CN1504484A - Preparation method of modified pig blood albumin for replacing human blood albumin - Google Patents

Preparation method of modified pig blood albumin for replacing human blood albumin Download PDF

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CN1504484A
CN1504484A CNA021446792A CN02144679A CN1504484A CN 1504484 A CN1504484 A CN 1504484A CN A021446792 A CNA021446792 A CN A021446792A CN 02144679 A CN02144679 A CN 02144679A CN 1504484 A CN1504484 A CN 1504484A
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peg
albumin
porcine
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cyanuric chloride
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王树岐
苟仕金
苟鸿鹰
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Gou Shijin
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SHENYANG NANKE PATENT TECHNOLOGY DEVELOPMENT CORP
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Abstract

The preparation method of the modified pig blood albumin for replacing human blood albumin comprises a, preparing anhydrous benzene; b. recrystallizing cyanuric chloride; c. porcine serum albumin nodules; activation of PEG1900; e. coupling of activated PEG to porcine serum albumin. PEG is used as a modifier to modify the porcine blood albumin, when the amino modification rate reaches 50 percent, the immunogenicity of the porcine blood albumin is completely relieved, and systematic research works such as toxicology and the like are carried out, which shows that the PEG modified porcine blood albumin becomes a substitute of human blood albumin.

Description

Modify the preparation method of porcine blood albumin replacement human serum albumin
Affiliated field
The present invention relates to albuminous preparation method, particularly modify the preparation method that porcine blood albumin replaces human serum albumin
Background technology
Recent years, medical development was found, blood donation personnel, usually have and be difficult for found hepatitis virus, as hepatitis B virus, hepatitis C virus, hepatitis A virus (HAV), even the AIDS virus carrier, blood products has the suspicion of propagating these virus diseases, therefore, the production of plasma albumin is restricted, and the shortage problem of human serum albumin will become clear day by day.Annual very big to albuminous requirement, annual output just about 200 tons, needs the hundreds of thousands of them to donate blood, so the price comparison costliness.
Summary of the invention
The objective of the invention is at the demand of human serum albumin greatly, cost an arm and a leg, and the deficiency of the toxicity disease that is easy to spread disease, provide a kind of the utilization to modify the preparation method that porcine blood albumin substitutes human serum albumin.
Blood is made up of hemocyte and blood plasma.The single peptide chain that the Plasbumin-25 is made up of 575 amino acid.Have unique effect and function in human body, it is the important carrier of multiple matter transportation in the blood, and also the internal metabolism with multiple medicine is relevant.People are by discovering, warfarin, stable and Myodigin, these three kinds of medicines have three binding sites at least on human serum albumin, many medicines play a role with detoxify all by with albumin bound after take place.
The molecular weight of human serum albumin is about 65.00 according to estimates, and content is many because molecular weight is less.So its main function is to keep plasma colloid osmotic pressure.Keep suitable moisture in blood plasma, keep blood circulation, human normal plasma's total protein concentration is between 6.5-7.5 gram %, and wherein albumin accounts for 3.9-4.5 gram %.Surplus is sphaeroprotein.
When albumin was lower than 3 gram %, hydrosarca often took place, liver cirrhosis and serious renal failure patient, because synthetic albumin dysfunction of liver or liver function are unusual, the blood oncotic pressure reduces, and causes ascites or oedema.As vein input albumin, improve its concentration in blood plasma, just can cause diuresis, oedema is disappeared.
The PEG of porcine blood albumin---modify and remove antigenic basis
Porcine blood albumin is a protein main in the porcine blood serum, and its content accounts for about half of total protein in the serum, molecular weight 67000, and iso-electric point is 4.7 to participate in keeping functions such as osmotic pressure and matter transportation in blood.Its character is more stable, and it is very abundant to originate, and has obtained at aspects such as molecular biology and cytobiologies using widely.But be mostly the application on diagnosis and detection method.Bovine serum albumin as a kind of amplification carrier, is come the sensitivity of enhancing immunity reaction, the useful trimerization ammonia of modifier commonly used cyanogen activated PEG, epoxy bromopropane activatory agarose ball.Epicyanohydrin activation sheep methoxy poly (ethylene glycol), studied about the epoxy chloropropane activation and revolved glucosides etc., the variation of immunological characteristic when Abraham etyal was with polyoxyethylene glycol covalent modification bovine serum albumin in 1977, its ultimate principle is earlier the OH of PEG to be connected with chlorine in the cyanuric chloride, and then links to each other with amino in the protein by cyanuric chloride.The variation of essence has taken place in the character of the bovine serum albumin after the modification, and as solubleness, the electrophoresis movability in the acrylamide electricity arteries and veins is higher than the displacement chromatography collection of illustrative plates and compares with natural bovine serum albumin with settling ratio, and tangible change is all arranged.In addition, polymer (polyoxyethylene glycol) is attached to has immunization barrier, causes the albumen non-immunogenicity.
In recent years, people to polyoxyethylene glycol bonded bovine serum albumin esterase activity immunoreactive variation done further exploratory development.The result shows, in adorned albumin molecule, and 60 wherein 15 active PEG couplings of quilt of amino, its immunoreactivity forfeiture as a result and become the non-immunogenicity bovine serum albumin, and have 63% esterase activity to be retained.
Along with bionic development, the widely-used possibility that becomes of numerous protein class medicine.Bovine serum albumin has 18 determinants, and fixed bunch of each wart by by one in fact the residue of quantity formed.Generally be that a kind of antigenic determinant has a kind of antigen-specific.For this reason, people's profit has the chemical substance modifying protein to cover heterologous protein at the intravital antigen of people position, and the immune response in avoiding using has in vivo obtained useful effect.Studies have shown that: the polyoxyethylene glycol effect is better, use polyethyleneglycol modified many enzymes, protein, antigenicity significantly reduces in using in vivo, obviously prolong action time in the body, drug effect has also had large increase, at present, the polyethyleneglycol modified adenosine deaminase of U.S. FDA approved (PEG---ADA) face treatment on the art because the serious comprehensively immunodeficiency symptoms (SCID) that this enzyme defect causes, thereby determined the status of chemically modified in medical research, therefore reduce its antigenicity with polyethyleneglycol modified bovine serum albumin, thereby replace the Plasbumin-25 to be applied to clinical.
Crowd virus carrying rate increases year by year, as hepatitis B virus carrying rate in the population of China up to 12%; The hepatitis C virus carrying rate is more up to 15%; Also have hepatitis A virus (HAV), hiv virus or the like in addition, blood donor's blood qualification rate is descended, and new blood donor's blood qualification rate only is 50%, and old blood donor's blood qualification rate is quite low, blood source difficulty makes and replaces the research of human serum albumin imperative, causes that various countries pay attention to.
Produce human serum albumin with genetic engineering means, but the purifying difficulty is very big.
Replace human serum albumin with the animal seralbumin, what this need solve is that the animal seralbumin is to human antigen's property problem.
Remove the foreign protein antigenicity with PEG.Up to the present U.S. FDA has been ratified seven kinds of PEG modified proteins (enzyme) medicine at least and has been used for clinical according to reports.
A, Abuchowski, A, Matsushima etc. modify the ox blood albumin with PEG, and its antigenicity forfeiture is because Britain's mad cow disease makes this research stranded.
With PEG is modifier, modifies porcine blood albumin, and when its amido modified rate reached 50%, its immunogenicity was removed fully, and has made toxicity etc. than systematic research work, shows that PEG modifies porcine blood albumin and becomes the human serum albumin surrogate.
The preparation of porcine blood albumin: get the aseptic pig blood of taking, make serum earlier, through biochemical separation and purification means, obtain electrophoretically pure porcine blood albumin again, lyophilize is standby.Specifically undertaken, and hold the raw materials quality pass by the calibrating project operation detailed rules and regulations of human serum albumin simultaneously by the requirement of national regulation human serum albumin cold ethanol method technological operation detailed rules and regulations.
Modify the preparation method of porcine blood albumin replacement human serum albumin, comprise
The preparation of a, dry-out benzene; The recrystallization of b, cyanuric chloride; C, porcine hemoglobin knot; D, PEG1900 activation; The coupling of e, activated PEG and porcine hemoglobin.
Concrete modification porcine blood albumin replaces the preparation method of human serum albumin, comprises
The preparation of a, dry-out benzene
Get flask at the bottom of the 1000ml garden, top grade purified petroleum benzin 700ml is inserted at the bottom of the garden in the flask, add about 20 of sodium Metal 99.5 sheets, on connect a cover backflow device, the gas outlet Calcium Chloride Powder Anhydrous (purpose prevents that moisture from entering) of packing into, below flask at the bottom of the garden was put in the electric mantle backflow after 3 hours, take off prolong, settle oblique prolong, right jointing temp meter, temperature keeps 30 ℃ of little boiling, treat that prolong one end has liquid to ooze, receive with Brown Glass Brown glass bottles and jars only, be dry-out benzene, standby.
The recrystallization of b, cyanuric chloride
Get cyanuric chloride 80g, place flask at the bottom of the 1000ml garden, add dry-out benzene 500ml again, mix dissolving, flask is put into and is connect a cover backflow condensing works on the electric mantle at the bottom of the garden, reflux 3 hours, take off prolong,, insert in the 500ml conical flask with stopper the liquid filtered while hot, put into 40 refrigerators and preserve more than 24 hours, can see the crystallization of cyanuric chloride.
With benzene incline after, take out the crystallization cyanuric chloride and place flask at the bottom of the garden, above method repetitive operation obtains the cyanuric chloride of recrystallization.
C, porcine hemoglobin crystallization
(1) 2g porcine hemoglobin → be dissolved in the 20ml distilled water (is measured volume V1 at last) → is added saturated (NH 4) 3SO 4V 3Ml (equal-volume) → room temperature is placed 20 minutes → filtration → filtrate V 2Continue to drip (NH 4) 2SO 4Constantly stir → when resolution of precipitate is very slow, stop to drip → use 0.2NH 2SO 4---4.8 → the place 24---48hr that transfers PH4.6 → have crystallization.
(2) recrystallization
To precipitate (crystallization) in 2000 rev/mins 10 minutes.Remove supernatant → drip down (NH with a small amount of dissolved in distilled water crystalline deposit → stirring 4) 2SO 4Solution → resolution of precipitate stops to drip when very slow → use 0.2NH 2SO 4Transfer PH4.6-4.8 to place 24---48hr → crystallization.
Crystallization is dissolved with a small amount of PBS liquid PH7.4.Pack in the dialysis tubing totally 4 into.With 100ml beaker dress 800ml PBS damping fluid, the common 24h that dialyses, intermittently changing the liquid time is to take out dialysis tubing 3.3.6.6.6 hour next day, and liquid is poured in the freeze-drying bottle, freeze-drying is standby.
d、PEG1900。5000 activation.
Get preparation recrystallization cyanuric chloride (5,7g0.03mol) be dissolved in 400ml and contain in the dry-out benzene solution of (in xeothermic case xeothermic 160 ℃ 2 hours then use) of 10g Carbon Dioxide, adding PEG---1900 (19g0.01mot is at room temperature 22-24 ℃) stir day and to stir liquid.
Get and stirred liquid (activated PEG) and filter (on add filter paper) with the triangle glass funnel and place Erlenmeyer flask.Filter the clear and bright solution 400ml in back and be placed on to stir and day go up stir about after 30 minutes, slowly add sherwood oil 600ml and white precipitate occurs and continue to stir after 20 minutes and use G 4The molten glass funnel of hammer filters.The sticking tube precipitation of adularescent filtrate clear on the filter, again white precipitate is moved into and add dry-out benzene 400ml dissolving in the Erlenmeyer flask, this process repetitive operation repeatedly, each operation is monitored with ultraviolet (UV-2201 type) spectrophotometer, till detecting less than free cyanuric chloride absorption peak, obtain activated PEG---1900 place moisture eliminator to vacuumize, and benzene is waved and dispersed, and is standby.PEG---5000 adopt above-mentioned identical method preparation feedback formula to be:
The coupling of e, activated PEG and porcine hemoglobin
Getting four recrystallization porcine blood albumins (400mg.6.04.umol) is dissolved in the 40ml0.1MPH9.2 sodium tetraborate solution, with this liquid be placed in 4 ℃ of refrigerators preserve 2 hours after, take out, add activated PEG-1000 (this amount of 3.55gl.74mol is 5 times of amino group), placing 4 ℃ of refrigerators after stirring 21 hours on the agitator, take off and stir liquid and pack in the dialysis tubing, with the dialysis of 0.01MPH7.4PBS damping fluid, dialysis time early 10.30 to inferior 10.30,24 hours, per intermittence 3,3,6,6,. hour replacing dialyzate, at early 9.30 minutes next day, take out dialysis tubing, bag liquid was inserted in the freeze-drying bottle freeze-drying after 10 hours, it is Powdered to be white in color, standby in the receiving flask.
Reaction formula is:
PEG---modify the condition and the influence degree of porcine blood albumin
PEG---1900 and two kinds of PEG of PEG500 all 25 ℃ of 18 hours modification rates for the highest, all can reach about 90%.And in the time of 37 ℃, take second place.About 50% 4 ℃ the time effect the poorest, can reach 30-40%.Show that reaction has apparent in view influence to temperature to PEG modification porcine blood albumin.(seeing Table 1)
Methoxy poly (ethylene glycol) and albuminous covalent coupling, as coupling agent, reaction formula is as follows with cyanuric chloride:
Figure A0214467900122
Figure A0214467900123
In this reaction, the chlorine atom in the cyanuric chloride molecule is an active atomic, can be replaced by nucleophilic reagent.One or two chlorine atoms can be provided electronics gene and replace.Forming stable C-C1 key] people such as John Son reports that cyanuric chloride and amido imide and hydroxy reaction should form stable keys.Near 40 o'clock, first chlorine atom reacts rapidly, and second ammonia atom is 25 ℃ of reactions, and the 3rd reacts at 80 ℃.This conforms to experimental result.In the time of 4 ℃, 30-40 ℃ of modification rate reaches 80-90% and modify during to 25 ℃, and the modification rate can improve more than 1 times.But in the time of 37 ℃, the modification rate but is lower than 25 ℃ of modification rates, this may be to have two kinds of reasons (1) temperature to mention 37 ℃ once, be unfavorable for delaying the reaction of a chlorine atom, so and end far away reaches the 3rd chlorine atom to participate in the best of reaction only is that second chlorine atom is active for 30 ℃ and strengthens, and totally modification rate descends, 37 ℃ be porcine blood albumin near the temperature of native conformation under this conformation situation, since the position of free amine group therewith in the activated PEG under the temperature position of chlorine atom be difficult for forming the C-C1 key, and the modification rate is reduced.
PEG---modify porcine blood albumin antigenicity, security, effectuation experiment and verification result.
PEG---modify porcine blood albumin molecular weight 5000, its amido modified rate is 50%.
1, PEG---albumin antigen.
(1) immunodiffusion(ID) agar plate method: qualified.
(2) hypersensitive test: laboratory animal is a cavy, injections of antigens every other week, and continuous four times, for qualified.(seeing Table 2)
The result shows that porcine blood albumin has obvious anaphylaxis, even causes animal dead, and PEG-albumin group is without any anaphylaxis.
(3) clearance test (see Table 3) of radio-labeling PEG---albumin on the rabbit body
Found that and have only the albumin immune animal that albumin is removed rapidly, (nonimmune animal is to albumin for the clearance rate of all the other each groups, PEG---albumin, albumin immune animal and PEG---the albumin immune animal is to PEG---albumin, PEG---the albumin immune animal is to albumin) similar substantially, there is no and be eliminated phenomenon rapidly, this explanation, albumin produces albumin antibody after being expelled to rabbit, albumin antibody rapidly and its generation immune response and being eliminated when reinjecting albumin, and PEG---the albumin immunizing rabbit does not promptly produce albumin antibody, do not produce PEG---albumin antibody yet, therefore as albumin or the PEG of reinjecting---do not produce immune response during albumin, do not observe yet and removed phenomenon rapidly, PEG also is described---antigen antibody reaction does not take place between albumin and the albumin antibody yet.
Above description of test PEG---albumin has been disengaged antigenicity fully.
2, toxicity test:
Press the Chinese Pharmacopoeia method, be PEG---albuminous acute toxicity test and chronic toxicity test.The weight of animals, white corpuscle, lymphocyte, red corpuscle, oxyphorase, uremic nitrogen, transaminase, internal organs (heart, liver, spleen, lung, kidney, brain) weight have been observed in long term toxicity test, check each internal organs pathological change of animal, there is no unusually, PEG be described---albumin is to the animal free of toxic effects.
3, proof test:
By the pharmacopeia requirement, be object with the cavy, made PEG---the albumin safety experiment.The result shows PEG---albumin meets proof test requirement in the biological products rules.
4, clearance rate and distribution thereof in the animal body:
The radio-label experiment shows that PEG---albumin clearance rate in animal body is similar substantially to native albumin, does not all have obviously long-pending folded effect; Be liver 3.8% in each internal organs retained percentage after 31 days, Tiroidina 0.95%, kidney 0.055%, spleen and thymus gland also have small amount of residual, and at brain and ovary essentially negative.
5, osmotic pressure test:
The porcine blood albumin of volumetric molar concentrations such as albumin that with the porcine blood albumin is contrast, has made PEG---albumin osmotic pressure test, the result shows PEG---is is isoosmotic, and PEG is described---albumin has the effect of the normal body of keeping osmotic pressure.
6, PEG---porcine blood albumin pH value, moisture content, toxoid, pyrogen test, sterility test are all qualified.
7, other:
The albuminous moisture determination of PEG---determining the protein quantity, free amine group mensuration, purity, PEG in the albumin molecule---.Meet the requirement of albumin index.
The chemically modified of porcine blood albumin will have using value preferably, and it has 3 advantages, and first, raw material sources are abundant, domestic most of meat packing plants all throw away pig blood as waste, so, with pig blood production albumin abundant raw material sources are arranged.Can reduce simultaneously the pollution of right of abandonment thing again; The second, cost is low, because cost of material is low, the chemical reagent PEC price and the cyanuric chloride price that are used to modify porcine blood albumin are not high yet, so the cost of the porcine blood albumin after the modification will be starkly lower than the human serum albumin cost.The finished product price is estimated the low 2-3 of comparable human serum albumin finished product price doubly; The 3rd, safe, be raw material with pig blood, can get rid of the pollution of viruses such as hepatitis A, hepatitis B, third liver, mad cow disease, acquired immune deficiency syndrome (AIDS) fully, can not propagate the transmissible disease that causes by these viruses.With the exception of this, the porcine blood albumin after the modification also can be used as the protective material of multiple bioactive macromolecule preparation.Have wide practical use and the potential economic benefit.
Embodiment
Embodiment 1:
1, healthy quarantine: new must physical examination the blood sampling simultaneously for the blood sampling pig done relevant zoonosis quarantine, the negative patient object of taking a blood sample.
2, blood sampling:
The selection of pig is advisable with the black pig.Blood collecting bottle be 180 ℃ xeothermic, each blood sampling (3 liter) bottle adds 4% and arrests rafter and receive and 300 milliliters in 2% sodium-chlor.Tampon with the glassine paper wrapping.Glass tubing and sebific duct after acid-alkali treatment and pure water rinsing, dry back wrapping, sterilization, standby.
With pig fixing after, clean neck cropping sterilization, after the wipes of alcohol examination, get the carotid artery otch, insert the blood sampling tube edge and shake edge joint blood, reach till 6 liters.Pull out sebific duct, bottle is tightly wrapped up, quiet being suitable in the warm water, slurry folds.Afterwards blood collecting bottle is put in to leave standstill in the freezer and makes the blood plasma layering.When putting into freezer, clean with lysol or carbasus carbolata.
The strictness of the isolated pig blood of blood collecting bottle blood plasma is made qualified freeze-drying porcine blood albumin by human albumin low temperature alcohol method technology detailed rules and regulations, the surplus anaphylactogen of quality standard meets human injection albumin quality standard outward fully, concrete protein content reaches more than 96%, one in purity electricity arteries and veins.Aseptic safety, pyrogen, endotoxin content, moisture, PH etc. will meet the requirements.
Embodiment 2:
A, PEG---1900 and PEG---5000 activation, get the cyanuric chloride 5.7g (0.03mol) of secondary recrystallization, being dissolved in 400ml contains in the dry-out benzene of 10g anhydrous sodium carbonate, add 19g (0.01mol) PEG---24 ℃ of 1900 or 50g (0.01mol) PEG---5000 room temperature (22---) stir the after-filtration that spends the night, getting clear filtrate 400ml is placed on and stirs 20min on the agitator, slowly add sherwood oil 600ml, occur continuing to stir 20min behind the white precipitate, filter with the molten glass funnel of G4 hammer, to precipitate again and move in the Erlenmeyer flask, add dry-out benzene 400ml dissolving, this process repetitive operation is repeatedly operated repeatedly at every turn, each operation is monitored with ultraviolet (UV-2201 type) spectrophotometer, till detecting, obtain activated PEG and place moisture eliminator to find time, drying for standby less than free cyanuric chloride absorption peak.
The porcine blood albumin 400mg (6.04umol) of 4 recrystallizations is got in the coupling of b, activated PEG and porcine blood albumin, be dissolved in 40ml0.1mol/L, in the sodium tetraborate solution of pH9.2, this solution is placed in 4 ℃ of refrigerators takes out after preserving 2h, add activated PEG---19003.55g (1.87mmol) or activated PEG-50009.35g (1.87mmol), in 25 ℃ of stirred in water bath 21h, with the PBS damping fluid dialysis of 0.01mol/LpH7.4.Dialysis is placed on freeze-drying 10h in the freeze-drying bottle, pulverous PEG must be white in color---1900---BSA and PEG---5000---BSA.
Embodiment 3:
Prepare PEG with the method for implementing 1 and 2---modify test agent in qualified three batches, PEG---the porcine blood albumin of modification is done every experiment and relevant calibrating by the new declaration material requirement of new biological product medicine to each 3000 gram.
Modify the result under the different PEG of table 1 porcine blood albumin and the different condition
    PEG——1900     4℃  25℃  37℃     PEG——5000     4℃  25℃  37℃
The modified amino acid per molecule     25    53    34     19    55    30
Modification rate %     42    89    56     32    91    52
Table 2PEG---albumin Hypersensitive tests result
The cavy numbering Colour code For the first time injection After one week After two weeks All around Allergic reaction
    1 Head is blue     10mg     10mg     10mg     20mg     ——
    2 Flower     10mg     10mg     10mg     20mg     ——
    3 The tail orchid     10mg     10mg     10mg     20mg     ——
    4 The waist orchid     10mg     10mg     10mg     20mg + death
    5 The leg orchid     10mg     10mg     10mg     20mg     +
    6 Black     10mg     10mg     10mg     20mg     +
Illustrate: 1,2, No. 3 injection PEG-albumin
4,5, No. 6 injection albumin
Table 3 PEG---I125---albumin clearance test in the rabbit body
Figure A0214467900181
Illustrate: 1, nonimmune animal; 2, albumin immune animal; 3, PEG-albumin is exempted from The epidemic disease animal.

Claims (2)

1. modify the preparation method that porcine blood albumin replaces human serum albumin, comprise the preparation of a, dry-out benzene; The recrystallization of b, cyanuric chloride; C, porcine hemoglobin knot; D, PEG activation; The coupling of e, activated PEG and porcine hemoglobin.
2. replace the preparation method of human serum albumin according to the described modification porcine blood albumin of claim 1, comprise
The preparation of a, dry-out benzene
Get flask at the bottom of the 1000ml garden, top grade purified petroleum benzin 700ml is inserted at the bottom of the garden in the flask, add about 20 of sodium Metal 99.5 sheets, on connect a cover backflow device, the gas outlet Calcium Chloride Powder Anhydrous (purpose prevents that moisture from entering) of packing into, below flask at the bottom of the garden was put in the electric mantle backflow after 3 hours, take off prolong, settle oblique prolong, right jointing temp meter, temperature keeps 30 ℃ of little boiling, treat that prolong one end has liquid to ooze, receive with Brown Glass Brown glass bottles and jars only, be dry-out benzene, standby;
The recrystallization of b, cyanuric chloride
Get cyanuric chloride 80g, place flask at the bottom of the 1000ml garden, add dry-out benzene 500ml again, mix dissolving, flask is put into and is connect a cover backflow condensing works on the electric mantle at the bottom of the garden, reflux 3 hours, take off prolong,, insert in the 500ml conical flask with stopper the liquid filtered while hot, put into 40 refrigerators and preserve more than 24 hours, can see the crystallization of cyanuric chloride;
With benzene incline after, take out the crystallization cyanuric chloride and place flask at the bottom of the garden, above method repetitive operation obtains the cyanuric chloride of recrystallization;
C, porcine hemoglobin crystallization
(1) 2g porcine hemoglobin → be dissolved in the 20ml distilled water (is measured volume V1 at last) → is added saturated (NH 4) 3SO 1V 3Ml (equal-volume) → room temperature is placed 20 minutes → filtration → filtrate V 2Continue to drip (NH 1) 2SO 4Constantly stir → when resolution of precipitate is very slow, stop to drip → use 0.2NH 2SO 4---4.8 → the place 24---48hr that transfers PH4.6 → have crystallization;
(2) recrystallization
To precipitate (crystallization) in 2000 rev/mins 10 minutes, remove supernatant → drip down (NH with a small amount of dissolved in distilled water crystalline deposit → stirring 4) 2SO 4Solution → resolution of precipitate stops to drip when very slow → use 0.2NH 2SO 4Transfer PH4.6-4.8 to place 24---48hr → crystallization;
Crystallization is dissolved in the dialysis tubing of packing into totally 4 with a small amount of PBS liquid PH7.4; With 100ml beaker dress 800ml PBS damping fluid, the common 24h that dialyses, intermittently changing the liquid time is to take out dialysis tubing 3.3.6.6.6 hour next day, and liquid is poured in the freeze-drying bottle, freeze-drying is standby;
D, PEG1900 activation
Get preparation recrystallization cyanuric chloride (5,7g 0.03mol) being dissolved in 400ml and containing in the dry-out benzene solution of (in xeothermic case xeothermic 160 ℃ 2 hours then use) of 10g Carbon Dioxide, add PEG---1900 (19g0.01mot is at room temperature 22-24 ℃) stir day and to stir liquid;
Get and stirred liquid (activated PEG) and filter (on add filter paper) with the triangle glass funnel and place Erlenmeyer flask, filter the clear and bright solution 400ml in back and be placed on to stir and day go up stir about after 30 minutes, slowly add sherwood oil 600ml and white precipitate occurs and continue to stir after 20 minutes and use G 4The molten glass funnel of hammer filters, the sticking tube precipitation of adularescent filtrate clear on the filter, again white precipitate is moved into and add dry-out benzene 400ml dissolving in the Erlenmeyer flask, this process repetitive operation repeatedly, each operation is monitored with ultraviolet (UV-2201 type) spectrophotometer, till detecting less than free cyanuric chloride absorption peak, obtains activated PEG---and 1900 place moisture eliminator to vacuumize, benzene is waved and dispersed, and is standby;
The coupling of e, activated PEG and porcine hemoglobin
Getting four recrystallization porcine blood albumins (400mg.6.04.umol) is dissolved in the 40ml0.1MPH9.2 sodium tetraborate solution, with this liquid be placed in 4 ℃ of refrigerators preserve 2 hours after, take out, add activated PEG-1000, placing 4 ℃ of refrigerators after stirring 21 hours on the agitator, take off and stir liquid and pack in the dialysis tubing, with the dialysis of 0.01MPH7.4PBS damping fluid, dialysis time early 10.30 to inferior 10.30,24 hours, per intermittence 3,3,6,6,. hour replacing dialyzate, at early 9.30 minutes next day, take out dialysis tubing, bag liquid was inserted in the freeze-drying bottle freeze-drying after 10 hours, it is Powdered to be white in color, standby in the receiving flask.
3. the preparation method who replaces human serum albumins according to claim 1 or 2 described modification porcine blood albumins, a, PEG---1900 activation, get the cyanuric chloride 5.7g (0.03mol) of secondary recrystallization, being dissolved in 400ml contains in the dry-out benzene of 10g anhydrous sodium carbonate, add 19g (0.01mol) PEG---1900 room temperature (22---24 ℃) stir the after-filtration that spends the night, getting clear filtrate 400ml is placed on and stirs 20min on the agitator, slowly add sherwood oil 600ml, occur continuing to stir 20min behind the white precipitate, filter with the molten glass funnel of G4 hammer, to precipitate again and move in the Erlenmeyer flask, add dry-out benzene 400ml dissolving, this process repetitive operation repeatedly, each operation is repeatedly operated with the monitoring of ultraviolet (UV-2201 type) spectrophotometer, till detecting less than free cyanuric chloride absorption peak at every turn, obtain activated PEG and place moisture eliminator to find time, drying for standby;
The porcine blood albumin 400mg (6.04umol) of 4 recrystallizations is got in the coupling of b, activated PEG and porcine blood albumin, be dissolved in 40ml0.1mol/L, in the sodium tetraborate solution of pH9.2, this solution is placed in 4 ℃ of refrigerators takes out after preserving 2h, add activated PEG---19003.55g (1.87mmol), in 25 ℃ of stirred in water bath 21h, with the PBS damping fluid dialysis of 0.01mol/LpH7.4; Dialysis is placed on freeze-drying 10h in the freeze-drying bottle, and pulverous PEG---1900---BSA must be white in color.
4. replace the preparation method of human serum albumin according to the described modification porcine blood albumin of claim 1, the activation of PEG can be PEG---5000 activation.
5. the preparation method who replaces human serum albumins according to claim 1 or 4 described modification porcine blood albumins, a, PEG---5000 activation, get the cyanuric chloride 5.7g (0.03mol) of secondary recrystallization, being dissolved in 400ml contains in the dry-out benzene of 10g anhydrous sodium carbonate, add 50g (0.01mol) PEG---5000 room temperature (22---24 ℃) stir the after-filtration that spends the night, getting clear filtrate 400ml is placed on and stirs 20min on the agitator, slowly add sherwood oil 600ml, occur continuing to stir 20min behind the white precipitate, filter with the molten glass funnel of G4 hammer, to precipitate again and move in the Erlenmeyer flask, add dry-out benzene 400ml dissolving, this process repetitive operation repeatedly, each operation is repeatedly operated with the monitoring of ultraviolet (UV-2201 type) spectrophotometer, till detecting less than free cyanuric chloride absorption peak at every turn, obtain activated PEG and place moisture eliminator to find time, drying for standby;
The porcine blood albumin 400mg (6.04umol) of 4 recrystallizations is got in the coupling of b, activated PEG and porcine blood albumin, be dissolved in 40ml0.1mol/L, in the sodium tetraborate solution of pH9.2, this solution is placed in 4 ℃ of refrigerators takes out after preserving 2h, add activated PEG-50009.35g (1.87mmol), in 25 ℃ of stirred in water bath 21h, with the PBS damping fluid dialysis of 0.01mol/LpH7.4; Dialysis is placed on freeze-drying 10h in the freeze-drying bottle, and pulverous PEG---500---BSA must be white in color.
CNA021446792A 2002-12-04 2002-12-04 Preparation method of modified pig blood albumin for replacing human blood albumin Pending CN1504484A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102707069A (en) * 2012-06-04 2012-10-03 南京巴傲得生物科技有限公司 Immunohistochemical double-antibody kit

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102707069A (en) * 2012-06-04 2012-10-03 南京巴傲得生物科技有限公司 Immunohistochemical double-antibody kit
CN102707069B (en) * 2012-06-04 2015-04-08 南京巴傲得生物科技有限公司 Immunohistochemical double-antibody kit

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