Duck gamma interferon mutant and preparation method and application thereof

A technology of interferon and mutants, applied in the fields of poultry interferon and its mutants, prevention or treatment of duck viral diseases, duck lambda interferon and its mutants, and can solve the problems of unreported activity of duck type III interferon

Active Publication Date: 2019-09-20
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

Yao et al. cloned and expressed Peking duck type III interferon IFN-λ in 293T cells in 2014, and their research showed that recombinant duck interferon λ could up-regulate the mRNA levels of OASL and Mx-1 in primary duck liver cells, which was comparable to anti- Virus defense and inflammatory response are closely related, but the activity of duck type III interferon IFN-λ has not been reported

Method used

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  • Duck gamma interferon mutant and preparation method and application thereof
  • Duck gamma interferon mutant and preparation method and application thereof
  • Duck gamma interferon mutant and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Expression and detection of duck interferon lambda gene in silkworm bioreactor

[0054] 1. Experimental method

[0055] 1.1 Synthesis of target gene and construction of recombinant plasmid

[0056] The present invention analyzes all amino acid sequences of duck interferon lambda on NCBI, performs sequence alignment and signal peptide analysis, and finally determines that the amino acid sequence shown in the accession number NP_001297721.1 is used as the original amino acid sequence of duck interferon lambda. The sequence is shown in SEQ ID NO.1, and the nucleotide sequence of its coding gene is shown in SEQ ID NO.2.

[0057] Use DNAman software to analyze the restriction enzyme cutting sites, according to the analysis results of the enzyme cutting sites and the multiple cloning sites on the transfer vectors pVL1393 and pUC57, add restriction enzyme cutting sites that do not exist in the target gene sequence at both ends of the target gene . According to the...

Embodiment 2

[0076] Example 2 Obtainment of the conserved amino acid sequence of DuIFN-λ and the expression and detection of the optimized sequence in the silkworm bioreactor 1.1 Obtainment of the conserved amino acid sequence of duck interferon lambda and construction of its codon-optimized mutant gene

[0077] All the amino acid sequences of duck lambda interferon were analyzed to generate its conservative sequence, its amino acid sequence is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.4.

[0078] The present invention utilizes OptimumGene TM Technology optimized the above-mentioned duck interferon lambda conserved sequence gene (SEQ ID NO.4), modified the gene sequence according to the codon preference of the silkworm in the bioreactor, and improved the GC content that affects gene transcription efficiency, translation efficiency and protein folding , CpG dinucleotide content, codon bias, mRNA secondary structure, mRNA free energy stability, RNA instability m...

Embodiment 3

[0097] Example 3 Expression and detection of DuIFN-λ-C-O in silkworm bioreactor after amino acid point mutation

[0098] 1. Experimental method

[0099] 1.1 Construction of duck interferon lambda mutant gene

[0100] The present invention uses the gene sequence of DuIFN-λ-C-O as a template, and designs multiple pairs of primers to perform site-directed mutation on the sequence. The site-directed mutation is carried out by fusion PCR, and the construction of the transfer vector of the mutant is carried out by homologous recombination. The specific method Refer to Ma Kai, et al. (Comparison of double enzyme digestion and homologous recombination method to construct pMIR-reporter vector [J]. Chinese Journal of Pathogenic Biology, 2015(6):495-499.) and sequence verification.

[0101] The mutation sites are A19V, S30T, Y41S, L48Q, K51R, N56S, P62S, T74K, L84I, I95V, D104H, F117L, G126D, Q136K, H143Y, S158G, V166L, H173N; the obtained duck lambda interferon mutants were named DuIF...

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Abstract

The invention discloses a duck gamma interferon mutant and a preparation method and application thereof. Firstly, an amino acid sequence shown in SEQ ID NO. 1 is determined as a duck gamma interferon original amino acid sequence, all amino acid sequences of the duck gamma interferon are compared to obtain a duck gamma interferon conserved sequence shown in SEQ ID No. 3, and codon optimization is carried out on a coding gene to obtain an optimized gene shown in SEQ ID No. 5. Amino acid single-site or multi-site mutation is carried out on the conserved sequence to obtain a plurality of duck gamma interferon mutants with improved antiviral activity. According to the invention, a silkworm baculovirus expression system is further utilized to express the duck gamma interferon or the mutant thereof in a silkworm bioreactor, so that the antiviral activity of the obtained mutant is greatly improved, and the mutant can be used for preventing or treating duck viral diseases, and has remarkable prevention and inhibition effects on viral infectious diseases with great threats in the duck breeding industry.

Description

technical field [0001] The present invention relates to poultry interferon and its mutants, in particular to duck lambda interferon and its mutants. The present invention also relates to a method for preparing the duck lambda interferon mutants using the silkworm baculovirus expression system and the application of the duck lambda interferon The interferon mutant prevents or cures duck viral diseases, and belongs to the field of preparation and application of duck lambda interferon mutants. Background technique [0002] Interferon is a cytokine with a wide range of biological activities such as broad-spectrum anti-virus, anti-parasitic bacteria in cells, anti-tumor, and regulation of immune function. The IFN protein family is divided into type I, type II and type III interferon according to the sequence of its coding gene, chromosomal location and receptor specificity. Type I interferons include IFN-α, IFN-β, IFN-ω, IFN-δ, IFN-ε, IFN-ζ, IFN-τ, etc., and mainly IFN-α and IFN...

Claims

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Application Information

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IPC IPC(8): C07K14/555C12N15/20C12N15/866A61K38/21A61P31/12
CPCA61K38/00A61P31/12C07K14/555C12N15/86C12N2710/14043Y02A50/30
Inventor 胡小元张志芳刘兴健易咏竹李轶女王朋
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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