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Optimized Expression of Duck Tembusu Virus E Gene Delivered by a Vectored Duck Enteritis Virus In Vitro

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Abstract

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.

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Acknowledgements

The study was supported by grants from the National Key Research and Development Program of China (2016YFD0500107); the National Natural Science Foundation of China (31670150); Zhejiang Provincial Natural Science Foundation of China (Grant No. LY15C180002); and  the Public Service Technology Application Research Project of Science and Technology Department of Zhejiang Province (2016C32070). We would like to thank Kui Yang, Department of Pathobiological Sciences, School of Veterinary Medicine Louisiana State University, Louisiana, USA, for critical reading of the manuscript.

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  1. Institute of Animal Husbandry and Veterinary Sciences, Zhejiang Academy of Agriculture Sciences, Hangzhou, 310021, China

    Liu Chen, Bin Yu, Jonggang Hua, Zheng Ni, Weicheng Ye, Tao Yun & Cun Zhang

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  1. Liu Chen
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  2. Bin Yu
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  3. Jonggang Hua
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Correspondence to Cun Zhang.

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The authors declare that they have no competing interests. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; and in the decision to publish the results.

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Chen, L., Yu, B., Hua, J. et al. Optimized Expression of Duck Tembusu Virus E Gene Delivered by a Vectored Duck Enteritis Virus In Vitro. Mol Biotechnol 61, 783–790 (2019). https://doi.org/10.1007/s12033-019-00206-1

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