China Animal Husbandry and Veterinary Medicine ›› 2023, Vol. 50 ›› Issue (7): 2923-2930.doi: 10.16431/j.cnki.1671-7236.2023.07.032

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Prokaryotic Expression and Polyclonal Antibody Preparation of Bluetongue Virus NS4 Protein

LUO Shimei1, ZHAO Yao1,2, MA Xianping1, ZHANG Jinyang3, YI Huashan1   

  1. 1. College of Veterinary Medicine, Southwest University, Rongchang 402460, China;
    2. Honghe Center for Animal Disease Prevention and Control of Yunnan Province, Mengzi 661199, China;
    3. Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China
  • Received:2022-12-12 Published:2023-06-30

Abstract: 【Objective】 The purpose of this study was to prepare polyclonal antibody against Bluetongue virus (BTV) NS4 protein and lay a foundation for studying the role of BTV NS4 gene in virus proliferation and antagonizing the innate immune response of host cells.【Method】 The pCzn1-NS4 prokaryotic expression vector was constructed, and the positive plasmid was transformed into Escherichia coli BL21(DE3) competent cells after identification by PCR and sequencing.The recombinant NS4 protein was induced by IPTG and purified with His-tag Ni-sepharose purification, and then identified by SDS-PAGE and Western blotting.Then, New Zealand White rabbits were immunized with the purified protein to prepare its polyclonal antibody.The titer of polyclonal antibody was determined by SDS-PAGE and indirect ELISA.The expression of NS4 protein in BHK-21 cells infected with BTV was detected by indirect immunofluorescence assay (IFA) using NS4 polyclonal antibody as the primary antibody.【Result】 The prokaryotic expression plasmid pCzn1-NS4 was successfully constructed.At 37 ℃ and the induced concentration of IPTG was 0.6 mmol/L, a large amount of recombinant NS4 protein was obtained when induced 8 h.SDS-PAGE and Western blotting results showed that the molecular weight of the recombinant protein was about 18 ku, which could detect by rabbit His-tag antibody.The titer of polyclonal antibody prepared by immunizing New Zealand white rabbits was more than 1:512 000 detected by indirect ELISA.SDS-PAGE and IFA results showed that the polyclonal antibody had good purity and could specifically recognize the NS4 protein expressed in BHK-21 cells infected by BTV.【Conclusion】 In this study, a specific polyclonal antibody was prepared using the prokaryotic expression of NS4 protein, and the polyclonal antibody was preliminarily used for the IFA, which provided a powerful tool for the function and application study of BTV NS4 gene.

Key words: Bluetongue virus (BTV); NS4 protein; prokaryotic expression; polyclonal antibody

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