阅读说明：本技术 一种高效筛选细菌感染特效药的细胞膜色谱柱制备方法 (Cell membrane chromatographic column preparation method for efficiently screening specific medicines for bacterial infection ) 是由 杨天逸 于 2020-12-29 设计创作，主要内容包括：一种高效筛选细菌感染特效药的细胞膜色谱柱制备方法,包括制备全细胞蛋白提取液、制备APTES共价修饰后的硅胶、细胞膜和硅胶键合及湿法装柱四个步骤。本发明以具有活性的败血症细菌细胞膜及受体为固定相,利用细胞膜自身的融合作用和硅胶表面硅羟基(Si—OH)相互吸附制备成细胞膜色谱固定相,该方法最大限度地保持了细胞膜的完整性、膜受体的四级空间结构以及膜受体的生物活性。比现有筛选方法和技术更加精减,制作成本更低,可规模化生产,质量可控,性能优良,成本合理,能填补市场空缺,市场需求大,生产成本低,制备工艺成熟,同时能完全保证生产质量安全,有效地降低了投资门槛和投资风险。(A method for preparing a cell membrane chromatographic column for efficiently screening specific drugs for bacterial infection comprises four steps of preparing a whole cell protein extracting solution, preparing APTES covalently modified silica gel, bonding cell membranes and the silica gel, and filling the column by a wet method. The invention takes the active septicemia bacteria cell membrane and receptor as the stationary phase, and prepares the cell membrane chromatographic stationary phase by utilizing the fusion of the cell membrane and the mutual adsorption of silicon hydroxyl (Si-OH) on the surface of silica gel, and the method furthest maintains the integrity of the cell membrane, the quaternary space structure of the membrane receptor and the biological activity of the membrane receptor. Compared with the existing screening method and technology, the method has the advantages of more precise reduction, lower manufacturing cost, large-scale production, controllable quality, excellent performance, reasonable cost, capability of filling the gap of the market, large market demand, low production cost and mature preparation process, can completely ensure the safety of the production quality, and effectively reduces the investment threshold and the investment risk.)

1. A cell membrane chromatographic column preparation method for efficiently screening specific drugs for bacterial infection is characterized by comprising four steps of preparing a whole cell protein extracting solution, preparing APTES covalently modified silica gel, bonding cell membranes with the silica gel and filling the column by a wet method; in the preparation of the whole-cell protein extracting solution, denatured lysate and non-denatured lysate are used as reagents, a centrifuge, a 1.5ml centrifugal test tube and a 50ml cylindrical polyethylene centrifugal test tube with a diaphragm in the middle are used as extraction tools, and the whole-cell protein extracting solution is prepared by the following steps: placing cells in a 1.5ml centrifugal test tube, adding 1ml PBS cell washing solution, centrifuging for 3 minutes by using a centrifuge, removing supernatant, repeating the procedures of centrifuging and removing supernatant for three times, adding precooled PBS cell washing solution according to the cell precipitation volume, carrying out cell resuspension, adding denatured lysate according to the cell amount in the centrifugal test tube, shaking for 15s, transferring the shaken lysate into a 50ml cylindrical polyethylene centrifugal test tube, centrifuging by using the centrifuge for 30s, and collecting the lysate at the bottom of the tube, namely whole-cell protein extracting solution; in the preparation of APTES covalence modified silica gel, preparing a cell membrane chromatographic column by using cells with high expression of specific protein, adding an inhibitor of the cell membrane chromatographic column, washing by using ph7.0 phosphate buffer solution to remove non-specifically bound impurities, eluting the buffer solution by using ph3.3 phosphate buffer solution, collecting eluent, concentrating, and performing high performance liquid detection; in the bonding of the cell membrane and the silica gel, mixing the cell membrane suspension and the silica gel at normal temperature for reaction for 5 rain under the vacuum vortex condition, then stirring for 50 rain under the ice bath condition to obtain the cell membrane, placing the silica gel suspension in a refrigerator at 4 ℃ and standing overnight; and (3) in the wet column packing, adopting a Waters 996.515 type liquid phase pump, and using a PBS buffer solution as a mobile phase to pack the cell membrane, wherein a silica gel suspension is filled in a cell membrane chromatographic column core, and the gradient is increased at the speed of 0.5 min: 0.2-1.0 mL/min, then equilibrating 30 rain at O.2 mL/min flow rate to obtain a stable column pressure; the cell membrane chromatographic column is soaked in PBS buffer solution and stored for standby at 4 ℃.

2. The method for preparing a cell membrane chromatographic column for efficiently screening specific drugs for bacterial infections according to claim 1, wherein the silica gel is prepared by covalent modification of APTES, wherein the silica gel surface contains abundant silicon hydroxyl (Si-OH), and the silica gel is reacted with (3. aminopropyl) triethoxysilane (APTES) to link an amino group, and then reacted with glutaraldehyde to link an aldehyde group; cell membranes are composed of abundant phospholipids and therefore contain a large number of amino groups; the aldehyde group-bonded silica gel and the amino group on the cell membrane undergo an amino-aldehyde condensation reaction, so that the silica gel and the cell membrane are covalently bonded.

3. The method for preparing the cell membrane chromatographic column for efficiently screening the specific drug for bacterial infection, according to claim 1, is characterized in that the preparation of the APTES covalent modified silica gel comprises the following steps of (1) accurately weighing 1 g of silica gel powder, weighing 0.5 mL of LAPTES, adding the silica gel powder into a 50mL toluene system, and heating and refluxing for 12 hours in an oil bath at constant temperature under the protection of N2; (2) collecting the suspension, centrifuging at 12009 × 5 min at room temperature, removing supernatant, and collecting precipitate; washing the precipitate with HEPES buffer solution (100 mM) for 3 times, and oven drying at 80 deg.C to obtain dry powder; (3) adding 500 mL of 5% glutaraldehyde methanol solution into the dried powder, and stirring for 2 h at normal temperature, wherein the solution is changed from white to brick red; centrifuging at 1200Gx 5 min at room temperature, removing supernatant, and collecting precipitate; washing the precipitate with HEPES buffer solution for 3 times; (4) the precipitate was added to 500 mL of sodium phosphate solution (100 mM, pH =8) and stirred at room temperature for 2 h to allow the reacted group to spread, the suspension was centrifuged at 12009 × 5 min at room temperature, and the supernatant was discarded to leave the precipitate.

4. Washing the precipitate with HEPES buffer solution for 3 times; (5) and drying the precipitate at the constant temperature of 80 ℃ to obtain dry brick red powder, namely the APTES covalently modified silica gel.

Technical Field

The invention relates to the field of biology, in particular to a preparation method of a cell membrane chromatographic column for efficiently screening specific drugs for bacterial infection.

Background

Bacterial infection is acute systemic infection caused by the invasion of pathogenic bacteria into the blood system and the growth and reproduction of pathogenic bacteria in the blood, and the generation of a large amount of toxins. The disease is dangerous and easy to cause death. Therefore, the specific medicine of the bacteria causing the infection is quickly screened out, the selection and the use of the antibiotic are determined, the blind medication can be reduced, and the treatment time of a patient is shortened. In the prior art, due to technical limitation, the specific medicine for quickly screening out bacteria causing infection has a great defect and cannot meet the actual requirement.

Disclosure of Invention

In order to overcome the defects in the prior art as background, the invention provides the preparation method of the cell membrane chromatographic column for efficiently screening the specific medicine for bacterial infection, which adopts the cell membrane of the sepsis bacteria with high expression as the main raw material, is more precise and less than the existing screening method and technology, has lower manufacturing cost, controllable quality, excellent performance and reasonable cost, can fill the market vacancy, has large market demand, low production cost and mature preparation process, can completely ensure the production quality safety, and effectively reduces the investment threshold and the investment risk.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a cell membrane chromatographic column preparation method for efficiently screening specific drugs for bacterial infection is characterized by comprising four steps of preparing a whole cell protein extracting solution, preparing APTES covalently modified silica gel, bonding cell membranes with the silica gel and filling the column by a wet method; in the preparation of the whole-cell protein extracting solution, denatured lysate and non-denatured lysate are used as reagents, a centrifuge, a 1.5ml centrifugal test tube and a 50ml cylindrical polyethylene centrifugal test tube with a diaphragm in the middle are used as extraction tools, and the whole-cell protein extracting solution is prepared by the following steps: placing cells in a 1.5ml centrifugal test tube, adding 1ml PBS cell washing solution, centrifuging for 3 minutes by using a centrifuge, removing supernatant, repeating the procedures of centrifuging and removing supernatant for three times, adding precooled PBS cell washing solution according to the cell precipitation volume, carrying out cell resuspension, adding denatured lysate according to the cell amount in the centrifugal test tube, shaking for 15s, transferring the shaken lysate into a 50ml cylindrical polyethylene centrifugal test tube, centrifuging by using the centrifuge for 30s, and collecting the lysate at the bottom of the tube, namely whole-cell protein extracting solution; in the preparation of APTES covalence modified silica gel, preparing a cell membrane chromatographic column by using cells with high expression of specific protein, adding an inhibitor of the cell membrane chromatographic column, washing by using ph7.0 phosphate buffer solution to remove non-specifically bound impurities, eluting the buffer solution by using ph3.3 phosphate buffer solution, collecting eluent, concentrating, and performing high performance liquid detection; in the bonding of the cell membrane and the silica gel, mixing the cell membrane suspension and the silica gel at normal temperature for reaction for 5 rain under the vacuum vortex condition, then stirring for 50 rain under the ice bath condition to obtain the cell membrane, placing the silica gel suspension in a refrigerator at 4 ℃ and standing overnight; and (3) in the wet column packing, adopting a Waters 996.515 type liquid phase pump, and using a PBS buffer solution as a mobile phase to pack the cell membrane, wherein a silica gel suspension is filled in a cell membrane chromatographic column core, and the gradient is increased at the speed of 0.5 min: 0.2-1.0 mL/min, then equilibrating 30 rain at O.2 mL/min flow rate to obtain a stable column pressure; the cell membrane chromatographic column is soaked in PBS buffer solution and stored for standby at 4 ℃.

Furthermore, in the silica gel after APTES covalent modification, the silica gel surface contains abundant silicon hydroxyl (Si-OH), amino groups are linked after the reaction with (3. aminopropyl) triethoxysilane (APTES), and then the reaction with glutaraldehyde is carried out to link an aldehyde group; cell membranes are composed of abundant phospholipids and therefore contain a large number of amino groups; the aldehyde group-bonded silica gel and the amino group on the cell membrane undergo an amino-aldehyde condensation reaction, so that the silica gel and the cell membrane are covalently bonded.

Further, the preparation of the APTES covalently modified silica gel comprises the following steps of (1) accurately weighing 1 g of silica gel powder, weighing 0.5 mL of LAPTES, adding into a 50mL toluene system, and heating and refluxing for reaction for 12 hours in an oil bath kettle at constant temperature under the protection of N2; (2) collecting the suspension, centrifuging at 12009 × 5 min at room temperature, removing supernatant, and collecting precipitate; washing the precipitate with HEPES buffer solution (100 mM) for 3 times, and oven drying at 80 deg.C to obtain dry powder; (3) adding 500 mL of 5% glutaraldehyde methanol solution into the dried powder, and stirring for 2 h at normal temperature, wherein the solution is changed from white to brick red; centrifuging at 1200Gx 5 min at room temperature, removing supernatant, and collecting precipitate; washing the precipitate with HEPES buffer solution for 3 times; (4) the precipitate was added to 500 mL of sodium phosphate solution (100 mM, pH =8) and stirred at room temperature for 2 h to allow the reacted group to spread, the suspension was centrifuged at 12009 × 5 min at room temperature, and the supernatant was discarded to leave the precipitate. Washing the precipitate with HEPES buffer solution for 3 times; (5) and drying the precipitate at the constant temperature of 80 ℃ to obtain dry brick red powder, namely the APTES covalently modified silica gel.

The invention has the beneficial effects that: the invention takes the active septicemia bacteria cell membrane and receptor as the stationary phase, and prepares the cell membrane chromatographic stationary phase by utilizing the fusion of the cell membrane and the mutual adsorption of silicon hydroxyl (Si-OH) on the surface of silica gel, and the method furthest maintains the integrity of the cell membrane, the quaternary space structure of the membrane receptor and the biological activity of the membrane receptor. Compared with the existing screening method and technology, the method has the advantages of more precise reduction, lower manufacturing cost, large-scale production, controllable quality, excellent performance, reasonable cost, capability of filling the gap of the market, large market demand, low production cost and mature preparation process, can completely ensure the safety of the production quality, and effectively reduces the investment threshold and the investment risk. Based on the above, the invention has good application prospect.

Drawings

The invention is further illustrated with reference to the following figures and examples.

FIG. 1 is a block flow diagram of the present invention.

Detailed Description

FIG. 1 shows a method for preparing a cell membrane chromatographic column for efficiently screening specific drugs for bacterial infection, which comprises three steps of preparing a whole cell protein extracting solution, preparing APTES covalently modified silica gel, bonding cell membranes with the silica gel and filling the cell membranes into the column by a wet method; in the preparation of the whole-cell protein extracting solution, denatured lysate and non-denatured lysate are used as reagents, a centrifuge, a 1.5ml centrifugal test tube and a 50ml cylindrical polyethylene centrifugal test tube with a diaphragm in the middle are used as extraction tools, and the whole-cell protein extracting solution is prepared by the following steps: placing cells into a 1.5ml centrifugal test tube, adding 1ml PBS cell washing solution, centrifuging for 3 minutes by using a centrifuge, removing supernatant, repeating the procedures of centrifuging and removing supernatant for three times, adding precooled PBS cell washing solution according to the cell precipitation volume, carrying out cell resuspension, adding denatured lysate according to the cell amount in the centrifugal test tube, shaking for 15s, transferring the shaken lysate into a 50ml cylindrical polyethylene centrifugal test tube, centrifuging by using the centrifuge for 30s, and collecting the lysate at the bottom of the tube, namely whole-cell protein extracting solution.

As shown in fig. 1, in the preparation of APTES covalently modified silica gel, a cell membrane chromatography column is prepared using cells highly expressing a specific protein, an inhibitor thereof is added, washing is performed using ph7.0 phosphate buffer to remove non-specifically bound impurities, then ph3.3 phosphate buffer is used to elute the buffer, the eluate is collected, and after concentration, high performance liquid detection is performed; in the bonding of the cell membrane and the silica gel, mixing the cell membrane suspension and the silica gel at normal temperature for reaction for 5 rain under the vacuum vortex condition, then stirring for 50 rain under the ice bath condition to obtain the cell membrane, placing the silica gel suspension in a refrigerator at 4 ℃ and standing overnight; and (3) in the wet column packing, adopting a Waters 996.515 type liquid phase pump, and using a PBS buffer solution as a mobile phase to pack the cell membrane, wherein a silica gel suspension is filled in a cell membrane chromatographic column core, and the gradient is increased at the speed of 0.5 min: 0.2-1.0 mL/min, then equilibrating 30 rain at O.2 mL/min flow rate to obtain a stable column pressure; the cell membrane chromatographic column is soaked in PBS buffer solution and stored for standby at 4 ℃. The surface of the silica gel contains rich silicon hydroxyl (Si-OH), amino is linked after the silica gel reacts with (3. aminopropyl) triethoxysilane (APTES), and then the silica gel reacts with glutaraldehyde to link an aldehyde group; cell membranes are composed of abundant phospholipids and therefore contain a large number of amino groups; the aldehyde group-bonded silica gel and the amino group on the cell membrane undergo an amino-aldehyde condensation reaction, so that the silica gel and the cell membrane are covalently bonded. The preparation method comprises the following steps of (1) accurately weighing 1 g of silica gel powder, weighing 0.5 mL of LAPTES, adding into a 50mL toluene system, and heating and refluxing for 12 h in an oil bath kettle at constant temperature under the protection of N2; (2) the suspension was collected and centrifuged at 12009X 5 min at room temperature, and the supernatant was discarded to leave a precipitate. Washing the precipitate with HEPES buffer solution (100 mM) for 3 times, and oven drying at 80 deg.C to obtain dry powder; (3) the dried powder was stirred at room temperature for 2 h by adding 500 mL of 5% glutaraldehyde in methanol, at which time the solution turned from white to brick-red. Centrifuging at 1200Gx 5 min at room temperature, discarding the supernatant, and collecting the precipitate. Washing the precipitate with HEPES buffer solution for 3 times; (4) the precipitate was added to 500 mL of sodium phosphate solution (100 mM, pH =8) and stirred at room temperature for 2 h to allow the reacted group to spread, the suspension was centrifuged at 12009 × 5 min at room temperature, and the supernatant was discarded to leave the precipitate. Washing the precipitate with HEPES buffer solution for 3 times; (5) and drying the precipitate at the constant temperature of 80 ℃ to obtain dry brick red powder, namely the APTES covalently modified silica gel.

The invention takes the active septicemia bacteria cell membrane and receptor as the stationary phase, and prepares the cell membrane chromatographic stationary phase by utilizing the fusion of the cell membrane and the mutual adsorption of silicon hydroxyl (Si-OH) on the surface of silica gel, and the method furthest maintains the integrity of the cell membrane, the quaternary space structure of the membrane receptor and the biological activity of the membrane receptor. Compared with the existing screening method and technology, the method has the advantages of more precise reduction, lower manufacturing cost, large-scale production, controllable quality, excellent performance, reasonable cost, capability of filling the gap of the market, large market demand, low production cost and mature preparation process, can completely ensure the safety of the production quality, and effectively reduces the investment threshold and the investment risk.

Having thus shown and described the fundamental principles and essential features of the invention, and its advantages, it will be apparent to those skilled in the art that it is not limited by the details of the foregoing exemplary embodiments, but is capable of numerous other specific forms of practicing the invention without departing from the spirit or essential characteristics thereof.

Furthermore, it should be understood that although the present description refers to embodiments, the embodiments do not include only one independent technical solution, and such description is only for clarity, and those skilled in the art should take the description as a whole, and the technical solutions in the embodiments may be appropriately combined to form other embodiments that can be understood by those skilled in the art.