China Animal Husbandry and Veterinary Medicine ›› 2023, Vol. 50 ›› Issue (3): 1160-1168.doi: 10.16431/j.cnki.1671-7236.2023.03.030

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Eukaryotic Expression and Subcellular Localization of Nonstructural Protein 7 of Porcine Reproductive and Respiratory Syndrome Virus

LI Huawei1, GUO Kewei2, WANG Xuying3, ZHANG Xiaoyu3, LU Ziwei3, LI Xinfeng1, MA Hui4, HOU Wenjing4   

  1. 1. Institute of Animal Product Quality and Safety Technology, Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China;
    2. College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China;
    3. College of Veterinary Medicine, Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China;
    4. College of Food and Bioengineering, Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China
  • Received:2022-08-24 Online:2023-03-05 Published:2023-03-02

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Abstract: 【Objective】 The purpose of this experiment was to express nonstructural protein 7 (NSP7) of Porcine reproductive and respiratory syndrome virus (PRRSV) in eukaryotic cells and analyze its bioinformatics,so as to speculate its function in the process of PRRSV replication.【Method】 The target gene was synthesized according to the sequence of NSP7 gene in GenBank database,and the P3×FLAG-CMV-NSP7 eukaryotic expression vector was constructed.The protein expression was verified by Western blotting after transient transfection of Marc-145 cells.The subcellular localization of NSP7 was performed by indirect immunofluorescence assay (IFA),and the physicochemical properties,structure and function of NSP7 were predicted by bioinformatics analysis software.【Result】 The eukaryotic expression vector P3×FLAG-CMV-NSP7 was successfully constructed.Western blotting results showed that the target band with a size of 2.7 ku was obtained,which confirmed that the recombinant vector was highly expressed in Marc-145 cells.IFA results confirmed that NSP7 protein was mostly distributed in the cytoplasm at the initial stage of PRRSV infection,and entered the nucleus from the cytoplasm with the extension of infection time.Bioinformatics analysis results showed NSP7 protein was encoded by 259 amino acids,the molecular formula was C1284H2020N348O379S8,the molecular weight was 28 652.75 u,the theoretical isoelectric point was 5.88,and it was an unstable and hydrophilic protein.Structural prediction found that the secondary structure of NSP7 protein included alpha helix and beta turn accounted for 35.91% and 5.41%, respectively.Nuclear localization sequence analysis showed that NSP7 protein had a transmembrane sequence RLNKKKRRRMEAVGIF.【Conclusion】 The eukaryotic expression vector of PRRSV NSP7 was successfully constructed.NSP7 was distributed in cytoplasm at the initial stage of PRRSV infection and in nucleus at the later stage of PRRSV infection.It was found that NSP7 had nuclear localization sequence.

Key words: Porcine reproductive and respiratory syndrome virus (PRRSV); nonstructural protein 7 (NSP7); subcellular localization

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