College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, China.
Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, 450002, China.
Appl Microbiol Biotechnol. 2020 May;104(9):3847-3857. doi: 10.1007/s00253-020-10483-5. Epub 2020 Mar 4.
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 2 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.
猪细小病毒（PPV）病毒样颗粒（VLPs）是预防怀孕母猪细小病毒引起繁殖失败的潜在疫苗候选物。目前，大肠杆菌（E. coli）表达系统是表达重组蛋白最具成本效益的方法。为了克服蛋白质错误折叠的限制，并制备可溶性高生物活性抗原和高产量的蛋白质，我们优化了 PPV-VP2 基因，将其亚克隆到 pET24a、pET26b、pET28a 和 pET30a 中，并转化到大肠杆菌 BL21(DE3)-Tf16 感受态细胞中。选择 pET28a 质粒进行进一步操作，因为它表达了高水平的有生物活性的 PPV-VP2 蛋白。在 70-L 发酵罐中优化高密度发酵条件下，湿重大肠杆菌细胞的总产量为 124.86 g/L，PPV-VP2 蛋白的产量为 2.5 g/L。经过 Triton X-114 两相萃取和活性炭粉末吸附的大规模纯化后，纯化的 PPV-VP2 蛋白的血凝（HA）效价达到 2，内毒素降低至 2500 EU/mL。动态光散射（DLS）和透射电子显微镜（TEM）结果表明，纯化的 PPV-VP2 蛋白自组装成 VLPs。豚鼠和猪的免疫原性试验表明，ISA-201 VG 佐剂的 PPV-VP2 VLP 疫苗诱导的血凝抑制（HI）和病毒中和（VN）抗体效价可与 PPV 商业灭活疫苗相媲美，而攻毒豚鼠脾脏和肝脏中的病毒载量明显降低。总之，我们的研究提供了一种在大肠杆菌中生产 PPV-VLP 疫苗以预防 PPV 感染的方法，也为其他疫苗抗原的可溶性表达提供了一种新策略。
