Precaution of classical swine fever (CSF) is an important mission for the worldwide swine industry. Glycoprotein E2 is the leading antigen candidate for subunit vaccine of classical swine fever virus (CSFV). In this study, two Spy-tagged E2 genes were synthesized in vitro and subcloned into pMCO-AOX vector for intracellular expression in Pichia pastoris after methanol induction. Western blot analysis and semi-quantitative analysis showed that the yield of recombinant E2 protein was improved 17.87 folds by using co-translocational signal peptide cSIG. After the construction of the tandem multiple copy expression vectors, further increase of E2 production was observed by repetitive transforming expression vectors into P. pastoris genome. Finally, the yeast transformants harboring 8 or 16 copies of cSIG-E2-Spy increased the E2 expression level by 27.01-fold or 30.72-fold, respectively. These results demonstrate that utilizing co-translocational signal peptide together with multi-copy integration strategy can increase the production of recombinant E2 protein efficiently.

中文翻译：

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通过组合策略在巴斯德毕赤酵母中优化经典猪瘟病毒E2蛋白的表达。

预防经典猪瘟（CSF）是全球养猪业的一项重要使命。糖蛋白E2是经典猪瘟病毒（CSFV）亚基疫苗的主要抗原候选物。在这项研究中，在体外合成了两个带有Spy标签的E2基因，并将其亚克隆到pMCO-AOX载体中，以便在甲醇诱导后在巴斯德毕赤酵母中进行细胞内表达。Western印迹分析和半定量分析表明，通过使用共易位信号肽cSIG，重组E2蛋白的产量提高了17.87倍。构建串联多拷贝表达载体后，通过将表达载体重复转化到巴斯德毕赤酵母基因组中，观察到E2产量的进一步增加。最后，带有8或16个拷贝的cSIG-E2-Spy的酵母转化子使E2表达水平提高了27。分别为01倍或30.72倍。这些结果表明，利用共易位信号肽与多拷贝整合策略一起可以有效地提高重组E2蛋白的产量。