Intracellular protein cell membrane stationary phase based on SNAP-tag technology, chromatographic column, preparation method of intracellular protein cell membrane stationary phase and chromatographic column, and application of intracellular protein cell membrane stationary phase and chromatographic column in blood concentration monitoring
By fusing SNAP-tag and GPI gene sequences on intracellular proteins and combining them with benzylguanine-modified silica gel, the problem of insufficient stability and specificity in fixing intracellular proteins is solved, and efficient expression and detection of intracellular proteins is achieved.
Patent Information
- Application Number
- CN202411973274.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-30
- Publication Date
- 2025-04-08
AI Technical Summary
Traditional cell membrane chromatography columns have problems with insufficient stability and specificity in fixing intracellular proteins, and it is difficult to efficiently express intracellular proteins on the cell membrane.
By fusing highly expressed SNAP-tag at the N-terminal end of the intracellular protein and adding GPI gene sequence, the intracellular protein is highly expressed on the cell membrane by genetic engineering technology, and then it is bound to benzylguanine-modified silica gel, and covalent bonds are formed through SNAP-tag reaction, and it is fixed on the surface of the silica gel.
It realizes autonomous high expression of intracellular proteins on the cell membrane, improves the stability and specificity of the cell membrane stationary phase, extends the service life of the chromatography column, and enhances the ability to detect blood drug concentrations of intracellular protein ligand drugs.
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Figure CN119771380A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of drug analysis, and specifically relates to an intracellular protein cell membrane stationary phase based on SNAP-tag technology, a chromatographic column and a preparation method thereof, and an application thereof in blood drug concentration monitoring. Background Art
[0002] Cell membrane chromatography (CMC) is a new affinity chromatography technology that can quickly identify target substances in complex components for separation and analysis. The cell membrane is fixed to a carrier to form a cell membrane stationary phase (CMSP), and then the cell membrane chromatography column is prepared by wet column packing.
[0003] Traditional CMC mainly adsorbs cell membranes on the surface of silica gel by physical adsorption. The cell membrane and silica gel are mainly bound by weak hydrophobic interaction and van der Waals force. The receptors contained in the cell membrane on the cell membrane chromatography column used are all membrane receptors. During use, the cell membrane falls off from the silica gel surface quickly, and the service life of the chromatography column is short. In addition, the types of cell surface receptors that have not been highly expressed by the target protein are complex, and it is difficult to determine the specific receptors of potential active drugs. The specificity of the cell membrane chromatography column is also low. At the same time, the current scope of application of the cell membrane chromatography column is limited to proteins expressed on the cell membrane, which will limit the research on intracellular proteins by the cell membrane chromatography column, thereby limiting the scope of application of the cell membrane chromatography column, making it difficult for traditional cell membrane chromatography columns to be widely used.
[0004] It has been reported that SNAP-tag labeling technology has been applied to cell membrane chromatography technology. SNAP-tag uses cysteine as a reaction site to specifically attack O 6 The benzyl group (BG) of the modified benzylguanine forms a stable thioether bond between them, so that the cell membrane and silica gel are bonded by covalent bonds, which improves the stability of the stationary phase. However, due to the limitations of biological and molecular mechanisms, this method cannot highly express intracellular proteins (such as FKBP1A protein present in the cytoplasm) on the cell membrane. Summary of the invention
[0005] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide an intracellular protein cell membrane stationary phase, a chromatographic column and a preparation method thereof based on SNAP-tag technology, and an application in blood drug concentration monitoring, so as to solve the technical problem that intracellular proteins cannot be highly expressed on the cell membrane autonomously.
[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention discloses a method for preparing an intracellular protein cell membrane stationary phase based on SNAP-tag technology, comprising the following steps: 1) A highly expressed SNAP-tag was fused to the N-terminus of the intracellular protein, and a GPI gene sequence was added to the 3' end of the intracellular protein gene sequence. Then, HEK293T cells were transfected, broken, and separated to obtain a SNAP-tag-intracellular protein highly expressed cell membrane; 2) The SNAP-tag-intracellular protein high-expression cell membrane obtained in step 1) is fixed on the benzylguanine-modified silica gel to obtain the intracellular protein cell membrane stationary phase based on the SNAP-tag technology.
[0007] Preferably, the intracellular protein is FKBP1A protein.
[0008] Preferably, in step 2), the SNAP-tag-intracellular protein high-expressing cell membrane obtained in step 1) is prepared into a SNAP-tag-intracellular protein high-expressing cell membrane suspension with physiological saline, which is then broken and then added to benzylguanine-modified silica gel to obtain an intracellular protein cell membrane stationary phase based on the SNAP-tag technology.
[0009] The second aspect of the present invention discloses an intracellular protein cell membrane stationary phase based on SNAP-tag technology obtained by the above preparation method.
[0010] The third aspect of the present invention discloses the application of the intracellular protein cell membrane stationary phase based on the SNAP-tag technology in the detection of blood drug concentration of intracellular protein ligand drugs.
[0011] The fourth aspect of the present invention discloses an intracellular protein cell membrane chromatography column based on SNAP-tag technology, wherein the intracellular protein cell membrane stationary phase based on SNAP-tag technology is wet packed to obtain an intracellular protein cell membrane chromatography column based on SNAP-tag technology.
[0012] The fifth aspect of the present invention discloses a blood drug concentration detection system for an intracellular protein ligand drug, comprising a first-dimensional separation detection system and a second-dimensional separation detection system, wherein the first-dimensional separation detection system comprises the above-mentioned intracellular protein cell membrane chromatography column based on the SNAP-tag technology, and the second-dimensional separation detection system comprises an HPLC / MS system.
[0013] Preferably, the first-dimensional separation and detection system is connected to the second-dimensional separation and detection system via a ten-way valve and an enrichment column.
[0014] The sixth aspect of the present invention discloses a method for detecting the blood concentration of an intracellular protein ligand drug, wherein the intracellular protein ligand drug and the mobile phase are sequentially injected into the intracellular protein cell membrane chromatography column based on the SNAP-tag technology, and then the enriched components are analyzed using an HPLC / MS system to achieve the blood concentration detection of the intracellular protein ligand drug.
[0015] Preferably, the column temperature of the intracellular protein cell membrane chromatography column based on SNAP-tag technology is 37°C, the mobile phase is water, and the flow rate is 0.2 mL / min.
[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for preparing a cell membrane stationary phase of intracellular proteins based on SNAP-tag technology, which combines glycosylated phosphatidylinositol (GPI) technology with SNAP-tag protein labeling technology, and uses genetic engineering technology to prepare a cell membrane with high expression of intracellular proteins; in this process, GPI and intracellular protein chimeric expression anchors the intracellular protein on the cell membrane surface through the glycosylated phosphatidylinositol structure at the carboxyl end of GPI, so that the intracellular protein can be autonomously highly expressed on the cell membrane. Then the cell membrane is mixed with silica gel modified with benzylguanine, and the SNAP-tag fusion receptor specifically reacts with benzylguanine to form a covalent bond, so that the cell membrane with intracellular proteins is covalently fixed on the silica gel surface. The cell membrane stationary phase finally formed not only makes it more convenient to use for membrane expression of intracellular proteins, but also can improve the specificity and stability of the cell membrane chromatographic column, prolong the life of the chromatographic column, and is better applied to the blood drug concentration detection of intracellular protein ligand drugs and subsequent drug screening.
[0017] The present invention provides a blood drug concentration detection system for an intracellular protein ligand drug, which combines an intracellular protein cell membrane stationary phase based on SNAP-tag technology with HPLC / MS. Through the separation and analysis of a tacrolimus solution, it is shown that the blood drug concentration detection system for an intracellular protein ligand drug can be used for subsequent monitoring of tacrolimus concentration in blood samples, providing technical support for further extensive application of cell membrane chromatography columns. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 is the plasmid map of the lentiviral expression vector containing hFKBP1A; Figure 2 is the plasmid map of the control lentiviral expression vector; Figure 3The figure is the result of virus titer determination of each group of concentrated virus solution; A is the number of fluorescence-positive cells in the concentrated virus solution containing hFKBP1A (0 time), B is the number of fluorescence-positive cells in the concentrated virus solution containing hFKBP1A (96 time), C is the number of fluorescence-positive cells in the control concentrated virus solution (0 time), and D is the number of fluorescence-positive cells in the control concentrated virus solution (96 time); Figure 4 This is a graph showing the protein bands and grayscale value statistics of FKBP1A and SNAP-tag in SNAP-tag-FKBP1A HEK293T cells detected by Western blot; wherein A is the protein band of FKBP1A expression, B is the grayscale value statistics of FKBP1A, C is the protein band of SNAP-tag expression, and D is the grayscale value statistics of SNAP-tag; Figure 5 Transmission electron micrographs of silica gel modified with benzylguanine and FKBP1A cell membrane chromatography column stationary phase based on SNAP-tag; the magnifications are 500 times and 40,000 times; wherein, transmission electron micrographs of silica gel modified with benzylguanine (a, c) and FKBP1A cell membrane chromatography column stationary phase based on SNAP-tag (b, d); the magnifications of (a, b) are 500 times, and the magnifications of (c, d) are 40,000 times; Figure 6 The chromatograms of quetiapine fumarate, prazosin hydrochloride and tacrolimus (FK506) solutions on SNAP-tag-FKBP1A / CMC columns, and the chromatograms of tacrolimus solutions on benzylguanine-modified silica gel columns and NC-HEK293T / CMC columns; wherein, (a) the chromatograms of quetiapine fumarate, prazosin hydrochloride and tacrolimus solutions on SNAP-tag-FKBP1A / CMC columns, (b) the chromatograms of tacrolimus solutions on benzylguanine-modified silica gel columns and NC-HEK293T / CMC columns; Figure 7 The results of the analysis of the tacrolimus reference solution on the SNAP-tag-FKBP1A / CMC-HPLC / MS two-dimensional system are shown in the figure; (a) the retention behavior of the tacrolimus reference solution on the SNAP-tag-FKBP1A / CMC column; (b) the chromatogram and main ions of the retained component R1 after enrichment switching on the HPLC-MS system; (c) the chromatogram of the tacrolimus reference solution directly injected into the HPLC. DETAILED DESCRIPTION
[0019] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.
[0020] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.
[0021] In this article, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values within the range (including integers and fractions).
[0022] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”
[0023] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.
[0024] The present invention provides an intracellular protein cell membrane stationary phase based on SNAP-tag technology, which is prepared by the following method: 1. Using genetic engineering technology, a highly expressed SNAP-tag was fused to the N-terminus of the intracellular protein, and then the GPI gene sequence was added to the 3' end of the intracellular protein gene sequence, and HEK293T cells were transfected to obtain SNAP-tag-intracellular protein highly expressed cells, which were cultured, broken, and separated to obtain SNAP-tag-intracellular protein highly expressed cell membranes. 2. Aminopropylated silica gel was prepared into cyanuric chloride modified silica gel, which was then reacted with 6-[[4-(aminomethyl)phenyl]methoxy]-7H-purine-2-amine to obtain benzylguanine modified silica gel (SiO2-BG).
[0025] 3. The SNAP-tag-intracellular protein highly expressed cell membrane was prepared into a SNAP-tag-intracellular protein highly expressed cell membrane suspension with physiological saline, and the SNAP-tag-intracellular protein highly expressed cell membrane suspension was added to the silica gel modified with benzylguanine to fix the intracellular protein indirectly on the silica gel surface in a direction, thereby obtaining the intracellular protein cell membrane stationary phase based on the SNAP-tag technology.
[0026] The present invention also provides an intracellular protein cell membrane chromatography column based on SNAP-tag technology, and the intracellular protein cell membrane stationary phase based on SNAP-tag technology is loaded into the column by a wet method to obtain an intracellular protein cell membrane chromatography column based on SNAP-tag.
[0027] The present invention also provides a blood drug concentration detection system for an intracellular protein ligand drug, comprising an HPLC / MS system, a ten-way valve, an enrichment column and the intracellular protein cell membrane chromatography column based on the SNAP-tag technology, wherein the HPLC / MS system is connected to the intracellular protein cell membrane chromatography column based on the SNAP-tag technology via the ten-way valve and the enrichment column.
[0028] The present invention also provides an application of the intracellular protein cell membrane chromatography column based on the SNAP-tag technology in conjunction with HPLC in the detection of blood drug concentration of intracellular protein ligand drugs.
[0029] The present invention is further described below in conjunction with specific examples, using FKBP1A as an intracellular protein and tacrolimus as a FKBP1A ligand drug. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.
[0030] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples are conventional commercial products unless otherwise specified, and their specifications are conventional specifications in the art. In the specification of the present invention and the following examples, unless otherwise specified, "%" means weight percentage, and ratio means weight ratio.
[0031] The experimental methods in the following examples where specific conditions are not specified are usually carried out according to the conventional conditions described in the Molecular Cloning Experiment Guide or the conditions recommended by the reagent manufacturers.
[0032] The backbone vector LV-EFS-CMV>EGFP / T2A / Puro used in the following examples was provided by Saiye (Guangzhou) Biotechnology Co., Ltd. The lentiviral expression vector containing hFKBP1A (LV-EFS>Kozak-MLS / SNAP tag / HumanFKBP1A-GPI-CMV>EGFP / T2A / Puro) and the control lentiviral expression vector LV-EFS>Kozak-MLS / SNAP tag / GPI-CMV>EGFP / T2A / Puro were both constructed by Saiye (Guangzhou) Biotechnology Co., Ltd.
[0033] 1. Construction of HEK293T cells with high expression of SNAP-tag-FKBP1A 1. Construction of lentiviral expression vector 1) Double digest the backbone vector LV-EFS-CMV>EGFP / T2A / Puro with restriction endonucleases XhoI and AscI to recover the linearized vector.
[0034] 2) According to the CDS sequence of Human FKBP1A (hFKBP1A; CDS: NM_000801.5) in the NCBI gene database, PCR primers were designed according to the primer design principles. The upstream primer was hFKBP1A-F, and the sequence was shown in SEQ ID NO.1. The downstream primer was hFKBP1A-R, and the sequence was shown in SEQ ID NO.2 to obtain Human FKBP1A. By fusion PCR, the SNAP-tag sequence was introduced into the N-terminus of the Human FKBP1A protein. According to the primer design principles, the upstream primer was MLS SNAPtag-F, and the sequence was shown in SEQ ID NO.3. The downstream primer was MLS SNAPtag-R, and the sequence was shown in SEQ ID NO.4 to obtain SNAPtag-Human FKBP1A. By fusion PCR, the SNAP-tag sequence was introduced into the 3' end of the Human FKBP1A protein gene sequence. GPI The gene, the sequence of which is shown in SEQ ID NO.5, was amplified by PCR to obtain the target gene SNAP tag-Human FKBP1A-GPI. The target gene fragment was amplified using primers with homologous recombination sequences, the Kozak sequence was introduced into the 5' end of the upstream primer, and the AscⅠ restriction site was introduced into the 5' end of the downstream primer. The upstream primer and the downstream primer respectively contained sequences homologous to the backbone vector LV-EFS-CMV>EGFP / T2A / Puro, and the amplified target gene fragment SNAP tag-Human FKBP1A-GPI was recovered by nucleic acid electrophoresis.
[0035] 3) Under the action of DNA ligase, the linearized vector recovered in step 1) and the amplified SNAP tag-Human FKBP1A-GPI were connected, and the ligation product was transformed into E. coli competent cells, coated with ampicillin (Amp) resistant LB plates, and cultured at 37°C overnight. The clones on the plate were picked for PCR identification, and the positive clones were identified by restriction digestion and sequencing. The correct clones were extracted to obtain a lentiviral expression vector containing hFKBP1A (LV-EFS>Kozak-MLS / SNAPtag / Human FKBP1A-GPI-CMV>EGFP / T2A / Puro) for subsequent virus packaging. The constructed plasmid map is shown below. Figure 1 shown.
[0036] Table 1 Sequence Listing
[0037] At the same time, LV-EFS>Kozak-MLS / SNAP tag / GPI-CMV>EGFP / T2A / Puro was used as the control lentiviral expression vector, and the plasmid map was as follows Figure 2 shown.
[0038] 2. Packaging lentivirus HEK293T cells were cultured to the logarithmic phase, and the lentiviral expression vector containing hFKBP1A and the packaging plasmid pMD2G were co-transfected into HEK293T cells; at the same time, the control lentiviral expression vector and the packaging plasmid pMD2G were co-transfected into HEK293T cells as a control; each group of cells was cultured for 72 hours, and the cell supernatant was collected, and impurities were removed by microfiltration and high-speed centrifugation to obtain concentrated virus solution containing hFKBP1A and control concentrated virus solution, respectively.
[0039] 3. Virus titer determination The concentrated virus solution of each group was diluted in series and infected with HEK293T cells. The number of fluorescent positive cells was calculated after 96 hours, and the number of active virus particles per milliliter of virus solution was converted according to the dilution multiple. The packaged lentivirus was infected with HEK293T cells, and the overexpression was verified by qPCR.
[0040] The results are as follows Figure 3 As shown, the viral transduction effect of each experimental group was good, and the fluorescence rate was more than 80%.
[0041] 4. Sensitivity determination The target HEK293T cells were cultured to the vigorous growth phase, and 1 μg / mL Puromycin (Puro) was used as the screening drug to determine the sensitivity of the cells to the antibiotic and determine the concentration for screening. After the drug screening was completed, the concentration was maintained at 0.5 μg / mL until the cells were frozen.
[0042] 5. Construction of HEK293T cells with high expression of SNAP-tag-FKBP1A The packaged lentiviral solution of each group was used to infect HEK293T cells at a known MOI value; the medium was changed and 1 μg / mL Pro was added for screening for 2-3 weeks (thereafter, resistance medium was used for screening) to obtain SNAP-tag-FKBP1A HEK293T cells and NC-HEK293T cells, respectively.
[0043] 6. Cell detection 1) qPCR validation During the qPCR verification process, the designed upstream primer of hGAPDH is hGAPDH-F, the sequence is shown in SEQ ID NO.6, and the downstream primer is hGAPDH-R, the sequence is shown in SEQ ID NO.7. Take some cells for the first qPCR test verification; maintain the screening and expand the culture, take some cells for the second qPCR test verification, and the second qPCR result is required to be no worse than the first 2 Ct, which is considered stable and qualified. Collect qualified cells and freeze them for seed preservation.
[0044] The results of qPCR are shown in Table 1. In the constructed cells, the expression level of the hFKBP1A gene was 14.51305 times that of the control group (NC-HEK293T cells), indicating that the SNAP-tag-FKBP1A high-expression HEK293T cells were successfully constructed.
[0045] Table 1 qPCR detection results of FKBP1A
[0046] 2) Western blot verification To further confirm that FKBP1A was also overexpressed at the protein level, membrane proteins of NC-HEK293T and SNAP-tag-FKBP1A overexpressing HEK293T cells were first extracted, and then Western blot experiments were performed. The statistical results of the obtained protein bands and their grayscale values are shown in Figure 2. Figure 4 shown.
[0047] Table 1 and Figure 4 The results showed that SNAP-tag-FKBP1A high-expression HEK293T cells were successfully constructed.
[0048] 2. Preparation of FKBP1A Cell Membrane Column Stationary Phase (CMSP) Based on SNAP-tag 1. Preparation of cell membrane SNAP-tag-FKBP1A HEK293T cells (no less than 2 × 10) in the logarithmic growth phase were collected by 0.25% trypsin digestion. 7 ), 4℃, 1500 rpm centrifugation for 5 min, then discard the culture medium, then wash the cells twice with normal saline by centrifugation, add 20 mmol / L Tris-HCl to the cell pellet, resuspend it on ice, and disrupt it with a cell disruptor (power: 150 W, 5 times), 4℃, 5000 rpm centrifugation for 10 min, transfer the supernatant cell membrane solution to a new centrifuge tube, 4℃, 12000g centrifugation for 20 min, and obtain SNAP-tag-FKBP1A HEK293T cell membrane pellet.
[0049] 2. Preparation of SiO2-BG 1.4 g of amino silica gel (SiO2-NH2) was added to 14 mL of tetrahydrofuran and cooled to 0°C in an ice bath. Then, 0.36 g of cyanuric chloride (TCT) and 1.4 mL of N,N-diisopropylethylamine were slowly added while stirring, and the reaction temperature was kept below 4°C. After 2 h of reaction, the product was washed with anhydrous ethanol to ensure that no excess cyanuric chloride was adsorbed on the silica gel to obtain a cyanuric chloride-modified silica gel stationary phase (SiO2-TCT). 2 g of SiO2-TCT was dissolved in 50 mL of DMF, and 50 mg of 6-[[4-(aminomethyl)phenyl]methoxy]-7H-purine-2-amine was added. After reflux at 95°C for 8 h, the product was filtered and repeatedly washed with DMF to obtain SiO2-BG.
[0050] 3. Preparation of FKBP1A cell membrane chromatography column stationary phase based on SNAP-tag The SNAP-tag-FKBP1A HEK293T cell membrane precipitate obtained in step 1 was prepared into a cell membrane suspension with physiological saline, placed on ice and disrupted with a cell disruptor (power: 200 W, 5 times), then added with 50 mg of the benzylguanine-modified silica gel obtained in step 2, shaken at 37°C for 30 min and allowed to stand overnight to obtain the SNAP-tag FKBP1A cell membrane chromatography column stationary phase.
[0051] Meanwhile, according to the aforementioned method, NC-HEK293T cells were used to prepare the NC cell membrane chromatography column stationary phase.
[0052] 4. Characterization of the stationary phase of FKBP1A cell membrane chromatography column based on SNAP-tag Transmission electron microscopy (TEM) was used to characterize the stationary phase of the FKBP1A cell membrane chromatography column modified with benzylguanine and SNAP-tag.
[0053] The results are as follows Figure 5 As shown, the surface of amino silica gel stationary phase after benzylguanine modification is rough ( Figure 5 a and c), and after reacting with the cell membrane solution, the surface of the stationary phase becomes significantly smoother and can be clearly observed to be covered by a layer of membrane. Since the silica gel not coated with the cell membrane is porous silica gel, the surface pores are obviously covered after bonding with the cell membrane, and the surface of the stationary phase appears smooth ( Figure 5 b and d), thus the prepared SNAP-tag FKBP1A cell membrane chromatography column stationary phase was successfully prepared.
[0054] 3. Establishment of SNAP-tag-based FKBP1A cell membrane chromatography system (SNAP-tag-FKBP1A CMC) 1. Establishment of SNAP-tag-FKBP1A CMC The SNAP-tag FKBP1A cell membrane chromatography column stationary phase obtained in step 2 was wet-packed into a 10 mm × 2.0 mm (ID) column core using a column packing machine, and a matching column sleeve was placed in it. The column sleeve nut was tightened to obtain a cell membrane chromatography stationary phase, which was then connected to a liquid chromatography system to obtain SNAP-tag-FKBP1A CMC.
[0055] 2. Verification of the effect of SNAP-tag-FKBP1A CMC The retention time of tacrolimus, a positive drug for FKBP1A receptor, was used as an indicator to investigate the selectivity, specificity, and intra-column and inter-column variability of the SNAP-tag-FKBP1A cell membrane chromatography column.
[0056] A benzylguanine-modified silica column without cell membrane, an NC cell membrane column, and a SNAP-tag-based FKBP1A cell membrane column were prepared respectively. After the three columns were fully balanced, 2 μL of 0.5 mg / mL positive drug reference solution was injected respectively. The mobile phase was water at a flow rate of 0.2 mL / min. The retention chromatograms and retention times of the positive drugs on the three columns were recorded. Subsequently, 2 μL of 0.5 mg / mL quetiapine fumarate and 2 μL of prazosin hydrochloride were injected on the SNAP-tag-FKBP1A cell membrane column respectively. The obtained chromatograms and drug retention times were compared with those of the positive drug tacrolimus.
[0057] The results are as follows Figure 6As shown, the results indicate that the prepared SNAP-tag-FKBP1A cell membrane chromatography column has good specificity and selectivity.
[0058] To detect intra-column variability, a SNAP-tag-FKBP1A cell membrane chromatography column was prepared, and 2 μL of 0.5 mg / mL tacrolimus solution was injected continuously for 6 times after equilibrium, and the retention time was recorded. To detect inter-column variability, three SNAP-tag-FKBP1A cell membrane chromatography columns were prepared simultaneously according to the same method, and 2 μL of 0.5 mg / mL tacrolimus solution was injected respectively, and the retention time of the positive drug was recorded.
[0059] The results are shown in Table 2. The prepared chromatographic column has good intra-column and inter-column variability and can be used for screening and analysis of active ingredients in complex systems.
[0060] Table 2 Intra-column and inter-column repeatability of SNAP-tag-FKBP1A / CMC column
[0061] IV. Establishment of SNAP-tag-FKBP1A CMC / HPLC / MS system The SNAP-tag-FKBP1A CMC system and HPLC / MS were coupled via a ten-way valve and an enrichment column (Shimpack VP-ODS; purchased from Shimadzu, Japan) to form an online two-dimensional coupling system. The operation of the system is as follows: In the first step, the analyte is injected onto the SNAP-tag-FKBP1A CMC column, and the retained components are cut into the enrichment column for enrichment, while the C18 column in the two-dimensional system is balanced, and then the components enriched in the enrichment column are further separated and identified by HPLC-MS after switching the ten-way valve. The workflow is shown below.
[0062] First-dimension cell membrane chromatography conditions: chromatographic column: SNAP-tag-FKBP1A CMC column (10 × 2.0 mm ID); mobile phase: water; flow rate: 0.2 mL / min; column temperature: 37°C.
[0063] Second-dimension reversed-phase conditions: chromatographic column: Waters SunFire® C18 (150 × 4.6 mm ID, 5.0 µm); 60% 0.1% formic acid in water, 40% acetonitrile, flow rate: 1 mL / min.
[0064] The specific parameters of the second-dimensional mass spectrometry detection are as follows: equipped with an electrospray ion source, using positive and negative ion scanning modes, with a mass scanning range of 200-1000 m / z, a nebulizer gas of high-purity nitrogen (purity>99.999%) with a flow rate of 3 L / min, a drying gas pressure of 109 kPa, an interface voltage of 4.5 kV, and a detector voltage of 1.57 kV; DL temperature: 250°C.
[0065] Inject 2 μL of 0.5 mg / mL tacrolimus solution into the first-dimension cell membrane chromatography column. When retention is observed on the cell membrane chromatography column, switch the ten-way valve to connect the cell membrane chromatography column to the enrichment column. After the enrichment is completed, switch the ten-way valve again to restore the enrichment column to the C18 column, so that the enriched components enter the HPLC / MS system for analysis. By analyzing the C18 column obtained in the second dimension and measuring the peak area of the chromatographic peak in the chromatogram, the specific amount of tacrolimus in the sample can be obtained.
[0066] Chromatogram and mass spectrum Figure 7 As shown, the results show that the established SNAP-tag-FKBP1A CMC / HPLC / MS system can be used for the separation and analysis of tacrolimus solution and can be used for the subsequent monitoring of tacrolimus concentration in blood samples.
[0067] The above contents are only for explaining the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.
Claims
1. A method for preparing an intracellular protein cell membrane stationary phase based on SNAP-tag technology, characterized in that: The following steps are involved: 1) A highly expressed SNAP-tag was fused to the N-terminus of the intracellular protein, and a GPI gene sequence was added to the 3' end of the intracellular protein gene sequence. Then, HEK293T cells were transfected, broken, and separated to obtain a SNAP-tag-intracellular protein highly expressed cell membrane; 2) The SNAP-tag-intracellular protein high-expression cell membrane obtained in step 1) is fixed on the benzylguanine-modified silica gel to obtain the intracellular protein cell membrane stationary phase based on the SNAP-tag technology.
2. The method for preparing an intracellular protein cell membrane stationary phase based on SNAP-tag technology according to claim 1, characterized in that: The intracellular protein is FKBP1A protein.
3. The method for preparing an intracellular protein cell membrane stationary phase based on SNAP-tag technology according to claim 1, characterized in that: In step 2), the SNAP-tag-intracellular protein high-expressing cell membrane obtained in step 1) is prepared into a SNAP-tag-intracellular protein high-expressing cell membrane suspension with physiological saline, which is then broken and added to benzylguanine-modified silica gel to obtain an intracellular protein cell membrane stationary phase based on the SNAP-tag technology.
4. An intracellular protein cell membrane stationary phase based on SNAP-tag technology obtained by the preparation method according to any one of claims 1 to 3.
5. Use of the intracellular protein cell membrane stationary phase based on SNAP-tag technology as claimed in claim 4 in the detection of blood drug concentration of intracellular protein ligand drugs.
6. An intracellular protein cell membrane chromatography column based on SNAP-tag technology, characterized in that: The intracellular protein cell membrane stationary phase based on the SNAP-tag technology described in claim 4 is packed by a wet method to prepare an intracellular protein cell membrane chromatography column based on the SNAP-tag technology.
7. A blood drug concentration detection system for intracellular protein ligand drugs, characterized in that: It comprises a first-dimensional separation and detection system and a second-dimensional separation and detection system, wherein the first-dimensional separation and detection system comprises the intracellular protein cell membrane chromatography column based on the SNAP-tag technology according to claim 6, and the second-dimensional separation and detection system comprises an HPLC / MS system.
8. A blood drug concentration detection system for intracellular protein ligand drugs according to claim 7, characterized in that: The first-dimension separation and detection system and the second-dimension separation and detection system are connected through a ten-way valve and an enrichment column.
9. A method for detecting the blood concentration of an intracellular protein ligand drug, characterized in that: The intracellular protein ligand drug and the mobile phase are sequentially injected into the intracellular protein cell membrane chromatography column based on the SNAP-tag technology as described in claim 6, and then the enriched components are analyzed using an HPLC / MS system to detect the blood concentration of the intracellular protein ligand drug.
10. The method for detecting the blood concentration of an intracellular protein ligand drug according to claim 9, characterized in that: The column temperature of the intracellular protein cell membrane chromatography column based on SNAP-tag technology was 37°C, the mobile phase was water, and the flow rate was 0.2 mL / min.