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Pig ω7 interferon mutant and its preparation method and application

A mutant and interferon technology, which is applied in the field of preparation and application of swine ω7 interferon mutants, can solve problems such as affecting daily life, viral diseases affecting pig breeding industry, etc.

Active Publication Date: 2020-09-29
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

Pork boiled soup can be used as a quick tonic for irritability, dry cough, constipation and dystocia caused by insufficient body fluid; however, some viral diseases affect the survival and development of the pig breeding industry, and also affect people's daily life

Method used

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  • Pig ω7 interferon mutant and its preparation method and application
  • Pig ω7 interferon mutant and its preparation method and application
  • Pig ω7 interferon mutant and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Expression and detection of porcine ω7 interferon and its signal peptide mutant gene in silkworm bioreactor

[0070] 1. Experimental method

[0071] 1.1 Target gene synthesis and construction of recombinant plasmids

[0072] The present invention analyzes all porcine ω7 interferon amino acid sequences on NCBI, performs sequence comparison and signal peptide analysis, and finally determines that the amino acid sequence with the accession number ACF17568.1 is the original sequence, and its amino acid sequence is shown in SEQ ID NO.1 , the nucleotide sequence of its coding gene is shown in SEQ ID NO.2. During the sequence analysis, it was found that the signal peptide of this sequence had multiple cleavage peaks. In view of the previous discovery that the cleavage site of the signal peptide affects the secretion efficiency of interferon, the present invention decided to mutate the amino acid sequence of porcine ω7 interferon. Using SignalP4.1 to predict the sig...

Embodiment 2

[0099] Example 2 Expression and detection of optimized SwIFN-ω7-S mutant in silkworm bioreactor

[0100] 1. Experimental method

[0101] 1.1 Construction of pig ω7-type interferon mutant gene

[0102] The present invention utilizes OptimumGene TM Technology optimized the above-mentioned porcine ω7 interferon mutants, modified the gene sequence according to the codon preference of the silkworm in the bioreactor, and improved the GC content, CpG dinucleotide content, CpG dinucleotide content, and Codon bias, mRNA secondary structure, mRNA free energy stability, RNA instability motif, repeat sequence and other related parameters are optimized and designed, which is conducive to improving the transcription efficiency and translation efficiency of the optimized gene in silkworm , and keep the final translated protein sequence unchanged.

[0103] In order to improve the translation initiation efficiency in the silkworm baculovirus eukaryotic expression system, the Kozak sequence ...

Embodiment 3

[0122] Example 3 Expression and Detection of SwIFN-ω7-S Mutant Optimization and Amino Acid Single Point Mutation in Bombyx mori Bioreactor

[0123] 1. Experimental method

[0124] 1.1 Construction of pig ω7-type interferon mutant gene

[0125] Based on the results of Example 2, the present invention uses the gene sequence of the codon-optimized SwIFN-ω7-S-O mutant as a template, and designs multiple pairs of primers to perform site-directed mutation on this sequence. The site-directed mutation is performed by fusion PCR. For the method of PCR, see "2. Experimental method" above.

[0126] The mutation sites are S27F, I33V, L61F, D67E, H70Q, A74T, I93T, K94E, D101N, I103T, H109C, Q114R, M138V, Q143E, H146R, I161T, E185V, H186D and P190S; The variant was named SwIFN-ω7-S-O-M (1-19) mutant.

[0127] Primers required for amino acid single-site, double-site and multi-site mutations in the nucleotide sequence of SwIFN-ω7-S-O mutant:

[0128] (1) Both upstream and downstream prime...

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Abstract

The invention discloses a pig omega 7 interferon mutant and a preparation method and application thereof. Pig omega 7 interferon with the amino acid sequence shown in SEQ ID NO.1 and the obtained mutant with improved antiviral activity and the sequence shown in SEQ ID NO.3 after the pig omega 7 interferon is subjected to single point mutation are disclosed. Mutant coding genes are subjected to codon optimization to obtain optimized genes with the significantly improved antiviral activity. The mutant shown in the SEQ ID NO.3 is subjected to amino acid single point mutation, dual point mutationand multi-point mutation respectively to obtain a plurality of pig omega 7 interferon mutants with the significantly improved antiviral activity. A silkworm-baculovirus expression system is utilized to express the pig omega 7 interferon mutants in a silkworm bioreactor, and the antiviral activity of the expressed products is greatly improved. The provided pig omega 7 interferon mutant can be usedfor preparing medicines or reagent for preventing or treating pig virosis.

Description

technical field [0001] The present invention relates to porcine ω7 interferon and a porcine ω7 interferon mutant with improved antiviral activity, and the present invention also relates to a method for preparing the porcine ω7 interferon or its mutant using a silkworm baculovirus expression system; the present invention further relates to The invention relates to the application of the porcine ω7 interferon or its mutants in the preparation of drugs or reagents for preventing or treating porcine viral diseases, and belongs to the field of preparation and application of porcine ω7 interferon mutants. Background technique [0002] Interferon is a family of proteins with anti-virus, anti-tumor, and immune-stimulating properties. It plays an important role in resisting virus invasion, killing tumor cells, stimulating humoral immunity and cellular immunity, and regulating physiological balance in humans and animals. The IFN protein family is divided into type I, type II and type ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/555C12N15/20C12N15/866C12N5/10A61K38/21A61P31/14A61P31/16A61P31/20
CPCA61K38/00A61P31/14A61P31/16A61P31/20C07K14/555C12N15/86C12N2710/14043
Inventor 张志芳李轶女胡小元易咏竹刘兴健王先翔赵璐璐赵泽王朋
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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