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卵形鲳鲹IRF7的原核表达、纯化及其多克隆抗体制备

Prokaryotic expression and polyclonal antibody preparation of Trachinotus ovatus interferon regulatory factor IRF7

  • 摘要: IRF7在免疫调节、抗病毒、抗细菌、细胞增殖和细胞凋亡等方面均发挥着重要作用。目前关于鱼类IRF7在抗病免疫应答方面的研究相对匮乏,本研究以海南深水网箱主要养殖鱼类卵形鲳鲹为研究对象,对卵形鲳鲹IRF7(TroIRF7)进行原核表达、纯化和多克隆抗体制备。首先,构建了TroIRF7的原核表达载体E. coli BL21(DE3)/pET32a-TroIRF7,然后加入0.1 mmol·L−1的IPTG于20 ℃振荡培养24 h,诱导重组蛋白rTroIRF7表达,其次,通过镍离子亲和层析柱对重组蛋白rTroIRF7进行纯化,得到电泳纯的rTroIRF7,最后将电泳纯的rTroIRF7与弗氏(不)完全佐剂混合,然后以此混合剂免疫小鼠,获得鼠抗rTroIRF7的多克隆抗体,Elisa的实验结果显示所制备的多克隆抗体能与重组蛋白结合,抗体效价为1︰25 600。

     

    Abstract: Interferon regulatory factor 7 (IRF7) plays important roles in immune regulation, antivirus, and antisepsis, cell proliferation and cell apoptosis, however, the role of IRFs in anti-disease response remains poorly understood. In the report, the recombinant Trachinotus ovatus IRF7(rTroIRF7) protein was prepared in Escherichia coli, purified, and used to produce polyclonal antibodies. The pET32a(+)/Escherichia coli BL21(DE3) expression system was used for the inducible expression of TroIRF7. Under the conditions, 0.1 mmol·L−1 IPTG, 24 h, 20 ℃, the soluble recombinant protein rTroIRF7 was obtained. The molecular mass of the rTroIRF7 protein was 50 kDa. The rTroIRF7 protein was purified by nickel affinity chromatography. The purified fusion protein rTroIRF7 was used as an antigen to immunize mouse for the preparation of polyclonal antibody. ELISA was performed, and the results indicated that the total titer of the polyclonal antibody of rTroIRF7 was 1∶25 600.

     

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