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Recombinant bovine consensus ω-interferon and its application

An interferon and gene technology, applied in the field of molecular biology, can solve problems such as differences in biological activity, unclearness, and lack of comparative research.

Active Publication Date: 2018-12-28
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The homology of these subtypes is more than 80%. Different subtypes may be secreted by different cells or the same cell at different stages. However, due to the lack of systematic comparative studies, it is unclear whether there are differences in their biological activities.

Method used

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  • Recombinant bovine consensus ω-interferon and its application
  • Recombinant bovine consensus ω-interferon and its application
  • Recombinant bovine consensus ω-interferon and its application

Examples

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Embodiment 1

[0039] The acquisition of embodiment 1 protein CoBoIFN-ω (recombinant bovine consensus ω-interferon)

[0040] 1. Design of CoBoIFN-ω and its gene optimization and synthesis

[0041] 1.1 Design of CoBoIFN-ω gene sequence

[0042] CoBoIFN-ω is a synthetic non-naturally occurring BoIFN-ω hybrid. Its amino acid sequence is designed by scanning all BoIFN-ω subtype sequences that have been published in GenBank and combining the most frequently occurring amino acids at each corresponding position. , all amino acids of which are derived from the gene sequences of each subtype of the BoIFN-ω family. The DNA sequence of CoBoIFN-ω in this example is a designed gene fragment directly synthesized by BGI, but the acquisition of CoBoIFN-ω DNA is not limited to this example.

[0043] 1.2 Addition of restriction sites and protease (Kex2) restriction sites

[0044] When designing the CoBoIFN-omega gene sequence, XhoI and EcoRI restriction endonuclease recognition sites were introduced at the...

Embodiment 2

[0130] Example 2 Antiviral Activity Analysis of Yeast Secreted Protein CoBoIFN-ω

[0131] Note: For the determination of its antiviral activity, the prokaryotic expressed BoIFN-omega24 in our laboratory (see literature for specific preparation methods, Characterization and antivirus activities of a novel bovine IFN-omega24, Xiuxin Luo, Yongli Guo, Jun Bao, Ying Liu, Dong An, Bo Ma. Molecular Immunology.05 / 2015; 66(2):357-363.) as its control. Rabbit anti-BoIFN-ω24, BoIFNR1 and BoIFNR2 polyclonal antibody serum was prepared in this laboratory.

[0132] 1 Detection of antiviral activity of yeast secreted protein CoBoIFN-ω

[0133] 1.1 Preparation of VSV

[0134] (1) Amplification and preservation of VSV

[0135] Dilute the VSV seed at 1:1000 with DMEM nutrient solution containing 10% superior fetal bovine serum, inoculate it on well-growing monolayer bovine kidney cells (MDBK) for proliferation, incubate until 80% of the cells are diseased, and freeze-thaw repeatedly for 3 T...

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Abstract

The invention relates to a recombinant common omega-interferon and an application thereof, and belongs to the field of molecular biology. The amino acid sequence of the recombinant common omega-interferon (CoBoIFN-omega) is represented by SEQ ID No.1, and a nucleotide sequence for encoding CoBoIFN-omega is represented by SEQ ID No.2. The CoBoIFN-omega realizes efficient and soluble secretion and expression in Pichia pastoris, and an antiviral activity experiment result confirms that the CoBoIFN-omega has strong VSV pathology formation inhibition ability in MDBK and PK-15 cells, the VSV pathology formation inhibition ability of the CoBoIFN-omega in the MDBK is 3.93 times higher than that of BoIFN-omega 24 in the MDBK cell, and the VSV pathology formation inhibition ability of the CoBoIFN-omega in the PK-15 cell is 12.8 times higher than that of the BoIFN-omega 24 in the PK-15 cell. The antiviral activity of the CoBoIFN-omega is greatly higher than that of other similar products, so the CoBoIFN-omega is more suitable for clinic application and industrial exploitation.

Description

technical field [0001] The invention relates to recombinant bovine consensus ω-interferon and application thereof, in particular to the nucleotide sequence of bovine consensus IFN-ω (CoBoIFN-ω) and its expressed protein, as well as its biological activity, belonging to the field of molecular biology. Background technique [0002] The bovine IFN-ω subfamily consists of 24 members with similar structures and functions. The bovine IFN-ω gene has 585 nucleotides, encodes 195 amino acids, and contains a signal peptide of 23 amino acids. Bovine IFN-ω has four conserved cysteines, located at positions 1, 29, 99 and 139, which can form two intramolecular disulfide bonds (Cys 1-Cys99, Cys29-Cys139). [0003] IFN-ω evolved from IFN-α about 130 million years ago, and its serological characteristics and its main structure are different from IFN-α. In 1985, Capon et al. used a non-stringent human cDNA probe to isolate BoIFN-ω, whose C-terminus has 6 more amino acids than IFN-α, and has ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/555C12N15/81C12N1/19A61K38/21A61P31/14
CPCA61K38/00C07K14/555
Inventor 王君伟高明春罗修鑫郭永丽曹翀刘莹安东周萍萍
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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