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Recombinant porcine IL-29 fusion protein and preparation method and application thereof

A fusion protein, pil-29-fc technology, applied in the field of recombinant porcine IL-29 fusion protein and its preparation, can solve the problems of lack, increase the difficulty of operation, insufficient yield, etc., and achieve the advantages of reducing side effects and reducing the number of administrations. Effect

Active Publication Date: 2020-10-30
BEIJING VJT BIO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, commercial porcine IFN-α is produced by porcine white blood cells induced by Newcastle virus (NDV), and the yield is insufficient and the quality is uneven.
The Escherichia coli system has been used to produce interferon protein successfully, but bacteria are prokaryotic systems, which cannot process and fold heterologous proteins correctly, and often appear in the form of inclusion bodies, which increases the difficulty of operation
[0007] In view of the lack of long-acting and safe anti-viral interferon products in pig breeding, this patent focuses on the use of gene recombination technology and mammalian protein expression technology to develop long-acting porcine IL-29 fusion protein for porcine viral diseases prevention and treatment of

Method used

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  • Recombinant porcine IL-29 fusion protein and preparation method and application thereof
  • Recombinant porcine IL-29 fusion protein and preparation method and application thereof
  • Recombinant porcine IL-29 fusion protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of pIL-29Fc and pIL-29-PSA fusion protein recombinant cell lines

[0044] Nucleotide search of porcine IL-29, porcine Fc and porcine PSA in UniProt and GenBank databases or amino acid sequence. According to the codon preference of hamsters, pIL-29-Fc and pIL-29-PSA were codon-optimized, and related nucleotides were artificially synthesized. The codon-optimized pIL-29-Fc nucleotide sequence is shown in SEQ ID NO Shown in: 1, the codon-optimized pIL-29-PSA nucleotide sequence is shown in SEQ ID NO: 3. The synthetic pIL-29-Fc and pIL-29-PSA nucleotides were respectively constructed in the pcDNA3.1 vector to obtain the recombinant expression vectors pcDNA3.1-pIL-29-Fc and pcDNA3.1-pIL-29-PSA. After the pcDNA3.1-pIL-29-Fc recombinant vector was linearized, it was electroporated into CHO cells, and the pIL-29-Fc stably transfected cell line pIL-29-Fc / CHO was obtained after pressurized screening; the pcDNA3. After the 1-pIL-29-PSA recombinant vector ...

Embodiment 2

[0045] Example 2 Purification of pIL-29Fc and pIL-29-PSA fusion protein

[0046] Recombinant cells pIL-29-Fc / CHO and pIL-29-PSA / CHO were fermented and cultured in a fermenter, and the fermentation broth was first passed through two-stage deep filter membrane bag to remove cells and cell debris, and then filtered with a 0.22 μm filter membrane , to obtain a clarified fermentation broth. pIL-29Fc and pIL-29-PSA were purified using the following methods, respectively.

[0047] Purification of pIL-29Fc: The fermentation broth was first purified by affinity chromatography Protein A (MabSelect SuReTM, GE Healthcare): first equilibrated to the baseline with an equilibrium solution (50 mM glycine, 0.15 M NaCl, pH 7.2), and then with the eluent ( 50 mM glycine, pH 3.0) and collect the eluate. The protein A collected solution was purified by anion exchange Capto Q (GE Healthcare) column chromatography: the collected solution was adjusted to pH 8.0 with 1M NaOH, equilibrated to baselin...

Embodiment 3

[0049] Example 3 Determination of biological activity of pIL-29Fc and pIL-29-PSA fusion protein

[0050] The titers of pIL-29-Fc and pIL-29-PSA were determined by cytopathic inhibition method. Dilute well-grown MDBK (bovine kidney cell) cell suspension by 2×10 5 The density of cells / mL was inoculated in a 96-well cell culture plate, 100 μL / well, and cultured overnight at 37°C in a 5% CO2 cell incubator. Dilute pIL-29, pIL-29-Fc and pIL-29-PSA in 4-fold gradients with 2% FBS-DMEM respectively, set 8 replicates for each gradient, add 100 μL protein / well, and store at 37°C, 5% CO2 Continue culturing for 24 h in the cell culture incubator. The cell culture medium was discarded, and the cells were infected with 100 TCID50 of VSV per well, 24h after infection. Discard the culture medium, add 50 μL of crystal violet staining solution to each well and stain at room temperature for 30 min, wash away the staining solution with running water, add 100 μL of destaining solution to each...

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Abstract

The invention discloses a recombinant porcine IL-29 fusion protein and a preparation method and application thereof. The fusion protein is formed by directly or indirectly connecting porcine IL-29 with an immunoglobulin Fc fragment or porcine serum albumin (PSA) through a connecting element. The porcine IL-29 fusion protein can be prepared by utilizing a mammalian cell expression system based on agene engineering technology. Compared with natural porcine IL-29, the recombinant porcine IL-29 fusion protein is longer in half-life period and higher in antiviral activity, and can be used for preparing medicines for preventing and treating porcine viral diseases.

Description

technical field [0001] The invention relates to the technical field of prevention and control of biological medicine and animal viral diseases, in particular to a recombinant porcine IL-29 fusion protein and its preparation method and application. Background technique [0002] Interleukin 29 (IL-29) is a cytokine with antiviral activity reported for the first time in 2003, which is highly similar to the IL-10 cytokine family in amino acid sequence; Belongs to the type III interferon or IFN-λ family, also known as IFN-λ1. [0003] IL-29, similar to IFN-α / β (type I interferon), binds to the corresponding receptor, activates the JAK-STAT signaling pathway, and induces the transcription of downstream interferon-stimulated genes (ISGs), thereby stimulating and antiviral Response-related protein expression, including antiviral proteins such as MxA, OASL, and PKR. MxA is a member of the antimyxovirus protein family, which can prevent viral nucleic acid from entering the cell. OA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K38/20A61K47/64A61K47/68A61P31/12A61P31/22A61P31/20A61P31/14
CPCC07K14/54A61K47/643A61K47/68A61P31/12A61P31/22A61P31/20A61P31/14C07K2319/30C07K2319/31A61K38/00
Inventor 罗昊澍师磊钟小荣于金库韩国熊剑胜
Owner BEIJING VJT BIO CO LTD
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