Production and use of high expression, high purity, high activity gene recombinant human lysozyme

Production and use of high expression, high purity, high activity gene recombinant human lysozyme

CN
  • CN 1,410,538 A
  • Filed: 06/13/2002
  • Published: 04/16/2003
  • Est. Priority Date: 06/13/2002
  • Status: Active Application
First Claim
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1. , the production and the purposes of a kind of high expression level, high purity, high reactivity gene recombinant human lysozyme, it is characterized in that:

  • the material of described production usefulness is;

    the smd1165 (his4.prb1) that adopts pichia spp, smd1163 (his.pep4.prb1), Gs115, km71 bacterial strain;

    The plasmid of its genetically engineered human lysozyme mainly is that plasmid integration with pPIC9k series is on above-mentioned bacterial strains karyomit(e);

    Production stage is;

    1, shake-flask seed preparation;

    to prepare 200 milliliters of substratum is benchmark, uses H 3PO 4(phosphoric acid) 4-8 milliliter, MgSO 4(sal epsom) 1-5 gram, K 2SO 4(vitriolate of tartar) 2-6 gram, KOH (potassium hydroxide) 1-3 gram, CaSO 42H 2O (calcium sulfate) 1-3.5 gram, adding distil water to 200 milliliter, inoculation glycerine pipe seed behind the autoclaving, the shaking table revolution is that per minute 250 changes, culture temperature is 20-35 ℃

    , cultivates 36-48 hour on the constant temperature bed.2, seed tank culture;

    be upgraded to benchmark with seeding tank maximum volume 1, preparation substratum H 3PO 4(phosphoric acid) 25-19 milliliter, MgSO 4(sal epsom) 13-17 gram, CaSO 42H 2O (calcium sulfate) 0.4-0.8 gram, K 2SO 4(vitriolate of tartar) 16-20 gram, KOH (potassium hydroxide) 2.12-6.12 gram, adding distil water to 500 milliliter, inoculation shaking table seed behind the autoclaving;

    Fermentation reactor operating parameters temperature is 20-32 ℃

    , and pH value is 2.0-8.0;

    Dissolved oxygen concentration is 10-60%, and when the cell density in the nutrient solution is increased to about OD200, seed culture finishes.3, produce a jar cultivation;

    be upgraded to 10 and produce a jar preparation substratum, use H 3PO 4(phosphoric acid) 93-97 milliliter, MgSO 4(sal epsom) 51-55 gram, CaSO 42H 2O (calcium sulfate) 19-23 gram, K 2SO 4(vitriolate of tartar) 61-65 gram, KOH (potassium hydroxide) 13-17 gram adding distil water to 7 liter, inoculation seeding tank seed behind the autoclaving, fermentation reactor operating parameters temperature is controlled at 25-32 ℃

    , and pH value is 4.0-8.0, and dissolved oxygen concentration is 10-60%;

    After cell in the nutrient solution reached certain density, stream added methanol induction in nutrient solution, the time length 50-60 in this stage hour, put a jar cultivation then and finished.Extraction and purification process;

    the pichia spp bacteria culture fluid of human lysozyme that will contain gene engineering expression is collected filtrate with the 10-50 ten thousand molecular weight membrane ultrafiltration that dams;

    With the 5000 molecular weight ultrafiltration of damming, collect dope;

    Carboxymethyl cellulose on the dope, the elution buffer wash-out is collected elutriant, mistake-50 post desalination, frost drying is measured protein content and lysozyme activity;

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