CN110354817A - A kind of affinity chromatographic column based on immobilized thrombin and its preparation method and application - Google Patents

Abstract

The invention discloses an affinity chromatographic column based on immobilized thrombin, a preparation method and application thereof, and belongs to the fields of chromatographic science, biology and the like. In the method, glutaraldehyde is first bonded to aminopropyl silica gel, and thrombin solution can form a covalent bond with glutaraldehyde, so that thrombin is fixed on the surface of aminopropyl silica gel to prepare a thrombin stationary phase. Wet packed in a chromatographic column to make a bioaffinity chromatographic column based on immobilized thrombin. The affinity column has good stability and high specificity, can be used for the screening application of direct thrombin inhibitors, and lays the foundation for the commercialization of bio-affinity chromatography columns.

Description

A kind of affinity chromatographic column based on immobilized thrombin and its preparation method and application

technical field

The invention belongs to the fields of chromatographic science, biology, etc., and relates to an affinity chromatographic column and a preparation method thereof, in particular to an affinity chromatographic column based on immobilized thrombin, a preparation method and application thereof.

Background technique

Current screening methods for thrombin inhibitors include spectroscopic methods, electrochemical methods, microfluidic chips, biosensors, chromatography, capillary electrophoresis, mass spectrometry, and molecular docking methods. Most of these methods are only suitable for the analysis of monomeric compounds, and chromatographic methods have a large workload and consume more organic solvents, which have some limitations.

(1) Screening method for monomeric compounds

Biosensors and microarray chips are currently the most commonly used methods for studying thrombin. This method has high sensitivity and good specificity, but lacks the function of "separation". It is more suitable for the screening of monomeric compounds with high purity. Computer-aided drug design can screen drugs by calculating the interaction force between thrombin and drugs. Obviously, this method also lacks the function of "separation", and is only suitable for monomer compounds with clear structures. There are certain artificial settings in the screening conditions. Only as an aid.

(2) Screening methods for complex systems

For the screening of complex systems of Chinese herbal medicines, the traditional research mode requires a large number of extraction, separation and activity verification experiments. Due to the complexity of traditional Chinese medicine ingredients and lack of activity screening steps in the early stage, the workload in the later stage is very large, and thousands of ingredients need to be separated and prepared for activity verification, which greatly increases the cost of research and development.

(3) Activity-based screening method

Adding an activity screening step with higher "specificity" in the early stage can achieve twice the result with half the effort for the later separation and identification. For example, using thrombin magnetic beads as a sample pretreatment method has the advantage of improving the specificity and screening efficiency of the screening, but the interaction process between the compound and thrombin cannot be observed, and the signal value is weak after the sample is diluted. less active ingredient.

Capillary electrophoresis affinity chromatography with thrombin as a ligand can be used for the screening of thrombin inhibitors. However, because the interface technology of capillary electrophoresis and mass spectrometry has not been popularized, this method is limited and is not suitable for the analysis and identification of unknown compounds. Thrombin-coupled agarose gel is used as the affinity chromatography medium, which has good biocompatibility and low non-specific adsorption of proteins, but has poor mechanical strength and is easily deformed, which limits the operating flow rate and has a short service life. Can be used in low- and medium-pressure chromatography, where the efficiency of separation and screening is limited.

Combining high performance liquid chromatography and affinity chromatography organically can greatly improve the separation efficiency of affinity chromatography. At present, no research work on screening thrombin inhibitors by thrombin high performance liquid phase affinity chromatography has been found.

Contents of the invention

In order to overcome the shortcomings of the above-mentioned prior art, the object of the present invention is to provide an affinity chromatography column (Thrombin affinity column, TAC) based on immobilized thrombin and its preparation method and application. The affinity chromatography column has activity recognition and Due to the dual characteristics of chromatographic separation, it has good screening specificity; the preparation method is simple to operate and has good repeatability.

In order to achieve the above object, the present invention adopts the following technical solutions to achieve:

The invention discloses an affinity chromatographic column based on immobilized thrombin. The thrombin stationary phase is made of thrombin wrapped with glutaraldehyde and bonded with aminopropyl silica gel, and the prepared thrombin stationary phase is packed by a wet method. An affinity column based on immobilized thrombin was obtained.

Preferably, the glutaraldehyde-bonded aminopropyl silica gel is to bond 3-aminopropyldimethylchlorosilane to the activated silica gel by silanization to obtain aminopropyl silica gel, and then glutaraldehyde is covalently Bonded to aminopropyl silica gel.

The invention also discloses a preparation method of an affinity chromatographic column based on immobilized thrombin, comprising the following steps:

1) Activate the silica gel for later use;

2) Take activated silica gel and 3-aminopropyl dimethyl chlorosilane, use toluene as a solvent, reflux reaction, after the reaction is completed, wash, filter and collect the filter cake, dry in vacuum to obtain aminopropyl silica gel;

3) stirring and reacting aminopropyl silica gel with glutaraldehyde at room temperature to obtain glutaraldehyde-bonded aminopropyl silica gel;

4) The thrombin freeze-dried powder is formulated into a thrombin solution, added to the glutaraldehyde-bonded aminopropyl silica gel prepared in step 3), stirred evenly and left to stand overnight to obtain glutaraldehyde-modified silica gel as a carrier. Thrombin stationary phase;

5) The thrombin stationary phase is packed into a column by a wet method to prepare an affinity chromatography column based on immobilized thrombin.

Preferably, in step 2), the mass ratio of activated silica gel to 3-aminopropyldimethylchlorosilane is 1:1-3:1.

Preferably, in step 2), the reflux reaction temperature is 110°C, and the reflux reaction time is 12h.

Preferably, in step 3), aminopropyl silica gel is added to a glutaraldehyde solution with a mass fraction of 2.5% at a dosage ratio of 1 g: 20 mL, stirred and reacted at room temperature for 2 h, filtered, and the filter cake is collected, washed to remove excess glutaraldehyde.

Preferably, in step 4), a sodium carbonate-sodium bicarbonate buffer solution with a pH value of 8.3 is used to prepare the lyophilized thrombin powder into a 10 U/ml thrombin solution.

The invention also discloses the application of the affinity chromatographic column based on immobilized thrombin in the screening of thrombin inhibitors.

Compared with the prior art, the present invention has the following beneficial effects:

In the affinity chromatography column based on immobilized thrombin disclosed in the present invention, glutaraldehyde is covalently bonded to silica gel, and the amino side chain on thrombin can specifically react with the free aldehyde group on the stationary phase to form covalent bond, so that thrombin is immobilized on the surface of silica gel, and then the affinity chromatography column based on immobilized thrombin is obtained by wet packing. Thereby further prolonging the column life of the affinity chromatographic column and improving its stability; on the other hand, since thrombin is a pure protein, it can specifically interact with the substrate, that is, there is only a single protein of thrombin on the affinity chromatographic column, and no Other interferences greatly improved its specificity.

The preparation method of the above-mentioned affinity chromatographic column for immobilized thrombin disclosed by the invention has simple operation, mild reaction conditions and good repeatability.

The affinity chromatographic column based on immobilized thrombin of the present invention can be used in high performance liquid chromatography, further expands the scope of application, and has a large market space; it can be used to quickly screen direct thrombin inhibitors from complex traditional Chinese medicine systems, and further expand research The object is not limited to the screening of monomeric compounds.

Description of drawings

Fig. 1 is the preparation flowchart of the affinity chromatographic column based on immobilized thrombin;

Figure 2 is a graph showing the selectivity investigation results of the TAC affinity chromatographic column.

Detailed ways

In order to enable those skilled in the art to better understand the solutions of the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only It is an embodiment of a part of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

It should be noted that the terms "first" and "second" in the description and claims of the present invention and the above drawings are used to distinguish similar objects, but not necessarily used to describe a specific sequence or sequence. It is to be understood that the data so used are interchangeable under appropriate circumstances such that the embodiments of the invention described herein can be practiced in sequences other than those illustrated or described herein. Furthermore, the terms "comprising" and "having", as well as any variations thereof, are intended to cover a non-exclusive inclusion, for example, a process, method, system, product or device comprising a sequence of steps or elements is not necessarily limited to the expressly listed instead, may include other steps or elements not explicitly listed or inherent to the process, method, product or apparatus.

The present invention is described in further detail below in conjunction with accompanying drawing:

Referring to Fig. 1, the preparation method of the affinity chromatographic column based on immobilized thrombin of the present invention, first prepare amino-bonded silica gel, then bond glutaraldehyde to aminated silica gel, then thrombin solution can form with aldehyde group covalent bond, so that thrombin is immobilized on the surface of silica gel to prepare thrombin stationary phase. Then it is loaded into the chromatographic column by a wet method to form a bio-affinity chromatographic column based on immobilized thrombin. The bio-affinity chromatographic column with immobilized thrombin prolongs the column life of the bio-affinity chromatographic column, improves its stability and specificity, and lays a foundation for the commercialization of the bio-affinity chromatographic column.

Specifically, the preparation method comprises the following steps:

1) Preparation of amino-bonded silica stationary phase

Weigh macroporous silica gel (model: ZEX-II, 5 μm, ) 10g in a 500mL round bottom flask, add 200mL of 1mol/L hydrochloric acid aqueous solution, after ultrasonic treatment for 30min, use a heating mantle to heat and reflux for 2h, transfer the silica gel and hydrochloric acid solution to a 1000mL beaker, add ultrapure water to 1000mL, Stir evenly, let the silica gel settle freely, remove the supernatant after the sedimentation is complete, add 1000mL of ultrapure water again, repeat 4-5 times, filter with a vertical fusing glass funnel, wash until the pH of the supernatant is neutral, transfer the silica gel to In a watch glass, dry at 105°C for 12 hours to obtain activated silica gel for use. Accurately weigh 10.0g of activated silica gel, 5.0g of 3-aminopropyldimethylchlorosilane and 100mL of toluene, place them in a 250mL three-neck flask, and stir and reflux at 110°C for 12h. Wash the obtained solid powder, filter and collect the filter cake, and dry it under vacuum drying condition at 100° C. to obtain the amino chromatography stationary phase with isopropyl side chain protection.

2) Add 1 g of aminopropyl silica gel to 20 ml of 2.5% glutaraldehyde, and stir magnetically at room temperature. After 2 hours, the mixture was filtered and washed several times with a large amount of deionized water to ensure that no excess glutaraldehyde was adsorbed on the silica gel to obtain a glutaraldehyde-modified silica gel stationary phase.

3) Use sodium carbonate-sodium bicarbonate buffer solution (pH 8.3) to prepare 10 U/ml thrombin solution with thrombin freeze-dried powder, add it to glutaraldehyde-modified aminopropyl silica gel, stir well and let it stand overnight. Obtain the thrombin stationary phase with the silica gel modified by glutaraldehyde as the carrier;

4) Establishment of thrombin affinity chromatographic column

The obtained immobilized thrombin affinity chromatographic stationary phase is packed into a column core of 10 mm×2.0 mm (I.D.) by wet method with RPL-ZD10 column packing machine, and the immobilized thrombin affinity chromatographic column is obtained.

The effect of the affinity chromatographic column based on immobilized thrombin prepared by the present invention is carried out as follows verification test:

Using the retention time of the positive control drug argatroban as an index, the intra-column precision, inter-column precision and column lifetime of the thrombin affinity chromatography column were investigated.

1. In-column precision inspection

Prepare a TAC affinity chromatographic column according to the above method, place it in liquid chromatography, and after fully equilibrating, inject 5 μL of 0.1 mg/mL argatroban sample continuously for 6 times, and record the argatroban in each injection The retention time, the results are shown in Table 1.

Table 1 Intraday reproducibility investigation of TAC affinity chromatography column

It can be seen from Table 1 that the retention time precision of the positive control drug argatroban on the TAC affinity chromatographic column is less than 3%, indicating that the intraday precision of the TAC affinity chromatographic column is good.

2. Inter-column precision inspection

Prepare three TAC affinity chromatographic columns according to the above method, place them in a liquid chromatograph and apply the same chromatographic conditions to fully balance, inject 5 μL 0.1mg/mL argatroban samples respectively, and record the bioaffinity of each thrombin The retention time of argatroban on the chromatographic column, the results are shown in Table 2.

Table 2 Investigation of the differences between TAC affinity chromatography columns

It can be seen from Table 2 that the retention time precision of the positive control drug argatroban on three different TAC affinity chromatography columns is 3%, indicating that the precision between the TAC affinity chromatography columns is good.

3. Column life investigation

Prepare a TAC affinity chromatographic column at the same time, and then place it in the liquid chromatograph. After fully equilibrating, inject 5 μL of 0.1 mg/mL argatroban sample every day, and use a blank column without thrombin as a control , to test for a retention time of three days. The results are shown in Table 3:

Table 3 Activity investigation of TAC affinity chromatography column

It can be seen from Table 3 that after 3 days, argatroban still has obvious retention on the thrombin bioaffinity chromatographic column. Compared with the retention time of the blank group, the difference in retention time is still about 6 minutes . Therefore, it shows that the TAC affinity column can be used as a tool for drug screening within 3 days.

4. Selective inspection

Prepare a TAC affinity chromatographic column, place it in the liquid chromatograph and equilibrate fully, inject 5 μ L of 0.1 mg/ml heparin sodium and argatroban sequentially. Observe the retention behavior of these drugs acting on different receptors on the chromatographic column, and study the selectivity of the TAC affinity chromatographic column. The result is shown in Figure 2. The results showed that the positive drug argatroban was retained on the TAC affinity chromatographic column, and heparin sodium was not retained on the TAC affinity chromatographic column as the negative control drug. The results show that the selectivity of the TAC affinity column is good.

The above content is only to illustrate the technical ideas of the present invention, and cannot limit the protection scope of the present invention. Any changes made on the basis of the technical solutions according to the technical ideas proposed in the present invention shall fall within the scope of the claims of the present invention. within the scope of protection.

Claims (8)

1. An affinity chromatographic column based on immobilized thrombin is characterized in that, the thrombin stationary phase is made by encapsulating glutaraldehyde bonded aminopropyl silica gel with thrombin, and the thrombin stationary phase is passed through a wet method The column was packed to obtain an affinity chromatography column based on immobilized thrombin. 2. the affinity chromatographic column based on immobilized thrombin according to claim 1, is characterized in that, glutaraldehyde bonded aminopropyl silica gel is to utilize silanization method to make 3-aminopropyl dimethyl chlorosilane bond Glutaraldehyde is then covalently bonded to aminopropyl silica to produce aminopropyl silica. 3. A method for preparing an affinity chromatographic column based on immobilized thrombin, comprising the following steps: 1) Activate the silica gel for later use; 2) Take activated silica gel and 3-aminopropyl dimethyl chlorosilane, use toluene as a solvent, reflux reaction, after the reaction is completed, wash, filter and collect the filter cake, dry in vacuum to obtain aminopropyl silica gel; 3) stirring and reacting aminopropyl silica gel with glutaraldehyde at room temperature to obtain glutaraldehyde-bonded aminopropyl silica gel; 4) The thrombin freeze-dried powder is formulated into a thrombin solution, added to the glutaraldehyde-bonded aminopropyl silica gel prepared in step 3), stirred evenly and left to stand overnight to obtain glutaraldehyde-modified silica gel as a carrier. Thrombin stationary phase; 5) The thrombin stationary phase is packed into a column by a wet method to prepare an affinity chromatography column based on immobilized thrombin. 4. the preparation method of the affinity chromatographic column based on immobilized thrombin according to claim 3, is characterized in that, in step 2), the mass ratio of activated silica gel and 3-aminopropyl dimethyl chlorosilane is 1 :1-3:1. 5 . The method for preparing an affinity chromatography column based on immobilized thrombin according to claim 3 , wherein in step 2), the reflux reaction temperature is 110° C., and the reflux reaction time is 12 hours. 6. the preparation method of the affinity chromatographic column based on immobilized thrombin according to claim 3, is characterized in that, in step 3), according to the consumption ratio of 1g:20mL, aminopropyl silica gel is added mass fraction to be 2.5 % glutaraldehyde solution, stirred at room temperature for 2 h, filtered, collected filter cake, and washed to remove excess glutaraldehyde. 7. the preparation method of the affinity chromatographic column based on immobilized thrombin according to claim 3, is characterized in that, in step 4), adopts the sodium carbonate-sodium bicarbonate buffer solution that pH value is 8.3 to freeze thrombin The dry powder was formulated into a 10U/ml thrombin solution. 8. Application of the affinity chromatography column based on immobilized thrombin according to claim 1 or 2 in the screening of thrombin inhibitors.