Technique for producing gene rHu(Cu/Zn-SOD) with one-step method

Technique for producing gene rHu(Cu/Zn-SOD) with one-step method

  • CN 101,245,342 A
  • Filed: 02/15/2007
  • Published: 08/20/2008
  • Est. Priority Date: 02/15/2007
  • Status: Active Application
First Claim
Patent Images

1. single stage method prepares gene recombinant human source copper, zinc superoxide dismutase technology is characterized in that:

  • (1) temperature control secretor type people source SOD bacterial classificationPRPL promotor and STII signal peptide and people source sod gene are cloned among the PBR322, made up secretive expression vector PBV/STII/SOD, this plasmid has temperature-regulated promoter PRPL, have the STII signal peptide, expressed foreign protein is secreted among the cell Zhou Jizhi with soluble form, secretive expression vector with making up is transformed into intestinal bacteria W3110, obtains the rhSOD-Cu/Zn bacterial classification;

    (2) engineering fermentation culture(1) bacterial screening technologyStreak culture →

    test tube is cultivated →

    is chosen single bacterium colony →

    lab scale expression →

    amplification cultivation →

    production and uses bacterium(2) preparation of seed liquorA. get frozen bacterial classification PBV/STII/SOD1 and prop up, get a small amount of bacterial classification with transfering loop and on the LB plate, rule, cultivated 24 hours at 30 ℃

    ,B. picking list colony inoculation is in LB substratum test tube, and 30 ℃

    , 170 rev/mins of shaking tables were cultivated 30 hours,(3), fermentor cultivationA. adopt the M9 substratum of improvement,B. the ullage of fermenting disappears;

    121 ℃

    of conditions, and 30 minutes,(4), inoculation, in the jar substratum portioning of sterilization separately injected jar, under sterilising conditions, the protection seed liquor inserted in the fermentor tank,(5), cultivateA. before the inducing culture, per hour detect the OD value one time, and do microscopy and observe bacterium shape,B. control oxygen dissolving value by revolution and air flow,C. when jar in the thermal induction that heats up when being 3.0 of bacterium liquid OD600 value, inducing temperature is controlled at 42 ℃

    ,D. induce the back to continue to cultivate two and a half hours, go out jar,(3), centrifugal collection thalline, on the GQ-105 whizzer, carry out,(4), adopt the composite breaking method;

    freeze thawing combines with homogeneous method or ultrasonic disruption method, freeze thawing is reduced to-15 ℃

    from room temperature, isothermal was freezing after apse rate was 10 ℃

    /hour, melt before broken, adopt homogeneous method two-stage fragmentation;

    one-level 800bar, secondary 200bar, the dilution in 1;

    3 of enzyme liquid, percentage of damage reaches more than 95%(5), the compound supernatant liquor of collectingComposite breaking bacterial cell disruption rate reaches more than 95%, and heating gradient is 20 ℃

    /hour, is warming up to 50 ℃

    , and after the temperature, again with the centrifugal supernatant of enzyme liquid, original enzyme liquid impurity and various impurity albumen are 8.4mg/ml, and supernatant liquor drops to 2.3-3.5mg/ml, and activity recovery reaches 130%,(6), filter, filtering and adopting mesh is 0.5 μ

    m, 0.2 μ

    m, two filtering element (cartridge)s filter,(7), ultrafiltration adopts two ultrafiltration net 10KD, 100KD ultrafiltration, activity reaches 6-10 ten thousand U/ml after the ultrafiltration, rate of loss<

    10%,(8), freeze-drying active requirement as required, carry out rare joining, desalination, sterilization, freeze-drying.

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