Long-acting canine interferon alpha fusion protein as well as preparation method and application thereof

An alpha interferon and fusion protein technology, applied in the field of biological genetic engineering, can solve the problems of unfavorable purification and activity measurement, and achieve the effects of not easy to break and enzymatic hydrolysis, moderate molecular size, and less background protein.

Pending Publication Date: 2021-01-29
广州源博医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing studies have used canine serum albumin CSA for the fusion expression of canine α-interferon, but the CSA used is a full-length sequence, and the fusion expression of the full-length CSA will cause different degrees of fragmentation and enzymatic degradation. Determination of purification and activity brings many adverse effects

Method used

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  • Long-acting canine interferon alpha fusion protein as well as preparation method and application thereof
  • Long-acting canine interferon alpha fusion protein as well as preparation method and application thereof
  • Long-acting canine interferon alpha fusion protein as well as preparation method and application thereof

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Experimental program
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Embodiment

[0032] Preparation of a long-acting canine interferon-alpha fusion protein:

[0033] 1. Construction of recombinant plasmids

[0034] After taking canine alpha interferon CaIFN-α and CSA truncated peptides (CSA1, CSA2, CSA3 and CSA4) for codon optimization (the canine alpha interferon CaIFN-α, CSA1~4 truncated peptides, Linker, His-Tag Before the fusion expression, Pichia pastoris codons were respectively modified, and the base sequences of canine alpha interferon CaIFN-α, CSA1-4 truncated peptide, Linker, and His-Tag after codon modification are shown in the sequence table SEQ ID NO. 13-19;), synthesized on the plasmid vector pUC57 to obtain CSA1L-CaIFN-α-pUC57, CSA2L-CaIFN-α-pUC57, CSA3L-CaIFN-α-pUC57 and CSA4L-CaIFN-α-pUC57, in the target gene A histidine tag and a stop codon TAA were introduced into the C-terminus of the C-terminus, and the four recombinant plasmids were digested by EcoRI and XbaI, and the target fragments were cloned into the same double-digested express...

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Abstract

The invention belongs to the technical field of biological gene engineering, and particularly relates to a long-acting canine interferon alpha fusion protein as well as a preparation method and an application thereof. The preparation method comprises the following steps: carrying out codon optimization on canine interferon alpha and CSA truncated peptide, synthesizing the canine interferon alpha and CSA truncated peptide on a plasmid vector, carrying out double enzyme digestion, cloning to obtain a target fragment, carrying out Linker connection to obtain a recombinant plasmid vector protein,carrying out enzyme digestion linearization on recombinant plasmids, and introducing the recombinant plasmids into expression host bacteria to obtain recombinant yeast; and carrying out induced expression, deglycosylation and chromatographic purification to obtain a target protein, namely the canine interferon-alpha fusion protein. The fusion protein is established by adopting an efficient pichiapastoris methanol induced secretory expression system, and meanwhile, CSA truncated peptide and Linker with high stability and long-acting canine interferon alpha with high activity are selected, so that the fusion protein can be used for preparing antiviral drugs.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and specifically relates to a long-acting canine alpha interferon fusion protein and its preparation method and application. Background technique [0002] Canine alpha interferon (CaIFN-α) is a protein with broad-spectrum antiviral, immune regulation, anti-tumor and other biological activities. Antiviral activity is the main biological activity of canine interferon alpha, and it is widely used in canine viral diseases Prevention and treatment, including: canine distemper, canine parvovirus enteritis, canine adenovirus, canine parainfluenza, canine coronavirus infection, canine herpes virus infection, canine viral keratitis and other viral diseases. Its mechanism of action is that canine alpha interferon stimulates the body to produce ISG (interferon stimulated gene) products through a series of signal transduction. ISG products include a variety of antiviral proteins, which can eff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/81A61K38/21A61P31/12
CPCA61K38/00A61P31/12C07K14/56C07K2319/31C12N15/81C12N15/815C12N2800/22
Inventor 刘昕赖强王弋谢汝祝段巧梅彭小珍覃伟恒
Owner 广州源博医药科技有限公司
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