识别天然抗原的活性。结果成功筛选出7株稳定分泌抗rBoTNF-αmAb的杂交瘤细胞株。结果证明7株mAb均具有良好的反应性及特异性。流式细胞术分析显示mAb 4G4具有良好的识别天然抗原的活性。结论成功制备了抗BoTNF-α的mAb。
Objective To prepare monoclonal antibodies(mAbs) against bovine tumor necrosis factor-alpha(BoTNF-α).Methods Recombinant BoTNF-α with His tag(rHis-BoTNF-α) was expressed in a prokaryotic system as immunogen,and recombinant BoTNF-α with GST tag(r GST-BoTNF-α) was expressed as detection antigen. The mAbs were developed by hybridoma cell technology. The reactivity of mAbs with r His-BoTNF-α and rGST-BoTNF-α was detected by Western blot analysis. Indirect ELISA was used to detect the titer and specificity of mAbs, and the reactivity to commercialized recombinant BoTNF-α(rBoTNF-α) was also evaluated. The reactivity of mAbs against natural antigens was identified by flow cytometry. Results Seven hybridomas stably secreting anti-rBoTNF-α mAbs were successfully obtained. The results showed that all seven mAbs had good reactivity and specificity. Flow cytometry analysis also showed that mAb 4G4 had good reactivity with natural BoTNF-α antigens. Conclusion The anti-rBoTNF-α mAbs are successfully achieved.
Objective To prepare monoclonal antibodies(mAbs) against bovine tumor necrosis factor-alpha(BoTNF-α).Methods Recombinant BoTNF-α with His tag(rHis-BoTNF-α) was expressed in a prokaryotic system as immunogen,and recombinant BoTNF-α with GST tag(r GST-BoTNF-α) was expressed as detection antigen. The mAbs were developed by hybridoma cell technology. The reactivity of mAbs with r His-BoTNF-α and rGST-BoTNF-α was detected by Western blot analysis. Indirect ELISA was used to detect the titer and specificity of mAbs, and the reactivity to commercialized recombinant BoTNF-α(rBoTNF-α) was also evaluated. The reactivity of mAbs against natural antigens was identified by flow cytometry. Results Seven hybridomas stably secreting anti-rBoTNF-α mAbs were successfully obtained. The results showed that all seven mAbs had good reactivity and specificity. Flow cytometry analysis also showed that mAb 4G4 had good reactivity with natural BoTNF-α antigens. Conclusion The anti-rBoTNF-α mAbs are successfully achieved.
引文
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[24] Gondaira S,Higuchi H,Iwano H,et al.Innate immune response of bovine mammary epithelial cells to Mycoplasma bovis[J].J Vet Sci,2018,19(1):79-87.
[25] Kwong L S,Thom M,Sopp P,et al.Production and characterization of two monoclonal antibodies to bovine tumour necrosis factor alpha (TNF-alpha) and their cross-reactivity with ovine TNF- alpha [J].Vet Immunol Immunopathol,2010,135(3/4):320-324.
[26] Paape M J,Rautiainen P M,Lilius E M,et al.Development of anti-bovine TNF-αlpha mAb and ELISA for quantitating TNF-αlpha in milk after intramammary injection of endotoxin[J].J Dairy Sci,2002,85(4):765-773.
[2] Bystrom J,Clancy F I,Taher T E,et al.TNF-α in the regulation of Treg and Th17 cells in rheumatoid arthritis and other autoimmune inflammatory diseases[J].Cytokine,2018,101:4-13.
[3] Jung M K,Lee J S,Kwak J E,et al.Tumor necrosis factor and regulatory T cells[J].Yonsei Med J,2019,60(2):126-131.
[4] Cludts I,Cleuter Y,Kettmann R,et al.Cloning and characterization of the tandemly arranged bovine lymphotoxin and tumour necrosis factor-alpha genes[J].Cytokine,1993,5(4):336-341.
[5] Qu Y,Zhao G,Li H.Forward and reverse signaling mediated by transmembrane tumor necrosis factor-alpha and TNF receptor 2:potential roles in an immunosuppressive tumor microenvironment[J/OL].Front Immunol,2017,8:1675.DOI:10.3389/fimmu.2017.01675.eCollection 2017.
[6] Elnaggar M M,Abdellrazeq G S,Elsisy A,et al.Evaluation of antigen specific interleukin-1β as a biomarker to detect cattle infected with Mycobacterium bovis[J].Tuberculosis (Edinb),2017,105:53-59.
[7] 郭金玉,张鹤晓,吴丹,等.牛肠道病毒2型单克隆抗体的制备及其应用研究[J].畜牧兽医学报,2015,46(11):2025-2031.Guo J,Zhang H,Wu D,et al.Preparation and application of the monoclonal antibodies against bovine enterovirus type 2[J].Xu Mu Shou Yi Xue Bao,2015,46(11):2025-2031.
[8] Kunz D J,Gomes T,James K R.Immune cell dynamics unfolded by single-cell technologies[J/OL].Front Immunol,2018,9:1435.DOI:10.3389/fimmu.2018.01435.eCollection 2018.
[9] 艾能,孟闯,徐正中,等.结核分枝杆菌TB10.4真核表达质粒的构建及免疫特性鉴定[J].扬州大学学报(农业与生命科学版),2016,37(2):1-6.Ai N,Meng C,Xu Z,et al.Construction of eukaryotic vectors expressing Mycobacterium tuberculosis TB10.4 and its immunoproperties[J].Yang Zhou Da Xue Xue Bao (Nong Ye Yu Sheng Ming Ke Xue Ban),2016,37(2):1-6.
[10] 陈祥,张成全,徐正中,等.重组牛白细胞介素4的原核表达及其单克隆抗体研制[J].细胞与分子免疫学杂志,2011,27(6):653-656.Chen X,Zhang C,Xu Z,et al.Prokaryotic expression of recombinant bovine IL-4 and development of monoclonal antibodies against bovine IL-4[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2011,27(6):653-656.
[11] 张成全,陈祥,牛中伟,等.重组牛γ-干扰素单克隆抗体的研制[J].中国动物传染病学报,2009,17(4):15-19.Zhang C,Chen X,Niu Z,et al.Development of monoclonal antibodies against recombinant bovine interferon-γ[J].Zhong Guo Dong Wu Chuan Ran Bing Xue Bao,2009,17(4):15-19.
[12] Maggioli M F,Palmer M V,Thacker T C,et al.Increased TNF-α/IFN-γ/IL-2 and decreased TNF-α/IFN-γ production by central memory T cells are associated with protective responses against bovine tuberculosis following BCG vaccination[J/OL].Front Immunol,2016,7:421.DOI:10.3389/fimmu.2016.00421.
[13] Mehta A K,Gracias D T,Croft M.TNF activity and T cells[J].Cytokine,2018,101:14-18.
[14] Tracey K J,Cerami A.Tumor necrosis factor:a pleiotropic cytokine and therapeutic target[J].Annu Rev Med,1994,45:491-503.
[15] Aggarwal B B.Natarajan K.Tumor necrosis factors:developments during the last decade[J].Eur Cytokine Netw,1996,7(2):93-124.
[16] Bemelmans M H,van Tits L J,Buurman W A.Tumor necrosis factor:function,release and clearance[J].Crit Rev Immunol,1996,16(1):1-11.
[17] 余娟,徐彦召,刘兴友.流式细胞术在兽医临床上的应用研究[J].现代农业科技,2018,731(21):249-250.Yu J,Xu Y,Liu X.Application research of flow cytometry in veterinary clinic[J].Xian Dai Nong Ye Ke Ji,2018,731(21):249-250.
[18] Soares R,Martins V C,Macedo R,et al.Go with the flow:advances and trends in magnetic flow cytometry[J].Anal Bioanal Chem,2019,411(9):1839-1862.
[19] Gascue A,Merino J,Paiva B.Flow cytometry[J].Hematol Oncol Clin North Am,2018,32(5):765-775.
[20] Imanishi J.Expression of cytokines in bacterial and viral infections and their biochemical aspects[J].J Biochem,2000,127(4):525-530.
[21] Benoit M,Desnues B,Mege J L.Macrophage polarization in bacterial infections[J].J Immunol,2008,181(6):3733-3739.
[22] Gondaira S,Higuchi H,Iwanob H,et al,Cytokine mRNA profiling and the proliferative response of bovine peripheral blood mononuclear cells to Mycoplasma bovis[J].Vet Immunol Immunopathol,2015,165(1/2):45-53.
[23] Gao R,Yang H,Jing S,et al.Protective effect of chlorogenic acid on lipopolysaccharide-induced inflammatory response in dairy mammary epithelial cells[J].Microb Pathog,2018,124:178-182.
[24] Gondaira S,Higuchi H,Iwano H,et al.Innate immune response of bovine mammary epithelial cells to Mycoplasma bovis[J].J Vet Sci,2018,19(1):79-87.
[25] Kwong L S,Thom M,Sopp P,et al.Production and characterization of two monoclonal antibodies to bovine tumour necrosis factor alpha (TNF-alpha) and their cross-reactivity with ovine TNF- alpha [J].Vet Immunol Immunopathol,2010,135(3/4):320-324.
[26] Paape M J,Rautiainen P M,Lilius E M,et al.Development of anti-bovine TNF-αlpha mAb and ELISA for quantitating TNF-αlpha in milk after intramammary injection of endotoxin[J].J Dairy Sci,2002,85(4):765-773.