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CN-121991201-A - Chicken interferon alpha polypeptide, encoding gene thereof, recombinant bacterium thereof and application thereof

CN121991201ACN 121991201 ACN121991201 ACN 121991201ACN-121991201-A

Abstract

The invention belongs to the technical field of microbial genetic engineering, and discloses chicken alpha interferon polypeptide, a coding gene thereof, recombinant bacteria thereof and application thereof, wherein the chicken alpha interferon polypeptide is any one of 1) leucine mutation at 35 th position is threonine, 2) leucine mutation at 35 th position is threonine, tyrosine mutation at 122 th position is glycine, 3) leucine mutation at 35 th position is threonine, glutamine mutation at 82 nd position is arginine, and tyrosine mutation at 122 th position is glycine, wherein the amino acid sequence of the chicken alpha interferon polypeptide is shown as SEQ ID NO.5, the amino acid sequence of the chicken alpha interferon polypeptide is shown as SEQ ID NO.7, the amino acid sequence of the chicken alpha interferon polypeptide is shown as SEQ ID NO. 9. The coding gene of chicken alpha interferon polypeptide is any one of 1) the nucleotide sequence of which is shown as SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.18, SEQ ID NO.20 or SEQ ID NO.22.

Inventors

  • LIU XIAOJING
  • XU XIAOXUE
  • ZHAO PENG
  • LIU ZHAOLU
  • YU LI

Assignees

  • 青岛英特凡恩生物科技有限公司

Dates

Publication Date
20260508
Application Date
20260209

Claims (9)

  1. 1. A chicken interferon-alpha polypeptide, characterized by any one of the following: 1) The amino acid sequence is shown as SEQ ID NO.5, and leucine at position 35 is mutated into threonine; 2) The amino acid sequence is shown as SEQ ID NO.7, the leucine at the 35 th position is mutated into threonine, and the tyrosine at the 122 th position is mutated into glycine; 3) The amino acid sequence is shown in SEQ ID NO.9, wherein leucine at position 35 is mutated into threonine, glutamine at position 82 is mutated into arginine, and tyrosine at position 122 is mutated into glycine.
  2. 2. A gene encoding a chicken interferon-alpha polypeptide according to claim 1, which is any one of the following: 1) The nucleotide sequence of the polypeptide is shown as SEQ ID NO.6, and the coded amino acid sequence of the polypeptide is shown as SEQ ID NO. 5; 2) The nucleotide sequence of the polypeptide is shown as SEQ ID NO.8, and the coded amino acid sequence of the polypeptide is shown as SEQ ID NO. 7; 3) The nucleotide sequence of the polypeptide is shown as SEQ ID NO.10, and the coded amino acid sequence of the polypeptide is shown as SEQ ID NO. 9.
  3. 3. The coding gene of chicken alpha interferon polypeptide according to claim 1, which is obtained by removing signal peptide from the coding gene according to claim 2 and optimizing according to pichia pastoris codon preference, wherein the nucleotide sequence is shown as any one of SEQ ID NO.18, SEQ ID NO.20 or SEQ ID NO. 22.
  4. 4. A recombinant vector carrying a gene encoding the chicken interferon-alpha polypeptide of claim 2.
  5. 5. A recombinant bacterium obtained by transforming a host strain with the recombinant vector according to claim 2 or 3.
  6. 6. The recombinant bacterium according to claim 5, wherein the host bacterium is pichia pastoris or agrobacterium.
  7. 7. Use of the chicken interferon-alpha polypeptide of claim 1, the gene encoding the chicken interferon-alpha polypeptide of claim 2 or 3, the recombinant vector of claim 4 or the recombinant bacterium of claim 5 for preparing chicken interferon-alpha polypeptide.
  8. 8. A method for preparing chicken interferon-alpha polypeptide according to claim 1, which is characterized by comprising the steps of fermenting the recombinant bacterium according to claim 5, inducing expression, separating and purifying to obtain chicken interferon-alpha polypeptide, and preferably, the recombinant bacterium is Pichia pastoris.
  9. 9. Use of the chicken interferon-alpha polypeptide of claim 1, the encoding gene of the chicken interferon-alpha polypeptide of claim 2 or 3, the recombinant vector of claim 4 or the recombinant bacterium of claim 5 in the preparation of a medicament for preventing and treating viral diseases of poultry.

Description

Chicken interferon alpha polypeptide, encoding gene thereof, recombinant bacterium thereof and application thereof Technical Field The invention relates to the technical field of microbial genetic engineering, in particular to chicken alpha interferon polypeptide, a coding gene thereof, recombinant bacteria thereof and application thereof. Background The first large country of global poultry breeding is over 120 hundred million chickens, accounting for over 35% of the world, however, the virus diseases such as avian influenza, newcastle disease and the like cause direct economic loss per year. Chicken interferon is widely used for preventing and treating viral diseases of poultry at present. Interferon has been included as an alternative to the "veterinary antibacterial use reduction regimen" since the general ban of somatotrophic antibiotics in 2020. Therefore, the research of chicken interferon is not only the strategic requirement of epidemic prevention and control, but also the industrial upgrading and international competition core gripper. Chicken alpha interferon (chifnα) is an important antiviral cytokine in the immune system of poultry, and through binding with a receptor (heterodimer formed by IFNAR1 and IFNAR 2) on a cell membrane, activates JAK-STAT signal pathway, induces expression of a series of antiviral proteins (such as 2',5' -oligoadenylate synthetase), thereby inhibiting proliferation of viruses such as avian influenza, newcastle disease and infectious bursal disease, and exerting antiviral effect. Researches show that the chicken alpha interferon can reduce the use amount of antibiotics by more than 50 percent, and simultaneously improve the culture benefit. The chicken alpha interferon is a green additive which is mainly promoted in Henan province, shandong province and the like, and enjoys research and development subsidies and tax benefits. However, the binding affinity of wild-type chicken interferon-alpha to the receptor is limited, which makes it difficult to achieve the desired level of antiviral efficiency, and the wild-type chicken interferon-alpha is easily degraded by proteases in the poultry body, thus limiting the application thereof in the production of breeding. In the prior art, more attempts are made to reform the ChIFNalpha by random mutation or truncation, but the problems of large blindness of mutation sites, insignificant activity improvement and the like exist. Thus, the existing chicken interferon alpha is still to be further improved. Disclosure of Invention Aiming at the problems, the invention provides a modified chicken alpha interferon polypeptide, a coding gene thereof, recombinant bacteria thereof and application thereof, wherein the chicken alpha interferon polypeptide is a wild mutant, has high specific binding force with IFNAR2 and obviously improves antiviral activity. In order to solve the problems, the application provides the following technical scheme: In a first aspect, the present application provides a chicken interferon-alpha polypeptide, which is any one of the following: 1) The amino acid sequence is shown as SEQ ID NO.5, and leucine (L) at position 35 is mutated into threonine (T); 2) The amino acid sequence is shown as SEQ ID NO.7, leucine (L) at position 35 is mutated into threonine (T), tyrosine (Y) at position 122 is mutated into glycine (G); 3) The amino acid sequence is shown in SEQ ID NO.9, leucine (L) at position 35 is mutated into threonine (T), glutamine (Q) at position 82 is mutated into arginine (R), and tyrosine (Y) at position 122 is mutated into glycine (G). In a second aspect, the present application provides a gene encoding the chicken interferon-alpha polypeptide, which is any one of the following: 1) The nucleotide sequence of the polypeptide is shown as SEQ ID NO.6, and the coded amino acid sequence of the polypeptide is shown as SEQ ID NO. 5; 2) The nucleotide sequence of the polypeptide is shown as SEQ ID NO.8, and the coded amino acid sequence of the polypeptide is shown as SEQ ID NO. 7; 3) The nucleotide sequence of the polypeptide is shown as SEQ ID NO.10, and the coded amino acid sequence of the polypeptide is shown as SEQ ID NO. 9. The above sequence is obtained by codon optimization according to codon preference of tobacco, then replacing self signal peptide with tobacco secretion signal peptide (PR 1 a) at N terminal, and removing terminator at the end of the sequence. The IFNAR2 ectodomain gene fragment (amino acids 30-243) was codon optimized according to the codon preference of tobacco, then a tobacco secretion signal peptide (PR 1 a) was added at the N-terminal, and the terminator was removed at the end of the sequence. In a third aspect, the application also provides another coding gene of chicken interferon alpha polypeptide, which is obtained by removing signal peptide from the coding gene and optimizing according to pichia pastoris codon preference, wherein the nucleotide sequence of the coding gene is sho