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.

中文翻译：

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猪细小病毒VP2 VLP疫苗高密度发酵大规模生产

摘要

猪细小病毒 (PPV) 病毒样颗粒 (VLP) 是预防妊娠母猪细小病毒引起的繁殖障碍的潜在候选疫苗。目前，大肠杆菌（E.coli）表达系统是最经济高效的重组蛋白表达系统。为了克服蛋白质错误折叠的局限性，制备可溶性高生物活性抗原和高产量蛋白质，我们优化了 PPV-VP2 基因，将其亚克隆到 pET24a、pET26b、pET28a 和 pET30a 中，并将其转化到大肠杆菌中BL21(DE3)-Tf16 感受态细胞。选择 pET28a 质粒进行进一步操作，因为它表达了高水平的生物活性 PPV-VP2 蛋白。在70-L发酵罐最佳高密度发酵条件下，大肠杆菌总湿重产量为124.86 g/L，PPV-VP2蛋白为2.5 g/L。经Triton X-114两相萃取和活性炭粉吸附大规模纯化后，纯化的PPV-VP2蛋白的血凝（HA）效价达到2 ¹⁹内毒素降至 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 VLP。豚鼠和猪的免疫原性测定表明，ISA-201 VG 佐剂的 PPV-VP2 VLP 疫苗引起的血凝抑制 (HI) 和病毒中和 (VN) 抗体滴度与 PPV 商业灭活疫苗相当，而脾脏和肝脏中的病毒载量受攻击的豚鼠显着减少。总之，我们的研究提供了一种在大肠杆菌中生产针对 PPV 感染的 PPV-VLP 疫苗的方法，并可能为其他疫苗抗原的可溶性表达提供一种新策略。