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Duck plague virus recombinant vaccine strain rDEVTK-EGFP for expressing enhanced green fluorescent protein genes and constructing method and application therefore

A green fluorescent protein, enhanced technology, applied in the field of recombinant virus vaccine strain, recombinant duck plague virus strain rDEV TK-EGFP and its construction, can solve the problem that the recombinant virus construction of foreign genes has not been reported and the like

Inactive Publication Date: 2015-01-28
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, the construction and application of recombinant viruses using DEV non-essential gene TK sites to carry out foreign genes have not been reported so far.

Method used

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  • Duck plague virus recombinant vaccine strain rDEVTK-EGFP for expressing enhanced green fluorescent protein genes and constructing method and application therefore
  • Duck plague virus recombinant vaccine strain rDEVTK-EGFP for expressing enhanced green fluorescent protein genes and constructing method and application therefore
  • Duck plague virus recombinant vaccine strain rDEVTK-EGFP for expressing enhanced green fluorescent protein genes and constructing method and application therefore

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The construction of embodiment 1 transfer vector

[0034] According to the DEV viral genome TK gene sequence and its flanking sequence (GenBank accession number is AY963569), a pair of primers were designed using Oligo 6.0 software, and the primer sequences are as follows:

[0035] T1 (upstream): 5'-GACGTGTTGGCATCGGTTC-3'

[0036] T4 (downstream): 5'-AAACAAATAGGGAGTAGCGAAGG-3'

[0037] First use primers T1 (upstream) and T4 (downstream) to amplify the DEV TK gene and its flanking sequence (shown in SEQ ID NO: 1), and clone it into the pMD18-T Simple vector to construct plasmid pTK; The EGFP expression cassette (shown in SEQ ID NO: 2) of the sCMV promoter was inserted into the Hpa I site in the vector pTK ( figure 1 shown), the transfer vector pTK-EGFP was constructed. Wherein, the sCMV promoter is derived from the pCS2+ plasmid, and Hind III and Stu I restriction endonuclease sites are added at both ends of the EGFP gene during amplification, through which EGFP can ...

Embodiment 2

[0038] The extraction of embodiment 2 duck plague virus genome

[0039] 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 Adsorbed for 2 hours, discarded the virus solution, and replaced the DMEM cell maintenance solution (containing 2% FBS), and when the cytopathy reached 80% to 90%, discarded the cell maintenance solution, and added cell digestion solution (1860 μL STE; 100 μL 10% SDS; 40μL proteinase K 20mg / 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-dr...

Embodiment 3

[0040] Example 3 Transfection

[0041] Day 1: Prepare cells

[0042] The CEF cells were subcultured in advance and spread in 5mL cell flasks, cultured in a 2% constant temperature incubator at 37°C.

[0043] Day 2: Transfection

[0044] (1). Change the cell culture medium 3-4 hours before transfection

[0045] (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: 150μL 2×Hepes Buffered Saline (HBS)

[0046] (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.

[0047] (4). Incubate the A and B mixture at room temperature for 30 minutes.

[0048] (5). Add the mixed solution into the cell culture medium.

[0049] (6). Place the cells in a 2% constant temperature incubator at 37° C. to continue culturing.

[0...

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Abstract

The invention discloses a duck plague virus recombinant vaccine strain rDEVTK-EGFP for expressing enhanced green fluorescent protein (EGFP) genes and a constructing method and application of the duck plague virus recombinant vaccine strain rDEVTK-EGFP. The microorganism preservation number of the vaccine strain rDEVTK-EGFP is CGMCC No. 9456. According to the duck plague virus recombinant vaccine strain rDEVTK-EGFP, the constructing method and the application, a recombinant clone technology is adopted; a gene segment sCMV-EGFP containing enhanced green fluorescent proteins (EGFP) and sCMV promoter sequences is inserted into a duck plague virus TK gene region; recombinant EGFP duck plague viruses with an sCMV-EGFP expression cassette inserted into the corresponding position of a TK gene is constructed; the EGFP genes are stably expressed by the recombinant viruses. The invention further relates to a method for constructing the recombinant duck plague virus vaccine strain for stably expressing other poultry pathogeny exogenous genes and the application of the recombinant duck plague virus vaccine strain to preparation of vaccines for preventing duck plagues and other poultry infectious diseases.

Description

technical field [0001] The invention relates to a recombinant virus vaccine strain, in particular to a recombinant duck plague virus strain rDEV TK-EGFP capable of stably expressing foreign genes and its construction method and application. The invention belongs to the field of biomedicine. Background technique [0002] Duck plague, also known as duck viral enteritis (Duck viral enteritis, DVE), is a kind of infection caused by duck plague virus (also known as duck enteritis virus (Duck enteritis virus, DEV)) Acute, febrile, and contact infectious diseases. Its main characteristics are widespread prevalence, rapid spread, high morbidity and mortality, and ducks of different ages are susceptible, causing huge economic losses to the duck industry and endangering the duck industry. one of the most important diseases. [0003] Natural infection of duck plague virus is limited to members of Anatidae (ducks, geese, swans, etc.). The main sources of infection are sick ducks, goos...

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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