Abstract
Porcine parvovirus (PPV) virus-like particles (VLPs) are a potential vaccine candidate for the prevention of parvovirus-induced reproductive failure in pregnant sows. Currently, the Escherichia coli (E. coli) expression system is the most cost-efficient to express recombinant proteins. To overcome the limitations of protein misfolding and to prepare soluble highly bioactive antigen and high yields of protein, we optimized the PPV-VP2 gene, subcloned it into pET24a, pET26b, pET28a, and pET30a, and transformed it into E. coli BL21(DE3)-Tf16 competent cells. The pET28a plasmid was selected for further manipulations because it expressed high levels of the bioactive PPV-VP2 protein. Under optimal high-density fermenting conditions in a 70-L fermenter, the total yield of wet weight E. coli cells was 124.86 g/L, and PPV-VP2 protein was 2.5 g/L. After large-scale purification with Triton X-114 two-phase extraction as well as activated carbon powder adsorption, hemagglutination (HA) titers in the purified PPV-VP2 protein reached 219 and endotoxin was reduced to 2500 EU/mL. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) results indicated that the purified PPV-VP2 protein self-assembled into VLPs. Immunogenicity assays in guinea pigs and pigs indicated that the ISA-201 VG adjuvanted PPV-VP2 VLP vaccine elicited hemagglutination inhibition (HI) and virus neutralization (VN) antibody titers comparable with PPV commercial inactivated vaccines, whereas viral loads in the spleen and liver of challenged guinea pigs were significantly reduced. In conclusion, our study provides a method for producing the PPV-VLP vaccine against PPV infection in E. coli and may offer a novel strategy for the soluble expression of other vaccine antigens.
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Funding
This work was supported by grants from the National Key R&D Program (2017YFD0501103) and “1125 talent gathering plan” of Zhengzhou, as well as the Special Fund for Scientific Research and Development of Henan Academy of Agricultural Sciences ([2017]76-21).
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All animal experiments were approved by the Animal Experiment Committee of Henan Academy of Agricultural Sciences with the approval number (LLSC1009605 and LLSC1009618). All the animals received humane care in compliance with good animal practice according to the animal ethics procedures and guidelines of the Institutional Animal Care and Use Committee (IACUC). All efforts were made to alleviate and minimize animal suffering.
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Wang, J., Liu, Y., Chen, Y. et al. Large-scale manufacture of VP2 VLP vaccine against porcine parvovirus in Escherichia coli with high-density fermentation. Appl Microbiol Biotechnol 104, 3847–3857 (2020). https://doi.org/10.1007/s00253-020-10483-5
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DOI: https://doi.org/10.1007/s00253-020-10483-5