Preparation method of thrombin
A technology for thrombin and prothrombin, which is applied in the field of thrombin preparation, can solve the problems of increasing production steps and increasing production costs, and achieves the effects of improving utilization rate, increasing titer, and reducing production costs.
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Embodiment 1
[0043] The preparation of embodiment 1 prothrombin
[0044] 1. Take fresh pig blood, add 38g / L trisodium citrate aqueous solution to anticoagulate, use a centrifuge to centrifuge at a speed of 12000r / min, and filter to obtain plasma;
[0045] 2. Add adsorbent DEAE-sephadex-A50 ion exchange resin to plasma, wash with 0.2M aqueous sodium chloride solution and 3g / L trisodium citrate aqueous solution, wash with 2M aqueous sodium chloride solution and 3g / L trisodium citrate aqueous solution Elution, and finally dialyze the eluate with a dialysis bag with a molecular weight of 7000 to obtain a prothrombin dialysate.
Embodiment 2
[0046] Example 2 Recombinant lipidated tissue factor activates prothrombin
[0047] Add 1,000 mL of the prothrombin dialysate prepared in Example 1 into a stainless steel container, heat it to 37°C in a water bath, and add recombinant lipidated human tissue factor at a concentration of 0.1 mg / mL purchased from Beijing Boersi Technology Co., Ltd. The solution was 1 mL, the temperature of the solution was controlled at 37° C., stirred for 10 min, filtered, and placed in a 4° C. freezer for 1.0 h to obtain a solution containing thrombin.
[0048] Adjust the solution containing thrombin to pH 6 with acetic acid, place it in a freezer at 1°C for 20 minutes, centrifuge and discard the precipitate, and collect the supernatant; adjust the supernatant to pH 6.5 with saturated aqueous sodium bicarbonate , placed in a freezer at 6°C for 30 minutes, centrifuged and discarded, and the supernatant was collected.
[0049] The detection was repeated three times, and the titers of the obtaine...
Embodiment 3
[0050] Example 3 Recombinant lipidated tissue factor activates prothrombin
[0051] Add 1000 mL of the prothrombin dialysate prepared in Example 1 into a stainless steel container, heat it to 37°C in a water bath, and add recombinant lipidated rabbit tissue factor at a concentration of 0.1 mg / mL purchased from Shanghai Guandong Biotechnology Co., Ltd. The solution was 1 mL, the temperature of the solution was controlled at 37° C., stirred for 10 min, filtered, and placed in a 4° C. freezer for 1.0 h to obtain a solution containing thrombin.
[0052] Adjust the solution containing thrombin to pH 6 with acetic acid, place it in a freezer at 1°C for 20 minutes, centrifuge and discard the precipitate, and collect the supernatant; adjust the supernatant to pH 6.5 with saturated aqueous sodium bicarbonate , placed in a freezer at 6°C for 30 minutes, centrifuged and discarded, and the supernatant was collected.
[0053] The detection was repeated three times, and the titers of the o...
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