Thymosin [alpha]1-porcine interferon [alpha] fusion protein gene and preparation method of recombinant protein of fusion protein gene

A porcine interferon and fusion protein technology, applied in the direction of microorganism-based methods, interferon, biochemical equipment and methods, etc., to achieve the effect of low cost and simple method

Active Publication Date: 2017-03-22
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report on the high-efficiency secretion and expression of Tα1-PoIFNα fusion protein by Pichia pastoris at home and abroad

Method used

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  • Thymosin [alpha]1-porcine interferon [alpha] fusion protein gene and preparation method of recombinant protein of fusion protein gene
  • Thymosin [alpha]1-porcine interferon [alpha] fusion protein gene and preparation method of recombinant protein of fusion protein gene
  • Thymosin [alpha]1-porcine interferon [alpha] fusion protein gene and preparation method of recombinant protein of fusion protein gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Synthesis of modified Tα1-PoIFNα fusion protein gene

[0056] The Tα1-PoIFNα fusion protein gene was modified after DNA analysis and RNA structure prediction, and the Tα1-PoIFNα fusion protein gene was artificially synthesized without changing the natural amino acid sequence, named Tα1-PoIFNα, the artificially synthesized Tα1-PoIFNα fusion The nucleotide sequence of the protein gene is shown in SEQ ID NO.1.

Embodiment 2

[0057] Example 2 Small amount preparation of Tα1-PoIFNα fusion protein

[0058] 1. Construction of genetically engineered yeast strains of Tα1-PoIFNα fusion protein

[0059] (1) Materials and methods:

[0060] Pichia pastoris Pichiapastoris GS115 and pPIC9K expression plasmids were purchased from Invitrogen, USA. DNA polymerase, restriction endonucleases EcoR I, Not I, and Sac I were purchased from TaKaRa, and T4 DNA ligase was purchased from NEB. For BMGY, BMMY, and YPD media, see Invitrogen Pichia pastoris operation manual. Plasmid extraction kit and PCR product recovery kit were purchased from Axgen. The primary antibody is an anti-pig interferon α monoclonal antibody, homemade, and the second antibody is a rabbit anti-mouse IgG-HRP antibody, purchased from Sigma;

[0061] (2) Construction of expression vector pPIC9K-Tα1-PoIFNα

[0062] (a) Add the EcoR I restriction enzyme site to the 5'end of the Tα1-PoIFNα fusion protein gene Tα1-PoIFNα synthesized in Example 1, and the Not I ...

Embodiment 3

[0068] Example 3 Mass preparation of Tα1-PoIFNα fusion protein

[0069] 1. Material:

[0070] Strain containing Tα1-PoIFNα fusion protein gene Tα1-PoIFNα: GS115 (pPIC9K-Tα1-PoIFNα) (prepared in Example 2);

[0071] Instruments: fermentation tank, electrophoresis instrument;

[0072] Medium: For the specific configuration method of YPD, BMGY, BSM and PTM1 fermentation medium, see Invitrogen Pichia pastoris operation manual;

[0073] 2. Method:

[0074] 2.1 Seed culture and accumulation of yeast cell biomass

[0075] Streak the frozen engineered bacteria on YPD agar plate and culture at 26℃. When the colony grows to 2mm, pick a single colony and add it to 10mL YPD culture medium (seed medium), and shake culture at 26℃, 250r / min for 24h. Inoculate 1mL of the above culture into 200ml YPD culture medium, culture with shaking at 26℃, 250r / min for 24h to make A600≈10. Prepare 2L BSM medium, add 5L fermenter, autoclave the medium and fermenter at 121℃ for 30min. When the culture medium in the...

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Abstract

The invention discloses a thymosin [alpha]1-porcine interferon [alpha] fusion protein gene and a preparation method of recombinant protein of the fusion protein gene. A nucleotide sequence of the reconstructed thymosin [alpha]1-porcine interferon [alpha] fusion protein gene provided by the invention is shown as SEQ ID NO.1. Meanwhile, the invention discloses a method for preparing the recombinant protein expressed by the gene and for conducting activity detection, wherein specifically, the method comprises such steps as reconstructing the thymosin [alpha]1-porcine interferon [alpha] fusion protein gene, cloning the gene and promoting secretory expression of the gene in pichia pastoris, preparing the recombinant protein and controlling fermentation culture conditions, improving such operating methods as the activity detection method and the like. The method provided by the invention is simple and easy to implement and is relatively low in cost; the efficient and stable secretory expression of the thymosin [alpha]1-porcine interferon [alpha] fusion protein in the pichia pastoris is achieved; and the expressed recombinant fusion protein is high in anti-viral activity and immune cell regulating activity levels; therefore, the invention lays a foundation for preparing green and no-residue biological products having functions of treating porcine viral diseases and immune regulation.

Description

Technical field [0001] The invention relates to the modification of a thymosin α1-pig interferon α fusion protein gene, and also relates to the construction of an expression vector containing the gene, the stable secretion expression in Pichia pastoris and the preparation of a recombinant thymosin α1-pig interferon α fusion protein The method, as well as the antiviral activity and immune regulation effect of the recombinant thymosin α1-porcine interferon α fusion protein, the present invention belongs to the technical field of animal genetic engineering and animal virology. Background technique [0002] Thymus hormone is the general name of polypeptide hormones produced by thymic epithelial cells, also known as thymopeptide. Most of the currently used thymosin peptides are peptides extracted and purified from calf or pig thymus. Its biological activity is mainly to induce T cell differentiation and maturity and regulate immune balance. In human medicine, it is mainly used for au...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/81C12N1/19C07K19/00A61P31/12A61P37/02C12R1/84
CPCC07K14/56C07K14/57581C07K2319/00C12N15/815C12N2800/102
Inventor 涂亚斌蔡雪辉王刚刘永刚
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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