Optimized Expression of Duck Tembusu Virus E Gene Delivered by a Vectored Duck Enteritis Virus In Vitro
- 34 Downloads
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.
KeywordsDuck Tembusu virus Duck enteritis virus Vectored vaccine Optimized expression E antigen
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.
Compliance with Ethical Standards
Conflict of interest
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.
- 5.Liu, P., Lu, H., Li, S., Moureau, G., Deng, Y. Q., Wang, Y., et al. (2012). Genomic and antigenic characterization of the newly emerging Chinese duck egg-drop syndrome flavivirus: Genomic comparison with Tembusu and Sitiawan viruses. Journal of General Virology, 93(Pt 10), 2158–2170.CrossRefGoogle Scholar
- 9.King, A.M.Q., Adams, M.J., Carstens, E.B., Lefkowitz, E.J. (2012) Virus taxonomy: Classification and nomenclature of viruses: Ninth report of the International Committee on Taxonomy of Viruses (http://ictvonline.org/virusTaxonomy.asp?version=2012&bhcp=1.2012)
- 15.Zou, Z., Hu, Y., Liu, Z., Zhong, W., Cao, H., Chen, H., et al. (2015). Efficient strategy for constructing duck enteritis virus-based live attenuated vaccine against homologous and heterologous H5N1 avian influenza virus and duck enteritis virus infection. Veterinary Research, 46, 42.CrossRefGoogle Scholar
- 16.Wang, J., Ge, A., Xu, M., Wang, Z., Qiao, Y., Gu, Y., et al. (2015). Construction of a recombinant duck enteritis virus (DEV) expressing hemagglutinin of H5N1 avian influenza virus based on an infectious clone of DEV vaccine strain and evaluation of its efficacy in ducks and chickens. Virology Journal, 12, 126.CrossRefGoogle Scholar
- 20.Chen, L., Yu, B., Ni, Z., Hua, J., Ye, W., Yun, T., et al. (2015). Construction and characterization of a recombinant duck enteritis virus expressing E protein of duck Tembusu virus. Acta Agriculturae Zhejiangensis, 27(11), 1889–1895. (in Chinese).Google Scholar
- 23.Yang, C., Li, J., Li, Q., Li, H., Xia, Y., Guo, X., et al. (2013). Complete genome sequence of an attenuated duck enteritis virus obtained by in vitro serial passage. Genome Announc, 1(5), e00685–e00685.Google Scholar
- 26.Chung, K. M., Nybakken, G. E., Thompson, B. S., Engle, M. J., Marri, A., Fremont, D. H., et al. (2006). Antibodies against West Nile Virus nonstructural protein NS1 prevent lethal infection through Fc gamma receptor-dependent and -independent mechanisms. Journal of Virology, 80(3), 1340–1351.CrossRefGoogle Scholar