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Siberian tiger alpha interferon and coding gene and application thereof

A technology of alpha interferon and Siberian tiger, applied in the direction of interferon, application, cytokine/lymphokine/interferon, etc., can solve the problem that the treatment effect of interferon virus disease is not very significant

Inactive Publication Date: 2017-01-18
NORTHEAST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are species differences between cats and Siberian tigers, and the therapeutic effect of interferon on the viral diseases of Siberian tigers is not very significant, so it is very necessary to study Siberian tiger interferon

Method used

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  • Siberian tiger alpha interferon and coding gene and application thereof
  • Siberian tiger alpha interferon and coding gene and application thereof
  • Siberian tiger alpha interferon and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Amplification of Amur tiger IFN-α family gene sequence

[0055] 1. According to the simple and rapid extraction method of whole blood genomic DNA, extract genomic DNA from the blood of Siberian tigers;

[0056] 2. Using Amur tiger genomic DNA as a template, primers mtIFN-αS and mtIFN-αA as primers, PCR amplified IFN-α gene, wherein the nucleotide sequence of primer mtIFN-αS is: 5'-GATCCCCAATGGCGCTGCCCT-3', primer The nucleotide sequence of mtIFN-αA is: 5'-TCCATGTTGAACAGGTCTCATTTCT-3'; the reaction system and reaction conditions are as follows:

[0057] PCR reaction system:

[0058]

[0059] Reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58.2°C for 30 s, extension at 72°C for 50 s, 30 cycles; final extension at 72°C for 10 min, and termination at 4°C. The results of electrophoresis of PCR products on 1% agarose gel were as follows: figure 1 shown.

[0060] The target fragment was recovered, and the p...

Embodiment 2

[0064] Example 2 Prokaryotic expression of Siberian tiger IFN-α family genes

[0065] In the process of prokaryotic expression, the signal peptide part of the Siberian tiger IFN-α family should be removed, so the primers TIFN-αS and TIFN-αA for expressing the mature peptide of the Siberian tiger IFN-α family should be designed first, TIFN-αS: 5'-ACG GGATCC TGTGACCTGCCTCAGACCCA-3' (underline: BamH I restriction site), TIFN-αA: 5'-CGT CTCGAG TCATTTCTCGCTCCTTAACCT-3' (underline: Xho I restriction site).

[0066] PCR amplifies the nucleotide sequence encoding the mature peptide of Amur tiger IFN-α family, and the reaction system is as follows:

[0067]

[0068] Reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58.2°C for 30 s, extension at 72°C for 50 s, 30 cycles; final extension at 72°C for 10 min, and termination at 4°C. The results of electrophoresis of PCR products on 1% agarose gel were as follows: figure 2 shown. ...

Embodiment 3

[0074] Example 3 Functional verification of the antiviral activity of the recombinant Siberian tiger IFN-α family

[0075] 1. Prepare cat kidney cells at 37°C, 5% CO 2 Cultivate for 24 hours;

[0076] 2. Change the cell culture medium, insert 100 μL of 0.3 mg / ml recombinant Siberian tiger IFN-α family protein (prepared in Example 2 of the present invention) into the cat kidney cells simultaneously, and cultivate for 24 h;

[0077] 3. Change the cell culture medium, add 100 μL vesicular stomatitis virus (VSV), canine distemper virus (CDV), avian influenza virus (AIV) respectively at the same time, 100 pfu / well, cultivate for 24 hours, observe under a microscope, and test the virus The received inhibition (inhibition rate) is counted, and the results are as follows Figure 4-Figure 9 shown.

[0078] The results show that the Siberian tiger IFN-α family protein prepared by the present invention has a common conserved domain in its structure, i.e. the IFabd domain (its amino ac...

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Abstract

The invention discloses a Siberian tiger alpha interferon and a coding gene and application thereof. A Siberian tiger alpha interferon family is obtained by cloning a Siberian tiger genome, an amino acid sequence of its mature peptide is shown as SEQ ID No.1-11, and the nucleotide sequence of the coding gene is shown as SEQ ID No.12-22. A Siberian tiger IFN-alpha family protein has a shared conservative structure domain, namely an IFabd structure domain. Antiviral activity functional verification results prove that the Siberian tiger IFN-alpha family protein has the functions of resisting vesicular stomatitis virus, canine distemper and bird flu virus. The invention further discloses an antiviral drug composition, including an effective dose of the Siberian tiger alpha interferon for prevention or treatment and pharmaceutically acceptable excipients or carriers. A solid theoretical basis is laid for the Siberian tiger alpha interferon for the further study of other viral diseases and appearing of Siberian tiger alpha interferon products.

Description

technical field [0001] The present invention relates to Siberian tiger α interferon, in particular to the Siberian tiger α interferon family, and the present invention also relates to the gene sequence encoding the Siberian tiger α interferon family and the use of the Siberian tiger α interferon family in the preparation of antiviral drugs The application belongs to the field of cloning of Siberian tiger alpha interferon family genes. Background technique [0002] The tiger (Panthera tigris) is recognized as the most endangered species in the world. Among the five subspecies of tigers, the Siberian tiger (Panthera tigris altaica) is distributed in the Far East of Russia, the eastern forest area of ​​Northeast China and northern Korea. Some studies in recent years have shown that the bacterial, viral and parasitic diseases of some Siberian tigers have greatly threatened wild Siberian tigers and artificially raised Siberian tigers, causing serious losses. Treatment cannot be ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/56C12N15/21A61K38/21A61P31/16A61P31/14
CPCC07K14/56A61K38/00
Inventor 邢明伟姜广顺孙潇侯志军杨思远柴洪亮
Owner NORTHEAST FORESTRY UNIVERSITY
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