D117L, F317L and EP364R co-expression recombinant adenovirus vector, recombinant adenovirus and construction and application

A technology of EP364R, F317L, applied in the direction of virus/phage, double-stranded DNA virus, application, etc.

Pending Publication Date: 2021-04-02
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But there is no recombinant adenovirus vaccine against these D117L, F317L and EP364R and a recombinant adenovirus vector for preparing the recombinant adenovirus vaccine

Method used

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  • D117L, F317L and EP364R co-expression recombinant adenovirus vector, recombinant adenovirus and construction and application
  • D117L, F317L and EP364R co-expression recombinant adenovirus vector, recombinant adenovirus and construction and application
  • D117L, F317L and EP364R co-expression recombinant adenovirus vector, recombinant adenovirus and construction and application

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Experimental program
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Embodiment 1

[0036] 1) Query the D117L gene (GI: 41902877), F317L gene (GI: 41902813) and EP364R gene (G: 41902829) included in the NCBI website and obtain the optimized and connected gene fragments through artificial synthesis.

[0037] 1. Modification and amplification of fragments

[0038] 1) Primer design

[0039]

[0040] 2) EGFP, EGFP-P2A-D117L-F317L-EP316R-HA fragment PCR

[0041] EGFP: EGFP-F(KpnI)+EGFP-R(+P2A)

[0042] D117L-F317L-EP316R: 117-317-364-F+117-317-364-R (template is PUC57-117-317-364)

[0043] EGFP-P2A-D117L-F317L-EP316R-HA: EGFP-F(KpnI)+HA-R(HindIII)

[0044] The PCR reaction system is as follows:

[0045]

[0046]

[0047] The PCR reaction conditions are as follows:

[0048]

[0049] After the PCR reaction, 1% agarose gel electrophoresis was used to detect the size of the digested band, and the gel recovery kit was used to recover the product.

[0050] 2. Restriction digestion and homologous recombination of pShuttle-CMV vector

[0051] 1) Digest th...

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Abstract

The invention provides a D117L, F317L and EP364R co-expression recombinant adenovirus vector, recombinant adenovirus and construction and application, and belongs to the technical field of gene engineering. Genes are inserted into a pShuttle-CMV eukaryotic expression vector and can be used for constructing the recombinant adenovirus expressing African swine fever virus D117L, F317L and EP364R proteins, and the recombinant adenovirus can directly infect eukaryotic cells, so that the purpose of co-expression of the D117L, F317L and EP364R genes in the eukaryotic cells is achieved, and a good foundation is laid for further research on recombinant adenovirus vector vaccines based on co-expression of the D117L, F317L and EP364R genes.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to D117L, F317L and EP364R co-expression recombinant adenovirus vector, recombinant adenovirus and its construction and application. Background technique [0002] African swine fever (ASF) is an acute, febrile, highly contagious disease of pigs caused by African swine fever virus (ASFV), which is characterized by a short course of disease and high mortality. Symptoms and pathological changes are similar to acute swine fever, showing high fever, skin congestion, abortion, edema and organ hemorrhage. p17 is a late membrane protein expressed by ASFV, encoded by the ORF D117L gene, and is a transmembrane protein located in the inner membrane of the virus. p17 is an intermediate required for icosahedral formation of the viral precursor membrane and is critical for viral viability. The proteins encoded by the two genes F317L and EP364R were relatively high in ASFV, which bel...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/34A61K39/12A61P31/20
CPCC12N15/86C07K14/005A61K39/12A61P31/20C12N2710/10343C12N2800/107C12N2710/12022C12N2710/12034A61K2039/5256A61K2039/552A61K2039/53
Inventor 张泉朱立麒殷俊
Owner YANGZHOU UNIV
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