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Recombinant African swine fever virus CD2V subunit protein as well as preparation method and application thereof

An African swine fever virus and subunit technology, applied in the field of veterinary biological products, can solve the problems of poor stability and difficult application of CD2V extracellular region, and achieve easy large-scale production, batch-to-batch stability, and high biosafety Effect

Pending Publication Date: 2020-07-31
NOVO BIOTECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no large amount of expression has been seen, indicating that the stability of the extracellular region of CD2V is poor, so it is difficult to apply in actual production

Method used

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  • Recombinant African swine fever virus CD2V subunit protein as well as preparation method and application thereof
  • Recombinant African swine fever virus CD2V subunit protein as well as preparation method and application thereof
  • Recombinant African swine fever virus CD2V subunit protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 CD2V protein expression and preparation

[0034] 1.1 Selection of African swine fever CD2V protein

[0035] The African swine fever structural protein CD2V is a polypeptide encoded by the EP402R gene. According to prediction analysis, there is a transmembrane region at 207Y-229L. Studies have shown that the CD2V protein can interact with red blood cells and play a role in the spread of the virus and the damage of lymphocytes. important role in the process. Therefore, using the CD2V protein as an antigen has a good prevention and control of African swine fever infection, and there is no report that the protein can be expressed and purified on a large scale in a eukaryotic expression system, which is also an important technology to be solved by the present invention question.

[0036] 1.2 African swine fever CD2V protein codon optimization

[0037] Based on the subtype of African swine fever strain reported in China in 2018 and referring to the full gene sequ...

Embodiment 2

[0039] Example 2: Construction of pEE12.4-OPTI-CD2V recombinant plasmid

[0040] 2.1PCR amplification of the target fragment OPTI-CD2V

[0041] 2.1.1 PCR reaction

[0042] (1) Primer design and synthesis

[0043] Upstream primer: 5'-cgAAGCTTGCCGCCACCATGATCATCCTG-3'

[0044] Downstream primer: 5'-CGCGAATTCTTAATGGTGATGGTG-3'

[0045] (2) Add 50 μL of the sample system, as shown in the table below:

[0046]

[0047] PCR amplification program:

[0048]

[0049] 2.1.2 Gel recovery of PCR products

[0050] (1) Mark the sample collection EP tube, adsorption column and collection tube;

[0051] (2) Take the weight of the marked empty EP tube, and record the value;

[0052] (3) Carefully cut out a single target DNA band from the agarose gel with a scalpel on a gel cutter and put it into a clean 1.5mL centrifuge tube;

[0053] (4) Add 600 μL PC buffer to the 1.5mL centrifuge tube in step (3), place in a 50°C water bath for about 5 minutes, and gently turn the centrifuge tu...

Embodiment 3

[0100] Example 3: Establishment of transfection of pEE12.4-OPTI-CD2V recombinant plasmid into CHO-K1 cells and monoclonal screening

[0101] 3.1 CHO-K1 cell transfection

[0102] (1) Preparation: UV sterilization in a biological safety cabinet for 30 minutes; DMEM / F12 (containing 10% serum, 1% double antibody), DMEM / F12 and PBS were placed in a 37°C water bath and preheated to 37°C.

[0103] (2) Take out the cells (10 cm cell culture dish) from the incubator at 37° C., discard the supernatant medium, wash the cells once with pre-warmed 8 mL PBS, and discard the PBS.

[0104] (3) Add 1-2mL 0.25% trypsin-EDTA to each 10cm cell culture dish, digest at room temperature for about 2 minutes, observe under the microscope that the cells shrink and become round, and appear as single cells.

[0105] (4) Add 4 mL of DMEM / F12 (containing 10% serum, 1% double antibody) to terminate the digestion reaction, and blow the cells away with a pipette.

[0106] (5) Transfer the digested cells to a...

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Abstract

The invention discloses a recombinant African swine fever virus CD2V subunit protein as well as a preparation method and application thereof. The protein comprises an extracellular region and an intracellular region of African swine fever virus surface envelope protein, and the amino acid sequence of the protein is shown as SEQ ID NO.3. The preparation method comprises the following steps: 1) cloning a codon-optimized gene sequence shown as SEQ ID NO.1 into an eukaryotic expression vector; 2) transfecting a recombinant expression vector containing the African swine fever virus subunit proteincoding gene into CHO cells; 3) culturing, screening and domesticating a CHO cell strain in the step 2) to obtain a highly-expressed cell strain; 4) fermenting and culturing the cell strain in the step3), and performing purifying to obtain the African swine fever virus CD2V subunit protein; and 5) mixing the CD2V protein with a pharmaceutically acceptable adjuvant to obtain a subunit vaccine. Theinvention can provide the African swine fever surface CD2V subunit protein which can be industrially produced on a large scale, the preparation method is simple and low in cost, and the prepared vaccine can reach the existing national standard.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products. The invention relates to a preparation method and application of African swine fever CD2V protein. Background technique [0002] African swine fever (ASF) is an acute, febrile and highly contagious infectious disease of pigs caused by African swine fever virus (ASFV), with a morbidity and mortality rate of up to 100%. Pigs infected with African swine fever virus have skin congestion, internal organ bleeding, and high fever as clinical symptoms. Pigs are the only mammalian hosts of ASFV natural infection, including domestic pigs and wild boars, especially domestic pigs. The industry has a huge impact. The World Organization for Animal Health classifies it as a Class A epidemic disease, and my country also classifies it as a Class I animal infectious disease. [0003] Since African swine fever was discovered on the African continent in 1927, it has dealt a severe blow to t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/01C12N15/34A61K39/12A61P31/20
CPCC07K14/005A61K39/12A61P31/20C12N2710/12022C12N2710/12034A61K2039/552Y02A50/30
Inventor 钱泓吴有强张强徐玉兰吴素芳车影
Owner NOVO BIOTECH CORP
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