DLL4蛋白真核表达及多克隆抗体制备

doi:10.13560/j.cnki.biotech.bull.1985.2018-0376

摘要/Abstract

摘要： 该研究旨为克隆、表达和纯化出人源DLL4蛋白,并制备出相对应的多克隆抗体。采用搭桥PCR,构建带有tPA信号肽的DLL4基因,并克隆到pGZX表达载体上,经过酶切和测序鉴定后,瞬时转染到HEK293F细胞中,转染48 h后对发酵上清液进行镍柱亲和纯化。利用Dot blotting、Western blotting和Fortebio大分子互作仪检测DLL4的生物活性;免疫兔子制备DLL4多克隆抗体,采用ELISA法检测抗体效价,Western blotting检测抗体特异性。PCR扩增出1.6 kb的tPA-DLL4-6His目的片段,测序结果表明,与NCBI数据库中的DLL4序列（NM_019074.3）相同。一步亲和纯化后,得到纯度为95%的DLL4蛋白,Dot blotting和Western blotting检测到发酵上清液中含有DLL4蛋白,DLL4蛋白和DLL4抗体之间的亲和力在1.848E-10,R²= 0.999,ELISA法获得DDL4抗体效价为1∶12 800,Western blotting检测DLL4抗体具有良好的特异性。成功制备出具有免疫原性的DLL4蛋白及其多克隆抗体。

关键词: DLL4, 真核表达, 亲和纯化, 多克隆抗体, 大分子互作仪

Abstract: This work is to clone,express and purify human DLL4 protein and to prepare corresponding polyclonal antibody. Bridging PCR was employed to construct a DLL4 gene with tPA signal peptide,and the gene then was cloned into pGZX expression vector. After identification by enzyme digestion and sequencing,it was transiently transfected into HEK293F cells. At 48 h after transfection,the supernatant was purified by nickel column affinity purification. Dot blotting,Western blotting,and Fortebio macromolecules were used to measure the biological activity of DLL4. Then,rabbits were immunized to prepare DLL4 polyclonal antibody. Antibody titer was detected by ELISA and specificity of antibody was tested by Western blotting. A 1.6 kb tPA-DLL4-6His target fragment was amplified by PCR and the sequencing result showed that it was consistent with DLL4 sequence（NM_019074.3）in the NCBI database. After one-step affinity purification,DLL4 protein with a purity of 95% was obtained. Dot blotting and Western blotting detected that the fermentation supernatant contained DLL4 protein,and the affinity between DLL4 protein and DLL4 antibody was 1.848E-10 and R² was 0.999. The titer of DDL4 antibody by ELISA was 1∶12 800,and the DLL4 antibody had solid specificity. In conclusion,the immunogenic DLL4 protein and its polyclonal antibody are successfully prepared.

Key words: DLL4, eukaryotic expression, affinity purification, polyclonal antibody, macromolecule interaction instrument

金巧, 甘志凯, 周鹏, 刘霞. DLL4蛋白真核表达及多克隆抗体制备[J]. 生物技术通报, 2018, 34(8): 175-180.

JIN Qiao, GAN Zhi-kai, ZHOU Peng, LIU Xia. Eukaryotic Expression of DLL4 Protein and Preparation of Polyclonal Antibody[J]. Biotechnology Bulletin, 2018, 34(8): 175-180.

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