鲤春病毒血症病毒糖蛋白的原核表达及单克隆抗体的制备

Prokaryotic expression of glycoprotein and preparation of monoclonal antibody in spring viremia of carp virus(SVCV)

利用原核表达系统表达纯化鲤春病毒血症病毒(SVCV)G蛋白,制备特异性单克隆抗体,扩增出鲤春病毒0504分离株G基因片段,并将其克隆至原核表达载体pET-28a(+)中,经诱导表达、裂解离心包涵体,包涵体经His Bind亲和层析柱纯化及尿素浓度梯度复性后,作为抗原免疫Balb/c小鼠,制备单克隆抗体。结果表明:成功筛选出2株稳定分泌G蛋白单克隆抗体的杂交瘤细胞株,命名为1C5、2D5,经Western-Blot检测表明,制备的单克隆抗体具有良好的特异性。本研究中成功制备的抗G蛋白单克隆抗体,可为建立新型鲤春病毒检测方法提供工具。

The fragment of G gene isolated from spring viremia of carp virus （ SVCV, 0504） was cloned into the pET-28a（＋） vector. The aimed protein was expressed after induction and lysed by concentration,then the G inclusion bodies were purified with Ni-NTA affinity column and renaturalized by the urea dialyzation way. The Balb/c was immuned by the recombinant glycoprotein and prepared for monoclonal antibody. Two hybridoma cell lines that secreted anti-G MAb were obtained successfully, named reacted specifically with recombinant glycoprotein, which as 1C5, and 2D5. Westeru-Blot showed that the MAbs laid foundation for the establishment of the new SVCV detection methods.