Biotechnology Bulletin ›› 2018, Vol. 34 ›› Issue (8): 175-180.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0376

Previous Articles     Next Articles

Eukaryotic Expression of DLL4 Protein and Preparation of Polyclonal Antibody

JIN Qiao, GAN Zhi-kai, ZHOU Peng, LIU Xia   

  1. Nanchang Institute of Technology,Nanchang 330000
  • Received:2018-04-19 Online:2018-08-26 Published:2018-09-04

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