CN108977498B - Method for determining enzyme inhibition activity of aminopeptidase N inhibitor and application thereof - Google Patents
Method for determining enzyme inhibition activity of aminopeptidase N inhibitor and application thereof Download PDFInfo
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Abstract
The invention provides a method for measuring the inhibition rate of a compound on the activity of aminopeptidase N, which takes a K562-CD13 cell strain expressing human-derived CD13 as an enzyme source to carry out the activity inhibition measurement of the compound. The invention also provides a kit capable of determining the inhibition rate of the compound on the aminopeptidase N activity, and the kit adopts the method for determination. The technical scheme of the invention adopts human-derived CD13 as an enzyme source to evaluate the inhibitory activity effect of the compound, has higher accuracy than the detection method adopting swine-derived CD13 in the prior art, and has more economic cost and more accurate result.
Description
Technical Field
The invention relates to a method for determining the enzyme inhibitory activity of an inhibitor, in particular to a method for determining the enzyme inhibitory activity of an aminopeptidase N inhibitor, a method for preparing an enzyme source and application thereof.
Background
Aminopeptidase N (Aminopeptidase N, APN/CD13, EC 3.4.11.2) is a zinc ion-dependent transmembrane metalloprotease that is localized to the outer surface of the cell membrane by non-covalent binding as a homodimer. Being able to hydrolyze oligodermal, and in particular degrading neutral amino acids from the N-terminus of the small skin chain, was first considered to be a surface marker of hematopoietic cells of the myeloid system, which are distributed in a variety of cells and tissues, including hematopoietic cells, fibroblasts, brain tissue cells, hepatocytes, osteoclasts, endometrial cells, epithelial cells of the liver and kidney intestine, synaptic membranes of the central nervous system, etc., and because of their wide distribution, their function depends mainly on their distribution.
Erythroleukemia K562 cells were the first human myeloid leukemia cultured cells derived from lymphoblasts of a female patient aged 53 years in the outbreak phase of chronic myeloid leukemia. Is used for the fields of tumor and leukemia treatment, drug target and the like.
There are many methods for determining enzyme activity, and since enzymes have high specific catalytic activity, the activity of an enzyme can be determined by measuring the change in the concentration of its corresponding substrate or product, or by using the change in the concentration of a reaction product. Examples of the method include reduction, chromogenic substrate, viscosity, high pressure liquid chromatography, immunological methods, and agar gel diffusion. Electrochemical and photophysical methods are generally used, i.e. the absorbance of the reactants or products is used, and the measurement is carried out by ultraviolet spectrophotometry or fluorescence. If the product or reactant has gas during the enzymatic reaction, it can be measured by a pressure gauge (Wagner's respirometer). If an acid is formed during the reaction, electrochemical methods may be used. The substrate labeled with an isotope can be subjected to a radiochemical method for measuring the change in the concentration of the substrate and calculating the enzyme activity. Some enzymes with stable properties can also be detected by high performance liquid chromatography.
The traditional method for determining the compound to inhibit the activity of the aminopeptidase N enzyme needs to continuously purchase enzyme standard products, is expensive, is easy to lose efficacy and is difficult to store.
Lentivirus (Lentivirus) vectors are gene therapy vectors developed based on HIV-1 (human immunodeficiency virus type I). The Lentivirus (Lentivirus) adopted by the invention is a suicide virus, the toxic gene of the Lentivirus is deleted and replaced by an exogenous target gene, the Lentivirus does not have the capability of infecting other cells after infecting target cells, and does not utilize host cells to generate new virus particles, thereby belonging to a pseudotype virus.
Disclosure of Invention
The invention aims to provide a method for determining the enzyme inhibition activity of a compound targeting aminopeptidase N, which is economical in cost and simple in operation. Most enzyme standards provided in the enzyme activity determination kit in the prior art are aminopeptidase N extracted from pig nephrosomes, and compared with human aminopeptidase N, the enzyme standards are greatly different and expensive. Compared with the prior art in which non-human aminopeptidase N is used as an evaluation tool, the method disclosed by the invention has the advantage that the measured enzyme inhibition activity data is more reliable.
An object of the present invention is to provide a method for measuring the inhibition rate of aminopeptidase N activity by a compound, which is characterized by measuring the activity of the compound against an enzyme source of K562-CD13 cell line capable of stably expressing human-derived CD 13.
The method for determining the inhibition rate of the compound on the aminopeptidase N activity comprises the following steps:
(1) inoculating the K562-CD13 cells washed by PBS into a 96-well plate;
(2) adding compounds of different concentration gradients to the wells;
(3) adding a CD13 substrate for culturing after the compound acts for a period of time;
(4) the absorbance value at 405nm was measured and the inhibition was calculated:
the average OD value of the added medicine group is the average OD value of the over-expressed added medicine group-the average OD value of the non-over-expressed added medicine group
Mean OD value of non-drug addition group-mean OD value of overexpression group-mean OD value of non-overexpression group
Preferably, the K562-CD13 cells are seeded in 96-well plate at density of 1 × 10 in the step (1) above 5 Per well.
Preferably, after the compound is added in step (3) for 5min, the CD13 substrate is added to 1.6mM, and the mixture is cultured at 37 ℃ for 30 min.
The second objective of the invention is to provide a method for constructing the K562-CD13 cell, which comprises the following steps:
(1) adding complete culture solution to resuspend K562 cells, and inoculating the cells in a 96-well plate for culture;
(2) adding a recombinant lentivirus LV-CD13 diluent into the hole, adding Polybrene serving as a virus infection auxiliary agent, and culturing overnight;
(3) three days after infection, puromycin was added to screen virus-infected K562 cells to obtain a K562-CD13 cell line stably expressing CD13 and puromycin resistance.
Preferably, in the above construction method, the K562 cells in step (1) are at a ratio of 5X 10 3 The density of each well was seeded in 96-well plates in a volume of 80. mu.L.
Preferably, in the above construction method, the preparation method of the recombinant lentivirus LV-CD13 diluent in step (2) comprises: the concentration is 2X 10 8 TU/ml stock solution of recombinant lentivirus LV-CD13, diluted with ENi.S to give an MOI of 40.
Preferably, in the above construction method, 10. mu.L of the recombinant lentivirus diluent and 10. mu.L of the viral infection helper solution Polybrene are added to each well in step (2).
Preferably, in the above construction method, the specific operation of step (3) is as follows: and replacing the fresh culture solution for continuous culture on the next day, replacing the fresh culture solution again on the third day until three days after infection, adding puromycin to screen virus-infected K562 cells, replacing puromycin with the culture solution with the maintenance concentration of 1.0mg/ml when all cells of a control group die after 2 weeks, and continuously performing passage amplification, thereby obtaining the K562-CD13 cell strain capable of stably expressing CD13 and puromycin resistance. Screening and identifying positive cell monoclonals from the successfully constructed K562-CD13 cells to obtain a K562-CD13 cell strain with high CD13 expression.
Preferably, the positive monoclonal identification method is bright field fluorescence imaging of K562-CD13 cells, enzyme activity trend comparison of K562-CD13 and K562 cells and Western Blotting detection of CD13 protein expression level.
The invention also aims to provide a detection kit for determining the inhibition of the aminopeptidase N activity by the compound, which comprises a PBS solution, a 96-well plate and a CD13 substrate, and is characterized by further comprising a K562-CD13 cell strain obtained by the construction method.
The fourth object of the present invention is to provide the use of the above-mentioned method for measuring the inhibitory activity of a compound in the evaluation of CD 13-inhibitor drugs. The method provided by the invention adopts a K562-CD13 cell strain expressing humanized CD13 as an enzyme source, and detection standard products do not need to be purchased repeatedly; and the enzyme source in the invention is human-derived CD13, so that the evaluation result is more accurate compared with the prior art in which pig-derived CD13 is adopted. Provides a more economic and more accurate tool for the evaluation of the CD13 inhibitor, and can be applied to the field of evaluation of CD13 inhibitor drugs.
The invention has the advantages of
1. The determination of the enzymatic inhibitory activity of the CD13 inhibitors in the prior art requires the purchase of kits for the evaluation of the enzymatic inhibitory activity. The enzyme activity kit in the prior art has the following problems:
(1) the enzyme provided by the kit is porcine-derived CD13, the porcine-derived CD13 has different structures and different protein sequences, and the inhibition effect of the compound on porcine-derived CD13 is different from the inhibition effect of human-derived CD13, so that the data of the enzyme inhibition activity measured by the kit in the prior art is not reliable, and the inventors have made relevant studies and published the above conclusion in SCI (Wang X, Zhang L, Yang K, et al.
The enzyme source provided by the method is human-derived CD13, and the enzyme source is more accurate as an evaluation tool of a compound inhibitor, and is economical in cost and convenient to use.
(2) In the prior art, the reagent kit is expensive and is imported by Sigma company, and the method for providing the enzyme source is more economic in cost.
And 2, the K562 cells are cells growing in suspension, and the operation is simple. Most cells belong to adherently growing cells, for example, as an enzyme source, the activity of a CD13 inhibitor is measured, and the culture is relatively complicated.
The CD13 belongs to membrane protein, is expressed on the outer surface of a cell membrane, can be combined with CD13 on the surface of the cell membrane only by adding an inhibitor into a culture solution, and evaluates the activity of the inhibitor.
4. K562 cells capable of stably expressing a certain protein are constructed in the prior art, are often used as basic tools for leukemia research, and can be used for screening diagnosis and prognosis judgment indexes, action mechanisms of targets on leukemia and the like. It can also be used to construct high purity protein controls, and the enzyme source of the compound of the present invention that inhibits enzyme activity has not been described in the prior art. Based on the suspension characteristics of K562 cells and the characteristics of CD13 in vitro expression, the inventors conjecture that K562-CD13 is used as an enzyme source for determining the activity of the compound in inhibiting the enzyme, and an unexpected technical effect is achieved. And the detection of the catalytic efficiency of different cells on the CD13 substrate shows that K562 cells have lower catalytic efficiency on the substrate used by the invention, and the K562 cells are used as carrier cells of a detection tool, have low background and have smaller influence on the detection precision.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, are included to provide a further understanding of the application, and the description of the exemplary embodiments and illustrations of the application are intended to explain the application and are not intended to limit the application.
FIG. 1 is a photograph of K562-CD13 cells imaged by bright field imaging, fluorescence imaging and superposition of the two;
FIG. 2 is a photograph of Western blots of K562-CD13 and K562 cells;
FIG. 3 shows the comparison of the enzyme activity trends of K562-CD13 and K562 cells;
FIG. 4 shows the inhibition rate of positive drug Bestatin on the activity of CD13 in K562-CD13 cells.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As introduced in the background art, the enzyme activity determination kit in the prior art has the defects of high manufacturing cost, accuracy and the like, and in order to solve the technical problems, the application provides a method for evaluating the activity of the compound enzyme inhibitor by taking K562-CD13 as an enzyme source.
In a typical embodiment of the present invention, there is provided a method for measuring the inhibition rate of aminopeptidase N activity by a compound, which comprises measuring the activity of the compound against an enzyme source K562-CD13 cell line capable of stably expressing human CD13, the method comprising the steps of:
(1) inoculating the K562-CD13 cells washed by PBS into a 96-well plate;
(2) adding compounds of different concentration gradients to the wells;
(3) adding a CD13 substrate for culturing after the compound acts for a period of time;
(4) the absorbance value at 405nm was measured and the inhibition was calculated:
the average OD value of the added medicine group is the average OD value of the over-expressed added medicine group-the average OD value of the non-over-expressed added medicine group
Mean OD value of non-drug addition group-mean OD value of overexpression group-mean OD value of non-overexpression group
In a preferred embodiment, the K562-CD13 cells are seeded in a 96-well plate at a seeding density of 1X 10 in step (1) above 5 Per well.
In a preferred embodiment, the compound is added in step (3) for 5min, and then the substrate CD13 is added to 1.6mM, and cultured at 37 ℃ for 30 min.
In another embodiment of the present invention, a method for constructing the K562-CD13 cell is provided, which comprises the following steps:
(1) adding complete culture solution to resuspend K562 cells, and inoculating the cells in a 96-well plate for culture;
(2) adding a recombinant lentivirus LV-CD13 diluent into the hole, adding Polybrene serving as a virus infection auxiliary agent, and culturing overnight;
(3) three days after infection, puromycin was added to select virus-infected K562 cells, thereby obtaining a K562-CD13 cell strain stably expressing CD13 and puromycin resistance.
In a preferred embodiment, the K562 cells in step (1) are present at 5X 10 3 The density of each well was seeded in 96-well plates in a volume of 80. mu.L.
In a preferred embodiment, the preparation method of the recombinant lentivirus LV-CD13 diluent in the step (2) comprises the following steps: the concentration is 2X 10 8 TU/ml stock solution of recombinant lentivirus LV-CD13, diluted with ENi.S to give an MOI of 40.
In a preferred embodiment, 10. mu.L of the recombinant lentivirus diluent and 10. mu.L of Polybrene as a virus infection helper are added to each well in step (2).
In a preferred embodiment, the specific operation of step (3) is as follows: replacing the fresh culture solution for continuous culture on the next day, replacing the fresh culture solution again on the third day until three days after infection, adding puromycin to screen virus-infected K562 cells, replacing the culture solution with puromycin maintaining concentration of 1.0mg/ml after 2 weeks until all cells of a control group die, and continuously performing generation and amplification to obtain the K562-CD13 cell strain capable of stably expressing CD13 and puromycin resistance. Screening and identifying positive cell monoclonals from the successfully constructed K562-CD13 cells to obtain a K562-CD13 cell strain with high CD13 expression.
In a preferred embodiment, the method for identifying the positive monoclonal is K562-CD13 cell bright field fluorescence imaging, enzyme activity trends of K562-CD13 and K562 cells are compared, and Western Blotting is used for detecting the expression level of the CD13 protein.
In another embodiment of the present invention, there is provided a test kit for determining inhibition of aminopeptidase N activity by a compound, comprising a PBS solution, a 96-well plate, a CD13 substrate, and a K562-CD13 cell line obtained by the above-described construction method.
In another embodiment of the present invention, there is provided a use of the aforementioned method for determining the activity of a compound aprotinin in the evaluation of a CD13 inhibitor drug.
The recombinant lentivirus LV-CD13 used in the present invention was purchased from Kjekay GeneChemicals, Inc. of Shanghai.
The present invention will be further illustrated with reference to the following specific examples, which are intended to illustrate the invention and not to limit the invention, and the simple modifications of the method for preparing the enzyme source and the method for determining the enzyme inhibitory activity of the compound according to the present invention based on the concept of the present invention are within the scope of the present invention as claimed.
The invention measures the enzyme inhibitory activity of the compound by taking lentivirus infected K562 cells to over-express CD13 as an enzyme source. The expression level of the CD13 protein in the K562-CD13 cells is increased through bright field fluorescence imaging of the K562-CD13 cells, enzyme activity trend comparison of the K562-CD13 cells and the K562 cells and Western Blotting detection of CD13 protein expression level changes.
The CD13 substrate in the following examples was purchased from Sigma under the trade name: l-leucoine-p-nitroanilide (L9125; Sigma, St Louis, MO, USA), Chinese name: l-leucine-p-nitroaniline.
Example 1: and (4) screening suitable cell vectors.
In order to improve the accuracy of enzyme activity determination, the invention inspects the carrier cells for constructing protein expression, and the inspected objects comprise K562, PLC, MCF7, A549 and EA.hy926. Cells in logarithmic growth phase were washed and counted at 1X 10 5 The number of each well was seeded in a 96-well plate, and CD13 substrate was added to 1.6mM, and the absorbance of each cell was measured in parallel in three wells at 0h, 0.5h, 1h, and 1.5h, respectively, with the results shown in table 1:
TABLE 1 ability of different cells to catalyze the luminescence of CD13 substrate
As can be seen from the results in Table 1, the K562 cell has the weakest catalytic ability on the CD13 substrate, and has the smallest influence on the color development result when being used as an enzyme activity detection tool. The prior art does not disclose the types and the quantity of the enzymes on the surface of the K562 cell membrane, and the invention proves that the application of the K562 to the determination of the CD13 enzyme activity has unobvious superiority through experiments.
Example 2: a K562 monoclonal cell line overexpressing CD13 was established using the methods described herein.
1. Resuspending K562 cells in complete medium, seeding K562 cells in 96-well plates, 5X 10 3 One/well, volume 80. mu.L.
2. The stock solution of the recombinant lentivirus LV-CD13 is 2 x 10 8 TU/mL, diluted with ENi.S to give an MOI of 40, 10. mu.L of recombinant lentivirus LV-CD13 dilution per well, and 10. mu.L of Polybrene as a viral infection adjuvant were added and incubated overnight.
3. And replacing the fresh culture solution for continuous culture on the next day, replacing the fresh culture solution again on the third day until the recombinant lentivirus LV-CD13 is infected for three days, adding puromycin to screen virus-infected K562 cells, replacing puromycin when all cells of a control group die after 2 weeks, maintaining the culture solution with the puromycin maintaining concentration of 1.0mg/mL, and continuously performing passage amplification to obtain the K562-CD13 cell strain stably expressing CD13 and puromycin resistance.
4. Screening positive monoclonal cells to obtain a K562-CD13 cell strain with high CD13 expression.
The results are shown in FIG. 1, with green fluorescence indicating successful infection of K562 cells (FIG. 1).
Example 3: the expression level of CD13 was detected by Western Blotting using the method described in the present invention.
Resuspending K562 and K562-CD13 cells, resuspending K562-CD13 cells and K562 cells, transferring into a centrifuge tube, centrifuging at 1000rpm to remove supernatant, placing on an ice box, adding a protein lysate, fully lysing the cells by adopting a freeze-thaw method, centrifuging at 4 ℃, 15min and 12000rpm, and taking the supernatant. mu.L of the supernatant was subjected to Bradford assay for protein concentration, and the remaining supernatant was subjected to an equal volume of 2 × loading buffer and heated to boil at 100 ℃ for 5min to denature the protein. Storing at-70 deg.C.
Adding 20 μ L, 40 μ L, 60 μ L, 80 μ L and 100 μ L BSA (1 μ G/ml) into 2.5 tubes, adding pure water to 1ml, adjusting to zero with 1ml pure water, adding 50 μ L of lysed whole-cell protein solution to 1ml with pure water, adding 1.4ml Coomassie brilliant blue G-250 solution into each tube, shaking, mixing, standing for 2min, measuring OD595nm absorbance with ultraviolet spectrophotometer, and calculating protein content by standard curve.
3. Preparing a glass plate, washing the glass plate with water, determining the volume of gel, carefully injecting separation gel into a gap of the prepared glass plate, adding a plurality of milliliters of double distilled water into the top layer to completely cover the liquid surface so as to prevent the inhibition effect of air on gel polymerization, standing the glass plate at room temperature for about 30 minutes, pouring the double distilled water after the gel polymerization is finished, absorbing residual liquid as much as possible, injecting upper layer concentrated gel, inserting a comb, adjusting the height of the comb to be proper so as to avoid generating bubbles, polymerizing at room temperature, taking out the comb after the gel polymerization, washing the comb holes with the double distilled water for a plurality of times, putting the gel into an electrophoresis tank, adding Tris-glycine electrophoresis buffer into the upper tank and the lower tank of the electrophoresis tank, checking whether the gel leaks or not, removing bubbles at the bottom of the gel, flushing the comb holes with the electrophoresis buffer before sampling, taking 15ul of samples, preparing electrophoresis, starting at a voltage of 80V, and after the dye enters the separation gel, increasing the voltage to 100V, performing electrophoresis until the dye reaches the bottom of the separation gel, and taking down the gel for protein immunoblot detection;
the following table was used to prepare 10% of separation gum and 5% of concentrated gum:
② 12 percent of separation glue and 5 percent of concentrated glue are configured according to the following table:
carefully taking down the gel after the gel electrophoresis is finished, soaking the gel in a transfer buffer solution for 15min, cutting 2 pieces of filter paper and a PVDF film, placing the gel in the transfer buffer solution for soaking for 10min, cutting the PVDF film, firstly soaking the film in methanol, then washing the film with triple distilled water, then placing the film in the transfer buffer solution for soaking, installing a transfer printing device, placing a plastic support in a tray containing the transfer buffer solution, placing a piece of sponge on the plastic support, placing a piece of filter paper on the sponge, and sequentially placing the gel, the PVDF film, the other piece of filter paper and the sponge. Placing a transfer membrane, removing bubbles when the transfer membrane is transferred one layer, clamping the layers by a plastic support, placing the transfer membrane into an electrophoresis tank, paying attention to the fact that the PVDF side is close to an anode and the glue side is close to a cathode, switching on a power supply, transferring the current of 300mA for about 2 hours, marking the upper edge of the membrane by scissors after the transfer is finished, placing the PVDF membrane into skim milk, sealing the membrane for 2 hours at room temperature, reacting with primary antibody, diluting the antibody by 5% sealing solution for 1: 1000-3000, putting PVDF film, adding primary anti-diluting solution according to 0.1ml of primary anti-solution per square centimeter, shaking overnight or room temperature for 1-2h after 4 ℃, removing reaction solution after reaction, washing 3 times with TBST, reacting with secondary antibody for 10min each time, diluting the secondary antibody in sealing solution, putting the film into a bag, adding secondary antibody diluting solution, removing all bubbles in the bag, sealing, shaking for 1-2h at room temperature, removing reaction solution after reaction, washing 3 times with TBST, placing the film on a preservative film, uniformly coating color development substrate solution, covering a preservative film, covering the film, placing the film in an exposure cassette, taking out the film, placing the film in developing solution, shaking slightly at room temperature, observing color development reaction until satisfaction, immediately washing to stop reaction, placing the film in fixing solution, shaking slightly, taking out, airing, scanning a gel imaging analysis system, the image is saved.
As shown in FIG. 2, the bands of the K562-CD13 cell line are obviously darker than those of the K562 cell line, indicating that the cell expression effect of the over-expressed strain is as expected (FIG. 2).
Example 4: the method of the invention is used for measuring the enzyme activity of K562 and K562-CD13 cell strains as enzyme sources.
1. The K562 and K562-CD13 cells were washed with PBS, transferred to a 96-well plate at 1X 10 5 Per well;
2. add CD13 substrate to 1.6mM, 30min, 37 ℃ to the wells;
3. and measuring the absorbance value at 405nm and processing the data.
The results are shown in FIG. 3, the slope of the enzyme activity trend line of the K562-CD13 strain is obviously higher than that of the K562 strain, which indicates that the cell expression effect of the over-expression strain is in line with the expectation (FIG. 3).
Example 5: the method of the invention is used for determining the inhibition rate of the compound Bestatin on the enzyme activity of CD13 in a K562-CD13 cell strain.
K562-CD13 cells were washed once with PBS and transferred to 96-well plates at 1X 10 5 Per well;
2. to the wells, compounds were added at different concentrations to a final concentration: 25. 100, 400 mu M;
after 3.5min, CD13 substrate was added to 1.6mM, 30min, 37 ℃;
4. measuring absorbance values at 405nm at intervals of 30 min;
5. calculating an inhibition rate:
the average OD value of the added medicine group is the average OD value of the over-expressed added medicine group-the average OD value of the non-over-expressed added medicine group
Mean OD value of non-drug group (mean OD value of over-expression group-mean OD value of non-over-expression group)
The result is shown in figure 4, the inhibition effect of the Bestatin on the K562-CD13 enzyme activity is increased along with the increase of the dosing concentration.
In the process of researching and developing new anti-tumor drugs, the influence of the compound on the enzyme activity of tumor cells in vitro is often required to be measured, and the method can be used for measuring the inhibition rate of the compound on the enzyme activity. The inhibition rate of the Bestatin on the CD13 generated by overexpression can be obtained according to an inhibition rate calculation formula, the result is shown in figure 4, and data show that the Bestatin compound can obviously inhibit the CD13 enzyme activity of K562-CD13 cells (figure 4).
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (1)
1. A method for measuring the inhibition rate of a compound on the activity of aminopeptidase N, which is characterized in that the enzyme inhibition activity of the compound is measured by taking a K562-CD13 cell line expressing human-derived CD13 as an enzyme source, and the method comprises the following steps:
(1) the K562-CD13 cells washed with PBS were seeded in a 96-well platePerforming the following steps; the seeding density of the K562-CD13 cells in a 96-well plate is 1 × 10 5 Per well;
(2) adding compounds of different concentration gradients to the wells;
(3) after 5min of compound action, CD13 substrate was added to 1.6mM and incubated at 37 ℃ for 30 min;
(4) the absorbance value at 405nm was measured and the inhibition was calculated:
mean OD value of additive medicine group (mean OD value of overexpressed additive medicine group-mean OD value of unexpressed additive medicine group)
Mean OD value of non-drug addition group-mean OD value of overexpression group-mean OD value of non-overexpression group;
the K562-CD13 cell line is constructed as follows:
(1) the K562 cells were resuspended at 5X 10 by adding complete medium 3 The density of each well is inoculated in a 96-well plate, and the volume is 80 mu L;
(2) adding a recombinant lentivirus LV-CD13 diluent into the hole, adding Polybrene serving as a virus infection auxiliary agent, and culturing overnight;
(3) three days after infection, puromycin is added to screen virus infected K562 cells, so as to obtain a K562-CD13 cell strain which stably expresses CD13 and puromycin resistance;
the preparation method of the recombinant lentivirus LV-CD13 diluent comprises the following steps: the concentration is 2X 10 8 TU/ml stock of recombinant lentivirus LV-CD13, diluted with ENi.S to give an MOI of 40.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101531613A (en) * | 2009-04-16 | 2009-09-16 | 山东大学 | Aminopeptidase N inhibitor bestatin dino ester, synthesis and application thereof |
| CN102382014A (en) * | 2011-09-08 | 2012-03-21 | 潍坊博创国际生物医药研究院 | Aminopeptidase N inhibitor, preparation method and application |
| CN103848778A (en) * | 2014-03-28 | 2014-06-11 | 潍坊高新生物园发展有限公司 | Aminopeptidase N inhibitor and preparation method and application thereof |
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| CN107384869A (en) * | 2017-06-26 | 2017-11-24 | 上海市食品药品检验所 | A kind of monoclonal cell strain and its application in IL 6R inhibitor relative biological activities are determined |
| CN107827820A (en) * | 2017-11-10 | 2018-03-23 | 山东大学 | Pyrazolines aminopeptidase N inhibitor and its preparation method and application |
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2018
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| CN101531613A (en) * | 2009-04-16 | 2009-09-16 | 山东大学 | Aminopeptidase N inhibitor bestatin dino ester, synthesis and application thereof |
| CN102382014A (en) * | 2011-09-08 | 2012-03-21 | 潍坊博创国际生物医药研究院 | Aminopeptidase N inhibitor, preparation method and application |
| CN104797557A (en) * | 2012-10-23 | 2015-07-22 | 领先药物公司 | Mixed inhibitors of aminopeptidase n and of neprilysine |
| CN103848778A (en) * | 2014-03-28 | 2014-06-11 | 潍坊高新生物园发展有限公司 | Aminopeptidase N inhibitor and preparation method and application thereof |
| CN104630273A (en) * | 2015-01-13 | 2015-05-20 | 王明丽 | Preparation method and application of K562 cell strain for stably expressing human cytomegalovirus pp65 protein in nucleus |
| CN107384869A (en) * | 2017-06-26 | 2017-11-24 | 上海市食品药品检验所 | A kind of monoclonal cell strain and its application in IL 6R inhibitor relative biological activities are determined |
| CN107827820A (en) * | 2017-11-10 | 2018-03-23 | 山东大学 | Pyrazolines aminopeptidase N inhibitor and its preparation method and application |
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| Leukemic Cell-Surface CD13/Aminopeptidase N and Resistance to Apoptosis Mediated by Endothelial Cells;Yuji Mishima等;《Journal of the National Cancer Institute》;20020703;第93卷(第13期);1020-1028 * |
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