Canine interferon mutant recombinant fusion protein as well as preparation method and application thereof

A technology of canine interferon and fusion protein, applied in the field of biological genetic engineering, to achieve the effect of long half-life, prolonging half-life and improving biological activity

Active Publication Date: 2020-03-17
JINLIN MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report on the study of improving the antiviral activity of the fusion protein and prolonging the half-life of the fusion protein by combining the canine IFN-α mutant and ricin B chain protein as a fusion protein

Method used

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  • Canine interferon mutant recombinant fusion protein as well as preparation method and application thereof
  • Canine interferon mutant recombinant fusion protein as well as preparation method and application thereof
  • Canine interferon mutant recombinant fusion protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Preparation of recombinant CaIFNα-Mut / RTB fusion protein

[0045] 1. Construction of CaIFNα mutant expression vector

[0046] Synthesize the CaIFNα-linker-RTB target gene, add canine interferon α downstream (G 4 S) 3Linker 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.

[0047] Design site-directed mutagenesis primers, the sequences of which are as follows:

[0048] SiteⅠ

[0049] Sense: 5'-TTATACCGACGATTTTGCATT-3';

[0050] Anti-sense: 5'-GGAATGCAAAATCGTCGGTATA-3';

[0051] SiteⅡ

[0052] Sense: 5'-GTGTTTCATCTGATTTGTCC-3';

[0053] Anti-sense: 5'-TATCCGGACAAATCAGATGAAAC-3';

[0054] SiteⅢ

[0055] Sense: 5'-TGCGCACCCGTTTTCAGCGCATTAG-3';

[0056] Anti-sense: 5'-TAATGCGCTGAAAACGGGTGCGCAG-3'.

[0057] Using the recombinant plasmid pUC57-CaIFNα / RTB as a tem...

Embodiment 2

[0071] Example 2 Antiviral Detection of Recombinant CaIFNα-Mut / RTB Fusion Protein

[0072] 1. Canine parvovirus infection

[0073] Select 18 Beagle dogs, fast before attacking the virus, and take the canine parvovirus liquid TCID orally 50 =10 7 . At 9 o'clock and 15 o'clock every day, the dog's body temperature, appetite, diarrhea and mental state were detected at fixed points. The success criteria of the model used in this test were: listlessness, elevated body temperature, vomiting, refusal to eat, diarrhea, feces with foul smell and mucus , and "tomato juice"-like bloody stools appeared, and the CPV colloidal gold test paper was strongly positive. Dogs that meet the above conditions are deemed to have successfully prepared the model. The normal group and the model control group were subcutaneously given normal saline 12 hours before the challenge, and the treatment group was subcutaneously given 10 million IU rCaIFNα-Mut / RTB to the neck and back of the dogs 12 hours be...

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Abstract

The invention discloses a canine interferon mutant recombinant fusion protein as well as a preparation method and application thereof and belongs to the field of biological gene engineering. The canine interferon mutant recombinant fusion protein is formed by connecting a canine interferon alpha mutant and a ricin B chain protein through a flexible linker, wherein the flexible linker is (Gly4Ser)3 connecting peptide. The preparation method of the canine interferon mutant recombinant fusion protein comprises the following steps that: step 1, a CaIFN alpha mutant expression vector is constructed; 2, an escherichia coli recombinant expression vector is constructed; 3, the CaIFN alpha and Mut / RTB fusion protein expression are recombined; and step 4, purification and renaturation are performed. The recombinant fusion protein can be applied to the preparation of antiviral drugs. Compared with common canine interferon alpha, the recombinant fusion protein of the invention has the advantagesthat longer half-life period and higher biological activity. The half-life period of the recombinant fusion protein is prolonged; the biological activity of the recombinant fusion protein is improved;the cost of an interferon preparation is reduced; and the recombinant fusion protein has a potential and value of being developed into broad-spectrum antiviral drugs and in veterinary clinical application.

Description

technical field [0001] The invention relates to the technical field of biogenetic engineering, in particular to a canine interferon mutant recombinant fusion protein and its preparation method and application. Background technique [0002] Interferon (Interferon, IFN), a glycoprotein produced by biological cells with multiple functions, has a broad-spectrum antiviral effect and is the main line of defense for the host to defend against pathogen invasion. Interferon itself cannot directly kill viruses, but by inducing host cells to synthesize antiviral proteins, enhancing the activity of natural killer cells (NK cells), macrophages and T lymphocytes, regulating host immune function, and exerting its antiviral effect. According to different sources of interferon production, different physical and chemical properties, different biological activities and different recognition receptors, IFN is divided into types I, II and III. Among them, type Ⅰ IFN has stronger antiviral activ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/36C07K1/34C07K1/22C07K1/18C12N15/62C12N15/70A61K38/21A61P31/12
CPCA61K38/00A61P31/12C07K14/56C07K2319/31C07K2319/55C12N15/70
Inventor 许娜孙成彪常影
Owner JINLIN MEDICAL COLLEGE
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