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Cat interferon omega, and coding gene and antiviral application thereof

A technology of interferon and antiviral drug, applied in the direction of interferon, antiviral agent, cytokine/lymphokine/interferon, etc., can solve the problems of limited sequence of cat interferon and delayed development, and achieve obvious antiviral activity. Effect

Active Publication Date: 2019-01-15
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Interferon is the most effective therapeutic drug for the treatment of viral infectious diseases, but the current research on feline interferon and the development of clinical preparations are still relatively lagging behind, and the published sequences of feline interferon are relatively limited

Method used

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  • Cat interferon omega, and coding gene and antiviral application thereof
  • Cat interferon omega, and coding gene and antiviral application thereof
  • Cat interferon omega, and coding gene and antiviral application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The acquisition of embodiment 1 novel cat interferon omega

[0038] 1. Primer design and sequence analysis

[0039] According to the published feline interferon ω gene sequence in GenBank, the gene sequence was compared by DNAMAN, and a pair of degenerate primers were designed using Oligo6 to amplify the full length of the feline interferon ω gene. BamHI and Kpn I restriction enzyme sites were introduced at the end (for constructing recombinant expression vectors expressing feline interferon). The primer sequences are shown in Table 1, and the primers were synthesized by Jilin Kumei Biotechnology Co., Ltd.

[0040] Table 1 Primer Sequence

[0041]

[0042] 2. Extraction of Feline Genomic RNA

[0043] The present invention adopts the method of virus stimulation (feline intestinal coronavirus) combined with interferon inducer stimulation (polymyocyte polyI:C) to stimulate pet cat body, and then induce cat interferon gene expression. The specific operation is as fol...

Embodiment 2

[0054] A large amount of expression of embodiment 2 novel feline interferon omega

[0055] 1. Construction of recombinant expression vector pCold-TF-ω

[0056] The PCR products of the expression vector pCold-TF and the novel feline interferon ω obtained in Example 1 were double-digested with restriction endonucleases Kpn I and BamH I, and the DNA gel recovery kit was used to recover the target gene fragment, and then the The PCR product of the novel feline interferon ω coding gene was connected into the expression vector pCold-TF by T4 ligase to construct the recombinant expression vector pCold-TF-ω.

[0057] 2. Construction of recombinant bacteria pCold-TF-ω / BL21

[0058] The recombinant expression vector was transformed into BL21 competent cells, spread on the LB agar plate containing ampicillin resistance and cultivated, the positive recombinant bacteria were selected, and the enzyme digestion method was used (results see image 3 Shown in A), PCR method (results see im...

Embodiment 3

[0061] The antiviral activity assay of embodiment 3 novel cat interferon omega

[0062] Taking feline enteric coronavirus and feline parvovirus as model viruses, the antiviral activity of the novel feline interferon ω obtained in the present invention is determined by a microcytopathic inhibition method. The specific operation is as follows:

[0063] Inoculate cat kidney cells F81 in 96-well cell plates and store at 37°C, 5% CO 2 Cultivate in the incubator until the cell monolayer, respectively add 2-fold dilution of the new cat interferon ω of the present invention to act for 16 hours, and then add 100TCID to each well 50 At the same time, a negative control group and a positive control group of known cat α, ω2 and ω9 interferon were set up, and the cytopathic situation was observed after 24 hours.

[0064] Table 3 Interferon antiviral activity comparison results

[0065]

[0066]

[0067] Note: + indicates no cytopathic effect (that is, has antiviral activity), - in...

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Abstract

The invention discloses a cat interferon omega, and a coding gene and antiviral application thereof. The amino acid sequence of the cat interferon omega is as shown in SEQ ID NO. 2. Preferably, a nucleotide sequence encoding the cat interferon omega is as shown in SEQ ID NO. 1. In addition, the invention also discloses a method for preparing the cat interferon omega and the application of the catinterferon omega to preparation of antiviral drugs. According to the invention, the manner of combined stimulation by a virus (a cat intestinal coronavirus) and an interferon inducer (poly I: C) is employed for stimulating the body of a pet cat so as to induce the expression of a cat interferon gene; and then, PCR amplification is carried out to obtain a cat interferon omega gene. The results of cell tests show that the cat interferon omega obtained in the invention has obvious antiviral activity and is superior to existing known cat interferons. The invention lays a foundation for further research, development and application of cat interferon products.

Description

technical field [0001] The present invention relates to a novel feline interferon omega, its encoding gene and its antiviral application. The invention belongs to the technical field of veterinary medicine. Background technique [0002] Interferon is an antiviral cytokine produced when the body is infected by a virus. It is released by infected cells to inhibit the proliferation of the virus. It has broad-spectrum antiviral and immune response enhancement effects. There are two types of interferons that have been identified: type I and type II. Type I interferon mainly includes IFN-α, IFN-β and IFN-ω; type II interferon mainly includes IFN-γ. At present, the research on human interferon is progressing rapidly. For example, recombinant human interferon α has been widely used in the treatment of human virus infection, and recombinant human interferon ω has also been used in the treatment of SARS. [0003] In recent years, the number of pet cats in my country has grown rapid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/555C12N15/20C12N15/70A61K38/21A61P31/14A61P31/20
CPCA61P31/14A61P31/20C12N15/70C07K14/555A61K38/00
Inventor 徐义刚范文禄王丽王紫微朱妍
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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