Duck alpha interferon and its mutant, preparation method and application

A technology of alpha interferon and mutant, which is applied in the preparation of the duck interferon alpha mutant by using the silkworm baculovirus expression system, the preparation and application of the duck interferon alpha mutant, and can solve the problem of late start of research and duck interference. Issues such as the lack of prime research

Active Publication Date: 2022-07-22
JIANGSU UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Interferon was first discovered in poultry, but compared with humans and other mammals, the study on the molecular level of duck interferon started relatively late, and compared with chicken, there are relatively few studies on duck interferon

Method used

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  • Duck alpha interferon and its mutant, preparation method and application
  • Duck alpha interferon and its mutant, preparation method and application
  • Duck alpha interferon and its mutant, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 Expression and detection of DuIFN-α original sequence in silkworm bioreactor

[0082] 1. Experimental method

[0083] 1.1 Obtaining the original sequence of duck interferon alpha

[0084] 1.1.1 In vitro stimulation of duck peripheral blood mononuclear cells (PBMC)

[0085] Ducks were collected aseptically for peripheral anticoagulation, and lymphocyte separation medium was used to separate peripheral blood mononuclear cells and red blood cells. Duck T cells were further isolated by the specific adsorption of nylon wool. In a 24-well cell culture plate, add 1 mL of 8×10 6 mL -1 The above-mentioned PBMCs were incubated at 37℃ for 1-2h to make antigen-presenting somatic cells (APCs) adherent. Separated T lymphocytes (8×10) were added to the above-mentioned 24-well plate with APC cells 6 mL / well) and red blood cells (4×10 6 mL / well) and phytohemagglutinin (PHA) at a final concentration of 5 μg / mL, incubated at 37 °C for 4 h, and collected the cells.

[008...

Embodiment 2

[0112] Example 2 Expression and detection of optimized sequences of DuIFN-α in silkworm bioreactors

[0113] 1.1 Construction of codon-optimized mutant gene of duck interferon alpha

[0114] By analyzing all the amino acid sequences of duck interferon alpha on NCBI, its consensus sequence was obtained. After comparing with the original amino acid sequence of duck interferon alpha cloned in Example 1, the amino acid sequence of the consensus sequence was consistent with the original amino acid sequence, and its amino acid sequence was consistent with the original amino acid sequence. The sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0115] The present invention utilizes OptimumGene TM The technology optimizes the above-mentioned duck alpha interferon consensus sequence gene, and modifies the gene sequence according to the codon preference of the bioreactor silkworm, which affects the GC content and CpG dinucleotid...

Embodiment 3

[0135] Example 3 Expression and detection of DuIFN-α-O in silkworm bioreactor after single amino acid site mutation

[0136] 1. Experimental method

[0137] 1.1 Construction of duck interferon alpha mutant gene

[0138] The present invention uses the gene sequence of DuIFN-α-O as a template, and designs multiple pairs of primers to carry out site-directed mutation of the sequence. The site-directed mutation is carried out by the method of fusion PCR. For the method of fusion PCR, see the aforementioned "2. Experimental method".

[0139] The mutation sites are P2A, L19T, P25L, R35P, D38N, N51D, H64N, L73I, Q80R, D95K, H108D, Q117R, H123Y, R127Q, L136S, R142C, I148T, L159F, D170E or R182C; the resulting duck alpha interference The mutants were named DuIFN-α-O-M1 (P2A, L19T, P25L, R35P, D38N, N51D, H64N, L73I, Q80R, D95K, H108D, Q117R, H123Y, R127Q, L136S, R142C, I148T, L159F, D170E or R182C) mutant.

[0140] Primers required for amino acid single-site, double-site and multi-s...

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Abstract

The invention discloses duck alpha interferon and its mutant, preparation method and application. The invention optimizes the duck alpha interferon and performs amino acid single-site mutation, double-site mutation and multi-site mutation on the basis thereof, and obtains a plurality of duck alpha interferon mutants with improved antiviral activity. The invention also relates to the application of the duck alpha interferon mutant in the preparation of medicines or reagents for preventing or treating duck viral diseases, and belongs to the field of preparation and application of the duck alpha interferon mutant. The present invention utilizes the silkworm baculovirus expression system to greatly improve the antiviral activity of the duck alpha interferon mutant expressed in the silkworm bioreactor, and can be used for preparing medicines or reagents for preventing or treating duck viral diseases, and is suitable for Viral infectious diseases, which are a major threat in the duck breeding industry, have defensive and inhibitory effects.

Description

technical field [0001] The present invention relates to a duck alpha interferon mutant, and also relates to a method for preparing the duck alpha interferon mutant by using a silkworm baculovirus expression system; Or the application in medicines or reagents for treating duck viral diseases, and belongs to the field of preparation and application of duck alpha interferon mutants. Background technique [0002] Interferon is a cytokine with broad-spectrum anti-virus, anti-parasitic bacteria, anti-tumor, and immune function regulation. The IFN protein family is divided into type I, type II and type III interferons according to the sequence, chromosomal location and receptor specificity of their encoded genes. Type I interferons include IFN-α, IFN-β, IFN-ω, IFN-δ, IFN-ε, IFN-ζ, IFN-τ, etc. In mammals, IFN-α and IFN-β are mainly. Type I interferon has strong antiviral activity, mainly inhibiting its proliferation by interfering with the replication of the virus, and also has th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/56C12N15/21C12N15/866A61K38/21A61P31/14A61P31/16A61P31/20
CPCC07K14/56C12N15/86A61P31/14A61P31/16A61P31/20A61K38/00Y02A50/30
Inventor 易咏竹王朋沈兴家唐顺明胡小元魏珍珍曾振
Owner JIANGSU UNIV OF SCI & TECH
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