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Method for constructing virus live vector recombinant vaccine by utilizing transposon

A technology of recombinant vaccines and transposons, applied in viruses/bacteriophages, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of easy mutation, low recombination efficiency, and scarcity

Inactive Publication Date: 2012-07-04
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages are: in Escherichia coli, the genetic selection pressure of the virus sequence is small, and the possibility of mutation leading to a decrease in virus activity is higher than that of recombination in eukaryotic cells; single enzyme cutting sites are relatively rare, and rare endonuclease expensive
Compared with the transposition mechanism involved in the present invention, the construction method of the above recombinant virus has the disadvantage of low recombination efficiency or prone to mutation

Method used

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  • Method for constructing virus live vector recombinant vaccine by utilizing transposon
  • Method for constructing virus live vector recombinant vaccine by utilizing transposon
  • Method for constructing virus live vector recombinant vaccine by utilizing transposon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preparation method of rabies-recombinant canine adenovirus type 2

[0030] 1. Construction of GFP and rabies glycoprotein gene expression cassettes

[0031] The commercially available eukaryotic expression plasmid vector pIRESneo ( figure 2 ) into the rabies virus glycoprotein gene (refer to GenBank: M31046 for the sequence) using the multiple cloning restriction sites EcoRV and BamHI, and insert the green fluorescent protein reporter gene (refer to the universal plasmid pEGPF-C1 for the sequence) using the Sma I and XbaI sites, and construct a parallel Expression cassettes expressing the glycoprotein and green fluorescent protein genes.

[0032] 2. Construction of recombinant transposons

[0033] The gene expression cassette was completely excised through the Nru I and Bst1107 I sites, and inserted into the shuttle vector pMOD-2 using T4 ligase The Sma I site between the two transposon sequences in figure 1 ), transform Escherichia coli DH5α...

Embodiment 2

[0045] Embodiment 2: the preparation method of rabies-recombinant herpes virus type I

[0046] 1. Construction of GFP and rabies glycoprotein gene expression cassettes

[0047] The commercially available eukaryotic expression plasmid vector pIRESneo ( figure 2 ) into the rabies virus glycoprotein gene (refer to GenBank: M31046 for the sequence) using the multiple cloning restriction sites EcoRV and BamHI, and insert the green fluorescent protein reporter gene (refer to the universal plasmid pEGPF-C1 for the sequence) using the Sma I and Xba I sites, and construct Expression cassette for parallel expression of glycoprotein and green fluorescent protein genes.

[0048] 2. Construction of recombinant transposons

[0049] The gene expression cassette was completely excised through the Nru I and Bst1107 I sites, and inserted into the shuttle vector pMOD-2 using T4 ligase The Sma I site between the two transposon sequences in figure 1 ), transform Escherichia coli D...

Embodiment 3

[0061] Embodiment 3: Preparation method of classical swine fever-recombinant canine adenovirus type 2

[0062] 1. Construction of gene expression cassettes for green fluorescent protein and classical swine fever virus E2 protein

[0063] The commercially available eukaryotic expression plasmid vector pIRESneo ( figure 2 ) into the E2 protein gene of classical swine fever virus (refer to GenBank: AF091507 for the sequence) using the multiple cloning restriction sites EcoRV and BamHI, and insert the green fluorescent protein reporter gene (refer to the universal plasmid pEGPF-C1 for the sequence) using the SmaI and XbaI sites, and construct a parallel Expression cassettes expressing the glycoprotein and green fluorescent protein genes.

[0064] 2. Construction of recombinant transposons

[0065] The gene expression cassette was completely excised through the Nru I and Bst1107 I sites, and inserted into the shuttle vector pMOD-2 using T4 ligase The Sma I site betwe...

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Abstract

The invention discloses a method for constructing virus live vector recombinant vaccine by utilizing transposon. Green fluorescent protein is taken as a report gene, expression boxes respectively expressing rabies virus glycoprotein and swine fever E2 protein genes are constructed and are cloned to the shuttle vector of the transposon, under the action of mediation of transposase, recombination with purified canine adenovirus type II virus and herpes virus type I entire genome are respectively carried out, then transfection agent (liposome and the like) is utilized to respectively transfect the recombination product with MDCK and Vero cells, thus obtaining four strains of recombinant viruses taking green fluorescent protein as report gene, namely recombinant canine adenovirus type II virus expressing glycoprotein, recombinant canine adenovirus type II virus expressing E2 protein, recombinant herpes virus type I expressing glycoprotein and recombinant herpes virus type I expressing E2 protein. Immunity test shows that the canine adenovirus type II virus expressing E2 gene and herpes virus type I live vector recombinant vaccine all can induce immunoreaction resistant to swine fever virus infection in swine and canine adenovirus type II virus expressing glycoprotein gene and herpes virus type I live vector recombinant vaccine all can induce immunoreaction resistant to rabies virus infection in dog.

Description

Technical field: [0001] The invention relates to a new method for constructing canine type 2 adenovirus and herpes virus type I live vector recombinant vaccines, in particular discloses a method for constructing virus live vector recombinant vaccines using transposons, which belongs to the technical field of vaccine preparation. Background technique: [0002] At present, there are more than ten kinds of vector systems used for genetic disease gene therapy and recombinant live vector vaccine construction, among which DNA virus vectors are the most commonly used in vaccine research, mainly including adenovirus, herpes virus, pox virus and baculovirus. [0003] In the past, the construction strategy of recombinant DNA viruses was mainly based on the principle of homologous recombination, including: ① intracellular homologous recombination, that is, recombination between two segments of genomic DNA molecules. Once homologous recombination occurs, the expected recombinant virus c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/205A61K39/187A61K39/295A61K48/00C12N15/47C12N15/34C12N7/01
Inventor 扈荣良刘晔范志强张菲张守峰
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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