Cat omega 2 interferon mutant as well as preparation method and application thereof

A technology of interferon and mutant, which is applied to cat ω interferon, to prepare medicines or reagents for preventing or treating cat viral diseases, and the field of cat ω2 interferon mutant, which can solve the problem of low expression level of cat interferon and anti-virus Insufficient activity, etc.

Active Publication Date: 2018-07-13
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no specific drug for the treatment of viral diseases, and there is an urgent need for high-quality and cheap feline interferon products to be launched, but the expression level of feline interferon is not high at present, and the antiviral activity is not strong

Method used

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  • Cat omega 2 interferon mutant as well as preparation method and application thereof
  • Cat omega 2 interferon mutant as well as preparation method and application thereof
  • Cat omega 2 interferon mutant as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1 Expression and detection of feline interferon ω2, 9, 11 and ω2 mutant gene sequence in silkworm bioreactor

[0051] 1. Experimental method

[0052] 1.1 Acquisition, synthesis and plasmid construction of feline ω-type interferon gene

[0053] 1.1.1 Gene acquisition

[0054] Search and obtain the gene sequence and amino acid sequence of FeIFN-ω2 (GenBank accession number: DQ420221), FeIFN-ω9 (GenBank accession number: DQ420228), FeIFN-ω11 (GenBank accession number: DQ420230.1) from NCBI.

[0055] Among them, the amino acid sequence of feline ω2 interferon is shown in SEQ ID NO.1, its gene sequence is shown in SEQ ID NO.3; the amino acid sequence of cat ω11 interferon is shown in SEQ ID NO.6.

[0056] After comparing the gene sequences and amino acid sequences of the 13 subtypes of feline ω-interferon, it was found that the amino acid composition of each subtype has very small mutations, such as amino acid changes from P to L, and G to A; cat ω-interferon ty...

Embodiment 2

[0074] Example 2 Expression and detection of mutated and optimized feline interferon ω11 gene in silkworm bioreactor

[0075] Based on the comparison results of Example 1, the mutation of the cat ω2 interferon gene is beneficial to the improvement of antiviral activity. Therefore, the cat ω11 type interferon gene was mutated according to the same method as ω2-M.

[0076] 1. Experimental method

[0077] 1.1 Mutation, optimization and synthesis of feline ω11-type interferon gene

[0078] Obtain the gene sequence and amino acid sequence of FeIFN-ω11 (GenBank accession number: DQ420230.1) from NCBI, according to the method of Example 1, introduce 4 amino acid site-directed mutations: R82Q, P105L, V141A, G194A, and obtain 196 The amino acid mutant sequence is shown in SEQ ID NO.7, and the deduced mutant gene sequence is shown in SEQ ID NO.8. The DNAman software was used to analyze the restriction enzyme cutting sites of the mutated gene sequence. According to the analysis results...

Embodiment 3

[0098] Embodiment 3 Mutation and optimization of cat interferon ω2 gene and expression and detection in silkworm bioreactor

[0099] Based on the results of Examples 1 and 2, cat ω-interferon gene mutation and optimization are conducive to the improvement of antiviral activity. For this reason, cat ω-2 type interferon gene and mutant gene are adjusted to ω2 and ω2-M according to the optimization method in Example 2. Gene optimization, the specific method is as follows:

[0100] 1. Experimental method

[0101]1.1 Construction of cat ω2 interferon mutant gene and optimization and synthesis of ω2 interferon gene and mutant gene sequence

[0102] 1.1.1 Construction of cat ω2-type interferon mutant gene

[0103] The present invention is based on the comparison results of the gene sequences and amino acid sequences of 13 subtypes of feline ω-interferon in Example 1. In order to improve the expression of feline ω-interferon in the baculovirus expression system, the present inventio...

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Abstract

The invention discloses a cat omega 2 interferon mutant as well as a preparation method and application thereof and belongs to the field of preparation and application of cat omega 2 interferon. The cat omega 2 interferon mutant is obtained by comparing gene sequences and amino acid sequences of 13 subtypes of the cat omega 2 interferon and performing amino acid site-directed mutation on the cat omega 2 interferon. The invention further discloses a method of preparing the cat omega 2 interferon mutant. The method comprises the following steps of performing site mutation on a cat omega 2 gene,performing optimization on a mutant gene, cloning a gene for encoding the cat omega 2 interferon mutant into a baculovirus transfer vector to be recombined with baculovirus to infect an insect host, and expressing a foreign gene to acquire a cat omega 2 interferon protein expression product, wherein the antiviral activity of the cat omega 2 interferon can be improved by 45 percent or above throughmutation and can be improved by 96 percent or above through optimization and mutation of the gene. The method provided by the invention is simple in technology and can efficiently and stably acquirethe cat omega 2 interferon which can be safely used, and the antiviral activity of the cat omega 2 interferon is remarkably improved.

Description

[0001] This application is a divisional application of the parent application with the application number 201510197296.5, the application date is April 23, 2015, and the invention title is "cat omega interferon mutant and its preparation method and application". technical field [0002] The present invention relates to feline omega interferon, in particular to cat ω2 interferon mutant, and the present invention also relates to a preparation method of the feline ω2 interferon mutant and its use in the preparation of medicines or reagents for preventing or treating feline viral diseases The invention belongs to the field of preparation and application of feline ω2 interferon. Background technique [0003] Interferon (IFN) is a highly biologically active glycoprotein produced by cells under the action of specific inducers, and has broad-spectrum antiviral activity in animal cells. It is currently known that interferon does not directly kill the virus, but interferes with the ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/555C12N15/20C12N15/866A61K38/21A61P31/12
CPCA61K38/00C07K14/555C12N15/866
Inventor 胡小元李轶女张志芳李浩洋刘兴键易咏竹陈晓妍
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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