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| The Expression of Senecavirus VP2 Protein Promoted by Molecular Chaperones TF in Escherichia coli |
| YANG Rui1,2,3, RU Yi2, HAO Rong-Zeng2, LI Ya-Jun2, YANG Yang2, REN Rui-Fang2, LU Bing-Zhou2, MAO Yu-Han2, ZHANG Yue2, ZHENG Hai-Xue2, ZHANG Yong1, ZHAO Xing-Xu1,* |
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China;
2 Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences/College of Veterinary Medicine, Lanzhou University/State Key Laboratory for Animal Disease Control and Prevention, Lanzhou 730046, China;
3 China Agricultural Veterinarian Biology Science and Technology Co., Ltd., Lanzhou 730046, China |
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Abstract Senecavirus A (SVA), an emerging RNA virus in recent years, which seriously endangers the development of global pig industry. Its viral structural protein 2 (VP2) plays an important role in inducing the body's immune response. To prepare a soluble structural protein VP2 of SVA and systematically analyze its immunogenicity, firstly, the SVA-VP2 protein recombinant expression plasmid pET28a-SVA-VP2 was constructed based on the gene sequence of the SVA/CH-FJ-2017 strain, and then transferred the pET28a-SVA-VP2 recombinant plasmid and the molecular chaperones pTf16 plasmid into Escherichia coli BL21 (DE3) competent cells. The soluble recombinant SVA-VP2 protein was induced to express by using the characteristics of molecular chaperones trigger factor (TF) to promote the soluble expression of protein in the prokaryotic expression system. BALB/c mice (Mus musculus) was immunized by the purified target protein which was identified by SDS-PAGE and Western blot. Serum antibody levels was detected by indirect ELISA, and neutralizing antibody titers was measured by virus neutralization experiments, and the CD4+ and CD8+ T lymphocyte subpopulations in the spleen and the proliferation and cytokine production of spleen lymphocytes were detected by flow cytometry. The results showed that the soluble recombinant VP2 protein was successfully prepared by using molecular chaperones TF16. The recombinant VP2 protein could react specifically with SVA positive serum. The serum antibody titer level of immunized mice could reach 1:32 000, and the neutralizing antibody level could reach 1:128. The percentage of CD4+ and CD8+ T lymphocytes in the spleen of immunized mice was very extremely significantly higher than that of the control group (P<0.001), and the expression levels of interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4 and IL-10 were very extremely significantly higher than those in the control group (P<0.001). In conclusion, this study successfully prepared soluble recombinant VP2 protein by co-expression of molecular chaperones TF, and the recombinant VP2 protein had good immunogenicity both in vivo and in vitro, which can trigger strong humoral immune response and cellular immune response of the body. This study provided experimental materials and theoretical basis for the development of SVA subunit vaccine and diagnostic agent.
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Received: 13 April 2023
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Corresponding Authors:
* zhaoxx@gsau.edu.cn
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