CN105950576A - Method for extracting multiple proteins from bovine blood - Google Patents

Method for extracting multiple proteins from bovine blood Download PDF

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Publication number
CN105950576A
CN105950576A CN201610364078.0A CN201610364078A CN105950576A CN 105950576 A CN105950576 A CN 105950576A CN 201610364078 A CN201610364078 A CN 201610364078A CN 105950576 A CN105950576 A CN 105950576A
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extraction step
filtrate
blood
filtering residue
cattle
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贾芳苗
熊川
邓飞
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CHENGDU YUANRUI BIOTECHNOLOGY Co Ltd
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CHENGDU YUANRUI BIOTECHNOLOGY Co Ltd
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Abstract

The invention discloses a method for extracting multiple proteins from bovine blood. The method comprises a blood separating step of taking bovine blood and adding an anticoagulant, and separating into blood cells and plasma; a blood cell extracting step of subjecting the blood cells to crushing, denaturation, pressure filtration and purification treatment to obtain at least one of superoxide dismutase, protoheme, and blood globulin powder; and a plasma extracting step of subjecting the plasma to pressure filtration and purification to obtain at least one of fibrinogen, prothrombin, thrombin, bovine IgM, bovine fibronectin , bovine IgG, and plasma protein powder. According to the method, by fully utilizing a biological separation technology, the superoxide dismutase, hemoglobin, fibrinogen, prothrombin, thrombin, IgM, bovine fibronectin and bovine IgG are extracted one by one in high purity according to a principle of separating similar components first and then finely separating, so that the potential value of blood is fully found and the utilization rate of bovine blood is improved.

Description

A kind of method extracting multiple protein from Sanguis Bovis seu Bubali
Technical field
The present invention relates to Sanguis Bovis seu Bubali processing technique field, carry from Sanguis Bovis seu Bubali in particular to one The method taking multiple protein.
Background technology
At present, Sanguis Bovis seu Bubali process technology refers mainly to extract the technique of various albumen from Sanguis Bovis seu Bubali, to carry For the surcharge of Sanguis Bovis seu Bubali, improve its utilization rate.But existing extraction process efficiency is low, extraction Few and extract component the content also low-purity of protein varieties the highest, and it is a large amount of to there is filtrate filtering residue The phenomenon abandoned, strongly limit the deep processing to Sanguis Bovis seu Bubali and utilization rate.
Summary of the invention
It is an object of the invention to provide a kind of method extracting multiple protein from Sanguis Bovis seu Bubali, this side Method can extract multiple proteins from bovine blood sample one by one, and the purity extracted is high, fills Divide the potential value having excavated bovine blood, improve the utilization rate of bovine blood.
The present invention solves it and technical problem is that and realize by the following technical solutions.
A kind of method extracting multiple protein from Sanguis Bovis seu Bubali, comprising:
Blood separation procedure: take fresh bovine blood, adds anticoagulant, separates, and collects hemocyte And blood plasma;
Hemocyte extraction step: hemocyte is difference after broken, degeneration, filter pressing, purification process Obtain superoxide dismutase, haemachrome and blood globulin powder at least one;
Blood plasma extraction step: blood plasma is through filter pressing, respectively obtain Fibrinogen, blood coagulation after purification Proenzyme, thrombin, IgM, cattle fibronectin, cattle IgG and spray-dried plasma protein are at least One of them.
A kind of method extracting multiple protein from Sanguis Bovis seu Bubali that the present invention provides provides the benefit that: Relative to traditional extraction process, the Sanguis Bovis seu Bubali extracting method of the present invention include blood separation procedure, Hemocyte extraction step and blood plasma extraction step, make full use of bioseparation technology, by first separating Close component, then carry out the principle of fine separation, can be expeditiously the super oxygen in bovine blood Compound dismutase, haemachrome, Fibrinogen, thrombinogen, thrombin, IgM, cattle fibre Dimension connects albumen, extracts to cattle IgG high-purity seriatim, and processing obtains hyperglobulinemia The effective ingredient such as powder and spray-dried plasma protein, fully excavates the potential value of blood, improves Sanguis Bovis seu Bubali The utilization rate of liquid.
Accompanying drawing explanation
In order to be illustrated more clearly that the technical scheme of the embodiment of the present invention, below will be in embodiment The required accompanying drawing used is briefly described, it will be appreciated that the following drawings illustrate only this Some bright embodiment, is therefore not construed as the restriction to scope, common for this area From the point of view of technical staff, on the premise of not paying creative work, it is also possible to according to these accompanying drawings Obtain other relevant accompanying drawings.
Fig. 1 is the flow chart of the method extracting multiple protein from Sanguis Bovis seu Bubali of the embodiment of the present invention.
Detailed description of the invention
For making the purpose of the embodiment of the present invention, technical scheme and advantage clearer, below by right Technical scheme in the embodiment of the present invention is clearly and completely described.In embodiment unreceipted Actual conditions person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or instrument Device unreceipted production firm person, being can be by the commercially available conventional products bought and obtain.
Below the method extracting multiple protein from Sanguis Bovis seu Bubali of the embodiment of the present invention is carried out specifically Explanation.
Refer to Fig. 1, the side extracting multiple protein from Sanguis Bovis seu Bubali that the embodiment of the present invention provides Method, comprises the following steps.
S1: blood separation procedure
Take fresh bovine blood, add anticoagulant, separated by blood cell seperator, and it is thin to collect blood Born of the same parents and blood plasma.Need explanation, when first processing hemocyte, can be by blood plasma in-20 DEG C of freezing guarantors Deposit, if first processing blood plasma, then can be by hemocyte in-20 DEG C of freezen protective.First process hemocyte The most first process blood plasma to select according to practical situation.
Wherein, it is preferred that anticoagulant is trisodium citrate.Further, press in blood Final concentration of 3.6~4% amount add trisodium citrate, prevent bovine blood from solidifying.
S2: hemocyte extraction step
Hemocyte can respectively obtain superoxides discrimination after broken, degeneration, filter pressing, purification process Change enzyme, haemachrome and blood globulin powder at least one.In other words, hemocyte extracts Step includes that superoxide dismutase extraction step, haemachrome extraction step and blood globulin powder carry Take step at least one.Hereinafter superoxide dismutase extraction step, haemachrome are extracted Step and blood globulin powder extraction step are specifically described.
(1) superoxide dismutase extraction step
With ultrasonic disruption hemocyte (KQ-100 type Ultrasound Instrument, power 100W, frequency 60K, Ultrasonic 10 minutes), discharge born of the same parents' inclusion, obtain hemocyte and crush liquid.Break toward hemocyte Broken liquid adds copper chloride and zinc chloride, after thermal denaturation processes, obtains hemocyte degeneration liquid. Filter pressing (model SHXB-Z, pressure 0.2Mpa, double-filtration, first order aperture be 6 μm, Aperture, the second level is 1.2 μm, first crosses the first order after the second level) hemocyte degeneration liquid, collect First filtrate and the first filtering residue.Purify the first filtrate with anion-exchange chromatography, collect super oxidation Thing dismutase, i.e. obtains highly purified superoxide dismutase.
Wherein, it is preferred that by final concentration of 0.48 in crushing liquid at hemocyte~0.52% (matter Amount than) amount add copper chloride and adding by the amount of final concentration of 0.28~0.32% (mass ratio) Enter zinc chloride.
Wherein, during it is preferred that thermal denaturation process hemocyte crushes liquid, under the conditions of 72~77 DEG C Heat treatment hemocyte crushes liquid 30~35min.
(2) obtained in haemachrome extraction step water or buffer solution above-mentioned steps One filtering residue, regulates pH to 1.8~2.2, adds sodium carboxymethyl cellulose, obtains the first filtering residue molten Solve liquid.Wherein, sodium carboxymethyl cellulose can adsorb the purification of haemachrome, beneficially haemachrome.
Filter pressing the first filtering residue lysate again, collects the second filtrate and the second filtering residue.In second filtering residue Containing haemachrome, with water or buffer solution the second filtering residue.Collect the residue after washing and be height The haemachrome of purity, and collect the cleaning mixture after washing the second filtering residue.
(3) blood globulin powder extraction step
It is spray-dried above-mentioned cleaning mixture and collects the mixed liquor of the second filtrate obtained, i.e. obtaining blood Globulin powder.
It should be noted that above-mentioned steps is optionally carried out, such as, super oxidation is only carried out Thing dismutase extraction step, obtains superoxide dismutase;Or only carry out superoxide dismutase Enzyme extraction step and haemachrome extraction step, obtain superoxide dismutase and haemachrome;Certainly, Superoxide dismutase extraction step, haemachrome extraction step and hyperglobulinemia can also be carried out Powder extraction step, obtains superoxide dismutase, haemachrome and blood globulin powder.
S3: blood plasma extraction step
Blood plasma through filter pressing, after purification respectively obtain Fibrinogen, thrombinogen, thrombin, Cattle IgM, cattle fibronectin, cattle IgG and spray-dried plasma protein at least one.Change Sentence talk about, blood plasma extraction step include Fibrinogen extraction step, thrombinogen extraction step, Thrombin extraction step, cattle IgM and cattle fibronectin extraction step, cattle IgG extract Step and spray-dried plasma protein extraction step at least one.Hereinafter Fibrinogen is extracted Step, thrombinogen extraction step, thrombin extraction step, cattle IgM and cattle fiber connect Protein extraction step, cattle IgG extraction step and spray-dried plasma protein extraction step at least within it One is specifically described.
(1) Fibrinogen extraction step
It is 0.85~the sodium chloride solution diluting plasma of 0.9% (mass ratio) by concentration, obtains blood Slurry diluent.If it is understood that blood plasma is frozen process, should first melt the blood of freezing Slurry, then it is diluted operation.
Filter pressing plasma extender, collects the 3rd filtrate and the 3rd filtering residue.Containing fibre in 3rd filtering residue Fibrillarin is former, with water or buffer solution the 3rd filtering residue, collects the residue after washing and is high-purity The Fibrinogen of degree.
Wherein, it is preferred that be by volume 4~6:1 amount toward blood plasma adds sodium chloride solution.
(2) thrombinogen extraction step
Take part the 3rd filtrate, and add potassium oxalate and barium chloride, stir 18~20min, make It fully dissolves.Wherein, it is preferred that by the 3rd filtrate final concentration of 0.08~0.12% The amount of (mass ratio) adds potassium oxalate and adds barium chloride by the amount of 0.18~0.22%.
This part the 3rd filtrate of filter pressing again, collects the 4th filtrate and the 4th filtering residue, with pH6.8~7.2 0.018~0.022M phosphate buffer dissolve the 4th filtering residue, obtain the 4th filtering residue lysate. (DEAE Beads 6FF, balance pH6.8~7.2 are purified with anion-exchange chromatography 0.018~0.022M phosphate buffer, eluting is with 0.1-1mol/L NaCl's and pH6.8~7.2 0.018~0.022M phosphate buffer, regeneration 2mol/LNaCl) the 4th filtering residue lysate, Collection obtains highly purified thrombinogen.
Mass ratio
(3) thrombin extraction step
Adding calcium chloride in thrombinogen and carry out activation process, after being activated, thrombinogen is molten Liquid, then chromatograph (SP Beads 6FF, balance pH5.2~the 0.018 of 6.0~0.022M phosphoric acid Salt buffer, the eluting 0.018~0.022M phosphorus of 0.1-1mol/L NaCl and pH5.2~6.0 Phthalate buffer, regeneration 2mol/LNaCl) prothrombin solution after this activation, collect To highly purified thrombin.
(4) cattle IgM and cattle fibronectin extraction step
Final concentration of 18~22% (mass ratio) ammonium sulfate are added, stirring in the 3rd filtrate 18~20min so that it is fully dissolve.This 3rd filtrate separately taken of filter pressing again, collects the 5th filter Liquid and the 5th filtering residue.The is dissolved with the 0.008~0.012M acetate buffer of pH5.0~5.5 Five filtering residues, obtain the 5th filtering residue lysate.
Then (SP Beads 6FF, balance pH5.0~5.5 are purified with cation-exchange chromatography 0.018~0.022M acetate buffer, eluting 0.1-1mol/L NaCl and pH5.0~5.5 0.018~0.022M acetate buffer, regeneration 2mol/LNaCl) the 5th filtering residue dissolve Liquid, collects and obtains cattle IgM and cattle fibronectin.
Wherein, it is preferred that add acid ammonium by the amount of final concentration of 20% in the 3rd filtrate.
(5) cattle IgG extraction step
Final concentration of 33~the ammonium sulfate of 37% (mass ratio) are added, stirring in the 3rd filtrate 18~22min so that it is fully dissolve.This 3rd filtrate the most separately taken of filter pressing, collects the 6th filter Liquid and the 6th filtering residue.Phosphate solution with the 0.008~0.012M of pH6.8~7.2 dissolves the Six filtering residues, obtain the 6th filtering residue lysate.
(SP Beads 6FF, balance pH6.8~7.2 are purified with cation-exchange chromatography 0.018~0.022M phosphate buffer, eluting 0.1-1mol/L NaCl and pH6.8~7.2 0.018~0.022M phosphate buffer, regeneration 2mol/LNaCl) the 6th filtering residue dissolve Liquid, collects and obtains cattle IgG.
Wherein, it is preferred that add acid ammonium by the amount of final concentration of 35% in the 3rd filtrate.
(6) spray-dried plasma protein extraction step
It is spray-dried the 3rd filtrate, the 4th filtrate, the 5th filtrate, the 6th filtrate and the 7th filtrate Mixed liquor, i.e. obtain spray-dried plasma protein.
Being also required to explanation, above-mentioned steps is optionally carried out, and such as, only carries out fiber Proteinogen extraction step, obtains Fibrinogen;Or only carry out Fibrinogen extraction step With thrombinogen extraction step, obtain Fibrinogen and thrombinogen;Or only carry out fiber Proteinogen extraction step, thrombinogen extraction step and thrombin extraction step, obtain fiber egg Bai Yuan, thrombinogen and thrombin;Or only carry out Fibrinogen extraction step, thrombin Former extraction step, thrombin extraction step and cattle IgM and cattle fibronectin are extracted Step, obtains Fibrinogen, thrombinogen extraction step, thrombin, cattle IgM and cattle Fibronectin;Or above-mentioned steps is all carried out, carry out Fibrinogen the most successively and carry Take step, thrombinogen extraction step, thrombin extraction step, cattle IgM and cattle fiber even Connect protein extraction step and cattle IgG extraction step, obtain Fibrinogen, thrombinogen Extraction step, thrombin, cattle IgM, cattle fibronectin and cattle IgG.
The method extracting multiple protein from Sanguis Bovis seu Bubali of the present invention, makes full use of bioseparation technology, Especially filter pressing isolation technics and ion-exchange chromatography, by first separating close component, then enter The principle of row fine separation, first isolates superoxide dismutase, blood red from hemocyte one by one The albumen such as element, and obtain blood globulin powder;Then isolate one by one from blood plasma Fibrinogen, The albumen such as thrombinogen, thrombin, cattle IgM, fibronectin, cattle IgG, and obtain Spray-dried plasma protein.Relative to traditional extraction process, the extracting method of the present invention can be from portion Blood sample goes out to extract multiple proteins, has saved the cost of raw material and time, has fully excavated The potential value of blood, improves the utilization rate of bovine blood.
Below in conjunction with embodiment, inventive feature and performance are described in further detail.
Embodiment
This example with from bovine blood extract superoxide dismutase, haemachrome, Fibrinogen, Thrombinogen, thrombin, IgM, cattle fibronectin, cattle IgG, blood globulin powder with And as a example by spray-dried plasma protein, describe present invention offer in detail extracts multiple protein from Sanguis Bovis seu Bubali The operating process of method.
First, blood separation procedure is carried out.
Take the fresh bovine blood of 10L, add trisodium citrate and make its final concentration in bovine blood It it is 3.8% anticoagulant.Utilize hemocyte and the blood of 5.2L of blood cell seperator isolated 5.0kg Slurry.In the present embodiment, first processing hemocyte, blood plasma is in-20 DEG C of freezen protective.
Secondly, from hemocyte, extract superoxide dismutase, haemachrome successively, and obtain blood Globulin powder.Its concrete extraction step is as follows.
(1) superoxide dismutase is extracted
After brine hemocyte, then with sonicator, hemocyte is carried out ultrasonic Ripple crushes so that it is discharges born of the same parents' inclusion, obtains hemocyte and crush liquid.
Filter pressing hemocyte crushes liquid, removes cell debris residue, after removing cell debris residue Hemocyte crush liquid and proceed to reactor is carried out heat treatment, and add final concentration of 0.5% Copper chloride and the zinc chloride of 0.3%, be warming up to 75 DEG C, is incubated 20min.
Hemocyte after filter pressing heat treatment crushes liquid and obtains the first filtrate and the first filtering residue.
First filtrate is accompanied with normal saline dilution 5 and is i.e. arrived by the volume ratio addition normal saline of 4:1 In first filtrate, and regulate its pH to 7.6, purify with anion-exchange chromatography after diluting First filtrate, collects after eluting and obtains superoxide dismutase sterling 200mg.
(2) haemachrome is extracted
The first filtering residue obtained by water or buffer solution above-mentioned steps, regulates pH to 2.0, Add sodium carboxymethyl cellulose absorption, obtain the first filtering residue lysate.
Filter pressing the first filtering residue lysate, collects and obtains the second filtrate and the second filtering residue.
Second filtering residue, with acid acetone extraction, reclaims acetone after backflow, and concentrate diluted alkaline is molten Xie Hou, obtains haemachrome lysate, regulates haemachrome lysate pH to 4.0, then sucking filtration, Obtain haemachrome filtering residue, wash this haemachrome filtering residue with water, obtain highly purified haemachrome 50g.And Collect the cleaning mixture after washing the second filtering residue.
(3) blood globulin powder is extracted
It is spray-dried cleaning mixture and the mixed liquor of the second filtrate that above-mentioned steps obtains, i.e. obtains blood Globulin powder.
Then, from blood plasma extract Fibrinogen, thrombinogen, thrombin, cattle IgM, Cattle fibronectin and cattle IgG.Its concrete extraction step is as follows.
(4) Fibrinogen is extracted
Melt the blood plasma of freezing, take 5L blood plasma, with the sodium chloride solution dilution that concentration is 0.9% It is that the amount of 5:1 adds sodium chloride solution in blood plasma the most by volume, obtains plasma extender. Filter pressing plasma extender, collects and obtains the 3rd filtrate and the 3rd filtering residue, wash the 3rd filtering residue with water, Collect the residue after washing and i.e. obtain highly purified Fibrinogen.
(5) thrombinogen is extracted
In the 3rd filtrate and add the potassium oxalate of final concentration of 0.01% and the barium chloride of 0.2%, Stirring 20min so that it is fully dissolve, filter pressing, collects and obtains the 4th filtrate and the 4th filtering residue. Dissolve the 4th filtering residue with the 0.02M phosphate buffer of pH7, obtain the 4th filtering residue lysate. (DEAE Beads 6FF, the 0.02M of balance pH7.0 is purified with anion-exchange chromatography Phosphate buffer, eluting 0.1-1mol/L NaCl and pH7.0 0.02M phosphate buffer, Regeneration 2mol/L NaCl) the 4th filtering residue lysate, collect and obtain highly purified thrombin Former.
(6) thrombin is extracted
In thrombinogen, add 0.05M calcium chloride carry out activation process, solidifying after being activated Hemase original solution, then chromatograph prothrombin solution after this activation, collect and obtain highly purified blood coagulation Enzyme 50mg.
(7) cattle IgM and cattle fibronectin are extracted
In the 3rd filtrate and add final concentration of 20% ammonium sulfate, stir 20min so that it is Fully dissolve, then filter pressing, collect and obtain the 5th filtrate and the 5th filtering residue.With the 0.01M of pH5.0 Acetate buffer dissolves the 5th filtering residue, obtains the 5th filtering residue lysate.
(SP Beads 6FF, the 0.01M vinegar of balance pH5.0 is purified with cation-exchange chromatography Phthalate buffer, the eluting 0.01M acetate salt buffer of 0.1-1mol/L NaCl and pH5.0 Liquid, regeneration 2mol/LNaCl) the 5th filtering residue lysate, collect and obtain cattle IgM10g and cattle Fibronectin 1 g.
(8) cattle IgG is extracted
In the 3rd filtrate, add the ammonium sulfate of final concentration of 35%, stir 20min so that it is fill Divide and dissolve.
Filter pressing the 3rd filtrate, collects and obtains the 6th filtrate and the 6th filtering residue.With the 0.01M of pH7 Phosphate salt solution the 6th filtering residue, obtain the 6th filtering residue lysate.
(SP Beads 6FF, the 0.01M phosphorus of balance pH7.0 is purified with cation-exchange chromatography Phthalate buffer, the eluting 0.018~0.022M phosphoric acid of 0.1-1mol/L NaCl and pH7.0 Salt buffer, regeneration 2mol/LNaCl) the 6th filtering residue lysate, collect and obtain cattle IgG40g.
(9) spray-dried plasma protein is extracted
It is spray-dried the 3rd filtrate, the 4th filtrate, the 5th filtrate and the mixed liquor of the 6th filtrate, I.e. obtain spray-dried plasma protein.
It should be noted that in other examples, above-mentioned steps can all be carried out, Can carry out with selectivity.Extract according to the actual requirements.
To sum up, the method extracting multiple protein from Sanguis Bovis seu Bubali of the present embodiment, make full use of biological point From technology, by first separating close component, then carry out the principle of fine separation, expeditiously cattle Superoxide dismutase in blood, haemachrome, Fibrinogen, thrombinogen, thrombin, Extract to IgM, cattle fibronectin, cattle IgG high-purity seriatim, and process To the effective ingredient such as blood globulin powder and spray-dried plasma protein, the filtrate of each of which step or filtering residue Can all be obtained by, do not have waste liquid or waste residue abandons situation, and it fully excavates blood Potential value, improves the utilization rate of bovine blood.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, For a person skilled in the art, the present invention can have various modifications and variations.All at this Within the spirit of invention and principle, any modification, equivalent substitution and improvement etc. made, all should Within being included in protection scope of the present invention.

Claims (10)

1. the method extracting multiple protein from Sanguis Bovis seu Bubali, it is characterised in that comprising:
Blood separation procedure: take fresh bovine blood, adds anticoagulant, is separated into hemocyte and blood Slurry;
Hemocyte extraction step: described hemocyte is after broken, degeneration, filter pressing, purification process Respectively obtain superoxide dismutase, haemachrome and blood globulin powder at least one;
Blood plasma extraction step: described blood plasma through filter pressing, after purification respectively obtain Fibrinogen, Thrombinogen, thrombin, cattle IgM, cattle fibronectin, cattle IgG and plasma protein Powder at least one.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 1, it is special Levying and be, described hemocyte extraction step includes superoxide dismutase extraction step, haemachrome Extraction step and blood globulin powder extraction step at least one, wherein
Described superoxide dismutase extraction step is with hemocyte described in ultrasonic disruption, obtains Hemocyte crushes liquid, crushes toward described hemocyte and adds copper chloride and zinc chloride in liquid, becomes through heat Property process after obtain hemocyte degeneration liquid, hemocyte degeneration liquid described in filter pressing, collect the first filtrate With the first filtering residue, purify described first filtrate with anion-exchange chromatography, obtain described super oxidation Thing dismutase;
Described haemachrome extraction step is with the first filtering residue described in water dissolution, regulates pH extremely 1.8~2.2, add sodium carboxymethyl cellulose, obtain the first filtering residue lysate, described in filter pressing first Filtering residue lysate, collects the second filtrate and the second filtering residue, washs described second filtering residue, obtains institute State haemachrome;
Described blood globulin powder extraction step is the second residue collection described in spray-dried laundry Cleaning mixture and the mixed liquor of described second filtrate, obtain described blood globulin powder.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 2, it is special Levy and be, be respectively 0.48~0.52% He by the final concentration crushed in liquid at described hemocyte 0.28~0.32% amount add described copper chloride and described zinc chloride.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 2, it is special Levying and be, described thermal deformation field crushes for hemocyte described in heat treatment under the conditions of 72~77 DEG C Liquid 30~35min.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 2, it is special Levying and be, described blood plasma extraction step includes that Fibrinogen extraction step, thrombinogen extract Step, thrombin extraction step, cattle IgM and cattle fibronectin extraction step, cattle IgG Extraction step and spray-dried plasma protein extraction step at least one, wherein
Described Fibrinogen extraction step be with concentration be 0.85~0.9% sodium chloride solution dilute Release described blood plasma, obtain plasma extender, plasma extender described in filter pressing, collect the 3rd filtrate With the 3rd filtering residue, wash described 3rd filtering residue, obtain described Fibrinogen;
Described thrombinogen extraction step is to add potassium oxalate and chlorination in described 3rd filtrate Barium, stirs 18~20min, then filter pressing, collects the 4th filtrate and the 4th filtering residue, with pH6.8~7.2 0.018~0.022M phosphate buffer dissolve described 4th filtering residue, obtain the 4th filtering residue molten Solve liquid, purify described 4th filtering residue lysate with anion-exchange chromatography, obtain described thrombin Former;
Described thrombin extraction step is to add calcium chloride to described thrombinogen to carry out at activation Reason, prothrombin solution after being activated, then chromatograph prothrombin solution after described activation, To described thrombin;
Described cattle IgM and cattle fibronectin extraction step are to add in described 3rd filtrate Enter final concentration of 18~22% ammonium sulfate, stir 18~20min, then the 3rd filtrate described in filter pressing, Collect the 5th filtrate and the 5th filtering residue, delay with the 0.008~0.012M acetate of pH6.8~7.2 Rush liquid and dissolve described 5th filtering residue, obtain the 5th filtering residue lysate, then use cation exchange layer 5th filtering residue lysate described in analysis purification, obtains described cattle IgM and described cattle fibronectin In vain;
Described cattle IgG extraction step is to add final concentration of 33~37% in described 3rd filtrate Ammonium sulfate, stirs 18~22min, three filtrates described in filter pressing, collects the 6th filtrate and the 6th filter Slag, the Acetate Solution with the 0.008~0.012M of pH6.8~7.2 dissolves described 6th filtering residue, Obtain the 6th filtering residue lysate, purify described 6th filtering residue lysate with cation-exchange chromatography, Obtain described cattle IgG;
Described spray-dried plasma protein extraction step be spray-dried collect obtain described 3rd filtrate, Described 4th filtrate, described 5th filtrate and the mixed liquor of described 6th filtrate, obtain described blood Slurry protein powder.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 5, it is special Levy and be, be that the amount of 4~6:1 adds described sodium chloride solution in described blood plasma by volume.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 5, it is special Levy and be, be respectively 0.08~0.12% He by the final concentration in described 3rd filtrate 0.18~0.22% amount be separately added into described potassium oxalate and described barium chloride.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 5, it is special Levy and be, add ammonium sulfate by the amount of final concentration of 20% in described 3rd filtrate.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 5, it is special Levying and be, the amount of final concentration of 35% in described 3rd filtrate adds ammonium sulfate.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 1, it is special Levying and be, described anticoagulant is trisodium citrate, final concentration of by described blood 3.6~4% amount add described trisodium citrate.
CN201610364078.0A 2016-05-26 2016-05-26 Method for extracting multiple proteins from bovine blood Pending CN105950576A (en)

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CN107326017A (en) * 2017-06-19 2017-11-07 南宁学院 A kind of method that SOD is extracted from ox blood
CN107699555A (en) * 2017-11-17 2018-02-16 浙江丰安生物制药有限公司 A kind of preparation method of pig blood fibrin ferment
WO2021108976A1 (en) * 2019-12-03 2021-06-10 Omrix Biopharmaceuticals Ltd. Pro-thrombin purification
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CN114558371A (en) * 2022-03-23 2022-05-31 长睿生物技术(成都)有限公司 Bacterium zymotic fluid separator
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Application publication date: 20160921