Duck tembusu virus (DTMUV) infectious clone attenuated vaccine strain and preparation method and application thereof

A technology of duck tembusu virus and infectious cloning, applied in the field of bioengineering, can solve the problem of lack of vaccines

Active Publication Date: 2015-12-30
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of efficient and safe vaccines

Method used

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  • Duck tembusu virus (DTMUV) infectious clone attenuated vaccine strain and preparation method and application thereof
  • Duck tembusu virus (DTMUV) infectious clone attenuated vaccine strain and preparation method and application thereof
  • Duck tembusu virus (DTMUV) infectious clone attenuated vaccine strain and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of full-length cDNA clone of duck Tembusu virus attenuated strain FX2010-180P genome

[0042] 1. Materials

[0043] 1.1 Viruses and Cells

[0044]Duck Tembusu virus FX2010-180P strain and DF-1 cells were preserved by Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0045] 2. Method

[0046] 2.1 Extraction of viral RNA

[0047] Use Takara’s RNAisoPlus reagent to extract viral RNA, and operate according to the instructions on the product manual. The specific method is as follows: draw 300 μl of virus culture solution, add 700 μl RNAisoplus Reagent, put it in a 1.5ml RNase-free centrifuge tube, pipette and mix, and then stand at room temperature for 5 minute. Add 200 μl chloroform to the mixture, vibrate vigorously for 15 seconds, and let stand at room temperature for 3 minutes. Centrifuge at 12000r / min at 4°C for 10min. Transfer the supernatant to another clean RNase-free centrifuge tube. Add an equal volume...

Embodiment 2

[0104] Example 2 Rescue and Identification of Duck Tembusu Virus Attenuated Strain FX2010-180P

[0105] 1. In vitro transcription

[0106] Using the full-length PCR product of the above virus as a template, use In vitro transcription kit for in vitro transcription to prepare infectious RNA.

[0107] React at 37°C for 5 hours, and the reaction is performed on a PCR machine.

[0108] Purify the transcription product by lithium chloride precipitation method, add 30 μl Nuclease-freeWater and 30 μl LiCl to the transcription termination product, and mix gently. Carefully remove the supernatant, add 1ml of 70% ethanol (prepared with DEPC water), centrifuge at 14,000g at 4°C for 15min, discard the supernatant to completely evaporate the ethanol, add 20μl Nuclease-freeWater to dissolve the RNA precipitate, and store at -70°C.

[0109] 2. Transfection

[0110] Use the LTX transfection kit to transfect the above RNA into DF-1 cells, spread the cells the day before, so that the cell ...

Embodiment 3

[0132] Example 3 Using duck Tembusu virus as a carrier to express foreign protein

[0133] 1. Materials

[0134] 1.1 Viruses and Cells

[0135] Duck Tembusu virus disease FX2010-180P strain is preserved by our laboratory; DF-1 cells are preserved by our laboratory.

[0136] 2. Method

[0137] 2.1 Selection of insertion sites and primer design for foreign genes

[0138] 2.1.1 Construction scheme of recombinant virus genome

[0139] Such as Figure 10 As shown, the sequence of the internal ribosome entry site (IRES) and the green fluorescent protein gene (EGFP) were inserted once between the NS5 gene and the 3' non-coding sequence of the FX2010-180P strain genome.

[0140] 2.1.2 Primer design

[0141] In order to insert the IRES+EGFP sequence into the Duck Tembusu virus genome, a specific sequence of Duck Tembusu virus FX2010-180P was introduced at the 5' end of the IRES sequence and the 3' end of the EGFP sequence by PCR method. The NS5 gene is the 3' end of the duck Tem...

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Abstract

The invention discloses a duck tembusu virus (DTMUV) infectious clone attenuated vaccine strain. The attenuated vaccine strain is a clone strain of a parent virus DTMUV attenuated vaccine strain FX2010-180P and has a whole-genome sequence shown as SEQ ID NO.1. The infectious clone attenuated vaccine strain is obtained through a reverse genetic operation method. The invention further discloses a preparation method and application of the DTMUV infectious clone attenuated vaccine strain. The DTMUV infectious clone attenuated vaccine strain not only can be used for developing a novel DTMUV disease vaccine but also can be used as a virus vector for expression of exogenous genes, and meanwhile the strain is an important tool for DTMUV molecular biology study.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a duck Tembusu virus infectious clone attenuated vaccine strain and a preparation method and application thereof. Background technique [0002] Duck Tembusuvirus (DTMUV) is a flavivirus that can cause duck egg production decline, growth retardation and death. Since the spring of 2010, a new disease has successively broken out in Shanghai, Jiangsu Province, Zhejiang Province, Anhui Province and other places, resulting in reduced production of laying ducks, growth retardation and death of meat ducks. Studies have proved that the pathogen causing the disease is Tembusu Virus (Tembusuvirus). The virus is pathogenic to both laying ducks and meat ducks. The main symptoms of sick ducks are high fever, movement disorders, loss of appetite or even extinction, decline or even cessation of egg production, and can lead to death in severe cases. The mortality rate can reach 5%-10%. ....

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N15/11C12N15/85A61K39/12A61P31/14C12N15/86C12N7/01C12R1/93
Inventor 李泽君吴晓刚李国新
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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