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
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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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