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Preparation method for bio-affinity chromatography column rich in P-glycoprotein and application thereof

A glycoprotein and chromatographic column technology, applied in the field of medicine, can solve the problems of being unsuitable for large-scale target drug screening, long experimental period, and cell models can only be used once, achieving good application prospects, long service life, strong exclusive effect

Inactive Publication Date: 2019-10-08
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing in vitro methods for screening P-gp substrates and inhibitors are commonly bidirectional transport, uptake, efflux, and ATPase activity assays, but these methods all have a long experimental cycle (generally cultured for more than 21 days) and cell models can only One-time use, not suitable for the screening of large quantities of target drugs

Method used

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  • Preparation method for bio-affinity chromatography column rich in P-glycoprotein and application thereof
  • Preparation method for bio-affinity chromatography column rich in P-glycoprotein and application thereof
  • Preparation method for bio-affinity chromatography column rich in P-glycoprotein and application thereof

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Experimental program
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Effect test

Embodiment 1

[0027] Cultivate Caco-2 cells: The culture conditions of Caco-2 cells are high-glucose medium containing 10% fetal bovine serum, 1% double antibody and 1% non-essential amino acids. 2 Count the cell flasks and collect the cells. Add the plasma membrane extract solution to the collected Caco-2 cells, the composition of the plasma membrane extract solution is: 1mL lysate (1%Triton-100, 10mmol / LTris, 150mmol / LNaCl, 2.5mmol / LEDTA), 10μL 100mmol / LPMSF and 75 μL aprotinin; gently beat the cells and then grind at 4°C, after grinding, let stand for 15 minutes, transfer the cell disruption solution to a special centrifuge tube, and then slowly add it in a thin stream along the wall at a volume ratio of 2: 2:1 80% sucrose solution, 30% sucrose solution, 5% sucrose solution, the interface of sucrose layer appears, let stand at 4°C for 20min, centrifuge at 4°C, 200000g for 16h, then take out; figure 1 It is the diagram of Caco-2 cell plasma membrane stratification after centrifugation, s...

Embodiment 2

[0036] Add 1mL lysis buffer (1%Triton-100, 10mmol / LTris, 150mmol / LNaCl, 2.5mmol / LEDTA), 10μL 100mmol / LPMSF and 75μL aprotinin to the collected Caco-2 cells. Grind at 4°C, stand still for 15 minutes after grinding, transfer the cell disruption solution to a special centrifuge tube, and then slowly add 80% sucrose solution and 30% sucrose solution with a volume ratio of 2:2:1 along the wall in a thin stream , 5% sucrose solution, the interface of the sucrose layer appeared, let stand at 4°C for 20min, centrifuged at 4°C, 200,000g for 16h, then collected and extracted the plasma membrane layer, the plasma membrane layer was taken as the 3-6 floc layer; the collected The obtained plasma membrane layer was dialyzed in PBS of pH 7.4 for 36 hours in a 3000Da dialysis bag to obtain a plasma membrane solution rich in P-glycoprotein, and stored in a -80°C refrigerator for later use; take 0.6mL rich in P-glycoprotein The protein plasma membrane solution was shaken with 1.2g of amino-bond...

Embodiment 3

[0038] The retention time of the P-glycoprotein bioaffinity chromatographic column for the P-glycoprotein positive drug verapamil was measured when the mobile phase concentration was 5mmol / L, 10mmol / L and 15mmol / L ammonium acetate, and the mobile phase at different concentrations was investigated. Relative adsorption performance. Chromatographic conditions: P-glycoprotein bioaffinity column 30mmx4.6mm; flow rate 0.3mL / min; column temperature 37°C; injection volume 20μL; injection concentration 200μg / mL; UV detector; detection wavelength 228nm. Figure 5 Be mobile phase ammonium acetate concentration respectively at 5mmol / L, when 10mmol / L and 15mmol / L, P-glycoprotein bioaffinity chromatographic column is to the adsorption performance contrast figure of verapamil; By Figure 5 Visible, ammonium acetate concentration is respectively 100min, 65min and 50min when the concentration of ammonium acetate is 5mmol / L, 10mmol / L and 15mmol / L time peak. It can be seen that as the concentra...

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Abstract

The invention, which belongs to the technical field of medicines, relates to a preparation method for a bio-affinity chromatography column rich in P-glycoprotein and application thereof. A plasma membrane rich in P-glycoprotein is extracted from a Caco-2 cell and, together with an amino-bonded silica gel, forms a P-glycoprotein bio-affinity chromatography column; the constructed P-glycoprotein bio-affinity chromatography column is applied to the chromatographic analysis to verify the specific binding with the drug using the P-glycoprotein as a target; and the P-glycoprotein bio-affinity chromatography column has the specific affinity adsorption function to the P-glycoprotein-positive drug verapamil but has no absorption function to the drug with the non-P-glycoprotein target. Therefore, anovel method is provided for screening the P-glycoprotein target drug. After performance evaluation, the prepared P-glycoprotein bio-affinity chromatography column is demonstrated to have advantages of high specificity and stability and long service life and thus has the broad application prospects.

Description

technical field [0001] The invention belongs to the technical field of medicine, and relates to a preparation method and application of a P-glycoprotein bio-affinity chromatographic column. Background technique [0002] P-glycoprotein (P-gp) is a transmembrane glycoprotein with a molecular weight of 170KD, which has the function of energy-dependent "drug pump". is an ATPase-dependent drug export pump, and is also a Ca 2+ and Cl - channel. It directly or indirectly pumps the drug out of the cell through ATP, so that the intracellular drug is pumped out of the cell, reducing the drug concentration in the cell and causing the cell to develop drug resistance. [0003] Substances that inhibit the transport of substrates by P-gp are known as P-gp inhibitors. P-gp inhibitors can improve the bioavailability of therapeutic drugs by blocking drug efflux, and in recent years, they have been widely used clinically to improve the efficacy of chemotherapy drugs. Many drugs and reagen...

Claims

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Application Information

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IPC IPC(8): G01N30/56G01N30/02
CPCG01N30/02G01N30/56G01N2030/562
Inventor 童珊珊陈昱初余江南徐希明
Owner JIANGSU UNIV
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