Kallikrein

Method 1. Extract the submandibular glands of pig as raw material. Take fresh or frozen submandibular glands of pigs, wash them, remove fat and other materials, and then mince them in a meat grinder. Add 5 times the water to extract per 100kg of porcine submandibular gland slurry, adjust Ph to 4.5 with acetic acid, and add 600ml xylene for preservative. After 12 hours of extraction, 600 ml of xylene was added, the Ph value was maintained at 4.5 with acetic acid, and the extraction was continued for 12 hours to remove the upper suspended fat, filtered with gauze, and filtered with nylon cloth to obtain the extract. Porcine submandibular gland H2O; HAc; xylene; Ph4.5 → The extract is adsorbed. Under constant stirring, add 3.4 kg of medicinal aluminum hydroxide xerogel that has been slurried with the extract in the extract, and continue to stir for 1 hour. Add 1.6kg of aluminum hydroxide dry gel, stir for 2h, and let stand overnight. Siphon and discard the supernatant, add water 5 times the weight of the slurry, stir and wash, let it stand, siphon and discard the supernatant. Repeat washing for 5-6 times until the upper layer is basically clear. The adsorption slurry is obtained. Extraction solution Al (OH03 dry gel → elution of adsorption adsorption slurry Weigh the adsorption slurry weight, add diammonium hydrogen phosphate according to 13.2g/L (1.32%) of the water content (aluminum hydroxide slurry weight minus aluminum hydroxide weight) , Adjust Ph to 8.4 with ammonia water (8.6%), eluting for 2h while stirring, at the initial stage of elution, Ph decreases, you can adjust Ph with ammonia water (8.6%) at any time to maintain 8.4. Then add 10g/L according to water content (1%) Sodium chloride, continue to stir for 10 minutes, and then add 49-50ml of ethanol with a purity of 92% or more to the eluent of each 100ml of eluent, let it stand overnight, filter through paper pulp the next day, and collect the filtrate. Adsorption slurry (NH4)2HPO4;NH^3^)·H2O;NH4·H2O;Ph8.4→The eluent is precipitated, the dried filtrate is adjusted to Ph 6.7 with acetic acid (2mol/L), under stirring, the 100th ml 80ml of ethanol with a purity of 92% or more was added to the filtrate. After the precipitation is complete, the supernatant is discarded by siphoning, the precipitate is separated by centrifugation, the precipitate is washed and dehydrated with acetone, and dried in vacuum to obtain a crude product. Eluent C2H5OH; Ph6.7→precipitation; dry crude product refining. Dissolve the crude product with 25 times the amount of ammonium acetate solution (10%) to make the Ph to 6.5～6.7, and add the same amount of ethanol as the solution (95%) Stir well, refrigerate, and let stand overnight. Filter the next day, add 80g (8%) of medicinal activated carbon to each liter of filtrate, stir for 15min, filter, add 3 times the amount of ethanol (95%) to the filtrate, leave it in the refrigerator, centrifuge, separate the precipitate, and wash the precipitate with acetone first , Dehydration and vacuum drying to obtain refined products. The total yield is 4500U/kg (for fresh submandibular glands). Crude product (NH4) Ac; C2H5OH; Ph6.5→purification; decontaminated protein clear liquid activated carbon→depyrogenated refined liquid C2H5OH→precipitated precipitated acetone→drying refined product Method two, extract and precipitate germanium pancreas as raw material After the acetone powder is made, add acetic acid (0.02mol/L) about 20 times the amount of porcine pancreas acetone powder, keep stirring at 100C, and extract for 12h. After the extraction is completed, centrifuge, add 10 times the amount of ethanol (0.02mol/L) to the filter residue. L) Continue to extract for 6 hours, combine the two filtrates, add 33ml of cold acetone per 100ml (33% volume fraction), and filter. The filtrate was supplemented with cold acetone to a volume fraction of 70%, allowed to stand for 4 hours, centrifuged, and the precipitate was separated, degreased and dehydrated with acetone and ether, and dried in vacuum to obtain a precipitate. Porcine pancreas acetone powder HAc; 100→extract filtrate acetone; ether→precipitate sediment graded precipitation Add 50 times the amount of cold sodium chloride (2g/L or 0.2%) to the precipitate, adjust the pH to 8 with ammonia, and stir to dissolve After the dissolution is complete, the pulp is filtered to obtain a clear filtrate. The filtrate is cooled to 2～30C, and 40ml of cold acetone is added to each 100ml filtrate to make the volume fraction reach 40%. Refrigerate at low temperature overnight. Centrifuge, separate the supernatant and add cold acetone to a volume fraction of 60%, let it stand for 4 hours, refrigerate at low temperature, wash the precipitate with acetone, ether, and vacuum dry to obtain a crude product. Precipitate NaCl; Ammonia; Acetone → Fractional precipitation crude product adsorption, elution, dialysis, freeze-drying The crude product is dissolved in a certain amount of pH 4.5 acetic acid buffer (0.001 mol/L), centrifuged, and the supernatant is separated. Add the weakly acidic cation exchange resin AmheriteCG-50 to the supernatant, m (resin): m (crude product)=50:1, make it absorb for 2h under stirring, collect the resin, and use pH4.5 acetic acid buffer (0.001mol /L) Rinse the resin until there is no foam. Stir and elute the resin with 2 times the amount of pH5 sodium chloride (1mol/L) solution for 1 hour, separate the resin, dialysis desalting the eluate, and freeze-dry to obtain the refined pancreatic kallikrein. Crude product acetic acid buffer: Amberlite CG-50; Ph4.5→adsorbent acetic acid buffer; NaCl→elution eluate dialysis; freeze-drying→refined product

Used in biochemical research, as a vasodilator in medicine, for the treatment of cerebral arteriosclerosis, occlusive arteritis, primary hypertension, angina pectoris, occlusive vasculitis, extremity skin ulcers, frostbite and other symptoms.