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Duck plague virus recombinant vaccine strain expressing enhanced green fluorescent protein gene, constructing method thereof and applications of the recombinant vaccine strain

A technology of green fluorescent protein and duck plague virus, applied in the field of biomedicine, can solve the problem that the construction of recombinant virus of foreign genes has not been reported.

Active Publication Date: 2014-10-29
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports on the construction and application of recombinant viruses using the US10 site of the non-essential gene of DEV at present.

Method used

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  • Duck plague virus recombinant vaccine strain expressing enhanced green fluorescent protein gene, constructing method thereof and applications of the recombinant vaccine strain
  • Duck plague virus recombinant vaccine strain expressing enhanced green fluorescent protein gene, constructing method thereof and applications of the recombinant vaccine strain
  • Duck plague virus recombinant vaccine strain expressing enhanced green fluorescent protein gene, constructing method thereof and applications of the recombinant vaccine strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The construction of embodiment 1 transfer vector

[0035] According to the flanking sequence of the US10 gene sequence of the DEV virus genome (GenBank accession number is EF524095), 2 pairs of primers were designed using Oligo6.0 software. The primer sequences are as follows:

[0036] VL1 (upstream): 5'-ATCGATTGACGATGAGCGATCGGAAT-3'

[0037] VL2 (downstream): 5'-CCTAGGGTTGCGCGTTGTGTATAAGT-3'

[0038] VR1 (upstream): 5'-ACGCGTGACTCTGACTGATACTCTAC-3'

[0039] VR2 (downstream): 5'-ATGCATCTAATCGGTTATTTGCTGCT-3'

[0040] First, primers VL1 (upstream) and VL2 (downstream) were used to amplify the left homology arm, primers VR1 (upstream) and VR2 (downstream) were used to amplify the right homology arm, and the left homology arm (Cla I+Bln I) and The right homology arms (Mlu I+Ava III) were respectively inserted into the pT-EGFP vector of the EGFP expression cassette (CMV-EGFP) with the CMV promoter constructed by our laboratory to construct the transfer vector pUS10-EGFP ...

Embodiment 2

[0041] The extraction of embodiment 2 duck plague virus genome

[0042] Inoculate DEV Clone-03 cytotoxicity at an MOI of 0.001 in a 5mL cell flask covered with a CEF monolayer (the preparation of chicken embryo fibroblasts refers to the operation of "Principles and Techniques of In Vitro Culture" (Xue Qingshan, 2001)), 37°C Adsorb for 2 hours, discard the virus solution, and replace the DMEM cell maintenance solution (containing 2% FBS), and when the cytopathy reaches 80% to 90%, discard the cell maintenance solution, and add the cell digestion solution (1860 μL STE; 100 μL 10% SDS; 40 μL Proteinase K20mg / mL), digest overnight at 37°C, add an equal volume of phenol for extraction once, an equal volume of phenol chloroform (1:1) for extraction once, add an equal volume of chloroform for extraction once; add 1 / 10 volume of NaAC (3M, pH5 .2), 2.5 times the volume of absolute ethanol, placed at -20°C for precipitation overnight, centrifuged at 4°C for 15 minutes, after air-drying,...

Embodiment 3

[0043] Example 3 Transfection

[0044] Day1: Prepare cells

[0045] The CEF cells were subcultured in advance and spread in 5mL cell flasks, cultured in a 2% constant temperature incubator at 37°C.

[0046] Day2: Transfection

[0047] (1) Change the cell culture medium 3-4 hours before transfection.

[0048] (2) Transfection system: Solution A: 18 μL 2M CaCl2, 10 μg DNA (the ratio of transfer vector to viral genome is 3:1), add deionized water to make up the volume to 150 μL. Solution B: 150 μL 2×Hepes Buffered Saline (HBS).

[0049] (3) Use a pipette to add liquid A to liquid B drop by drop, while adding liquid A, use another pipette to slowly blow air into liquid B. This process should be completed within 1-2 minutes.

[0050] (4) Incubate the A and B mixture at room temperature for 30 min.

[0051] (5) Add the mixed solution into the cell culture medium.

[0052] (6) Place the cells in a 2% constant temperature incubator at 37°C for further culture.

[0053] Day3: Ch...

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Abstract

The invention discloses a duck plague virus recombinant vaccine strain expressing an enhanced green fluorescent protein (EGFP) gene, a constructing method thereof and applications of the recombinant vaccine strain. In particular, by utilization of a recombinant clone technology, a gene fragment CMV-EGFP containing an enhanced green fluorescent protein (EGFP) and a CMV promoter sequence replaces a US10 gene of a duck plague virus, and a recombinant EGFP duck plague virus lacking the US10 gene with a CMV-EGFP expression cassette being inserted into the corresponding position is constructed. The recombinant virus can stably express the EGFP gene. The duck plague virus recombinant vaccine strain is named as rDEVUS10-EGFP. The microbial accession number is CGMCC No.8660. The invention also relates to constructing methods of duck plague virus recombinant vaccine strains capable of stably expressing other poultry pathogeny exogenous genes, and applications of the recombinant vaccine strains in preparation of vaccines preventing duck plague and other poultry infectious diseases.

Description

technical field [0001] The invention relates to a recombinant virus vaccine strain and its construction method and application, in particular to a recombinant duck plague virus strain rDEVUS10-EGFP capable of stably expressing exogenous genes and its construction method and application. The invention belongs to the technical field of biomedicine. Background technique [0002] Duck plague, also known as duck viral enteritis (Duck viral enteritis, DVE), is a disease caused by duck plague virus (also known as duck enteritis virus (Duck enteritis virus, DEV)) and a variety of anseriformes such as ducks and geese. An acute, febrile, contact infectious disease. Its main characteristics are widespread prevalence, rapid spread, high morbidity and mortality, and ducks of different ages are susceptible to it, causing huge economic losses to the duck industry. It is one of the important diseases that endanger the duck industry. [0003] Natural infection of duck plague virus is limite...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/85A61K39/245A61K39/295A61K39/29A61K39/215A61K39/17A61P31/14A61P31/22C12R1/93
Inventor 刘胜旺李慧昕韩宗玺孔宪刚
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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