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Interferon recombinant fusion protein and application thereof

A technology of fusion protein and interferon, which is applied in the direction of interferon, fusion with toxin, cytokine/lymphokine/interferon, etc., which can solve problems such as interference with antiviral function, chronic virus infection, and short half-life

Active Publication Date: 2018-11-23
ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, viruses can "interfere" with the antiviral function of interferon to escape immune clearance and cause chronic viral infection
The development of genetically engineered recombinant interferon makes interferon still very attractive as an antiviral drug choice. The treatment effect is greatly reduced due to the occurrence and species differences

Method used

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  • Interferon recombinant fusion protein and application thereof
  • Interferon recombinant fusion protein and application thereof
  • Interferon recombinant fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Preparation of CaIFNα-linker-RTB target protein

[0035] 1.1 Acquisition of CaIFNα-linker-RTB gene

[0036] Synthesize the CaIFNα-linker-RTB target gene, add canine interferon α downstream (G 4 S) 3 Linker sequence, add ricin B chain sequence, and add NdeI and XhoI restriction enzyme sites at both ends of the target gene. The synthesized target gene nucleotide sequence was connected to the pUC57 vector, and the plasmid was named pUC57-CaIFNα / RTB.

[0037] The synthesized pUC57-CaIFNα / RTB was identified by double enzyme digestion and sequencing to ensure the correct sequence of the target gene.

[0038] 1.2 Construction of Escherichia coli recombinant expression vector

[0039] After the target fragment was identified correctly by sequencing and double enzyme digestion, the pUC57-CaIFNα / RTB plasmid and PET28a sequence were double digested with NdeI and XhoI, and the CaIFNα / RTB target fragment was connected to the PET28a vector fragment by DNA gel recovery a...

Embodiment 2

[0049] Example 2 Activity detection of CaIFNα-linker-RTB fusion protein

[0050] 2.1 Cytotoxicity of CaIFNα / RTB fusion protein

[0051] Using the MTT method to detect the toxic effect of the recombinant CaIFNα / RTB fusion protein obtained in the experiment on MDCK cells, the main steps are as follows:

[0052] (1) Digest well-grown MDCK cells with trypsin, centrifuge and resuspend to make a cell suspension, count on a counting plate, and dilute with 1×10 5 cells / mL to inoculate a 96-well plate, 100 μL per well. 37°C, 5% CO 2 Incubate overnight in an incubator to allow the cells to adhere to a monolayer.

[0053] (2) The CaIFNα / RTB fusion protein obtained in the experiment, according to 10 2 、10 1 、10 0 、10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 μg / mL for dilution.

[0054] (3) Take out the pre-spread 96-well culture plate, add the diluted protein sample into the corresponding well, 100 μL per well, make 3 duplicate wells for each concentration, 37°C, 5% CO 2 Cultiv...

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PUM

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Abstract

The invention discloses an interferon recombinant fusion protein and application thereof. The fusion protein is formed by connecting canine interferon alpha and ricin B chain by (G4S)3 flexible linker. Compared with the common dog interferon alpha, the dog interferon fusion protein has the advantages that the anti-virus activity is improved; the broad-spectrum anti-virus function is realized, andthe self immune response of the dog body is improved.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and specifically relates to a fusion protein composed of canine interferon alpha and ricin B chain protein and a preparation method thereof. Background technique [0002] Interferon (IFN), a glycoprotein with strong antiviral activity, is one of the first lines of defense for the host to defend against pathogen invasion. Interferons are involved in many immune interactions during viral infection and contribute to the induction and regulation of innate and adaptive antiviral mechanisms. However, viruses can "interfere" with the antiviral function of interferon to escape immune clearance and cause chronic viral infection. The development of genetically engineered recombinant interferon makes interferon still very attractive as an antiviral drug choice. The treatment effect is greatly reduced due to reasons such as generation and species differences. [0003] Ricin toxin (R...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K38/21A61K47/42A61P31/12
CPCA61K38/00A61P31/12C07K14/415C07K14/56C07K2319/55
Inventor 刘文森许娜孙成彪
Owner ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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