CN103288953B - Method for separating and purifying functional protein in plasma - Google Patents
Method for separating and purifying functional protein in plasma Download PDFInfo
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- CN103288953B CN103288953B CN201310216023.1A CN201310216023A CN103288953B CN 103288953 B CN103288953 B CN 103288953B CN 201310216023 A CN201310216023 A CN 201310216023A CN 103288953 B CN103288953 B CN 103288953B
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 67
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 57
- 238000000605 extraction Methods 0.000 claims abstract description 72
- 239000007788 liquid Substances 0.000 claims abstract description 65
- 239000012071 phase Substances 0.000 claims abstract description 54
- 239000008346 aqueous phase Substances 0.000 claims abstract description 40
- 238000000746 purification Methods 0.000 claims abstract description 32
- 238000000926 separation method Methods 0.000 claims abstract description 30
- 239000003960 organic solvent Substances 0.000 claims abstract description 29
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 24
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 13
- 239000002904 solvent Substances 0.000 claims abstract description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 72
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- 238000013016 damping Methods 0.000 claims description 22
- 239000012530 fluid Substances 0.000 claims description 22
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 20
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 18
- 239000001509 sodium citrate Substances 0.000 claims description 17
- 239000000243 solution Substances 0.000 claims description 16
- 238000010828 elution Methods 0.000 claims description 15
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 14
- 229940038773 trisodium citrate Drugs 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 12
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 10
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 10
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 9
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 claims description 8
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 8
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 8
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 4
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- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 4
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- 238000005342 ion exchange Methods 0.000 claims description 3
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 3
- 229940093916 potassium phosphate Drugs 0.000 claims description 3
- 235000011009 potassium phosphates Nutrition 0.000 claims description 3
- OTNVGWMVOULBFZ-UHFFFAOYSA-N sodium;hydrochloride Chemical compound [Na].Cl OTNVGWMVOULBFZ-UHFFFAOYSA-N 0.000 claims description 3
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 2
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- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 2
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 claims description 2
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- 150000003016 phosphoric acids Chemical class 0.000 claims description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 2
- 235000015320 potassium carbonate Nutrition 0.000 claims description 2
- 239000001508 potassium citrate Substances 0.000 claims description 2
- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 claims description 2
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 2
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 2
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- SOBHUZYZLFQYFK-UHFFFAOYSA-K trisodium;hydroxy-[[phosphonatomethyl(phosphonomethyl)amino]methyl]phosphinate Chemical compound [Na+].[Na+].[Na+].OP(O)(=O)CN(CP(O)([O-])=O)CP([O-])([O-])=O SOBHUZYZLFQYFK-UHFFFAOYSA-K 0.000 description 3
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- KCIDZIIHRGYJAE-YGFYJFDDSA-L dipotassium;[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] phosphate Chemical compound [K+].[K+].OC[C@H]1O[C@H](OP([O-])([O-])=O)[C@H](O)[C@@H](O)[C@H]1O KCIDZIIHRGYJAE-YGFYJFDDSA-L 0.000 description 1
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention provides a method for separating and purifying functional protein in plasma, which comprises the following steps of: adding soluble inorganic salt and a hydrophilic organic solvent into a plasma solution to form a two-aqueous phase extraction system, and obtaining the upper-phase extraction liquid rich in plasma functional protein; and performing further separation and purification of the extraction liquid through hydrophobic chromatography and ion exchange chromatography to obtain the plasma functional protein of which the electrophoresis purity is higher than 95%. The method provided by the invention simplifies the purifying steps of the plasma functional protein; the organic solvent/salt two-aqueous phase extraction has the advantages of convenience in solvent recovery, low cost and short phase separating time, does not need back-extraction operation and can selectively remove glucose and partial protein in the plasma; and the extraction liquid is directly separated and purified by hydrophobic chromatography and ion exchange chromatography, the complicated steps of desalination are avoided, and the separation and purification effects of the plasma functional protein are remarkably improved. The method provided by the invention solves the problems of complicated separation steps, high cost, low purity and the like in the separation and purification of functional protein in the plasma protein.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to a kind of separation purification method of protein, particularly relate to the method for functional protein in a kind of separation and purification blood plasma.
Background technology
Blood plasma by the water of 90%, the albumen of 7% and 3% carbohydrate organism form, this albumen of 7% contains the protein that hundreds of have extensive physiological function, make blood plasma become the starting material of the fractionated products for the treatment of product or bio-pharmaceutical mostly, the functional protein that separation and Extraction obtains from blood plasma comprises the kinds such as albumin, immunoglobulin (Ig), blood clotting factors (scleroproein, former blood coagulation factor VIII), protein inhibitor class.Wherein albumin content is the highest, accounts for the over half of its total amount, is strand, non-glycosylated protein, and main participation maintains osmotic pressure, shipped material lipid acid and assist the functions such as human body removing toxic substances.Albuminous blood products is the plasma protein products be most widely used at present, is mainly used in the treatment of the disease such as hemorrhagic shock, hypoproteinemia clinically.Immunoglobulin (Ig) degree of enriching in blood is only second to albumin, reach 15% of Tot Prot, the most important with immunoglobulin G, the humoral immunization of main participation higher organism, protection body is from the infringement of the antigens such as pathogenic agent, in vivo, immunoglobulin (Ig) has and is combined with pathogenic agent or toxin, complement activation, lethal effect is engulfed in strengthening, produce anaphylaxis and slow down the biological functions such as rejection in organ transplantation, it is main force's effector molecule of humans and animals Acute toxicity, and there is treatment epilepsy, the effect of inhibition tumor cell propagation and inducing tumor cell early apoptosis, Application Areas is very extensive.
Some main human plasma functional proteins of table 1.
At present, in blood plasma, the separation purification method of functional protein mainly adopts CohnShi chilled alcohol precipitation method, the method is having people's inventions such as Cohn during the Second World War, improved by people such as Kistler and Nitschmann subsequently, the method is mainly through under cryogenic (-7-5 DEG C), in serum, substep adds the ethanol of different concns (8-25%), makes the fractional precipitation such as IgG, albumin, then collects the functional protein in plasma proteins by centrifugal method.The method has and is easy to industrialized advantage, but also there is many problems in actual applications, and first, the method needs to operate at low ambient temperatures, and need to temperature controlled accuracy requirement compared with high, equipment investment large, energy consumption is large; Secondly, the method complex operation step, yield are low, purity is low, need more precipitation and centrifugal, cause a large amount of losses of functional protein in plasma proteins.Research shows, only the loss of IgG just reaches 50%, and the purity being separated the blood plasma functional protein obtained through the method also cannot meet clinical needs.Many investigators attempt new separation method in recent years, Chinese patent mandate publication number CN1281617C discloses a kind of method adopting filter-pressing process separated plasma albumen, the yield of plasma proteins can be improved, but still the method for fractional precipitation need be adopted, and the plasma proteins purity that not mentioned the method obtains.Chinese patent Authorization Notice No. CN1259338C disclose a kind of can sealing pipeline in carry out plasma proteins separation method, the method is improved Cohn method, press filtration is adopted to substitute the centrifugally operated of solid-liquid separation part in Cohn method, production cycle can shorten half, but still does not overcome Cohn method Problems existing.WO9805685A1 discloses a kind of method adopting functional protein in large pore anion exchang medium purified plasma, can avoid more precipitation and centrifugal, but the purity of IgG only has 36-66%.Chinese patent Authorization Notice No. CN1089609C discloses a kind of method adopting affinity chromatography to obtain high purity IgG, but the high cost of affinity media, limit the application of the method.
Plasma protein composition is complicated, and there is the residual of the viruses such as HIV, HBV, the health of blood plasma functional protein confrontation user is made to there is potential threat, the preparation method of the high purity blood plasma functional protein low, easy and simple to handle in the urgent need to discovery cost, yield is high.
Aqueous two phase extraction technique comes across the sixties in 20th century, research is the earliest based on two kinds of polymkeric substance or a kind of polymkeric substance and a kind of salt, as polyoxyethylene glycol/dextran and polyoxyethylene glycol/inorganic salt, the molecule space inhibition under finite concentration, and form immiscible two aqueous phases.Vargas etc. find that the double-aqueous phase system be made up of polyoxyethylene glycol/potassiumphosphate, sodium-chlor can be separated IgG and albumin (Biotechnology progress, 2012,28,1005-1011), but water soluble polymer viscosity is large in the method, need to strip, later separation complex steps, and also expensive superpolymer reclaims difficulty, high (the foodstuffs industry science and technology of separation costs, 2007,28,235 ~ 238).The method of the plasma proteins that WO2013039449 adopts polyprotonic acid polymkeric substance to precipitate after disclosing the phase-splitting of a kind of EOPO of use polymkeric substance, can separated plasma functional protein at normal temperatures, but still there is the shortcomings such as cost is high, complex steps.By the double water-phase of hydrophilic organic solvent and salt formation, can extracting metals complex compound, (research and development of natural products, 2002,21 (3): 75 ~ 77 such as Effective Component of Chinese Medicine and low-molecular-weight diol; " analytical test journal ", 2006,18,647 ~ 649; CN101012151A; CN101012152A), be a kind of new extraction and separation method.The part contriver of the application submitted two pieces Chinese invention patent application (CN101481403 and CN102070714A) to, with recombination human serum albumin in this double-aqueous phase system extraction yeast fermentation broth.But compared with yeast fermentation broth, blood plasma not only differences in shape is large, and wherein protein kind, also difference is huge for content.How from blood plasma, separation and purification quickly and efficiently obtains high purity functional protein, becomes emphasis and the difficult point of research at present.
Summary of the invention
The invention provides the method for functional protein in a kind of simple to operate, blood plasma that low cost, purity are high, when the double-aqueous phase system using hydrophilic organic solvent and inorganic salt to be formed extracts blood plasma functional protein, significantly can be simplified the operation step, raising yield, and contain the aqueous two-phase extraction liquid of blood plasma functional protein, adopt the column chromatography means different with prior art, highly purified blood plasma functional protein can be obtained.
Technical scheme of the present invention is as follows.
In blood plasma, a separation purification method for functional protein, comprises the steps:
(1) in plasma solutions, add soluble inorganic salt I and hydrophilic organic solvent, stir, form two phase aqueous extraction system, after leaving standstill, phase extraction liquid in collection;
(2) the upper phase extraction liquid of step (1) is after distillating recovering solvent, crosses Phenyl Sepharose6FF
tMhydrophobic chromatography post, is first eluted to balance liquid I and flows out without albumen, then use elutriant I gradient elution, collecting function plasma proteins cut;
(3) the functional plasma protein fraction of step (2) is through protein sample solution of dialysing to obtain, cross Q SephroseFF or DEAE Sephrose FF ion exchange column, first be eluted to balance liquid II and flow out without albumen, then use elutriant II gradient elution, obtain high purity blood plasma functional protein;
Wherein said soluble inorganic salt I is one or more the mixture in phosphoric acid salt, carbonate, vitriol, halogenide, Citrate trianion; Described hydrophilic organic solvent is the one in ethanol, methyl alcohol, n-propyl alcohol, Virahol, isopropylcarbinol, ethylene glycol or acetone; Described balance liquid I is containing 0.2 ~ 3mol/L soluble inorganic salt II and 5 ~ 50mmol/L damping fluid; Described elutriant I is containing 0 ~ 1mol/L soluble inorganic salt II and 5 ~ 50mmol/L damping fluid; Described balance liquid II is containing 5 ~ 50mmol/L damping fluid; Described elutriant II is containing 0 ~ 1mol/L soluble inorganic salt II and 5 ~ 50mmol/L damping fluid; Described soluble inorganic salt II is one or more the mixture in dipotassium hydrogen phosphate, Trisodium Citrate, sodium-chlor; Described damping fluid is the one in phosphate buffered saline buffer, Tris-HCl damping fluid, Veronal sodium-hydrochloride buffer.
In technique scheme, the two phase aqueous extraction system of step (1), after leaving standstill, is layered as upper and lower phase, wherein goes up in phase extraction liquid and be rich in blood plasma functional protein, be mainly albumin and immunoglobulin (Ig) (IgG).The extraction mode of two phase aqueous extraction system of the present invention can adopt single-stage extraction, but protein recovery is lower than the extraction system employing multi-stage solvent extraction of 80%.
In technique scheme, described plasma solutions is that blood is through the supernatant liquor of centrifugal segregation hemocyte or the partially purified cut of employing Cohn method; Soluble inorganic salt I or soluble inorganic salt II can be solid or dense saline solution.
Described in step of the present invention (1), soluble inorganic salt I accounts for 1% ~ 45% of double-aqueous phase system quality, and preferably 5% ~ 35%, more preferably 15% ~ 25%.
Described in step of the present invention (1), hydrophilic organic solvent accounts for 3% ~ 50% of double-aqueous phase system quality, and preferably 5% ~ 40%, more preferably 12% ~ 25%.
Soluble inorganic salt I described in step of the present invention (1) be one or more in dipotassium hydrogen phosphate, potassiumphosphate, sodium carbonate, salt of wormwood, sodium-chlor, ammonium sulfate, sodium sulfate, potassium sulfate, Trisodium Citrate, Tripotassium Citrate with the mixture of arbitrary proportion, one or more in preferably phosphoric acid hydrogen dipotassium, sodium carbonate, sodium-chlor, Trisodium Citrate are with the mixture of arbitrary proportion.
The time left standstill of two phase aqueous extraction system described in step of the present invention (1) is 0.5 ~ 36 hour, preferably 0.8 ~ 8 hour, more preferably 1 ~ 4 hour; The temperature of double-aqueous phase system is 0 ~ 37 DEG C, preferably 2 ~ 30 DEG C, more preferably 4 ~ 25 DEG C; The pH value of double-aqueous phase system is 3 ~ 13, preferably 5 ~ 10, more preferably 7 ~ 9.
The temperature of step of the present invention (2) described distillation is 20 ~ 45 DEG C, to be distilled in phase extraction liquid organic solvent content lower than 20%.
Reclaim the upper layer of extraction liquid of organic solvent in step of the present invention (2), add soluble inorganic salt II, its final concentration in extraction liquid is 0.3 ~ 4.0mol/L, preferably 0.4 ~ 3mol/L, more preferably 0.5 ~ 2mol/L; Adjust pH to 5.5 ~ 9.0, then add in hydrophobic chromatography post and be separated.
Described in step of the present invention (2), the pH value of balance liquid I or elutriant I is 5.5 ~ 9.0; The pH value of balance liquid II or elutriant II is 5.5 ~ 9.0 in step (3).
From blood plasma, be separated the blood plasma functional protein obtained by method of the present invention, especially albumin and immunoglobulin (Ig), its protein electrophoresis purity is higher than 95.0%, and compared with standard substance, its protein-active is more than 95%, and its protein structure is unchanged.
The present invention in plasma solutions, add soluble inorganic salt and hydrophilic organic solvent also fully stirs, by regulating kind and the ratio of salt, hydrophilic organic solvent, and the pH value of system makes solution form double water-phase, glucose and Partial Protein impurity in selective removal blood plasma, improve the extraction efficiency of blood plasma functional protein.With the blood plasma functional protein that organic solvent/salt two phase aqueous extraction system reclaims, be rich in the feature of salt on rear according to extraction mutually, select hydrophobic chromatography to carry out purification experiment, avoid the tedious steps of desalination; After hydrophobic chromatography, available blood plasma functional protein cut, utilizes the iso-electric point difference of blood plasma functional protein, selects ion-exchange to be separated, and can obtain highly purified blood plasma functional protein, and it is large that whole chromatography process has treatment capacity, low cost and other advantages.
Beneficial effect of the present invention: the purification step that this invention simplifies blood plasma functional protein, organic solvent/salt aqueous two-phase extraction has convenient solvent reclaiming, cost is low, the phase-splitting time is short, operation of need not stripping, can glucose and Partial Protein impurity in selective removal blood plasma, be easy to from laboratory scale to advantages such as industrial production amplifications, the separation of blood plasma functional protein is without the need to low temperature environment and multiple centrifugation steps operation.The electrophoresis purity of the blood plasma functional protein after column chromatography purification of the plasma protein solution after separation is greater than 95.0%.The present invention, solves the problems such as in functional protein separation and purification, separating step is loaded down with trivial details, cost is high, purity is low in restriction plasma proteins.
Accompanying drawing explanation
Fig. 1. the method separation of embodiment 1, the human plasma functional protein IgG of purifying and albuminous non-reduced SDS-PAGE electrophorogram;
Fig. 2. the method for embodiment 1 is separated, the human plasma functional protein IgG of purifying and the fluorescence spectrum figure of IgG standard substance;
Fig. 3. in dipotassium hydrogen phosphate-ethanol double-aqueous phase system, ethanol, dipotassium hydrogen phosphate concentration extract the impact of yield to IgG;
Fig. 4. in Trisodium Citrate-ethanol double-aqueous phase system, ethanol, sodium citrate concentration extract the impact of yield to IgG;
Fig. 5. in sodium carbonate-ethanol double-aqueous phase system, ethanol, concentration of sodium carbonate extract the impact of yield to IgG;
Fig. 6. in sodium carbonate-ethanol, dipotassium hydrogen phosphate-ethanol double-aqueous phase system, time of repose extracts the impact of yield to IgG.
Embodiment
Following embodiment only for describing the separation method of blood plasma functional protein fermented liquid of the present invention in detail, should not be construed as limitation of the present invention.In following embodiment, if no special instructions, the experimental technique used is ordinary method, and agents useful for same etc. all can chemically Reagent Company be bought.Chemical reagent used is analytical pure.
Material and instrument:
Fresh pig blood is provided by Subsidiary Second Hospital, Dalian Medical Univ. purchased from battledore island, Dalian meat processing combine of limited-liability company, human plasma.Human serum albumin, IgG standard substance are respectively purchased from Shanghai Vaccine and Serum Institute and Equitech Bio company of the U.S..Glucose content detection kit is purchased from ShangHai RongSheng Biology Pharmacy Co., Ltd.Instrument: microplate reader is Tecan company of Switzerland Sunrise16039400, spectrophotofluorometer is U.S. Varian Cary Eclipse FL0812M018.Phenyl Sepharose6FF
tM, Q Sephrose FF
tM, DEAESephrose FF
tMfor GE healthcare life Science Products.
Experimental technique:
Protein concentration and rate of recovery analysis:
Adopt Bradford method to measure in plasma sample total protein content in total protein content and upper phase extraction phase, in blood sample, the concentration of functional protein is according to following formulae discovery:
The total protein content (mg/ml) of percentage composition (the %) × sample of functional protein in concentration (the mg/ml)=sample of functional protein; Wherein in sample, the percentage composition of functional protein is: after sample reduction SDS-PAGE running gel is separated, uses Genetools scanning, calculate and obtain after area integral.
The rate of recovery of the aqueous two-phase extraction functional protein of soluble inorganic salt/organic solvent calculates as follows:
The functional protein rate of recovery (%)=(functional protein concentration (mg/ml) in upper phase extraction phase × upper phase extraction phase volume (ml))/(functional protein concentration (mg/ml) in plasma solutions × volume (ml))
Electrophoresis method
Reduction SDS-PAGE electrophoresis and non-reduced SDS-PAGE electrophoresis method adopt the method for biotechnology laboratory manual, and (Chemical Industry Press, p23-35), resolving gel concentration is 8% and 12%, and concentrated gum concentration is 3%.
IgG activation analysis
ELISA method is adopted to measure (food and biotechnology journal, 2006,25,97-101).
The structural analysis of IgG
Adopt fluorescence spectrum method for measuring, light path is 1cm excitation wavelength: 280nm; Emission wavelength: 295nm ~ 400nm.Exciting light slit: 5nm; Utilizing emitted light slit: 5nm; Each Sample Scan three times, and and standard substance contrast.
Glucose clearance measuring method
Adopt glucose oxidase method, use the content of glucose in the sample of kit measurement stoste and aqueous two-phase extraction, calculate clearance according to the following formula:
Glucose clearance (%)=(glucose sugar concentration (mM) in the upper phase extraction phase of 1-(× upper phase extraction phase volume (ml)/(glucose concn (mM) in stoste × stoste volume (ml)) × 100%.
The purifying of embodiment 1 human plasma functional protein
(1) double water-phase solid-liquid separation process
Preparation 40%K
2hPO
4solution, adjusts pH7.0 with phosphoric acid, under room temperature (24 DEG C), takes the 40%K of 2.5gpH7.0 successively
2hPO
4solution, 0.8g water, 1.0g human plasma, 0.7g ethanol, stirring and evenly mixing, leaves standstill 2 hours at 4 DEG C, forms double water-phase, phase extraction liquid in collection, and protein concentration analysis shows, and be rich in IgG and albumin in upper phase extraction liquid, yield is respectively 81.18% and 87.61%.
(2) hydrophobic chromatography purifying
After the upper layer of extraction liquid of step (1) is adjusted pH6.0, carry out underpressure distillation and reclaim ethanol, distillation temperature is 37 DEG C, and distillation time is 30min, and in upper layer of extraction liquid, ethanol content is lower than 10%.
By Phenyl Sepharose 6FF
tMhydrophobic medium loads in XK16/20 post, and column volume is 5ml, balances chromatographic column with the balance liquid I of 2 times of column volumes, and balance liquid I is the 20mmol/L sodium phosphate buffer of 0.5mol/L Trisodium Citrate, pH7.0.
Get the upper phase extraction liquid after 10ml recovery ethanol, adding 1.47g Trisodium Citrate makes Trisodium Citrate final concentration in solution be 0.5mol/L, adjusts pH7.0, make the conductance of solution close with balance liquid I with pH value with HCl, cross film with the ultra-filtration membrane in 0.45 μm of aperture, obtain loading sample solution.By the Phenyl Sepharose 6FF that loading sample solution balances with the flow velocity inflow balance liquid I of 2ml/min
tMhydrophobic chromatography post carries out loading.After end of the sample, with balance liquid I wash-out of 2 times of column volumes in conjunction with component.Then elutriant I gradient elution is used, described elutriant I is Trisodium Citrate-sodium phosphate buffer, by such as Gradient wash-out successively: first Trisodium Citrate-20mmol/L sodium phosphate buffer (pH7.0) wash-out of the 0.175mol/L of 2 times of column volumes, abandon elutriant (except foreigh protein removing); Then Trisodium Citrate-20mmol/L the sodium phosphate buffer (pH7.0) of the 0.075mol/L of 2 times of column volumes and 20mmol/L sodium phosphate buffer (pH7.0) stepwise elution of 2 times of column volumes is used, detect according to 280nm and collect each several part elution peak, ultrafiltration and concentration rear electrophoresis detects, and the cut containing albumin and IgG merges, for subsequent use.
(3) anion-exchange chromatography purifying
By Q Sepharose FF
tManionic exchange medium loads in XK16/20 post, and column volume is 2ml, balances chromatographic column with the balance liquid II of 2 times of column volumes, and balance liquid II is 20mmol/L Tris-HCl damping fluid, pH8.5.The albumin obtained in step (2) and IgG mixed fraction balance liquid II to be dialysed desalination, obtain albumin and IgG mixing dialysis cut 1ml, flowed into the Q Sepharose FF balanced
tManion-exchange column carries out loading.After end of the sample, with balance liquid II wash-out of 2 times of column volumes in conjunction with component.After end of the sample, with balance liquid II wash-out of 2 times of column volumes in conjunction with component, then binary gradient system elutions is used, elutriant A is the 20mmol/L Tris-HCl damping fluid containing 1mol/L NaCl, and pH8.5, elutriant B are 20mmol/L Tris-HCl damping fluid, pH8.5, gradient is 0% elutriant A-50% elutriant A, and elution time is 50min, and flow velocity is 0.5ml/min.Detect according to 280nm and collect each elution peak, electrophoresis detection result shows, can obtain albumin and IgG single component by anion-exchange chromatography.
IgG and albumin analysis after purifying:
I. purity check:
The IgG that collection anion-exchange chromatography obtains and albumin cut, be 10 with molecular weight cut-off respectively, the super filter tube of 000 is concentrated into 250 μ l, obtain IgG purification sample and purification of albumin sample, the non-reduced SDS-PAGE of 8% is adopted to analyze, applied sample amount 10 μ g, in single tape after coomassie brilliant blue staining, Gene-Tools software analysis, the purity of IgG purification and purification of albumin is all greater than 99.0%.After being separated, the electrophorogram of sample as shown in Figure 1.
The activity of II .IgG and structural analysis:
It is 700ng that IgG purification sample and human IgG standard substance are all configured package amount, and ELISA method measures active, and the activity of IgG purification sample is 96.4% of standard substance.
The standard substance of IgG purification sample and IgG are carried out fluorescence spectrometry, and as shown in Figure 2, both spectrum coincide, show that the structure of the IgG with method separation and purification of the present invention does not change to fluorescence spectrum figure.
The separation and purification of embodiment 2 porcine blood plasma functional protein
(1) double water-phase solid-liquid separation process
Under room temperature (24 DEG C), get fresh pig blood 1,000ml, 3000r/min, 15min centrifugal twice, remove hemocyte, get supernatant, 0.9% physiological saline equal-volume dilution, obtains porcine blood plasma solution.Take 20gK
2hPO
4be dissolved in 46g water, slowly add 20g blood plasma, 14g ethanol, fully stir, the pH value measuring system is 9.6.Room temperature leaves standstill 2 hours, forms double water-phase, phase extraction liquid in collection, and protein concentration analysis shows, and be rich in IgG and albumin in upper phase extraction liquid, yield is respectively 95.7% and 93.0%.
(2) hydrophobic chromatography purifying
After the upper layer of extraction liquid of step (1) is adjusted pH7.0, carry out underpressure distillation and reclaim ethanol, distillation temperature is 30 DEG C, and distillation time is 40min, and in upper layer of extraction liquid, ethanol content is lower than 10%.
By Phenyl Sepharose 6FF
tMhydrophobic medium loads in XK16/20 post, and column volume is 5ml, balances chromatographic column with the balance liquid I of 2 times of column volumes, and balance liquid I is 0.5mol/L dipotassium hydrogen phosphate-20mmol/L sodium phosphate buffer, pH7.0.
Being 0.5mol/L by reclaiming the dipotassium hydrogen phosphate solution that adds 1mol/L in the upper phase extraction liquid after ethanol to the final concentration of dipotassium hydrogen phosphate in solution, adjusting pH7.0 with HCl.With the Phenyl Sepharose 6FF that the flow velocity inflow of 2ml/min has balanced with balance liquid I
tMdrainage column carries out loading.After end of the sample, with balance liquid I wash-out of 2 times of column volumes in conjunction with component.Then elutriant I gradient elution is used, described elutriant I is dipotassium hydrogen phosphate-sodium phosphate buffer (pH7.0), by such as Gradient wash-out successively: the dipotassium hydrogen phosphate-20mmol/L sodium phosphate buffer wash-out first using the 0.4mol/L of 4 times of column volumes, abandon elutriant, then dipotassium hydrogen phosphate-20mmol/L the sodium phosphate buffer (elutriant A) of the 0.25mol/L of 2 times of column volumes is used respectively, dipotassium hydrogen phosphate-20mmol/L the sodium phosphate buffer (elutriant B) of 0.1mol/L and 20mmol/L sodium phosphate buffer (elutriant C) wash-out successively, detect according to 280nm and collect each several part elution peak, ultrafiltration and concentration rear electrophoresis detects, cut containing albumin and IgG merges, for subsequent use.
In another embodiment of above-mentioned steps (2), when containing 0.1mol/L dipotassium hydrogen phosphate, 0.9mol/ Trisodium Citrate in balance liquid I, or 0.9mol/L Trisodium Citrate, 0.05mol/L NaCl, or during the damping fluid of the 10mmol sodium phosphate of 0.3mol/L Trisodium Citrate, 0.3mol/L dipotassium hydrogen phosphate, also can utilize Phenyl Sepharose 6FF
tMhydrophobic chromatography obtains the cut of albumin and IgG; The buffer solution system of hydrophobic chromatography is changed into phosphoric acid buffer, the Tris-HCl damping fluid of pH8.0, the Veronal sodium-hydrochloride buffer of pH7.5 of pH6.0, also do not affect and obtain IgG and albumin cut.
(3) anion-exchange chromatography purifying
By DEAE Sepharose FF
tManionic exchange medium loads in XK16/20 post, and column volume is 2ml, balances chromatographic column with the balance liquid II of 2 times of column volumes, and balance liquid II is 20mol/L Tris-HCl damping fluid, pH8.8.The albumin obtained in step (2) and IgG mixed fraction balance liquid II to be dialysed desalination, obtain albumin and IgG and to dialyse cut 1ml, flowed into the DEAE Sepharose FF that above-mentioned balance is good
tManion-exchange column carries out loading.After end of the sample, remove in conjunction with component with balance liquid II wash-out of 2 times of column volumes, then binary gradient system elutions is used, elutriant A is the 20mmol/LTris-HCl damping fluid containing 1mol/L NaCl, and pH8.5, elutriant B are 20mmol/L Tris-HCl damping fluid, pH8.5, gradient is 0% elutriant A-50% elutriant A, and elution time is 50min, and flow velocity is 0.5ml/min.Detect according to 280nm and collect each elution peak, electrophoresis detection result shows, can obtain albumin and IgG single component by anion-exchange chromatography.
The IgG that collection anion-exchange chromatography obtains and albumin cut, be 10 with molecular weight cut-off respectively, the super filter tube of 000 is concentrated into 250 μ l, obtain IgG purification sample and purification of albumin sample, adopt the reduction SDS-PAGE electrophoretic analysis of 12%, the purity of IgG purification and purification of albumin is respectively 97.2% and 95.8%, and the activity that ELISA method measures IgG purification is 95.2% of standard substance.
In another embodiment of above-mentioned steps (3), when elutriant A is for containing sodium-chlor with sodium citrate concentration than 8:2 mixing salt, or sodium-chlor is 1:1 mixing salt with sodium citrate concentration ratio, or dipotassium hydrogen phosphate and sodium chloride concentration than 9:1 mixing salt 30mmol/L Tris-HCl damping fluid (pH8.5), and buffer salt system is adjusted to the Tris-HCl damping fluid (pH9.5) of 40mmol/L, Veronal sodium-the hydrochloride buffer (pH9.0) of 10mmol/L, during phosphoric acid buffer (pH7.0) of 20mmol/L, IgG and albuminous electrophoresis purity also can reach more than 95%.
Embodiment 3 organic solvent, salt concn are on the impact of aqueous two-phase extraction IgG yield
Choose ethanol and the suitable quantity of water proportioning of the dipotassium hydrogen phosphate of different concns, Trisodium Citrate and sodium carbonate and different concns, form double-aqueous phase system, the pH value of system is 7.0, investigates organic solvent, salt concn in different double-aqueous phase systems and human IgG is extracted to the impact of yield.In all systems, the addition of IgG is 1mg, and time of repose is 8 hours, phase extraction liquid in collection, and Bradford method measures protein content wherein, and calculates the IgG rate of recovery (%).
In dipotassium hydrogen phosphate-ethanol system, the final concentration of ethanol is 14-16%(w/w), the concentration of dipotassium hydrogen phosphate is 18-24% (w/w), and temperature is 15 ° of C; IgG rate of recovery result as shown in Figure 3.
In Trisodium Citrate-ethanol system, the final concentration of ethanol is 22-25%(w/w), the concentration of Trisodium Citrate is 18-24% (w/w), and temperature is 30 ° of C; IgG rate of recovery result as shown in Figure 4.
In sodium carbonate-ethanol system, the final concentration of ethanol is 13-17%(w/w), the concentration of Trisodium Citrate is 12-21% (w/w), and temperature is 30 ° of C; IgG rate of recovery result as shown in Figure 5.
From Fig. 3 ~ 5 result, in the double-aqueous phase system be made up of salt and the organic solvent of different sorts, different concns, under the salt concn of proper range and the condition of organic solvent concentration, all can obtain higher IgG yield, in the IgG rate of recovery of most of double-aqueous phase systems of the present embodiment more than 90%.Wherein, the yield of the system of 20% dipotassium hydrogen phosphate-14% ethanol is the highest, and the rate of recovery is 97.4%.Above result display, organic solvent of the present invention/salt double-aqueous phase system can obtain higher IgG and extract yield.
In addition, also use the double-aqueous phase system extraction IgG of 3% sodium carbonate (w/w)-42% ethanol (w/w) and 5% ethanol (w/w)-40% dipotassium hydrogen phosphate, the rate of recovery is respectively 18.5% and 21.5%, and organic solvent of the present invention/salt double-aqueous phase system can extract IgG within the scope of wider salt, alcohol.
Embodiment 4 pH value is on the impact of aqueous two-phase extraction IgG yield
According to the method for embodiment 3, prepare 3 groups of dipotassium hydrogen phosphates-ethanol double-aqueous phase system, wherein ethanol final concentration is 14%(w/w), dipotassium hydrogen phosphate final concentration is 20%(w/w), with phosphoric acid, the pH value of 3 groups of double-aqueous phase systems is adjusted to 7.0 and 8.0 respectively, pH(pH is not adjusted to be 9.6), in the double-aqueous phase system of above three kinds of pH, the rate of recovery of extraction human IgG (add-on is 1mg) is respectively 88.4%, 92.1% and 97.4%.
According to the method for embodiment 3, prepare 3 groups of Trisodium Citrates-ethanol double-aqueous phase system, wherein ethanol final concentration is 19%(w/w), dipotassium hydrogen phosphate final concentration is 25% (w/w), with citric acid, the pH value of 3 groups of double-aqueous phase systems is adjusted to pH6.0 and 7.0 respectively, pH(pH is not adjusted to be 8.8), in the double-aqueous phase system of above three kinds of pH, the rate of recovery of extraction human IgG (add-on is 1mg) is respectively 69.4%, 92.9% and 90.2%.
Above result display, in identical organic solvent/salt double-aqueous phase system, the pH value of system affects the extraction yield of human IgG albumen, but can obtain higher IgG extraction yield under suitable pH scope.
Embodiment 5 organic solvents/mixing salt aqueous two-phase extraction IgG.
Take appropriate dipotassium hydrogen phosphate, sodium-chlor, water and ethanol to mix, form mixing salt-ethanol double-aqueous phase system, in system, ethanol final concentration is 14%(w/w), dipotassium hydrogen phosphate final concentration is 20%(w/w), sodium chloride concentration is 2.5%.Add 1mg IgG in described system, mix, room temperature places 8h, and phase extraction liquid in collection, recording the IgG rate of recovery is 93.9%.
Take proper amount of sodium carbonate, sodium-chlor, water and ethanol to mix, form mixing salt-ethanol double-aqueous phase system, in system, ethanol final concentration is 13%(w/w), sodium carbonate final concentration is 19%(w/w), sodium chloride concentration is 2.5%.Add 1mg IgG in described system, mix, room temperature places 8h, and phase extraction liquid in collection, recording the IgG rate of recovery is 81.5%.
The display of above result, organic solvent/mixing salt double-aqueous phase system, in the scope of suitable salt concn and organic solvent concentration, can obtain higher IgG and extract yield.
Embodiment 6 time of repose is on the impact of human IgG yield
According to the method for embodiment 3, prepare three kinds of double-aqueous phase systems, be respectively:
Dipotassium hydrogen phosphate-ethanol system 1: in system, ethanol final concentration is 14%(w/w), dipotassium hydrogen phosphate final concentration is 20%(w/w);
Dipotassium hydrogen phosphate-ethanol system 2: in system, ethanol final concentration is 14%(w/w), dipotassium hydrogen phosphate final concentration is 24%(w/w);
Sodium carbonate-ethanol system: in system, ethanol final concentration is 13%(w/w), sodium carbonate final concentration is 19%(w/w).
1mg IgG is all added in above-mentioned three kinds of systems, after stirring, at room temperature leave standstill 2-24 hour, phase extraction liquid in collection, detects IgG content, investigates time of repose to the impact of the IgG rate of recovery, as shown in Figure 6, in the double-aqueous phase system of dipotassium hydrogen phosphate-ethanol, time of repose affects the extraction efficiency of IgG, but still keeps higher extraction efficiency in suitable scope.Select the time of repose of suitable double-aqueous phase system in the present invention, the extraction efficiency of IgG can be improved.
Embodiment 7 aqueous two-phase extraction is to the removal of glucose in blood plasma
According to the method for embodiment 3, preparation dipotassium hydrogen phosphate-ethanol double-aqueous phase system, in system, ethanol final concentration is 14%(w/w), dipotassium hydrogen phosphate final concentration is 20%(w/w), adjust pH to be respectively 7.0,8.0, do not adjust the pH of pH group to be 9.6.Described three individual system, total mass is 5g.
1g porcine blood plasma solution (preparation method of porcine blood plasma solution is with embodiment 2) is added in above-mentioned three individual system, stir, 8h is placed in room temperature, phase extraction liquid in collection, mensuration obtains porcine blood plasma glucose clearance in three individual system and is respectively 51.8%, 52.2% and 52.6%, shows that two aqueous phase extraction method in the present invention is while extraction blood plasma functional protein, remove a large amount of impurity, reduce the removing burden of impurity in later separation purifying.
The present invention to plasma solutions with an organic solvent/extraction liquid that obtains of salt aqueous two-phase extraction, after removing ethanol, the medium of commodity in use, carry out hydrophobic and anion-exchange chromatography in a jointed manner, can obtain albumin and IgG that purity is greater than 95%, aqueous two-phase extraction can remove the impurity such as glucose in blood plasma.Compare with known references, simplify purification step, improve purifying purity, not only shorten purification time, and improve the quality of product.
Although when the present invention is in order to DETAILS SECTIONExample, limit experiment condition, the staff of this area can select different conditions in the scope not violating spirit of the present invention, or adjusts experimental technique and order.
Claims (3)
1. a separation purification method of albumin and IgG in blood plasma, is characterized in that, comprise the steps:
(1) in plasma solutions, add soluble inorganic salt I and hydrophilic organic solvent, stir, form two phase aqueous extraction system, after leaving standstill, phase extraction liquid in collection;
(2) the upper phase extraction liquid of step (1) is after distillating recovering solvent, adds soluble inorganic salt II, and its final concentration in extraction liquid is 0.3 ~ 4.0mol/L, adjusts pH to 5.5 ~ 9.0, crosses Phenyl Sepharose6FF
tMhydrophobic chromatography post, is first eluted to balance liquid I and flows out without albumen, then use elutriant I gradient elution, collect the cut containing albumin and IgG;
(3) cut containing albumin and IgG of step (2) is through protein sample solution of dialysing to obtain, cross QSephrose FF or DEAE Sephrose FF ion exchange column, first be eluted to balance liquid II and flow out without albumen, use elutriant II gradient elution again, obtain the single component of albumin and IgG;
Wherein said soluble inorganic salt I is one or more the mixture in phosphoric acid salt, carbonate, vitriol, halogenide, Citrate trianion; Described hydrophilic organic solvent is the one in ethanol, methyl alcohol, n-propyl alcohol, Virahol, isopropylcarbinol, ethylene glycol or acetone; Described balance liquid I is containing 0.2 ~ 3mol/L soluble inorganic salt II and 5 ~ 50mmol/L damping fluid; Described elutriant I is containing 0 ~ 1mol/L soluble inorganic salt II and 5 ~ 50mmol/L damping fluid; Described balance liquid II is containing 5 ~ 50mmol/L damping fluid; Described elutriant II is containing 0 ~ 1mol/L soluble inorganic salt II and 5 ~ 50mmol/L damping fluid; Described soluble inorganic salt II is one or more the mixture in dipotassium hydrogen phosphate, Trisodium Citrate, sodium-chlor; Described damping fluid is the one in phosphate buffered saline buffer, Tris-HCl damping fluid, Veronal sodium-hydrochloride buffer;
Be rich in blood plasma functional protein in described upper phase extraction liquid, be mainly albumin and IgG; Described in step (1), soluble inorganic salt I accounts for 15% ~ 25% of double-aqueous phase system quality, and hydrophilic organic solvent accounts for 12% ~ 25% of double-aqueous phase system quality;
The time left standstill described in step (1) is 0.5 ~ 36 hour; The temperature of double-aqueous phase system is 0 ~ 37 DEG C; The pH value of double-aqueous phase system is 5 ~ 10;
Described in step (2), the pH value of balance liquid I or elutriant I is 5.5 ~ 9.0;
Described in step (3), the pH value of balance liquid II or elutriant II is 5.5 ~ 9.0.
2. separation purification method according to claim 1, it is characterized in that, soluble inorganic salt I described in step (1) is one or more the mixture in dipotassium hydrogen phosphate, potassiumphosphate, sodium carbonate, salt of wormwood, sodium-chlor, ammonium sulfate, sodium sulfate, potassium sulfate, Trisodium Citrate, Tripotassium Citrate.
3. separation purification method according to claim 1, is characterized in that, the temperature of step (2) described distillation is 20 ~ 45 DEG C, is distilled to organic solvent content lower than 20%.
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