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Orf virus virulence gene VIR-deleted (interferon-resistance gene deleted) mutant and its preparing method and application

A technology of oral ulcer virus and virulence gene, which is applied in the field of veterinary biology in genetic engineering, can solve the problems of easily changing immunogenicity and long cycle of attenuated strain of oral ulcer virus, and achieves a short production cycle, rapid and reliable preparation method Effect

Inactive Publication Date: 2015-09-02
SHIHEZI UNIVERSITY
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Problems solved by technology

[0005] The purpose of the present invention is to address the shortcomings of the existing artificially domesticated attenuated strains of aphthus virus, such as long period and easy change in immunogenicity, by adopting genetic engineering methods to pass the virulence gene VIR (interferon resistance gene, interferon resistance factor of aphthus aphthus virus) ) is deleted to achieve a rapid reduction in its virulence, providing a preparation method for attenuated vaccines with significantly reduced virulence but still maintaining good immunogenicity

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  • Orf virus virulence gene VIR-deleted (interferon-resistance gene deleted) mutant and its preparing method and application
  • Orf virus virulence gene VIR-deleted (interferon-resistance gene deleted) mutant and its preparing method and application
  • Orf virus virulence gene VIR-deleted (interferon-resistance gene deleted) mutant and its preparing method and application

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Embodiment Construction

[0025] The present invention provides a construction method for preparing a VIR virulence gene-deleted strain of aphthus virus, which is used to prepare attenuated vaccine candidate strains with significantly reduced virulence but still maintaining good immunogenicity, including the following aspects:

[0026] 1. Cell culture of ORFV

[0027] Vero cells were used to cultivate the popular strain of ORFV SHZ1, the growth medium was DMEM solution containing 8% calf serum, and the maintenance solution was DMEM solution containing 2% calf serum. The poison was received when the cells grew into a monolayer, and the poison was received when 80% of the cells showed typical CPE. Freezing and thawing was repeated three times, centrifuged at 5000rpm for 10min, and the supernatant was taken as the material for extracting total DNA.

[0028] 2. Extraction of ORFV genomic DNA

[0029] Take 200μL of ORFV SHZ1 strain virus solution and add it to a 1.5mL centrifuge tube; then add 200μL Solut...

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Abstract

The invention relates to an orf virus virulence gene VIR-deleted (interferon-resistance gene deleted) mutant and its preparing method and application. The preparing method includes: cloning a gene segment containing an ORFV-SHZ1 VIR virulence gene and a virulence gene by means of PCR (polymerase chain reaction), deleting the VIR virulence gene by means of enzyme digestion, inserting an expression cassette carrying vaccinia virus late promoter P11 and a LacZ reporter gene into a deletion site, and building a recombinant shuttle vector using the LacZ gene as a selection marker; transfecting the recombinant shuttle vector to ORFV infected Vero cells to allow homologous recombination, and acquiring the VIR virulence gene deleted recombinant virus ORFV-DeltaVIR-LacZ by means of plaque screening and purifying. The mutant has the advantages that the ORFV virulence is lowered quickly by means of gene engineering, technical support is provided for the research and development of ORF vaccines, the preparing method features short production cycle, avoidance of domestication, operational convenience and quickness and the like, and the preparing method is widely applicable.

Description

technical field [0001] The invention relates to the field of genetic engineering applied to veterinary biotechnology, in particular to an attenuated oral ulcer virus vaccine and a preparation method thereof. Background technique [0002] Sheep aphthous sore is also called sheep contagious pustular (Contagious Ethyma, CE), contagious pustular dermatitis (Contagious pustular dermatitis, CPD), sheep contagious pustular (Ecthyma Contagiosum) etc., is caused by sheep oral ulcer virus (Orf virus, ORFV) is a contagious infectious disease of sheep and goats. Clinically, erythema, papules, nodules, blisters, pustules, ulcers, and warty thick scabs are formed on the skin and mucous membranes of the lips, tongue, nose, and breasts. as a feature. The disease is transmitted through direct contact, and lambs aged 3 to 6 months are the most susceptible, and are often popular in groups; deer, camels, musk oxen, and humans can also be infected. The disease is widely prevalent in countries ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N7/01A61K39/275A61P31/20
Inventor 乔军孟庆玲杨海波彭叶龙陈诚刘田莉王国超马玉胡政香张星星陈创夫
Owner SHIHEZI UNIVERSITY
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