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DNA molecule for coding porcine alpha interferon and expression and purification method of recombinant protein of porcine alpha interferon

An interferon and coding technology, applied in the field of protein purification in the large-scale production of recombinant interferon, can solve the problems of complex steps and low antiviral activity of recombinant porcine alpha-interferon, and achieve a short purification period and disulfide bond. Correct pairing, cost-saving effect

Inactive Publication Date: 2016-07-13
北京大北农科技集团股份有限公司动物医学研究中心 +3
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The method steps are complicated, and the recombinant porcine interferon alpha-interferon prepared has higher antiviral activity and lower

Method used

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  • DNA molecule for coding porcine alpha interferon and expression and purification method of recombinant protein of porcine alpha interferon
  • DNA molecule for coding porcine alpha interferon and expression and purification method of recombinant protein of porcine alpha interferon
  • DNA molecule for coding porcine alpha interferon and expression and purification method of recombinant protein of porcine alpha interferon

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Experimental program
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Embodiment 1

[0046] Construction, expression and purification of embodiment 1 recombinant porcine interferon alpha

[0047] (1) Optimization of porcine interferon-α gene and construction of seed bacteria: The porcine interferon-α gene was optimized and transformed according to the codon preference of Escherichia coli, and the transformed gene was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and constructed into pET21a On the vector, the recombinant expression plasmid pET-21a-rPoIFNα was obtained. Its nucleotide sequence is shown in SEQIDNo.1, including 519 bases, the 1st-516th base encodes 172 amino acids, and its theoretical molecular weight is 19.8kDa. The expression plasmid pET-21a-rPoIFNα was transformed into Escherichia coli BL21 competent cells to obtain the expression seed strain BL21 / pET-21a-rPoIFNα.

[0048] (2) Seed fungus recovery: Inoculate the seed fungus frozen at -20°C on the ampicillin-resistant LB solid medium with an inoculation loop, and cultivate it in a con...

Embodiment 2

[0059] Antiviral biological activity assay of embodiment 2 recombinant porcine alpha interferon protein

[0060] Biological activity was determined using a cytopathic (CPE) inhibition-based microinhibition assay. Using the Marc-145 cell-porcine reproductive and respiratory syndrome virus (PRRSV) system, the highest dilution of interferon that inhibited 50% cytopathic changes was defined as 1 interferon activity unit.

[0061] (1) Inoculate Marc-145 cells into a 96-well plate (100 μl / well), culture for 6-8 hours until all adhere to the wall, and make it reach 10 4 pcs / hole or so;

[0062] (2) Discard the culture medium, dilute the prepared recombinant porcine interferon alpha protein sample for 8 gradients, the dilution solution is DMEM medium containing 10% FBS, set 8 replicates for each gradient, and add 100 μl virus to each well Diluent.

[0063] (3) After culturing at 37°C for 18 hours, add PRRSV virus solution (300TCID50 / well) diluted with serum-free DMEM medium, and se...

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Abstract

The invention relates to the field of biological pharmacy, and mainly relates to a DNA molecule for coding porcine alpha interferon and an expression and purification method of a recombinant protein of the porcine alpha interferon. Recombinant escherichia coli BL21 / pET-21a-rPoIFN[alpha] is taken as a production bacterial strain, an expression protein of which has a coding sequence optimized through a codon bias in escherichia coli and is suitable for expression in escherichia coli. According to the expression and purification method, plenty of target protein in an inclusion body form can be obtained in precipitation of broken bacteria, and a large amount of recombinant porcine alpha interferon with high purity can be obtained through inclusion body denaturation, renaturation, affinity chromatography, and dialysis displacement of a buffer. The recombinant porcine alpha interferon is high in biological activity, and is convenient to apply with reduced production cost.

Description

technical field [0001] The present invention relates to the field of biopharmaceuticals, and mainly relates to the expression, purification and application of a DNA molecule encoding porcine alpha interferon, recombinant E. Protein purification during the scale-up production of interferon. Background technique [0002] Interferon (interferon, IFN) is a protein produced by the body stimulated by virus infection or other biological inducers. It is a broad-spectrum antiviral agent. The body function makes cells produce antiviral proteins, inhibits viral gene transcription and degrades viral RNA, and at the same time can enhance the activity of natural killer cells (NK cells), macrophages and T lymphocytes, thereby playing an immune regulation role and strengthening the body. Antiviral ability, its essence is a cytokine with immune regulation function. According to different sources of interferon, it can be divided into three types: α, β, and γ. Among them, α-interferon is a ...

Claims

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Application Information

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IPC IPC(8): C12N15/21C12N15/70C12N1/21C12P21/02C07K14/56C07K1/36C07K1/22
Inventor 史忠玉王贵华张国庆于萍萍刘培培赵亚荣
Owner 北京大北农科技集团股份有限公司动物医学研究中心
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