Recombinant adenovirus carrier for expressing African swine fever virus E183L genes, creation method of recombinant adenovirus carrier and preparation method of recombinant adenovirus

A technology of African swine fever virus and genetic recombination, applied in the field of genetic engineering

Pending Publication Date: 2018-09-14
YANGZHOU UNIV
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

So far, there is no vaccine that can prevent African swine fever virus infection in the world. In the pri

Method used

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  • Recombinant adenovirus carrier for expressing African swine fever virus E183L genes, creation method of recombinant adenovirus carrier and preparation method of recombinant adenovirus
  • Recombinant adenovirus carrier for expressing African swine fever virus E183L genes, creation method of recombinant adenovirus carrier and preparation method of recombinant adenovirus
  • Recombinant adenovirus carrier for expressing African swine fever virus E183L genes, creation method of recombinant adenovirus carrier and preparation method of recombinant adenovirus

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Experimental program
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Effect test

Embodiment 1

[0069] Directly synthesize target gene fragments, and add enzyme cutting sites AsisI / MLuI.

Embodiment 2

[0071] The gene synthesis sequence E183L and the vector pKO-FH( Figure 4 ) to obtain the linearized E183L gene fragment and the pKO vector fragment respectively. After the reaction, use 1% agarose gel electrophoresis to detect the size of the digested band, and use a gel recovery kit to recover the target fragment. The double enzyme digestion system is shown in Table 1.

Embodiment 3

[0073] The linearized E183L gene fragment and the pKO vector fragment were ligated with T4 ligase to obtain pKO-E183L. The ligation system is shown in Table 2. The ligation product was transformed into Escherichia coli DH5α competent cells. Spread on the LB plate containing kanamycin resistance, pick a single colony, extract the plasmid after expanding the culture, AsisⅠ and MLuⅠ double enzyme digestion to identify positive clones, and sequence verification to obtain the correct pKO-E183L clone, using CMV- F (SEQ No.2:5'CAATGGGAGTTTGTTTTGGCACCA-3') and FH-R (SEQ No.3:5'CTTATTAGTGGTGGTGGTGGTGGTGCTCG-3') were sequenced.

[0074] The conversion process is as follows:

[0075] (1) Take out the competent DH5a prepared in advance from -80°C and place it in an ice bath.

[0076] (2) After the DH5a competent cells are thawed, take 1 μL of the ligation product in 20 μL of DH5a competent cells, mix well, and let stand in an ice bath for 30 minutes.

[0077] (3) Put the centrifuge tub...

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Abstract

The invention provides a recombinant adenovirus carrier for expressing African swine fever virus E183L genes, a creation method of the recombinant adenovirus carrier and a preparation method of a recombinant adenovirus, and belongs to the technical field of genetic engineering. A pAD-EF1alpha-GFP adenovirus expression carrier serves as a base for an E183L-gene-expressing recombinant adenovirus plasmid pAD-E183L, and an E183L gene is introduced to a multiple cloning site. According to the preparation method of the E183L-gene-expressing recombinant adenovirus, the created plasmid pAD-E183L is mixed with an adenovirus packaging system PEI, and an adenovirus packaging process is achieved to obtain the recombinant adenovirus capable of directly infecting eukaryocytes, so that the E183L gene isexpressed normally in the eukaryocytes, and a foundation is laid for research on candidate vaccines for the E183L-gene-expressing recombinant adenovirus carrier.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant adenovirus vector expressing the African swine fever virus E183L gene, a construction method and a preparation method of the recombinant adenovirus. Background technique [0002] African swine fever (African swine fever, ASF) is an acute and severe infectious disease of pigs caused by African swine fever virus (African swine fever virus, ASFV). Clinically, it is characterized by high fever, systemic hemorrhage and high mortality. The disease is very harmful to the pig industry and is listed as a class A disease by OIE. Although my country has not yet introduced the disease, it is always facing the risk of introduction. Therefore, it is of great significance to carry out reserve research on this disease for the prevention of this disease. [0003] ASFV is a regular 20-hedron with a diameter of about 175nm to 215nm. It consists of five comp...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/66C12N15/34C12N7/01C12R1/93
CPCC07K14/005C12N7/00C12N15/66C12N15/86C12N2710/10343C12N2710/10352C12N2710/12022
Inventor 张泉曹舒扬钱淼王涛
Owner YANGZHOU UNIV
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