China Animal Husbandry and Veterinary Medicine ›› 2021, Vol. 48 ›› Issue (2): 631-639.doi: 10.16431/j.cnki.1671-7236.2021.02.025

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Expression of Pseudorabies Virus gE Protein and Preparation of Its Polyclonal Antibody

QU Zhehui1, ZHAO Yu1, ZHANG Xiwen1, LIAN Huixiang1,2, ZHAO Yunhuan1,2,3, CHEN Bin1, GUO Xiaoqiu1   

  1. 1. College of Animal Science and Veterinary Medicine, Xinyang Agriculture and Forestry University, Xinyang 464000, China;
    2. Engineering Research Center for Health Production of Ecological Livestock and Poultry of Henan Dabieshan District, Xinyang 464000, China;
    3. Xinyang Key Laboratory of Animal Husbandry and Environmental Control, Xinyang 464000, China
  • Received:2020-08-10 Online:2021-02-20 Published:2021-02-23

PDF (PC)

69

Abstract: The purpose of this study was to obtain recombinant PRV gE protein and polyclonal antibody against PRV gE protein.The PRV gE gene was amplified from PRV-infected pig tissues (lung,brain and tonsil) by PCR,and then ligated to the cloning vector pMD18-T (pMD-gE) for sequencing and phylogenetic tree analysis.Using pMD-gE as a template,part of the gene encoding gE protein extramembrane domain (gE-outside) was amplified by PCR,then connected to the prokaryotic expression vector pET-30a(+),transformed into E.coli DH5α competent cell to construct recombinant plasmid pET-gE-outside.The recombinant plasmid pET-gE-outside was transformed into the E.coli RosettaTM(DE3) pLysS competent cell.After induction by IPTG,the expressed product was analyzed and identified by SDS-PAGE and Western blotting,respectively.The recombinant PRV gE protein was purified by affinity chromatography and used to immunize mice.The titers measure and identification of the mouse anti-PRV gE polyclonal antibody in the serum from the immunized mice after the third immunization for 2 weeks were detected by indirect ELISA and Western blotting,respectively.The PCR and sequencing results showed that the PRV gE gene was successfully cloned in this study,which belonged to the same branch as the domestic virus strain after 2011.The SDS-PAGE and Western blotting results confirmed that the PRV gE-outside protein was correctly expressed by the prokaryotic expression system,with a molecular weight of about 55 ku,and could react with porcine anti-PRV polyclonal antibody.The concentration of gE-outside protein purified by affinity chromatography was 1.23 mg/mL.3 weeks after immunizing mice with gE-outside protein,the mouse anti-PRV gE-outside polyclonal antibody was prepared with a titer of 1:204 800,and had an immune reaction with gE-outside protein.In this study,recombinant PRV gE protein and mouse anti-PRV gE polyclonal antibody were prepared,which could provide technical guidance and material for the study on the mechanism of PRV infection and the establishment of rapid and efficient immunological detection technology.

Key words: Pseudorabies virus (PRV); gE protein; prokaryotic expression; polyclonal antibody

CLC Number: