CN100333828C - Biologic chromatography gelatin and producing texhnique - Google Patents
Biologic chromatography gelatin and producing texhnique Download PDFInfo
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- CN100333828C CN100333828C CNB2003101082142A CN200310108214A CN100333828C CN 100333828 C CN100333828 C CN 100333828C CN B2003101082142 A CNB2003101082142 A CN B2003101082142A CN 200310108214 A CN200310108214 A CN 200310108214A CN 100333828 C CN100333828 C CN 100333828C
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Abstract
The present invention relates to biologic chromatography gelatin and a production process thereof, which belongs to a biochemical field and solves the disadvantages of expensive price, complex synthetic regeneration, etc. existing in gelatin for the existing biologic chromatography. The biologic chromatography gelatin is mainly composed of poly amino sugar whose molecular weight is from 100, 000 to 12 0, 000. The present invention has the production process that a certain amount of chitosan is dissolved in an acidic medium and is degraded, and degradation time is not less than 3 hours; the poly amino sugar obtained by degradation is dripped in sodium hydroxide solution and is then washed to neutrality; the poly amino sugar after being washed is moved to a container, stirred, dripped with a crosslinking agent at the same time, and sufficiently linked in a cross mode; after crosslinking reaction, the poly amino sugar is reduced by a reducing agent; after reduction, the poly amino sugar is washed to neutrality, partial samples of 60 to 200 meshes are sieved to a sample, and the biologic chromatography gelatin is obtained. The poly amino sugar is used as main raw materials of the biologic chromatography gelatin by the present invention which has the advantages of good biocompatibility, rich surface group, wide use range of pH values, etc.
Description
Technical field
The invention belongs to the biochemical technology field, particularly a kind of biological chromatography gel and preparation method thereof.
Background technology
Biological chromatography gel is the necessary reagent of biological substance purifies and separates, and it plays ion exchange, has obtained extensive use in biochemical field.Flourish along with biological-pharmacy, the demand of biological chromatography gel is increasing, and is also more and more to the requirement of specifications and models.For the prepared protein medicaments of genetic engineering, be mixed with more foreign protein in its expression product especially, but and chromatography is the method for only purifying gene engineering medicine, therefore too busy to get away especially biological chromatography gel.
The gel that present biological chromatography is used is used glucan, agarose, DEAE cellulose always as carrier, as Pharmacia company product: Sepharose CL-4B, Sepharose CL-6B etc.But exist many shortcomings with agarose as carrier, as cost an arm and a leg, synthetic regenerated complicated, need to use hypertoxic CNBr activation etc.This situation has had a strong impact on the raising of biochemical isolation technics, has directly restricted the bio-pharmaceuticals industrialization process.Therefore, seeking, research and develop a kind of new type gel is very necessary and has huge economic results in society.
Summary of the invention
The purpose of this invention is to provide a kind of novel biological chromatography gel, to substitute the prior biological chromatography gel, make it not only have the advantage of existing biological chromatography gel, but also have some other distinctive advantage, its products material wide material sources, product cost are low simultaneously.
Another object of the present invention provides the preparation method of this new bio chromatography gel.
The objective of the invention is to realize by following technical proposal: this biological chromatography gel mainly is made up of the poly-ammonia sugar of molecular weight between 100,000 to 120,000.
The present invention adopts the primary raw material of poly-ammonia sugar as biological chromatography gel, be to consider that poly-ammonia sugar has and the agarose confers similar advantages in some aspects, as it is wide etc. to have excellent biological compatibility, surface group rich, the pH value scope of application.Simultaneously, as the chromatography gel carrier, it does not also have some shortcoming of agarose.
The preparation method of above-mentioned biological chromatography gel is as follows:
1, certain amount of chitosan is dissolved in the acid medium, and degrades, degradation time is not less than 3 hours;
2, the poly-ammonia sugar of degraded gained drips in concentration is 0.3%～3% sodium hydroxide solution, is washed to neutrality then;
3, the poly-ammonia sugar after the washing moves on in the container, stirs, and drips crosslinking agent simultaneously, and is full cross-linked, and described crosslinking agent is a kind of in formaldehyde, glutaraldehyde, glyoxal, the ethylene glycol bis glycidyl ethers;
4, after the cross-linking reaction, with the reducing agent reduction, described reducing agent is a sodium borohydride;
5, the reduction after washing divides sample to neutral with 60 order to 200 purpose sub-sieves, obtains biological chromatography gel.
Usually, the biological chromatography gel of gained is stored in 20% the ethanol.
This biological chromatography gel is to be the poly Glucosamine gel that primary raw material is prepared from the shitosan, abbreviates poly-ammonia sugar gel as.Shitosan (chitosan) has another name called chitin, chitosan, chitosan, polydextrose amine, is widely used in industries such as medicines and health protection, water treatment, daily use chemicals.
This preparation method utilizes 2 of poly-ammonia sugar to go up free-NH
2With on the glutaraldehyde-Schiff formula reaction takes place in CHO in weak acid medium, use sodium borohydride reduction then, makes it to become the weak base anion exchanger.
In this preparation method, the strand of the poly-ammonia sugar of HMW is than long, and glutaraldehyde is difficult for its generation crosslinked, therefore for improving the stability of synthesising biological chromatography gel, must carry out appropriateness to poly-ammonia sugar and degrade, and carries out crosslinked to it again.Degradation time is 3 to 6 hours.
In above-mentioned preparation method, utilize glutaraldehyde to shitosan carry out crosslinked after, two aldehyde radicals in the glutaraldehyde have only one to participate in reaction, also have an aldehyde radical not participate in reaction.
In above-mentioned preparation method, the effect that adds reducing agent is: 1, Zhi Bei biological chromatography gel color becomes milky by yellow; 2, owing to contain the aldehyde radical structure in its structure, absworption peak is arranged, therefore add reducing agent and can prevent from the detection of protein is exerted an influence in ultraviolet.
In above-mentioned preparation method, the crosslinked time is an important factor.The crosslinking time deficiency influences the acid pH stability and the mechanical strength of gel, wastes raw material; Crosslinking time is long, and some negative reactions easily take place, and influences the adsorption capacity of gel.Usually, crosslinking time is between 4～10 hours.
At room temperature, crosslinking time is between 6～8 hours.
In above-mentioned preparation method, described cross-linking reaction should be carried out under stirring condition.
In above-mentioned preparation method, reaction medium is not obvious to the absorption property influence of product, and benzinum, ether and water do not dissolve each other, but is difficult for the poly-ammonia sugar after the dispersion washing in benzinum and ether, and the washing of crosslinked back also bothers, so selection water is reaction medium.
In above-mentioned preparation method, the concentration range of described sodium hydroxide solution is 1%～2%.Select suitable concentration, will help separating out of poly-ammonia sugar, and make its amount of separating out more.
Affinity chromatography is the most effective isolation technics of protein and enzyme purification.Chromatography media easy compression as pearl sephadex and Ago-Gel commonly used is not easy for high pressure liquid chromatography; And have rigidity with the silica gel of modification, but the silica gel particle modification ratio is difficult, and bioaffinity is relatively poor.By comparison, the existing rigidity of poly-ammonia sugar of the present invention has hydrophily and antibiotic property again, can be used for the adsorbing separation of protein, enzyme fully.
Poly-ammonia sugar has good adsorptivity to blood plasma low-density lipoprotein bletilla VLDL, can be applicable to the blood perfusion of hyperlipidemia patient.Poly-ammonia sugar all has tangible suction-operated to the cholesterol triglyceride, and adsorption rate increases along with the raising of deacetylation.
Those can cause relevant immunoglobulin (Ig)s such as autoimmunity upset, allergy and tumour by the absorption of the high deacetylized poly-ammonia sugar of the pearl of high deacetylized poly-ammonia sugar of the crosslinked pearl of butanediol diglycidyl ether or glutaraldehyde cross-linking, be used for clinical, the poly-ammonia sugar of being modified by phenylalanine and tryptophan immobilization of pearl selectively adsorbs immune protein, the poly-ammonia sugar of this modification is higher to the compatibility of alpha-globulin than the poly-ammonia sugar of not modifying, and to fibrinous adsorbance reduction, can also reduce platelet adhesion reaction, be good adsorbent to blood perfusion.
The poly-ammonia sugar of cross-linked carboxymethyl has the amphion exchange capacity, and the albumin or the gamma globulin aqueous solution to containing carbohydrate do not adsorb carbohydrate, but can adhesion protein.When being solvent, can adsorb 9.4mg/g albumin or 58.4mg/g gamma globulin respectively with the PBS.Change the ionic strength of solvent, can also from gamma globulin, separate albumin, or from bovine serum albumin, isolate albumin.
Aspect the separation and purification enzyme, poly-ammonia sugar has good effect from plurality of enzymes such as Purification of Lysozyme, amyloglucosidase, cellulases.The effect substrate of these enzymes is structurally similar to poly-ammonia sugar, and poly-ammonia sugar can adsorb these enzymes securely in adsorption process, by also form the complex compound of fixedly forming.
The amount of poly-ammonia sugar absorption starch glycosidase was more much bigger than the adsorbance of other gel adsorbers, and the pH value had maximal absorptive capacity at 4.0～4.5 o'clock.The poly-ammonia sugar of pearl also adsorbs AMS and lactase consumingly, and optimal pH is near 6.0, and the maximal absorptive capacity of absorption AMS reaches the 286mg/ml gel, but the buffer solution of usefulness LIS is with regard to wash-out.
Directly chicken egg protein is carried out affinity chromatography with the poly-ammonia sugar of the carboxymethyl of pearl, with 0.1mol/L acetate wash-out, can obtain highly purified lysozyme then.
Poly-ammonia sugar all has decline to the absorption of proteins amount, but the adsorbance of beta-glycosidase, AMS alkali starch enzyme acid starch enzyme and soybean amylopsin inhibitor is increased on the contrary, at 2.4mol/L (NH behind glutaraldehyde cross-linking
4)
2SO
4In the solution, the adsorption rate of alkaline phosphatase reaches 99%, when 50% ethylene glycol wash-out, but the enzyme quantitative recovery, the purity of enzyme improves 4.5 times, reaches 4.70un/mg than motility rate.During the cellulose hydrolyzation cellulose crossed with poly-ammonia sugar purifying, can make the cellobiose content in the solution bring up to 95% by 42%.
The shitosan that the present invention can abound with China is processed as the biological chromatography gel products of high added value, and its product cost is lower, application surface is very wide, has higher utility.
The specific embodiment
Get the shitosan degraded earlier 4 hours of 2% (w/v), drip in concentration is 2% sodium hydroxide solution and separate out, be washed to neutrality; Move to then in the there-necked flask, drip 0.6% glutaraldehyde solution, stirred simultaneously crosslinked 6 hours; Add sodium borohydride and make free aldehyde radical adequate closure,, obtain the lily biological chromatography gel of 60 orders～200 orders with the sub-sieve washing.
In the present embodiment, described crosslinking agent adopts glutaraldehyde.The concentration of glutaraldehyde is usually between 0.5%～2.5%.In the phosphate buffer of certain ionic strength, along with the raising of glutaraldehyde concentration, the degree of cross linking of gel rises, and its adsorption capacity will progressively descend.This be since the glutaraldehyde consumption take when excessive cross polyamino on the chitosan molecule, make participate in adsorbing amino reduce cause.And we wish that the chromatography gel that synthesizes has bigger ion exchanging function, and crosslinked only is mechanical strength and the stability that increases biological chromatography gel, so dosage of crosslinking agent should be few as much as possible under the prerequisite that guarantees gel stability.Our experiments show that the concentration of glutaraldehyde the best is 0.6%.
The biological chromatography gel outward appearance that present embodiment synthesized is that pure white is fibrous.
This biological chromatography gel is all very stable to common organic solvents, and especially under the pH value was 1.5 acid condition, gel did not dissolve, and reaches good stable, and this point obviously is better than the DEAE cellulose.Concrete stability test the results are shown in Table shown in 1.
The biological chromatography gel stability result of the test of the poly-ammonia sugar of table 1
| Solvent | Action time (min) | Result of the test | ||
| Dissolubility | The solvent change in color | Shape | ||
| Distilled water | 60 | - | No change | No change |
| Hydrochloric acid (pH 1.5) | 30 | - | No change | No change |
| Hydrochloric acid (pH 1.0) | 30 | Be partly dissolved | Thin out yellow | Powder |
| NaOH (2%) | 60 | - | No change | No change |
| Ethanol (95%) | 60 | - | No change | No change |
| Glycerine (cp) | 60 | - | No change | No change |
| Acetone (cp) | 60 | - | No change | No change |
| Oxolane (cp) | 60 | - | No change | No change |
| Benzene (cp) | 60 | - | No change | No change |
| Carrene (cp) | 60 | - | No change | No change |
| Chloroform (cp) | 60 | - | No change | No change |
Annotate: "-" expression is insoluble, at room temperature test.
The biological chromatography gel dress post of present embodiment gained, last sample, wash-out, receive elution fraction respectively, measure the enzyme (neutral proteinase) and the protein content (bovine serum albumin) of each component, and make comparisons, the results are shown in Table 2, table 3, table 4 and table 5 with DEAE cellulose chromatography.
The poly-ammonia sugar of table 2 chromatography gel is to the separation of neutral proteinase
| Volume (ml) | Total protein (mg) | Total activity (U) | Than vigor (U/mg) | The purification multiple | Enzyme yield (%) alive | Protein yield (%) | |
| Sample | 1.0 | 20.00 | 109.10 | 5.46 | - | - | - |
| Component 1 | 12.0 | 2.74 | 29.42 | 10.74 | 1.97 | 26.97 | 13.70 |
| Component 2 | 12.0 | 2.40 | 21.68 | 9.03 | 0.84 | 19.87 | 12.00 |
| Component 3 | 11.0 | 2.51 | 26.97 | 11.54 | 1.28 | 24.72 | 12.56 |
| Component 4 | 8.5 | 1.94 | 20.84 | 10.74 | 0.93 | 19.10 | 9.70 |
| Add up to | 42.2 | 9.59 | 98.91 | - | - | 90.66 | 47.96 |
Annotate: with water is equilibrium liquid, the buffer solution elution of trishydroxymethylaminomethane-hydrochloric acid (pH=9).
Table 3 DEAE cellulose chromatography separates the result of neutral proteinase
| Volume (ml) | Total protein (mg) | Total activity (U) | Than vigor (U/mg) | The purification multiple | Enzyme yield (%) alive | Protein yield (%) | |
| Sample | 1.0 | 20.00 | 109.10 | 5.46 | - | - | |
| Component 1 | 21.0 | 4.22 | 47.03 | 11.14 | 2.04 | 43.11 | 21.10 |
Annotate: with water is equilibrium liquid, the buffer solution elution of trishydroxymethylaminomethane-hydrochloric acid (pH=9)
The poly-ammonia sugar of table 4 gel is to the bovine serum albumin chromatography
| Volume (ml) | Total protein (mg) | Protein yield (%) | |
| Sample | 1.0 | 20 | - |
| Peak 1 | 21.5 | 5.6 | 28.2 |
| Peak 2 | 25.0 | 8.4 | 42.0 |
| Add up to | 46.5 | 14.0 | 70.2 |
Annotate: with water is equilibrium liquid, the buffer solution elution of trishydroxymethylaminomethane-hydrochloric acid (pH=9)
Table 5 DEAE cellulose is to bovine serum albumin chromatography result
| Volume (ml) | Total protein (mg) | Protein yield (%) | |
| Sample | 1.0 | 20 | - |
| Peak 1 | 30.0 | 10.6 | 64.4 |
By table 2 and table 3 as can be known, synthetic poly-ammonia sugar chromatography gel carries out purifying to neutral proteinase, can obtain purification effect preferably, the enzyme rate of recovery alive reaches 90.66%, enzyme is lived and is mainly had peak 1, peak 3, and enzyme loss alive is less, and peak 2, peak 4 also have a spot of enzyme to live in addition.By table 4 and table 5 as can be known, under identical conditions, poly-ammonia sugar chromatography gel and DEAE cellulose show that to the absorption property experiment of bovine serum albumin poly-ammonia sugar chromatography gel performance is excellent slightly.Contrast by two groups of experiments can draw to draw a conclusion:
1, synthetic poly-ammonia sugar chromatography gel is similar to the absorption situation of neutral proteinase, bovine serum albumin with the DEAE cellulose, and to the rate of recovery of bovine serum albumin even than DEAE cellulose height, separating effect is excellent slightly;
2, synthetic poly-ammonia sugar chromatography gel is good to the bioaffinity of enzyme, is better than the DEAE cellulose, and the active conservation rate of chromatography is greater than 90%.
Therefore, poly-ammonia sugar has and the agarose confers similar advantages in some aspects, as it is wide etc. to have excellent biological compatibility, surface group rich, the pH value scope of application, is complete feasible as the chromatography gel material.
Claims (6)
1, a kind of biological chromatography gel is characterized in that this biology chromatography gel is made up of the poly-ammonia sugar of molecular weight between 100,000 to 120,000.
2, a kind of preparation method of biological chromatography gel as claimed in claim 1 is characterized in that this preparation method is as follows:
(1), certain amount of chitosan is dissolved in the acid medium, and degrade, degradation time is not less than 3 hours;
(2), the poly-ammonia sugar of degraded gained drips in concentration is 0.3%～3% sodium hydroxide solution, is washed to neutrality then;
(3), the poly-ammonia sugar after the washing moves on in the container, stirs, and drips crosslinking agent simultaneously, and is full cross-linked, described crosslinking agent is a kind of in formaldehyde, glutaraldehyde, glyoxal, the ethylene glycol bis glycidyl ethers;
(4), after the cross-linking reaction, with the reducing agent reduction, described reducing agent is a sodium borohydride;
(5), the reduction after washing is to neutral, divides sample with 60 order to 200 purpose sub-sieves, obtains biological chromatography gel.
3, the preparation method of biological chromatography gel according to claim 2 is characterized in that described degradation time is 3 to 6 hours.
4, the preparation method of biological chromatography gel according to claim 2 is characterized in that crosslinking time is between 4～10 hours.
5, the preparation method of biological chromatography gel according to claim 4 is characterized in that crosslinking time is between 6～8 hours.
6, the preparation method of biological chromatography gel according to claim 2, the concentration range that it is characterized in that described sodium hydroxide solution is 1%～2%.
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| CNB2003101082142A CN100333828C (en) | 2003-10-25 | 2003-10-25 | Biologic chromatography gelatin and producing texhnique |
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| CNB2003101082142A CN100333828C (en) | 2003-10-25 | 2003-10-25 | Biologic chromatography gelatin and producing texhnique |
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| CN100333828C true CN100333828C (en) | 2007-08-29 |
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| CN105602717A (en) * | 2015-12-28 | 2016-05-25 | 山东润泽制药有限公司 | Refining method of horse oil |
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| GB1243027A (en) * | 1967-08-30 | 1971-08-18 | Pharmacia Fine Chemicals Ab | A separating medium for gel filtration and a process for the preparation thereof |
| JPH02301A (en) * | 1988-12-29 | 1990-01-05 | Seiko Epson Corp | Permanent magnet |
| WO1997029132A1 (en) * | 1996-02-09 | 1997-08-14 | Merck Patent Gmbh | Cross-linking products of amino-group-containing biopolymers |
| CN1316450A (en) * | 2001-03-24 | 2001-10-10 | 天津大学 | Process for preparing magnetic sepharose microspheres by both-phase (oil and water) method |
| CN1108376C (en) * | 2000-05-17 | 2003-05-14 | 华南农业大学 | Process for preparing gel medium of lysozyme affinity chromatography |
-
2003
- 2003-10-25 CN CNB2003101082142A patent/CN100333828C/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1243027A (en) * | 1967-08-30 | 1971-08-18 | Pharmacia Fine Chemicals Ab | A separating medium for gel filtration and a process for the preparation thereof |
| JPH02301A (en) * | 1988-12-29 | 1990-01-05 | Seiko Epson Corp | Permanent magnet |
| WO1997029132A1 (en) * | 1996-02-09 | 1997-08-14 | Merck Patent Gmbh | Cross-linking products of amino-group-containing biopolymers |
| CN1108376C (en) * | 2000-05-17 | 2003-05-14 | 华南农业大学 | Process for preparing gel medium of lysozyme affinity chromatography |
| CN1316450A (en) * | 2001-03-24 | 2001-10-10 | 天津大学 | Process for preparing magnetic sepharose microspheres by both-phase (oil and water) method |
Non-Patent Citations (4)
| Title |
|---|
| "壳聚糖为载体金属亲和吸附剂的制备及性质" 王雪峰等，功能高分子学报，第16卷第3期 2003;"壳聚糖衍生物吸附剂在蛋白质分离纯化中的应用" 冯长根等，离子交换与吸附，第19卷第3期 2003;"两种不同的亲和吸附剂对猪凝血酶纯化效果的比较" 肖丽霞等，扬州大学学报（自然科学版），第4卷第3期 2001 * |
| "两种不同的亲和吸附剂对猪凝血酶纯化效果的比较" 肖丽霞等，扬州大学学报（自然科学版），第4卷第3期 2001 * |
| "壳聚糖为载体金属亲和吸附剂的制备及性质" 王雪峰等，功能高分子学报，第16卷第3期 2003 * |
| "壳聚糖衍生物吸附剂在蛋白质分离纯化中的应用" 冯长根等，离子交换与吸附，第19卷第3期 2003 * |
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