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Subunit fusion protein CD2V-Fc, preparation method and application thereof

A fusion protein and subunit technology, applied in biochemical equipment and methods, fusion polypeptides, chemical instruments and methods, etc., can solve the problems of difficult application and poor stability of the extracellular region of CD2V, and achieve batch-to-batch stability and ease of use. Mass production, highly controllable effects

Active Publication Date: 2020-07-10
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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  • Subunit fusion protein CD2V-Fc, preparation method and application thereof
  • Subunit fusion protein CD2V-Fc, preparation method and application thereof
  • Subunit fusion protein CD2V-Fc, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032]Example 1 Expression and preparation of CD2V-Fc protein

[0033] 1.1 Selection of African swine fever CD2V-Fc protein

[0034] 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 207-229aa. 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, the use of CD2V protein as an antigen has good prevention and control of African swine fever infection. There is no report that the protein can be expressed and purified on a large scale in a eukaryotic expression system. This may be caused by the instability of the CD2V protein. The present invention In order to solve this important technical problem, a porcine antibody Fc fragment was introduced at the carboxy-terminus of CD2V to ensure the structure and protein stability of CD2V.

[0...

Embodiment 2

[0041] Example 2: Construction of pEE12.4-OPTI-CD2V-Fc recombinant plasmid

[0042] 2.1 PCR amplification of the target fragment OPTI-CD2V-Fc

[0043] 2.1.1 PCR reaction

[0044] (1) Primer design and synthesis

[0045] Upstream primer: 5'-acgaAGCTTGCCGCCACCATGATCAT-3'

[0046] Downstream primer: 5'-GCGGAATTGAATTCTTAATGGTGATG-3'

[0047] (2) Add 50 μL of the sample system, as shown in the table below:

[0048]

[0049] PCR amplification program:

[0050]

[0051] 2.1.2 Gel recovery of PCR products

[0052] (1) Mark the sample collection EP tube, adsorption column and collection tube;

[0053] (2) Take the weight of the marked empty EP tube, and record the value;

[0054] (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;

[0055] (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 centrif...

Embodiment 3

[0119] Example 3: Establishment of transfection of pEE12.4-OPTI-CD2V-Fc recombinant plasmid into CHO-K1 cells and monoclonal screening

[0120] 3.1 CHO-K1 cell transfection

[0121] (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.

[0122] (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.

[0123] (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.

[0124] (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.

[0125] (5) Transfer the digested cells...

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Abstract

The invention provides a subunit fusion protein CD2V-Fc, a preparation method and application thereof. The subunit fusion protein CD2V-Fc contains an extracellular region of an African swine fever virus surface envelope protein CD2V and an antibody Fc protein of a pig, and the amino acid sequence of the subunit fusion protein CD2V-Fc is as shown in SEQ ID NO. 1. The CD2V-Fc can be subjected to soluble expression in a large amount, the protein is stable, a plurality of problems in the prior art are overcome, and the preparation method is simple and low in cost.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products. It relates to a subunit fusion protein CD2V-Fc and its preparation method and application. Background technique [0002] African swine fever (African swine fever, ASF) is an acute, febrile, highly contagious infectious disease of pigs caused by African swine fever virus (ASFV). Pigs are the only mammalian hosts of ASFV natural infection, including domestic pigs and wild boars, especially domestic pigs, which are highly susceptible. Pigs infected with African swine fever virus are clinically characterized by skin congestion, internal organ bleeding, and high fever, and the morbidity and mortality are as high as 100%. 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/85C12N5/10A61K39/187A61K39/385A61P31/20
CPCC07K14/005C12N15/85A61K39/12A61K39/385A61P31/20C12N2710/12022C07K2319/30C12N2710/12034A61K2039/6056
Inventor 钱泓吴有强张强徐玉兰吴素芳车影
Owner NOVO BIOTECH CORP
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