Recombinant duck plague virus of expressing duck tembusu virus E protein as well as construction method and application of recombinant duck plague virus

A technology of duck Tembusu virus and duck plague virus, which is applied in the fields of application, antiviral agent, virus/bacteriophage, etc., and can solve problems such as losses in the breeding industry

Inactive Publication Date: 2015-11-11
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Abstract
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  • Claims
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Problems solved by technology

The epidemic of the disease has caused huge losses to the breeding industry, and

Method used

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  • Recombinant duck plague virus of expressing duck tembusu virus E protein as well as construction method and application of recombinant duck plague virus
  • Recombinant duck plague virus of expressing duck tembusu virus E protein as well as construction method and application of recombinant duck plague virus
  • Recombinant duck plague virus of expressing duck tembusu virus E protein as well as construction method and application of recombinant duck plague virus

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Embodiment Construction

[0038]Unless otherwise specified, the experimental methods used in the following implementations are conventional conditions, such as "Refined Molecular Biology Experiment Guide" (F.M. Osper, R.E. Kingston, J.G. Seidman, etc., edited by Ma Xuejun, Shu Yuelong, etc. , Beijing: Science Press, 2004).

[0039] 1. Sequence optimization, primer design and synthesis

[0040] The duck Tembusu virus E gene sequence refers to GenBank (JF270480.1), and is optimized and synthesized by GenScript Company using chicken as a host; Shanghai Sangong Synthetic Co., Ltd.;

[0041] Primers EF1-ep1 and EF1-ep2 were used to replace the promoter Pcmv of the gfp gene in pDEV-vac with the EF1 promoter to construct pDEV-EF1. The underlined part is homologous to the pEP-EF1-in plasmid sequence, and the italic sequence is homologous to the upstream and downstream sequences of the promoter Pcmv of the gfp gene in the pDEV-vac sequence;

[0042] Primers pDEVvac-in-s and pDEVvac-in-as were used to insert ...

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Abstract

The invention discloses a recombinant duck plague virus of expressing duck tembusu virus E protein as well as a construction method and application of the recombinant duck plague virus, wherein the gene of the duck tembusu virus E protein is interpolated inside the genome of the recombinant duck plague virus; and the nucleotide sequence of the duck tembusu virus E protein gene is as shown in SEQ ID NO. 9. The construction method comprises the following steps: (1) substituting the CMV promoter of gfp gene in pDEV-vac with an EF1 promoter so as to obtain pDEV-EF1; (2) interpolating a Pcmv-E-BGH-pA expression cassette into the pDEV-EF1 so as to obtain pDEV-E; and (3) transfecting the pDEV-E with chicken embryo fibroblasts and rescuing so as to obtain the recombinant duck plague virus. The recombinant duck plague virus, compared with a parent strain, has no significant difference in the size of virus plaque, showing that the diffusion of the duck plague virus on the chicken embryo fibroblasts is not affected by the interpolation of the duck tembusu virus E protein gene. After transfecting with the chicken embryo fibroblasts, the recombinant duck plague virus can successfully express E protein, so as to lay a foundation for developing duck plague virus-duck tembusu virus bivalent vaccine.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant duck plague virus expressing duck Tembusu virus E protein and its construction method and application. Background technique [0002] Since April 2010, a new infectious disease has broken out in laying ducks and breeding ducks in the main duck production areas of my country. The main clinical features of the disease are rapid spread, sudden drop in egg production, and hemorrhagic oophoritis. . Through pathogen isolation and identification and full sequence determination, it is clear that the pathogen belongs to Flaviviridae, Flavivirus, Mosquito-borne, and Ntaya virus group, and has similarities with this group of Tembusu virus (TMUV). The recent genetic evolution relationship can be considered as a new member of TMUV, tentatively named Duck Tembusuvirus (DTMUV) (SuJ, LiS, HuX, et al. Duckegg-dropsyndromecausedbyBYDvirus, anewTembusu-relatedflavivirus[J].PLoSOne , 2011,...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/869A61K39/295A61K39/245A61K39/12A61P31/14A61P31/22
Inventor 陈柳张存倪征叶伟成余斌云涛华炯钢
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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