CN103303933B - The method of modifying of attapulgite and attapulgite fix the preparation method of ligand albumin A or Protein G - Google Patents
The method of modifying of attapulgite and attapulgite fix the preparation method of ligand albumin A or Protein G Download PDFInfo
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- CN103303933B CN103303933B CN201310212065.8A CN201310212065A CN103303933B CN 103303933 B CN103303933 B CN 103303933B CN 201310212065 A CN201310212065 A CN 201310212065A CN 103303933 B CN103303933 B CN 103303933B
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Abstract
The invention discloses the preparation method that the method for modifying of attapulgite and attapulgite fix ligand albumin A or Protein G, comprise the following steps: first attapulgite is carried out purifying, then thermal activation is carried out to attapulgite, then attapulgite is carried out organically-modified, again the attapulgite after organically-modified is carried out glutaraldehyde process, again attapulgite and albumin A or Protein G are fixed, finally close the unnecessary aldehyde radical of attapulgite, complete preparation.From said process, carry out the preparation of the attapulgite for fixing ligand albumin A or Protein G with the present invention, attapulgite resource is enriched, and all kinds of chemical articles used in preparation process are common articles for use, and preparation cost is relatively low; In addition, attapulgite has more activated hydroxo (-OH), improves the combination rate of albumin A or Protein G; Attapulgite matrix stability containing ligand albumin A or Protein G after fixing is good, have good antibody purification effect, can bear larger pressure simultaneously, prevent the loss of albumin A or Protein G; And preparation process is relatively simple and convenient, be suitable for large-scale popularization and production practical application.
Description
Technical field
The present invention relates to attapulgite field, be specifically related to the preparation method that a kind of method of modifying of attapulgite and attapulgite fix ligand albumin A or Protein G.
Background technology
Containing abundant immunoglobulin (Ig) in blood plasma, the Main Function of immunoglobulin (Ig) participates in immune response, thus block pathogenic agent to the harm of body, makes pathogenic agent lose pathogenic effects.Immunoglobulin (Ig), as important biochemical medicinal product, both can be widely used in clinical treatment, as additive application in feed industry, daily chemical industry and foodstuffs industry, can have higher economic value added again.Albumin A is a kind of cell wall protein be isolated from A type streptococcus aureus, and Protein G is the cell wall protein be isolated from G type suis.Their character is similar, is can region outside immunoglobulin (Ig) antigen binding site, forms the mixture containing Protein A-antibody or Protein G-antibody, with the protein of antibodies.They have higher binding ability to panimmunity sphaeroprotein and subclass thereof.Based on these characteristics of albumin A or Protein G, using albumin A or Protein G as ligand, the stationary phase of affinity chromatography can be made, and for the preparation of antibody and purifying.At present, market there is the multiple different matrix being combined with albumin A or Protein G ligand, these matrix being combined with albumin A or Protein G are after being subject to certain pressure, the loss of albumin A or Protein G can be caused, simultaneously when preparation, not only complex operation, process are complicated, and the effect of antibody purification also makes us being satisfied with not.
Summary of the invention
The object of the invention is to: overcome the deficiencies in the prior art, a kind of method of modifying of attapulgite and attapulgite is provided to fix the preparation method of ligand albumin A or Protein G, preparation technology is simple, better to the fixed effect of ligand albumin A or Protein G, the attapulgite of fixing ligand albumin A or Protein G is obvious for the refining effect of antibody.
The technical solution used in the present invention is:
The method of modifying of attapulgite, comprises the following steps:
1) purifying: attapulgite is mixed with water, and carry out shaking the abundant aquation dispersion of the attapulgite particle be stirred in suspension at the temperature of 55 ~ 65 degrees Celsius;
2) after stratification, pour out upper strata suspension, centrifugation is carried out to it and obtains solid attapulgite, afterwards heat drying is carried out to attapulgite;
3) thermal activation: ground by dried attapulgite, heats the attapulgite after grinding 2 ~ 4 hours at the temperature of temperature 280 ~ 320 degrees Celsius;
4) modification: after the attapulgite after to be heated is cooled to room temperature, then attapulgite is joined in coupling agent aqueous solution, regulate pH value to be 3 ~ 4 with mineral acid, be fully uniformly mixed, carry out solid-liquor separation afterwards;
5) attapulgite after step 4) solid-liquor separation is washed with water, carry out vacuum drying afterwards;
6) attapulgite after oven dry is ground, the attapulgite after grinding is added abundant stirring reaction in glutaraldehyde water solution, carries out solid-liquor separation afterwards; Glutaraldehyde is a kind of reagent with bifunctional, and attapulgite containing-NH2, adopts glutaraldehyde process through silane resin acceptor kh-550 modified showing, the end-NH2 of grafting on carrier and glutaraldehyde is reacted, forms Schiff (C=N); Meanwhile ,-the CHO of the other end of glutaraldehyde can react with free amino group, and Schiff reaction also can occur, thus it is fixing to realize albumin A or Protein G;
7) attapulgite after step 6) solid-liquor separation is washed with water, carry out vacuum lyophilization afterwards and obtain attapulgite modified;
The further improvement project of the present invention is, in step 1), the mass mixing ratio of attapulgite and water is 1:15 ~ 25.
The present invention further improvement project is, step 2), drying temperature is respectively 105 ~ 115 degrees Celsius, 70 ~ 90 degrees Celsius ,-85 ~-75 degrees Celsius in step 5), step 7)
The present invention further improvement project is, step 3), 6) in grinding after attapulgite order number be at least 200 orders.
The present invention further improvement project is, the concentration of coupling agent aqueous solution described in step 4) is the silane resin acceptor kh-550 aqueous solution of 5%; Described mineral acid is hydrochloric acid soln.
The present invention further improvement project is, in step 6), the concentration of glutaraldehyde water solution is 0.5%.
Attapulgite fixes the preparation method of ligand albumin A or Protein G, comprises the following steps:
8) by above-mentioned prepare attapulgite modifiedly add in albumin A or Protein G solution, slowly concussion at least 12 hours in ice bath, carries out vacuum lyophilization and obtains attapulgite and fix ligand albumin A or Protein G after taking-up; Described albumin A or Protein G solution are the phosphate buffered saline buffer that albumin A or Protein G are dissolved in the pH7.2 containing 0.05% tween 20, namely obtain in PBS buffered soln, and the volume ratio of albumin A or Protein G weight and PBS buffered soln is 1:1;
9) dried for step 8) attapulgite is fixed ligand albumin A or Protein G joins in lysine solution, under the envrionment temperature of 0 ~ 6 degree Celsius, place at least 4 hours, centrifugation afterwards, is placed in 4 DEG C, for subsequent use; Due to attapulgite also having unnecessary aldehyde radical, so unnecessary aldehyde radical must be closed before use, use Methionin to close residual aldehyde radical.
The present invention further improvement project is, albumin A described in step 8) or Protein G strength of solution are every milliliter and contain 1 milligram of albumin A or Protein G.
The present invention further improvement project is, the temperature of vacuum lyophilization described in step 8) is-85 ~-75 degrees Celsius.
The present invention further improvement project is, the concentration of lysine solution described in step 9) is 0.2mol/L.
Beneficial effect of the present invention is:
The first, carry out the preparation of the attapulgite for fixing ligand albumin A or Protein G with the present invention, attapulgite resource is enriched, and all kinds of chemical articles used in preparation process are common articles for use, and preparation cost is relatively low.
The second, with the attapulgite for fixing ligand albumin A or Protein G prepared by the present invention, there is more activated hydroxo (-OH), improve the combination rate of albumin A or Protein G.
Three, with the attapulgite for fixing ligand albumin A or Protein G prepared by the present invention, attapulgite matrix stability containing ligand albumin A or Protein G after fixing is good, have good antibody purification effect, larger pressure can be born simultaneously, prevent the loss of albumin A or Protein G.
Four, with the attapulgite for fixing ligand albumin A or Protein G prepared by the present invention, preparation process is relatively simple and convenient, is suitable for large-scale popularization and production practical application.
accompanying drawing illustrates:
Fig. 1 is the XRD figure before and after attapulgite is purified.
Fig. 2 is the infrared spectrum before and after attapulgite is purified.
Fig. 3 is the scanning electron microscope (SEM) photograph of attapulgite before and after purifying.
Fig. 4 is the EDS data plot of attapulgite before and after purifying.
Fig. 5 is the XRD figure before and after attapulgite activation.
Fig. 6 is the infrared spectrum before and after attapulgite activation.
Fig. 7 is the XRD figure of attapulgite modified front and back.
Fig. 8 is the infrared spectrum of attapulgite modified front and back.
Fig. 9 is the scanning electron microscope (SEM) photograph of attapulgite modified front and back.
Figure 10 is the infrared spectrum that albumin A or Protein G fix before and after attapulgite.
Embodiment
The method of modifying of attapulgite in the present invention, comprises the following steps:
1) purifying: be that 1:15 ~ 25 mix with water according to mass mixing ratio by attapulgite, and carry out shaking the abundant aquation dispersion of the attapulgite particle be stirred in suspension at the temperature of 55 ~ 65 degrees Celsius;
2) after stratification, pour out upper strata suspension, carry out centrifugation and obtain solid attapulgite, carry out heat drying afterwards to attapulgite to it, described drying temperature is 105 ~ 115 degrees Celsius;
3) thermal activation: dried attapulgite is ground, the attapulgite order number after grinding is at least 200 orders, is heated 2 ~ 4 hours by the attapulgite after grinding at the temperature of temperature 280 ~ 320 degrees Celsius;
4) modification: after the attapulgite after to be heated is cooled to room temperature, then attapulgite is joined in coupling agent aqueous solution, regulate pH value to be 3 ~ 4 with mineral acid, be fully uniformly mixed, carry out solid-liquor separation afterwards; The concentration of described coupling agent aqueous solution is the silane resin acceptor kh-550 aqueous solution of 5%; Described mineral acid is hydrochloric acid soln;
5) wash with water the attapulgite after step 4) solid-liquor separation, carry out vacuum drying afterwards, described bake out temperature is 70 ~ 90 degrees Celsius;
6) ground by the attapulgite after oven dry, the attapulgite order number after grinding is at least 200 orders, and the attapulgite after grinding is added abundant stirring reaction in glutaraldehyde water solution, and carry out solid-liquor separation afterwards, the concentration of described glutaraldehyde water solution is 0.5%; Glutaraldehyde is a kind of reagent with bifunctional, and attapulgite containing-NH2, adopts glutaraldehyde process through silane resin acceptor kh-550 modified showing, the end-NH2 of grafting on carrier and glutaraldehyde is reacted, forms Schiff (C=N); Meanwhile ,-the CHO of the other end of glutaraldehyde can react with free amino group, and Schiff reaction also can occur, thus it is fixing to realize albumin A or Protein G;
7) wash with water the attapulgite after step 6) solid-liquor separation, carry out vacuum lyophilization afterwards and obtain attapulgite modified, the temperature of described vacuum lyophilization is-85 ~-75 degrees Celsius;
Attapulgite fixes preparation method's (in the implementation case, for attapulgite fixes the preparation method of ligand albumin A) of ligand albumin A or Protein G, it is characterized in that comprising the following steps:
8) by above-mentioned prepare attapulgite modifiedly add in Protein A solution, slowly concussion at least 12 hours in ice bath, carries out vacuum lyophilization and obtains attapulgite and fix ligand albumin A after taking-up; The temperature of described vacuum lyophilization is-85 ~-75 degrees Celsius; Described Protein A solution concentration is every milliliter and contains 1 milligram of albumin A; Described Protein A solution is the phosphate buffered saline buffer that albumin A is dissolved in the pH7.2 containing 0.05% tween 20, namely obtains in PBS buffered soln, and the volume ratio of albumin A weight and PBS buffered soln is 1:1; 1.5mg albumin A is taken out from-20 DEG C of refrigerators, dissolves with the PBS buffered soln (pH=7.2) of 1.5mL, be made into the Protein A solution that concentration is 1mg/mL;
9) dried for step 8) attapulgite being fixed ligand albumin A joins in lysine solution, places at least 4 hours, centrifugation afterwards, be placed in 4 DEG C under the envrionment temperature of 0 ~ 6 degree Celsius, for subsequent use; The concentration of described lysine solution is 0.2mol/L; Due to attapulgite also having unnecessary aldehyde radical, so unnecessary aldehyde radical must be closed before use, use Methionin to close residual aldehyde radical.
Before attapulgite preparation, after attapulgite purifying, after attapulgite activation, carry out the sampling of attapulgite after Organic Surface Modification of Attapulgite respectively, and do X-ray diffraction analysis (XRD), make Fig. 1, Fig. 5 and Fig. 7;
Before attapulgite preparation, after attapulgite purifying, after attapulgite activation, after Organic Surface Modification of Attapulgite, after attapulgite fixed protein A, carry out the sampling of attapulgite respectively, and do Fourier infrared spectrum analysis (FT-IR), make Fig. 2, Fig. 6, Fig. 8 and Figure 10;
Before attapulgite preparation, after attapulgite purifying, after Organic Surface Modification of Attapulgite, carry out the sampling of attapulgite respectively, and do scanning electron microscope (SEM), make Fig. 3 and Fig. 9;
Before attapulgite preparation, after attapulgite purifying, carry out the sampling of attapulgite respectively, and do energy spectrum analysis (EDS), make Fig. 4;
Shown in composition graphs 1, Fig. 2 figure, 3 and Fig. 4, after purifying, in attapulgite quartz basic without, carbon impurity hydrochlorate also considerably reduces, and crystal grain is evenly distributed, dispersiveness has improved, and Ti, Zr element is neither present in;
Shown in composition graphs 5 and Fig. 6, after overactivation, the crystalline structure of attapulgite does not have considerable change, decreases the crystal water of internal structure;
Shown in composition graphs 7, Fig. 8 and Fig. 9, through modified, the crystalline structure of attapulgite self is not destroyed, and attapulgite's surface combines organic group;
As shown in Figure 10, albumin A has been fixed on attapulgite.
To sum up described in money, known attapulgite is fixed ligand Protein A matrix antagonist and is had higher adsorptive capacity, and reach 24.51mg/mL through calculating its Dynamic binding capacities, the antibody rate of recovery is 94.1%, close to or be better than similar product.
Claims (8)
1. attapulgite fixes the preparation method of ligand albumin A or Protein G, it is characterized in that comprising the following steps:
1) purifying: attapulgite is mixed with water, and carry out shaking the abundant aquation dispersion of the attapulgite particle be stirred in suspension at the temperature of 55 ~ 65 degrees Celsius;
2) after stratification, pour out upper strata suspension, centrifugation is carried out to it and obtains solid attapulgite, afterwards heat drying is carried out to attapulgite;
3) thermal activation: ground by dried attapulgite, heats the attapulgite after grinding 2 ~ 4 hours at the temperature of temperature 280 ~ 320 degrees Celsius;
4) modification: after the attapulgite after to be heated is cooled to room temperature, then attapulgite is joined in coupling agent aqueous solution, regulate pH value to be 3 ~ 4 with mineral acid, be fully uniformly mixed, carry out solid-liquor separation afterwards;
5) attapulgite after step 4) solid-liquor separation is washed with water, carry out vacuum drying afterwards;
6) attapulgite after oven dry is ground, the attapulgite after grinding is added abundant stirring reaction in glutaraldehyde water solution, carries out solid-liquor separation afterwards;
7) attapulgite after step 6) solid-liquor separation is washed with water, carry out vacuum lyophilization afterwards and obtain attapulgite modified;
8) attapulgite modifiedly add in albumin A or Protein G solution by what prepare, slowly concussion at least 12 hours in ice bath, carry out vacuum lyophilization after taking-up and obtain attapulgite and fix ligand albumin A or Protein G; Described albumin A or Protein G solution are the phosphate buffered saline buffer that albumin A or Protein G are dissolved in the pH7.2 containing 0.05% tween 20, namely obtain in PBS buffered soln, and the volume ratio of albumin A or Protein G weight and PBS buffered soln is 1:1;
9) dried for step 8) attapulgite is fixed ligand albumin A or Protein G joins in lysine solution, under the envrionment temperature of 0 ~ 6 degree Celsius, place at least 4 hours, centrifugation afterwards, is placed in 4 DEG C, for subsequent use;
In step 1), the mass mixing ratio of attapulgite and water is 1:15 ~ 25.
2. attapulgite as claimed in claim 1 fixes the preparation method of ligand albumin A or Protein G, it is characterized in that: step 2), drying temperature is respectively 105 ~ 115 degrees Celsius, 70 ~ 90 degrees Celsius ,-85 ~-75 degrees Celsius in step 5), step 7).
3. attapulgite as claimed in claim 1 fixes the preparation method of ligand albumin A or Protein G, it is characterized in that: step 3), 6) in attapulgite order number after grinding be at least 200 orders.
4. attapulgite as claimed in claim 1 fixes the preparation method of ligand albumin A or Protein G, it is characterized in that: the concentration of coupling agent aqueous solution described in step 4) is the silane resin acceptor kh-550 aqueous solution of 5%; Described mineral acid is hydrochloric acid soln.
5. attapulgite as claimed in claim 1 fixes the preparation method of ligand albumin A or Protein G, it is characterized in that: in step 6), the concentration of glutaraldehyde water solution is 0.5%.
6. attapulgite as claimed in claim 1 fixes the preparation method of ligand albumin A or Protein G, it is characterized in that: albumin A described in step 8) or Protein G strength of solution are every milliliter and contain 1 milligram of albumin A or Protein G.
7. attapulgite as claimed in claim 1 fixes the preparation method of ligand albumin A or Protein G, it is characterized in that: the temperature of vacuum lyophilization described in step 8) is-85 ~-75 degrees Celsius.
8. attapulgite as claimed in claim 6 fixes the preparation method of ligand albumin A or Protein G, it is characterized in that: the concentration of lysine solution described in step 9) is 0.2mol/L.
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Effective date of registration: 20201211 Address after: 223000 wisdom Valley, Huaian science and Education Industrial Park, No.19, Meigao Road, Huaian Economic Development Zone, Jiangsu Province Patentee after: Huai'an Kechuang Intellectual Property Operation Co.,Ltd. Address before: School of bioengineering and chemical engineering, Huaiyin Institute of technology, No.1, Meicheng East Road, Qingpu District, Huai'an City, Jiangsu Province Patentee before: Hu Yihuai Effective date of registration: 20201211 Address after: 211600 No. 47, Jianshe Road, Jinhu County, Jiangsu, Huaian Patentee after: Jinhu comprehensive inspection and Testing Center Address before: 223000 wisdom Valley, Huaian science and Education Industrial Park, No.19, Meigao Road, Huaian Economic Development Zone, Jiangsu Province Patentee before: Huai'an Kechuang Intellectual Property Operation Co.,Ltd. |