Med. Weter. 81 (11), 581-589, 2025
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| KAIWEI ZHAO, DIANGANG HAN, TONG REN, LINJUAN XIE, SHANGLIAN YIN, YANLIN LI, YUANYI YAN, RUI LI, JUN AI, JIGE XIN |
| Bioinformatics Analysis and Expression of Erns Protein of Bovine Viral Diarrhea Virus |
| To understand the immunogenicity and biological functions of the Erns protein of Bovine Viral Diarrhea Virus (BVDV) and express its protein, bioinformatics software was initially used for the analysis of BVDV Erns protein (physical and chemical properties, hydrophilicity, B-cell antigen epitopes, transmembrane regions, signal peptides, secondary and tertiary structures). Subsequently, using BVDV nucleic acid as a template, the BVDV-Erns gene was amplified by PCR and cloned into the pMD18-T vector, then transformed into DH5α competent cells to obtain the T-B-Erns recombinant plasmid. After that, both the T-B-Erns plasmid and the pFastBacHTB vector were subjected to double digestion with EcoR I and Hind III, and after ligation, they were again transformed into DH5α competent cells to obtain the H-B-Erns recombinant plasmid. After verification through double digestion, the H-B-Erns plasmid was transformed into DH10Bac competent cells to construct the recombinant shuttle plasmid B-B-Erns. After validation with M13 primer sequencing, it was transfected into Sf9 cells for expression and amplification of the target gene. The expressed recombinant protein was analyzed by Western blot. Results showed that the theoretical molecular weight of the BVDV Erns protein is 25,658.04 Da, with an isoelectric point of 7.77, classifying it as a stable protein. It exhibits strong hydrophilicity. Predictions on transmembrane domains and signal peptide analysis indicate that it is not a channel protein and lacks signal peptide characteristics. Prediction of antigenic epitopes revealed 11 dominant B-cell epitopes, suggesting potential application value in antibody production. Secondary structure analysis indicates that the Erns protein consists mainly of α-helices (41.85%) and random coils (39.21%), supplemented by β-sheets (6.61%) and extended strands (12.33%). The tertiary structure model of the Erns protein matches well with the secondary structure analysis. The Erns protein expressed through the baculovirus system shows a target band around 32 kDa, indicating successful expression of the BVDV-Erns protein, which possesses good immunogenicity, providing a theoretical foundation and material support for further research on this protein and preparation of antibodies.. |
| Key words: Bovine viral diarrhea virus, bioinformatic analysis, baculovirus expression; Erns protein; |