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Canine interferon-alpha6 alpha7 recombinant protein and preparation method and application thereof

A canine interferon and recombinant protein technology, which is applied in the fields of genetic engineering and biological products, and can solve the problems such as the expression of canine interferon tandem recombinant protein that has not yet been seen.

Pending Publication Date: 2018-09-04
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the expression of canine interferon-α6, α7 tandem recombinant protein using yeast eukaryotic expression system

Method used

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  • Canine interferon-alpha6 alpha7 recombinant protein and preparation method and application thereof
  • Canine interferon-alpha6 alpha7 recombinant protein and preparation method and application thereof
  • Canine interferon-alpha6 alpha7 recombinant protein and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The optimization of embodiment 1 gene sequence and the design and synthesis of primer

[0035] According to the cDNA sequence of canine interferon-α6 (NM_001007128.1) and the cDNA sequence of canine interferon-α7 (NM_001006654.1) in GenBank, after codon optimization, the target gene sequence was synthesized. The amplification primers designed for canine interferon-α6 are GN7PA-F and GN7PA-R1, and the sequences are shown in SEQ ID NO:7 and SEQ ID NO:8; the amplification primers GN7PA-F1 and GN7PA-R for canine interferon-α7, SEQ ID NO:9 and SEQ ID NO:10; Pichia pastoris general primer 5'AOX and 3'AOX sequences are shown in SEQ ID NO:11 and SEQ ID NO:12.

[0036] Table 1 Primer Sequence

[0037]

[0038] The optimization process of canine interferon-α6 and canine interferon-α7 gene sequences is as follows:

[0039] By analyzing the rare codons of Pichia pastoris in the canine interferon-α6 and canine interferon-α7 gene sequences, according to the codon usage preferenc...

Embodiment 2

[0040] Example 2 Construction of pPICZαA-CaIFN-α6-α7 Vector

[0041] Using the gene synthesized by the company as a template, the canine interferon-α6 gene fragment is amplified by polymerase chain reaction (PCR). The reaction system is as follows:

[0042]

[0043] PCR reaction program: pre-denaturation at 94°C for 8 min; denaturation at 94°C for 35 s, annealing at 52°C for 30 s, extension at 72°C for 45 s, a total of 35 cycles; final extension at 72°C for 5 min, and storage at 4°C. After mixing 5 μL of PCR product with 1 μL of loading buffer, use 1.0% agarose gel electrophoresis for nucleic acid, 120V, 32min, and observe the electrophoresis results under a UV gel imager. The PCR product CaIFN-α6 was recovered and purified with Cycle-Pure Kit.

[0044] Using the gene synthesized by the company as a template, the canine interferon-α7 gene fragment was amplified by polymerase chain reaction (PCR). The reaction system is as follows:

[0045]

[0046] PCR reaction progra...

Embodiment 3

[0048] Example 3 Preparation and linearization of pPICZαA-CaIFN-α6-α7 plasmid

[0049] After the pPICZαA-CaIFN-α6-α7 plasmid was sequenced correctly, it was transformed into Escherichia coli DH5a. The specific operation is as follows:

[0050] 1. Thaw a tube of 100 μl E.coli DH5a competent cells on ice, flick the tube wall to resuspend the cells. Add 10 μl of plasmid to the competent cells, flick a few times, and incubate on ice for 30 minutes.

[0051] Heat shock in a 2.42°C water bath for 90 seconds, then quickly place on ice for 5 minutes.

[0052] 3. Add 500 μl LB liquid medium and incubate at 37°C for 45-60 minutes.

[0053] 4. Centrifuge at 5000g for 3 minutes to collect the bacterial cells, spread a certain amount of bacterial cells evenly on low-salt LB plates containing 25 μg / ml Zeocine, and culture overnight.

[0054] Pick positive clones for expansion culture, and use the Plasmid Midi Preparation Kit (Plasmid Midi Preparation Kit) of Beyontian Biotechnology Co.,...

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Abstract

The invention provides a canine interferon-alpha6 alpha7 recombinant protein and a preparation method and an application thereof. An amino acid sequence of the canine interferon-alpha6 alpha7 recombinant protein is shown as SEQ ID NO:6, the preparation method comprises the following steps: performing codon optimization on a canine interferon-alpha6 gene and a canine interferon-alpha7 gene, splicing the optimized gene sequence, cloning the gene to pPICZalphaA for construction of recombinant expression plasmids, converting pichia yeast, and preparing recombinant bacteria, and performing steps offermentation, inducible expression, purifying to obtain the canine interferon-alpha6 alpha7 recombinant protein. The canine interferon-alpha6alpha7 recombinant protein can be used as a wide spectrumantiviral preparation, has obvious prevention and treatment effects for canine distemper, canine parvovirus disease, canine parainfluenza virus infection, and canine infectious hepatitis virus infection, and provides an effective technical means for realizing recombinant canine interferon-alpha6 alpha7 series-connection recombinant protein production and further broad-spectrum prevention and canine viral epidemic disease treatment.

Description

technical field [0001] The invention relates to the field of genetic engineering and biological products, in particular to a canine interferon-α6α7 recombinant protein and its preparation method and application. Background technique [0002] Interferon (IFN) is a cytokine with broad-spectrum anti-virus, anti-tumor, immune regulation and other biological activities. A secreted glycoprotein. Interferon can be divided into type I, type II, and type III according to the cell, receptor, activity, etc. in which interferon is produced. Type I interferons are mainly produced by white blood cells, including: IFN-α, IFN-β, IFN-ε, IFN-ω, IFN-κ, IFN-δ. There are many subtypes of IFN-α, and each subtype of mature IFN-α contains 165-166 amino acid residues, has a similar structure, and has a molecular weight of about 18kD. IFN-α contains four conserved cysteines, which form two pairs of disulfide bonds (Cys1-Cys99 and Cys29-Cys139), so that the IFN-α molecule folds into a spherical sha...

Claims

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Application Information

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IPC IPC(8): C12N15/21C12N15/81C12N15/66C07K14/56A61K38/21A61P31/14A61P31/20
CPCA61K38/00A61P31/14A61P31/20C07K14/56C12N15/815
Inventor 贾红朱鸿飞陈美荣侯绍华鑫婷姜一曈
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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