In our previous study, a recombinant duck enteritis virus (DEV) delivering codon-optimized E gene (named as E-ch) of duck Tembusu virus (DTMUV) optimized referring to chicken's codon bias has been obtained based on the infectious bacterial artificial chromosome (BAC) clone of duck enteritis virus vaccine strain pDEV-EF1, but the expression level of E-ch in recombinant virus rDEV-E-ch-infected cells was very low. To optimize DTMUV E gene expression delivered by the vectored DEV, different forms of E gene (collectively called EG) including origin E gene (E-ori), truncated E451-ori gene, codon-optimized E-dk gene optimized referring to duck's codon bias, as well as the truncated E451-ch and E451-dk, Etpa-ori and Etpa-451-ori, which contain prefixing chick TPA signal peptide genes, were cloned into transfer vector pEP-BGH-end, and several recombinant plasmids pEP-BGH-EG were constructed. Then the expression cassettes pCMV-EG-polyABGH amplified from pEP-BGH-EG by PCR were inserted into US7/US8 gene intergenic region of pDEV-EF1 by two-step Red/ET recombination, 7 strain recombinant mutated BAC clones pDEV-EG carrying different E genes were constructed. Next, the recombinant viruses rDEV-EG were reconstituted from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Western blot analysis showed that E or E451 protein is expressed in rDEV-E-ori, rDEV-E-ch, rDEV-Etpa-ori, rDEV-E451-ori, rDEV-E451-dk, and rDEV-E451-ch-infected CEFs, and protein expression level in rDEV-E451-dk-infected CEFs is the highest. These studies have laid a foundation for developing bivalent vaccine controlling DEV and DTMUV infection.

中文翻译：

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通过载体鸭肠道炎病毒体外递送的鸭Tembusu病毒E基因的优化表达。

在我们之前的研究中，已经基于传染性细菌人工染色体（A）获得了重组鸭肠道炎病毒（DEV），该重组腺病毒提供了经过优化的，针对鸡的密码子偏倚而得到的经优化的鸭Tembusu病毒（DTMUV）的经密码子优化的E基因（称为E-ch）。 BAC）鸭肠炎病毒疫苗株pDEV-EF1的克隆，但重组病毒rDEV-E-ch感染的细胞中E-ch的表达水平很低。为了优化由载体DEV传递的DTMUV E基因表达，不同形式的E基因（统称为EG）包括起源E基因（E-ori），截短的E451-ori基因，经密码子优化的E-dk基因（参考鸭的密码子进行了优化）将带有前缀鸡TPA信号肽基因的Etap-ori和Etpa-ori和Etpa-451-ori以及截短的E451-ch和E451-dk克隆到转移载体pEP-BGH-end，并构建了几种重组质粒pEP-BGH-EG。然后通过两步Red / ET重组将通过PCR从pEP-BGH-EG中扩增得到的表达盒pCMV-EG-polyABGH插入到pDEV-EF1的US7 / US8基因间区域中，携带7个菌株的重组BAC克隆pDEV-EG构建了不同的E基因。接下来，通过磷酸钙沉淀从鸡胚成纤维细胞（CEF）中重组重组病毒rDEV-EG。Western印迹分析表明E或E451蛋白在感染的rDEV-E-ori，rDEV-E-ch，rDEV-Etpa-ori，rDEV-E451-ori，rDEV-E451-dk和rDEV-E451-ch中表达CEF和rDEV-E451-dk感染的CEF中的蛋白表达水平最高。这些研究为开发控制DEV和DTMUV感染的二价疫苗奠定了基础。然后通过两步Red / ET重组将通过PCR从pEP-BGH-EG中扩增得到的表达盒pCMV-EG-polyABGH插入到pDEV-EF1的US7 / US8基因间区域中，携带7个菌株的重组BAC克隆pDEV-EG构建了不同的E基因。接下来，通过磷酸钙沉淀从鸡胚成纤维细胞（CEF）中重组重组病毒rDEV-EG。Western印迹分析表明E或E451蛋白在感染的rDEV-E-ori，rDEV-E-ch，rDEV-Etpa-ori，rDEV-E451-ori，rDEV-E451-dk和rDEV-E451-ch中表达CEF和rDEV-E451-dk感染的CEF中的蛋白表达水平最高。这些研究为开发控制DEV和DTMUV感染的二价疫苗奠定了基础。然后通过两步Red / ET重组将通过PCR从pEP-BGH-EG中扩增得到的表达盒pCMV-EG-polyABGH插入到pDEV-EF1的US7 / US8基因间区域中，携带7个菌株的重组BAC克隆pDEV-EG构建了不同的E基因。接下来，通过磷酸钙沉淀从鸡胚成纤维细胞（CEF）中重组重组病毒rDEV-EG。Western印迹分析表明E或E451蛋白在感染的rDEV-E-ori，rDEV-E-ch，rDEV-Etpa-ori，rDEV-E451-ori，rDEV-E451-dk和rDEV-E451-ch中表达CEF和rDEV-E451-dk感染的CEF中的蛋白表达水平最高。这些研究为开发控制DEV和DTMUV感染的二价疫苗奠定了基础。