CN1212884C - Affinity adsorption medium and its preparing medium - Google Patents
Affinity adsorption medium and its preparing medium Download PDFInfo
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- CN1212884C CN1212884C CN 01103114 CN01103114A CN1212884C CN 1212884 C CN1212884 C CN 1212884C CN 01103114 CN01103114 CN 01103114 CN 01103114 A CN01103114 A CN 01103114A CN 1212884 C CN1212884 C CN 1212884C
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Abstract
The present invention discloses an affinity adsorption medium and a preparation method thereof. The affinity adsorption medium is formed by compounding and fixing a gene recombination protein A with agarose after the grafting reaction of the agarose. The preparation method has the advantages of high safety and no toxin, and the affinity adsorption medium has the characteristics of strong specificity, high selectivity, good biologic compatibility and homogeneous structure. The present invention can be used for clinical immunoadsorption therapy.
Description
The present invention relates to the immune absorption material field, especially relate to affine absorption medium of a kind of adsorbable protein and preparation method thereof.
Since the seventies, some have the material of two valencys or multivalence adhesion, and as phytolectin, biotin and SP etc. is applied to immunocyte tissue chemical technology, and these materials have the affinity of height to certain morphological element.In recent years, in the clinical treatment immunity disease, adopt immunocyte tissue chemical technology to develop a kind of new therapy, i.e. immunity absorption is to remove endogenous and exogenous virulence factor by absorption, purifies the blood, thereby reaches the purpose that smelting is treated.What the sorbing material effect that is wherein adopted was best is SP (being called for short SPA).
Introduction in the 213rd page-216 pages of " blood purification " books that Beijing science tech publishing house published in December, 1992, SPA is a kind of protein ingredient that separates from some staphylococcus aureus wall, be a kind of single chain polypeptide structure, form by the 7-10 seed amino acid.Confirmed that with immunofluorescence, radio-immunity and enzyme mark method SPA has the ability that combines with the Fc section of people and other mammalian blood serum IgG.Find that the SPA amino terminal has 4 Fc lands highly roughly the same, there are 60 left and right sides amino acid in each district, and amino-terminal end has an active part, can combine with the Fc section of IgG, and its carboxyl terminal is connected with cell membrane.If digest whole SPA molecule with trypsase, its nubbin still links to each other with bacteria wall.Another part makes it to rive with staphylococcin, treated D, A, B, C and the X fragment of resolving into.SPA and combining of IgG have high specificity, characteristics that sensitiveness is high.Confirm that after deliberation different IgG fragments and different types of immunoglobulin (Ig) are different with SPA in conjunction with rate, simultaneously, SPA combines with IgG, but can be by separation such as urea, rhodanate, acid, guanine hydrochlorates.People utilize this specific character of SPA, make adsorbing medium, are used for immunity absorption therapy clinically.
The present adsorbing medium that uses clinically combines SPA with certain carrier.Sweden Gambro company produces the SPA immunoabsorbent column, under certain condition SPA and agarose grafting is formed.Adopt potassium cyanide as activator in its process of grafting.Potassium cyanide is toxic articles, this utmost point is unfavorable for being used for three class medical device product, can cause SPA very easily to come off with potassium cyanide in clinical again as the crosslinked adsorbing medium of activator simultaneously, in a single day the SPA that comes off enters the blood of human body circulation, can produce huge side effect even threat to life.
The SPA that selects for use at present obtains by extract purifying on the cell membrane of staphylococcus aureus, exists purity not high, have many fragments, and in clinical, these fragments can be treated again and bring side effect to smelting, and promptly specificity is not high.
The object of the present invention is to provide a kind of affine adsorbing medium, it is good that this affine adsorbing medium has a high specificity, selectivity height, bio-compatibility, the characteristic of structure homogeneous.
Another object of the present invention is to provide a kind of synthetic method of affine adsorbing medium, this synthetic method safety, nontoxic, product structure is stable.
The structure of affine adsorbing medium of the present invention is:
S-RPA
Here S represents agarose, and RPA represents gene recombinant protein A.
Gene recombinant protein A is synthetic by artificial gene, commercialization.
The preparation method of above-mentioned affine adsorbing medium comprises successively:
1. agarose (S) carries out grafting
(1) react in aqueous medium with epoxychloropropane, NaOH and agarose, reaction is 2-4 hour under 45-55 ℃ of temperature, cleans to neutrality after having reacted and drains;
(2) add ammoniacal liquor in above-mentioned product, reaction is 2-4 hour under temperature 45-55 ℃ temperature, cleans to neutrality after having reacted and drains;
(3) add glutaraldehyde again in above-mentioned product, reaction is 2-4 hour under temperature 25-35 ℃ temperature, cleans to neutrality after having reacted and drains.
2. gene recombinant protein A aglucon is immobilized
(4) agarose of above-mentioned grafting and gene recombinant protein A solution reacted 16-20 hour under temperature 15-25 ℃ temperature, drained after cleaning after having reacted;
(5) above-mentioned product is added in the BAS that contains glycine ethyl ester hydrochloride, adds sodium borohydride simultaneously and carry out end-block, reduction.Reacted 16-20 hour down at temperature 15-25 ℃, get affine adsorbing medium after the cleaning.
Affine adsorbing medium preparation process of the present invention does not contain Toxic matter, albumin A is immobilized efficient height on agarose, gene recombinant protein A purity height, be not with fragment, prepared affine adsorbing medium is measured by anti-enzyme-linked method, the amount of its adhesion protein is bigger than common adsorbing medium, the adsorption efficiency height, thus reach the purpose for the treatment of some relevant disease.
The invention will be further described below in conjunction with embodiment
Embodiment 1:
Preparation method of the present invention comprises successively:
1. react in aqueous medium with epoxychloropropane, NaOH and agarose (Sepharose CL-4B), reaction is 2 hours under 45 ℃ of temperature, has reacted back the cleaning to neutrality with distilled water and has drained;
2. add ammoniacal liquor (pH value is 11.0) in above-mentioned reactant, reaction is 2 hours under 45 ℃ of temperature of temperature, has reacted back the cleaning to neutrality with distilled water and has drained;
3. add glutaraldehyde in above-mentioned product again, reaction is 2 hours under 25 ℃ of temperature of temperature, cleans to neutrality after having reacted and drains;
4. agarose and the gene recombinant protein A solution with above-mentioned grafting reacted 16 hours under 15 ℃ of temperature of temperature, had reacted and had drained after the back is cleaned with BAS;
5. above-mentioned product is added in the BAS that contains glycine ethyl ester hydrochloride, adds sodium borohydride simultaneously and carry out end-block, reduction.Reacted 16 hours down for 15 ℃ in temperature, get affine adsorbing medium after cleaning with distilled water.
Embodiment 2:
According to the method for embodiment 1, under different temperature and reaction time the preparation affine adsorbing medium of the present invention, differential responses chronogeometry recombinant protein A in conjunction with rate as shown in Figure 1 and Figure 2; Gene recombinant protein A's in conjunction with rate such as Fig. 3, Fig. 4, shown in Figure 5 under the differential responses temperature.
Embodiment 3:
Utilize affine adsorbing medium of the present invention to be filled to affine absorber, utilize the characteristic of the materials such as efficient affine adhesion protein of gene recombinant protein A to be applied to clinical smelting treatment disease.
Affine absorber can take off suction with following two kinds of solution:
1.PH value is citric acid-hydrochloric acid solution of 2.2;
2.PH value is 7.0 the phosphate buffer that contains citric acid trisodium, sodium acetate, sodium chloride.
Affine adsorbing medium of the present invention can repeatedly recycle in clinical practice, the utilization rate height.
Claims (1)
1. one kind is prepared as follows the formula structure:
S-RPA
Here S represents agarose, and RPA represents gene recombinant protein A
The method of affine adsorbing medium, comprise successively:
(1) agarose is carried out grafting, the grafting process is:
(a) epoxychloropropane, NaOH and agarose react in aqueous medium, and reaction is 2-4 hour under 45-55 ℃ of temperature, cleans to neutrality after having reacted and drains;
(b) add ammoniacal liquor in the product, reaction is 2-4 hour under temperature 45-55 ℃ temperature, cleans to neutrality after having reacted and drains;
(c) add glutaraldehyde in the product again, reaction is 2-4 hour under temperature 25-35 ℃ temperature, cleans to neutrality after having reacted and drains;
(2) agarose after the above-mentioned grafting and gene recombinant protein A aglucon is immobilized, immobilized process is:
(d) agarose of above-mentioned grafting and gene recombinant protein A solution reacted 16-20 hour under temperature 15-25 ℃ temperature, drained after cleaning after having reacted;
(e) above-mentioned product is added in the BAS that contains glycine ethyl ester hydrochloride, adds sodium borohydride simultaneously and carry out end-block, reduction, reacted 16-20 hour down, get affine adsorbing medium after the cleaning at temperature 15-25 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01103114 CN1212884C (en) | 2001-01-17 | 2001-01-17 | Affinity adsorption medium and its preparing medium |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01103114 CN1212884C (en) | 2001-01-17 | 2001-01-17 | Affinity adsorption medium and its preparing medium |
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| CN1365853A CN1365853A (en) | 2002-08-28 |
| CN1212884C true CN1212884C (en) | 2005-08-03 |
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| CN 01103114 Expired - Fee Related CN1212884C (en) | 2001-01-17 | 2001-01-17 | Affinity adsorption medium and its preparing medium |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101050464B (en) * | 2006-03-17 | 2011-07-06 | 上海抗体药物国家工程研究中心有限公司 | Method for preparing A gene of recombined protein, and expressed products and application |
| CN101935665B (en) * | 2006-03-17 | 2013-08-28 | 上海抗体药物国家工程研究中心有限公司 | Preparation method and application of recombinant protein A gene and expression product thereof |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101069751B (en) * | 2006-05-10 | 2011-02-09 | 广州康盛生物科技有限公司 | Protein A immuo adsorption material and preparing method |
| CN101185878B (en) * | 2006-11-17 | 2010-05-26 | 广州康盛生物科技有限公司 | Protein A immunoadsorption material for eliminating pathogenic antibody and its complexes, and synthesizing method and application thereof |
| CN101190409B (en) * | 2006-11-18 | 2010-12-15 | 广州康盛生物科技有限公司 | Blood purifying protein A immunoadsorption material and synthesizing method thereof |
| CN101185881B (en) * | 2007-09-12 | 2011-07-20 | 天津大学 | Chromatogram medium for immunoglobulin class protein separation purification and preparation method thereof |
| CN100577283C (en) * | 2008-06-16 | 2010-01-06 | 厦门大学 | Method for preparing poly-N-2-carboxyethyl pyrrole metallic chelate nano-tube used as affinity adsorbing medium |
| CN102179237A (en) * | 2010-04-26 | 2011-09-14 | 无锡加莱克色谱科技有限公司 | Affinity chromatographic packing for separating and purifying monoclonal antibody and antibodyglobulin and preparation method thereof |
| CN107051396A (en) * | 2016-12-30 | 2017-08-18 | 重庆希尔康血液净化器材研发有限公司 | A kind of immune absorption material for pinpointing fixed protein A and preparation method thereof |
| CN110624274B (en) * | 2019-08-27 | 2021-04-13 | 苏州赛分科技有限公司 | Separation medium, preparation method and application thereof |
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2001
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101050464B (en) * | 2006-03-17 | 2011-07-06 | 上海抗体药物国家工程研究中心有限公司 | Method for preparing A gene of recombined protein, and expressed products and application |
| CN101935665B (en) * | 2006-03-17 | 2013-08-28 | 上海抗体药物国家工程研究中心有限公司 | Preparation method and application of recombinant protein A gene and expression product thereof |
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| CN1365853A (en) | 2002-08-28 |
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