猪IL-21的真核表达及其与CD40L共刺激抗体分泌的活性研究
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摘要
为研究猪白介素21 (IL-21)的生物学功能,本研究在猪IL-21基因(456 bp)的3'端加上猪Ig G Fc标签序列后,将其克隆至真核表达载体pcDNA3.1(-)中,构建重组质粒pcDNA-IL-21-Fc。将该重组质粒转染293T细胞瞬时表达后,利用protein A亲和纯化介质纯化转染上清中的目的蛋白,并检测其纯度与生物学活性。结果显示,构建的重组质粒pcDNA-IL-21-Fc能够在293T细胞中表达相对分子量约为44 ku的猪IL-21融合蛋白;亲和纯化后的猪IL-21融合蛋白能够与CD40L协同呈剂量依懒性地刺激猪外周血淋巴细胞(PBMCs)分泌Ig G。本研究为进一步探究猪IL-21在适应性免疫应答中的作用提供依据。
To investigate the biological function of porcine interleukin-21(IL-21), the porcine IL-21 sequence(456 bp) fused with porcine Ig G Fc tag at its 3' end was inserted into pcDNA3.1(-) to construct the recombinant plasmid pcDNA3.1(-)/IL-21-Fc.This recombinant plasmid was subsequently transfected into 293 T cells for transient expression and the recombinant porcine IL-21 fusion protein was purified using protein A affinity chromatography, the purity and biological activity of purified IL-21 fusion protein was determined. Results showed that recombinant plasmid pcDNA3.1(-)/IL-21-Fc was constructed and porcine IL-21 fusion protein with molecular mass of 44 ku was expressed in 293 T cells; The preliminary activity analysis of the purified recombinant porcine IL-21 protein demonstrated that the IL-21 in concert with CD40 L stimulated Ig G secretion in porcine peripheral blood lymphocytes(PBMCs) in a dose-dependent manner, indicating that the recombinant IL-21 has the biological activity. Our study provides a robust tool for further explorations of porcine IL-21 role in adaptive immune responses.
To investigate the biological function of porcine interleukin-21(IL-21), the porcine IL-21 sequence(456 bp) fused with porcine Ig G Fc tag at its 3' end was inserted into pcDNA3.1(-) to construct the recombinant plasmid pcDNA3.1(-)/IL-21-Fc.This recombinant plasmid was subsequently transfected into 293 T cells for transient expression and the recombinant porcine IL-21 fusion protein was purified using protein A affinity chromatography, the purity and biological activity of purified IL-21 fusion protein was determined. Results showed that recombinant plasmid pcDNA3.1(-)/IL-21-Fc was constructed and porcine IL-21 fusion protein with molecular mass of 44 ku was expressed in 293 T cells; The preliminary activity analysis of the purified recombinant porcine IL-21 protein demonstrated that the IL-21 in concert with CD40 L stimulated Ig G secretion in porcine peripheral blood lymphocytes(PBMCs) in a dose-dependent manner, indicating that the recombinant IL-21 has the biological activity. Our study provides a robust tool for further explorations of porcine IL-21 role in adaptive immune responses.
引文
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[6] Yeste A, Mascanfroni I D, Nadeau M, et al. IL-21 induces IL-22 production in CD4+T cells[J]. Nat Commun, 2014, 5:3753.
[7] Xue Mei, Zhao Jing, Ying Lan, et al. IL-22 suppresses the infection of porcine enteric coronaviruses and rotavirus by activating STAT3 signal pathway[J]. Antiviral Res, 2017, 142(2017):68-75.
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[9] Muneta Y, Kikuma R, Uenishi H, et al. Molecular cloning,chromosomal location, and biological activity of porcine interleukin-21[J]. J Vet Med Sci,2004, 66(3):269-275.
[10] Uddin M J, Nuro-Gyina P K, Islam M A, et al. Expression dynamics of Toll-like receptors mRNA and cytokines in porcine peripheral blood mononuclear cells stimulated by bacterial lipopolysaccharide[J]. Vet Immunol Immunopathol, 2012, 147(3-4):211-222.
[11] Ding B B, Bi E, Chen Hong-shan, et al. IL-21 and CD40L syn-ergistically promote plasma cell differentiation through upregulation of Blimp-1 in human B cells[J]. J Immunol, 2013, 190(4):1827-1836.
[12] Jue Z, Jane C, Sophia S, et al. Fusion partners as a tool for the expression of difficult proteins in mammalian cells[J]. Curr Pharm Biotechnol, 2010, 11(3):241-245.
[13] Yi J S, Du Ming, Zajac A J. A vital role vor IL-21 in the control of a chronic viral infection[J]. Science, 2009, 324(5934):1572-1576.
[14] Tang Li-bo,Chen Cheng-cong, Gao Xue-ping, et al. Interleukin-21 reinvigorates the antiviral activity of hepatitis B virus(HBV)-specific CD8+T cells in chronic HBV Infection[J]. J Infect Dis, 2019, 219(5):750-759
[15]鞠斌.白介素-21调节细胞免疫反应的研究及HIV-1单克隆广谱中和抗体的鉴定[D].北京:中国疾病预防控制中心,2016.
[2] Bryant V L, Ma C S, Avery D T, et al. Cytokine-mediated regulation of human B cell differentiation into Ig-secreting cells:Predominant role of IL-21 produced by CXCR5+T follicular helper cells[J]. J Immunol, 2007, 179(12):8180-8190.
[3] Ettinger R, Sims G P, Fairhurst A M, et al. IL-21 induces differentiation of human naive and memory B cells into antibody-secreting plasma cells[J]. J Immunol, 2005, 175(12):7867-7879.
[4] Kuchen S, Robbins R, Sims G P, et al. Essential role of IL-21in B cell activation, expansion, and plasma cell generation during CD4+T cell-B cell collaboration[J]. J Immunol, 2007, 179(9):5886-5896.
[5] Spolski R, Leonard W J. Interleukin-21:a double-edged sword with therapeutic potential[J]. Nat Rev Drug Discov, 2014, 13(5):379-395.
[6] Yeste A, Mascanfroni I D, Nadeau M, et al. IL-21 induces IL-22 production in CD4+T cells[J]. Nat Commun, 2014, 5:3753.
[7] Xue Mei, Zhao Jing, Ying Lan, et al. IL-22 suppresses the infection of porcine enteric coronaviruses and rotavirus by activating STAT3 signal pathway[J]. Antiviral Res, 2017, 142(2017):68-75.
[8]应兰,薛美,李麟,等.猪IL-22的原核表达及其活性研究[J].中国预防兽医学报,2016, 38(06):489-492.
[9] Muneta Y, Kikuma R, Uenishi H, et al. Molecular cloning,chromosomal location, and biological activity of porcine interleukin-21[J]. J Vet Med Sci,2004, 66(3):269-275.
[10] Uddin M J, Nuro-Gyina P K, Islam M A, et al. Expression dynamics of Toll-like receptors mRNA and cytokines in porcine peripheral blood mononuclear cells stimulated by bacterial lipopolysaccharide[J]. Vet Immunol Immunopathol, 2012, 147(3-4):211-222.
[11] Ding B B, Bi E, Chen Hong-shan, et al. IL-21 and CD40L syn-ergistically promote plasma cell differentiation through upregulation of Blimp-1 in human B cells[J]. J Immunol, 2013, 190(4):1827-1836.
[12] Jue Z, Jane C, Sophia S, et al. Fusion partners as a tool for the expression of difficult proteins in mammalian cells[J]. Curr Pharm Biotechnol, 2010, 11(3):241-245.
[13] Yi J S, Du Ming, Zajac A J. A vital role vor IL-21 in the control of a chronic viral infection[J]. Science, 2009, 324(5934):1572-1576.
[14] Tang Li-bo,Chen Cheng-cong, Gao Xue-ping, et al. Interleukin-21 reinvigorates the antiviral activity of hepatitis B virus(HBV)-specific CD8+T cells in chronic HBV Infection[J]. J Infect Dis, 2019, 219(5):750-759
[15]鞠斌.白介素-21调节细胞免疫反应的研究及HIV-1单克隆广谱中和抗体的鉴定[D].北京:中国疾病预防控制中心,2016.