Recombinant adenovirus vector for expressing African swine fever virus p72 and B602L proteins, recombinant adenovirus and construction method

A technology of African swine fever virus, B602L, applied in the field of genetic engineering

Inactive Publication Date: 2020-08-18
SICHUAN HUASHEN ANIMAL BIOLOGICAL PRODS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After the outbreak of African swine fever, many domestic units have carried out research on African swine fever vaccines, including subunit vaccines, peptide vaccines, and gene deletion vaccines, but so far no effective vaccines have come out

Method used

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  • Recombinant adenovirus vector for expressing African swine fever virus p72 and B602L proteins, recombinant adenovirus and construction method
  • Recombinant adenovirus vector for expressing African swine fever virus p72 and B602L proteins, recombinant adenovirus and construction method
  • Recombinant adenovirus vector for expressing African swine fever virus p72 and B602L proteins, recombinant adenovirus and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1 Construction of recombinant adenovirus vector

[0019] The amino acid sequence of the expressed African swine fever virus p72 protein is shown in SEQ ID NO: 2, the amino acid sequence of the expressed African swine fever virus B602L protein is shown in SEQ ID NO: 3, a histidine tag HHHHHH is added to the end of the p72 protein, and the added A T2A self-cleavage signal peptide is added between the p72 protein with the histidine tag HHHHHH and the B602L protein. The amino acid sequence of T2A is shown in SEQ ID NO:4.

[0020] According to the above amino acid sequence, the nucleotide sequence capable of expressing p72 and B602L proteins was artificially designed. The nucleotide sequence is shown in SEQ ID NO: 1. The designed nucleotide sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd. The synthesized sequence was inserted between the NheI and HindIII restriction sites of the pDC316-mCMV-EGFP vector, and the constructed vector was named pDC...

Embodiment 2

[0021] Embodiment 2 Preparation of recombinant adenovirus

[0022] Passage 293A cells to 12-well plates according to conventional methods, and use transfection reagent ( HD, purchased from Promega) was transfected with pDC316-p72-B602L plasmid and pBHGloxdelE13cre plasmid, and the transfection reagent used was 6ul, pDC316-p72-B602L plasmid and pBHGloxdelE13cre plasmid were each lug. After transfection, the cells were cultured in a 37°C, 5% carbon dioxide incubator. Cell status was observed daily. The expression of green fluorescent protein can be observed 12 hours after transfection, and green fluorescent spotting can be observed 7 days after transfection (result figure see figure 2 ). Continue culturing for 48 hours after the appearance of fluorescent spots, freeze and thaw the cells three times at -80°C, centrifuge at 12,000 rpm, store at -80°C, and count them as ADV p72 / B602L P1 generation virus.

[0023] Subculture 293A cells into 96-well plates according to the conv...

Embodiment 3

[0024] Example 3 Small-scale preparation and purification of ADV p72 / B602L

[0025] Passage 293A cells to T175 cell flasks according to the conventional method, remove the cell culture medium after the cells grow into a dense monolayer, inoculate 0.5ml ADV p72 / B602L P3 generation virus, absorb in a cell incubator at 37°C for 30 minutes, and then pour the inoculated cells 30 ml of 2% calf serum (purchased from GIBCO) in DMEM (purchased from GIBCO) culture solution was added to the bottle. Cultured in a 5% carbon dioxide incubator at 37°C. Harvest the virus liquid after more than 90% of the cells appear cytopathic, centrifuge at 12,000 rpm for 10 minutes, take the supernatant and add it to a 100KD ultrafiltration centrifuge tube (purchased from MERCK), centrifuge at 5,000 rpm to 1 / 10 of the original volume, and then add 0.1M Phosphate buffer to the original volume, centrifuged at 5000rpm to 1 / 100 of the original volume, and finally added glycerol at a ratio of 5:1, then aliquot...

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Abstract

The invention discloses a recombinant adenovirus vector for expressing African swine fever virus p72 and B602L proteins, a recombinant adenovirus and a construction method. The recombinant adenoviruscan synchronously express the African swine fever p72 and B602L proteins after infecting cells, and the B602L is serially expressed with the p72 through self-splitting decomposition 2A signal peptide.According to the invention, a recombinant adenovirus vector capable of simultaneously expressing p72 and B602L proteins is constructed; the recombinant adenovirus capable of expressing African swinefever virus p72 and B602L proteins is obtained after cells are co-transfected by using the vector and an adenovirus skeleton system, a test animal can be stimulated to generate a specific antibody after the recombinant adenovirus is used for immunizing the test animal, and new test data is provided for the development of African swine fever vaccines.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant adenovirus vector expressing African swine fever virus p72 and B602L proteins, a recombinant adenovirus and a construction method. Background technique [0002] African swine fever virus (ASFV) is a large nucleoplasmic DNA virus and the only arbovirus-borne DNA virus. It can infect domestic pigs and wild boars, and has strong infectivity and pathogenicity. The clinical symptoms of pigs infected with African swine fever are similar to those of swine fever and swine erysipelas. The severity of the symptoms is different from the virulence, infection rate and infection route of ASFV. According to the severity of clinical symptoms, it can be divided into acute infection , subacute infection and invisible infection. The main features of acute infection are loss of appetite, high fever, leukopenia, and hemorrhage of the skin and internal organs, and the probab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/34C12N15/861A61K39/12A61P31/20
CPCA61K39/12A61K2039/552A61P31/20C07K14/005C12N15/86C12N2710/10043C12N2710/12022C12N2710/12034C12N2800/107
Inventor 周远成邝声耀阴文奇
Owner SICHUAN HUASHEN ANIMAL BIOLOGICAL PRODS
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