Recombinant adenovirus vector for expressing African swine fever virus (ASFV) EP402R gene, construction method of recombinant adenovirus vector and preparation method of recombinant adenovirus

A technology of EP402R and African swine fever virus, applied in the field of recombinant adenovirus vector expressing African swine fever virus EP402R gene, recombinant adenovirus preparation

Inactive Publication Date: 2018-09-07
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the prior art, there is a lack of research on vaccines

Method used

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  • Recombinant adenovirus vector for expressing African swine fever virus (ASFV) EP402R gene, construction method of recombinant adenovirus vector and preparation method of recombinant adenovirus
  • Recombinant adenovirus vector for expressing African swine fever virus (ASFV) EP402R gene, construction method of recombinant adenovirus vector and preparation method of recombinant adenovirus
  • Recombinant adenovirus vector for expressing African swine fever virus (ASFV) EP402R gene, construction method of recombinant adenovirus vector and preparation method of recombinant adenovirus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

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

Embodiment 2

[0071] The gene synthesis sequence EP402R and the vector pKO-FH( Figure 4 ) to obtain a linearized EP402R gene fragment and a 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 EP402R gene fragment and the pKO vector fragment were ligated with T4 ligase to obtain pKO-EP402R. 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-EP402R clone, using CMV- F (SEQ No. 2: 5' CAATGGGAGTTTGTTTTGGCACCA-3') and FH-R (SEQ No. 3: 5' CTTATTAGTGGTGGTGGTGGTGGTGCTCG-3') were sequenced. The conversion process is as follows:

[0074] (1) Take out the competent DH5a prepared in advance from -80°C and place it in an ice bath.

[0075] (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.

[0076] (3) Put the centrifuge tub...

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Abstract

The invention provides a recombinant adenovirus vector for expressing an African swine fever virus (ASFV) EP402R gene, a construction method of the recombinant adenovirus vector and a preparation method of a recombinant adenovirus, belonging to the technical field of genetic engineering. The method provides recombinant adenovirus plasmids pAD-EP402R for expressing the EP402R gene, a pAD-EF1alpha-GFP adenovirus expression vector is used as basis, and the EP402R gene is introduced at a multiple cloning site. The invention also provides the preparation method of the recombinant adenovirus for expressing the EP402R gene; the contracted plasmids pAD-EP402R are mixed with an adenovirus packaging system PEI so as to realize the packaging process of the adenovirus, so that the recombinant adenovirus capable of directly infecting eukaryotic cells is obtained; therefore, the aim of the normal expression of the EP402R gene in the eukaryotic cells is realized, and a foundation is laid for the study of a recombinant adenoviral vector candidate vaccine based on expression of the EP402R.

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 EP402R gene, a construction method and a preparation method of the recombinant adenovirus. Background technique [0002] CD2 is a surface molecule of T cells and NK cells, B cells and macrophages. Its ligand is the CD58 molecule expressed on the surface of various types of cells. The combination of the two has a stabilizing effect on the combination between T cells and antigen-presenting cells, and thus participates in the activation of T cells. The EP402R gene encodes CD2v, which has a signal peptide sequence and a transmembrane region. The extracellular region contains two immunoglobulin-like domains. There is no similarity, so it is simply called CD2v. CD2v can bind to the conjugation protein of the host cell, interfere with the endocytosis and protein transport of the host cell,...

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 张泉朱辰钱淼王涛
Owner YANGZHOU UNIV
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