CN112858673A

CN112858673A - Unmarked arginine vasopressin receptor cell model construction and ligand screening method

Info

Publication number: CN112858673A
Application number: CN201911187957.0A
Authority: CN
Inventors: 梁鑫淼; 曲腊腊; 侯滔; 刘艳芳; 王纪霞
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2019-11-28
Filing date: 2019-11-28
Publication date: 2021-05-28

Abstract

The invention relates to a method for constructing and screening a cell model of an unlabeled arginine vasopressin receptor, in particular to a subtype (V1a receptor) of the arginine vasopressin receptor, which carries out model establishment and ligand screening on different cell lines by using an unlabeled cell dynamic mass resetting technology. The V1a receptor model is constructed on an HEK293 cell line for endogenously expressing a V1a receptor and a transfected CHO-V1a cell line respectively. The method has the characteristics of being close to the real environment in vivo, no mark, real-time monitoring, high flux and simple operation, and can screen the ligand with the activity of the V1a receptor, including agonist and antagonist.

Description

Unmarked arginine vasopressin receptor cell model construction and ligand screening method

Technical Field

The invention belongs to the field of pharmacological research and drug screening research, and particularly relates to establishment of a model of an arginine vasopressin receptor subtype, namely an arginine receptor subtype 1a (V1a) receptor on endogenous cells and transfected cells, and a ligand screening method of a V1a receptor.

Background

Arginine Vasopressin (AVP), a 9-peptide cyclic hormone produced by the supraoptic and paraventricular nuclei of the hypothalamus, is stored in the neurohypophysis and mediates a series of physiological actions such as regulation of circulation and water balance by interacting with arginine vasopressin receptor (AVPR). The arginine vasopressin receptor belongs to rhodopsin A family in G protein coupled receptors, and has three subtypes of V1a, V1b and V2 in human body according to different conduction mechanisms. The V1a receptor is mainly distributed in vascular smooth muscle cells, platelets, liver cells and myometrium, and mediates arterial vasoconstriction and release of vascular endothelial growth factor by binding to arginine vasopressin. The V1b receptor is distributed primarily in the anterior pituitary and mediates the release of adrenocorticotropic hormone (ATCH). The V2 receptor is located in the cells of the kidney collecting duct and is primarily involved in regulating the permeability of the collecting duct to water. AVPR is also involved in raynaud's syndrome, dysmenorrhea, early childbirth, anxiety, depression, and other processes. Therefore, AVPR inhibitors have been the focus of pharmaceutical research. In recent studies, V1a receptor antagonists have shown superior effects for the treatment of heart failure. In 2019, researchers found that the V1a receptor can help activate a signal path involved in castration resistance generation in prostate cancer, and can be used as a therapeutic target of castration resistant prostate cancer. Representative drugs targeting the V1a receptor include Cochinvaptan (YM078) and Recocovaptan (Relcovaptan), which are currently used for treating isosoluble hyponatremia, but have certain disadvantages in the safety evaluation of long-term administration. Thus, the study AND discovery of novel, highly potent AND safe, non-peptidic V1a receptor antagonists are of great interest for both biomedical research AND drug development (ZHao, N., Peacock, S.O., Lo, C.H., Heidman, L.M., Rice, M.A., Fahrenenthltz, C.D.et al. (2019) Arginine vasopression 1a thermal target for mapping-resistance regulatory reactor, Science transaction Medicine,11(498), Koshimizu, T.a., Nakamura, K., Egashira, N.Hiyama, M.noguchi, H.406, Tanoe.A. 1869, V.56, V.1, S.H., E.1, P.J., ethylene, P.11, C.11, C.S.11, C. 11, C.S. 11, C. 1, S. 11, C. 11, S. 11, C. 11, S. 1, S. 4, E. 1, E. E.

Model construction and ligand screening methods for GPCR receptors are generally established on transfected cell models. And for endogenous cells, the influence of the compound on intracellular functional factors such as interleukin, NF-kB and the like is more detected. DMR technology is characterized by the use of endogenous cells for model construction and ligand screening of GPCR receptors, in addition to transfected cells for model construction. The principle of the DMR technology is that mass change at the bottom of a cell caused by the binding of a probe molecule or a molecule to be detected with a cell receptor is converted into an optical signal by a remote waveguide grating chip to be output, so that signal elevation is monitored in real time within a certain time, and the binding condition of the probe molecule or the molecule to be detected with the receptor is determined. Therefore, DMR signals obtained on endogenous cells can cover a wider signal path, and also make the detection environment closer to the real environment of the organism. However, since the expression level and functional status of the receptor in each cell are different, the model construction of a specific GPCR on the DMR technology platform is under investigation. On the basis of previous research, the technology constructs a target model of a V1a receptor on HEK293 cells and CHO-V1a cell lines through a plurality of technical means, and is used for ligand screening and application.

Disclosure of Invention

The invention relates to establishment and application of a cell screening model of a unlabelled ligand of a V1a receptor of arginine vasopressin in a GPCR (GPCR), and aims to establish the cell screening model of the unlabelled ligand of the V1a receptor by adopting an unlabelled cell integration pharmacology technology; the other purpose is to use the established model for receptor activity screening of a sample library to find the V1a receptor agonist or antagonist with high activity and strong selectivity for subsequent drug development and the like.

The technical scheme of the invention is as follows:

cells endogenously expressing or transfecting and expressing a V1a receptor are used as a vector, a real-time response signal is obtained through an optical biosensor microplate, and the pharmacological parameters of a V1a receptor ligand are characterized through the processing result of the real-time response signal, so that the construction and the screening of a label-free V1a receptor cell model are realized.

A V1a receptor is constructed on two cell lines by using a marker-free cell dynamic mass resetting technology to establish a model and construct a ligand screening system, and specifically, a V1a receptor model is constructed on HEK293 for endogenously expressing a V1a receptor and a CHO-V1a cell line for transfecting and expressing a V1a receptor respectively.

The V1a receptor model is constructed by the following method:

A. culturing HEK293 cells carrying the V1a receptor or CHO-V1a cells carrying the V1a receptor;

B. harvesting the HEK293 or CHO-V1a cells cultured in the step A, inoculating the cells into an Epic optical biosensor microplate, and placing the microplate in an incubator for culture;

C. removing the culture medium for culturing the cells in the microporous plate in the step B, adding a certain volume of HBSS buffer solution, and placing on an Epic instrument for balanced incubation until the cell state is stable (the change of reflection wavelength detected by the instrument is +/-5 pm);

D. establishing a base line on an Epic instrument system, adding V1a receptor agonist or antagonist with different doses into different holes of a microplate as probe molecules, continuously monitoring cell response signals for designated time, and recording the time as step S1 to obtain a DMR signal curve of the probe molecules;

E. reestablishing a base line on the system of the Epic instrument, adding the V1a receptor agonist again into different holes of the microplate, continuously monitoring the appointed time, and marking as a step S2;

F. mapping the probe molecule dosage in S1 to the DMR signal of S1 or the DMR response signal caused by the V1a receptor agonist in S2 (respectively as abscissa and ordinate), drawing a model dose-effect relationship curve, and calculating the pharmacological parameters of the agonist or antagonist on the V1a receptor (binding strength model EC)₅₀～EC₁₀₀Value and model IC₅₀value-IC₁₀₀Value). The construction of the V1a receptor model on HEK293 or CHO-V1a cell lines was completed.

The V1a receptor screening system is constructed by the following method:

D. establishing a base line on an Epic instrument system, adding a certain amount of samples to be detected into different holes of the microporous plate, continuously monitoring cell response signals until the specified time is reached, and recording the time as step S1 to obtain a DMR signal curve of the samples to be detected;

E. re-establishing a base line on the system of the Epic instrument, adding the V1a receptor agonist with the required concentration into different holes of the microplate, and continuing monitoring until the specified time is recorded as step S2;

F. superposing the horizontal and vertical coordinates of the DMR signal curve of the sample to be detected and the DMR signal curve of the model, and if the DMR signal curve of the sample to be detected is positioned below the DMR signal curve of the model, indicating that the sample to be detected can be an agonist or antagonist of a V1a receptor; (ii) if coincident or above, indicates that it is not a ligand for the V1a receptor;

G. if the sample to be tested is an agonist or antagonist of the V1a receptor, drawing a dose-effect relationship curve according to a corresponding relationship (respectively serving as an abscissa and an ordinate) between the dosage of the sample to be tested in S1 and the DMR signal of S1 or the DMR response signal caused by the V1a receptor agonist in S2, and calculating the pharmacological parameter (the binding strength EC) of the sample to be tested on the V1a receptor (the binding strength EC)₅₀Value sum IC₅₀Value), completing the screening and binding strength determination of the tested sample on the V1a receptor model.

The V1a receptor agonist is one of the following: arginine vasopressin, lysine vasopressin.

The advantages of the invention are mainly: the model and the screening strategy of the V1a receptor established by the method are characterized in that the model and the screening strategy are on an endogenous surface or cells expressing the V1a receptor by transfection, and have the characteristic of being close to the real environment in vivo; meanwhile, the cell dynamic quality resetting technology is applied, and the method has the characteristics of no mark, no damage, real-time monitoring, high flux and simplicity in operation. The technology can be used for screening the receptor activity of samples (including Chinese medicinal material fractions or compound libraries), and can also be used for finding agonists and antagonists of receptor downstream signal paths. In addition, the extension of this method allows the measurement and analysis of a variety of pharmacological parameters for the ligand of the receptor.

Drawings

Figure 1 is a characterization of the V1a receptor on HEK293 cell line: (a) real-time DMR curves of arginine vasopressin on HEK293 cell line; (b) desensitization DMR profile of 20nM arginine vasopressin on HEK293 cell line; (c) desensitization DMR profile of 20nM arginine vasopressin on gradient SR49059 on HEK293 cell line; (d) dose-response curves for arginine vasopressin and SR49059 on HEK293 cell line.

FIG. 2 is a characterization of the V1a receptor on a CHO-V1a cell line transfected with V1a receptor: (a) real-time DMR curves of vasopressin arginine on the CHO-V1a cell line; (b) desensitization DMR profile of 50nM arginine vasopressin on CHO-V1a cell line; (c) desensitization DMR profile of 50nM arginine vasopressin on gradient SR49059 on CHO-V1a cell line; (d) arginine vasopressin and SR49059 dose-response curves on the CHO-V1a cell line.

FIG. 3 shows the screening of 20 fractions of Physalis alkekengi (abbreviated as JDL in the diagram) on CHO-V1a cell line: (left) histogram of S1 signal (20min) on CHO-V1a cell line of 20 fractions of Lantern calyx (JDL); effect of 20 fractions of (right) calyx seu fructus physalis (JDL) on 50nM arginine vasopressin signal (10min) histogram.

FIG. 4 is a chromatogram of the preparation of calyx seu fructus physalis.

Detailed Description

The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.

Various chemical reagents used in the examples of the present invention are obtained by a conventional commercial route unless otherwise specified; human embryonic kidney cell line HEK293 and hamster ovary cellsThe cell line CHO is purchased from Shanghai cell bank of Chinese academy of sciences, and the V1a receptor is transfected into the CHO cell line by means of plasmid stable transformation, which is called CHO-V1a cell line. Arginine vasopressin (arginin vasopressin) was purchased from the company Limited in the American biotech, Beijing, SR49059 was purchased from the company Limited in the Sigma Aldrich trade, and 20 fractions of JDL were separated by HPLC.

The 384-well biosensor microplate was purchased from Corning, Inc., and the detection platform was the third generation of Corning

An imager.

Example 1

Modeling of V1a receptor on HEK293 cell line

HEK293 cells were seeded into Epic optical biosensor 384 microwell plates at approximately 1.5X 10 per well⁴And (3) culturing the cells for 24 hours, then removing the culture medium by suction, adding incubated 30 mu LHBSS buffer solution, standing for 1 hour until the cell state is stable, and placing the cells at a signal detection position of an Epic instrument. After establishing a baseline of 2min on an Epic instrument system, adding a gradient V1a receptor agonist (such as argininol, starting from a final concentration of 80nM, with the concentration gradually decreasing from 1/2 times to 0.15625nM, for a total of 10 concentration points) or antagonist (such as SR49059, wherein SR49059 starts from a final concentration of 200nM, with the concentration gradually decreasing from 1/2 times to 0.390625nM, for a total of 10 concentration points), continuously monitoring a cell response signal for 1h, and recording as a step S1 to obtain a DMR signal curve of the probe molecule. The baseline was re-established on the Epic instrumentation system, arginine vasopressin (10 μ L) was added to each well to a final concentration of 20nM, and the cellular response signal was monitored for 1h, as indicated in step S2, to obtain the desensitized DMR signal curve for the probe molecule.

The original file of the signal response obtained by the Epic instrument was converted into data export using Imager Beta v3.7 software, and the export data was processed using Microsoft Excel 2010 and GraphPad Prism 6 software. After blank correction, DMR signals for agonists were obtained (fig. 1)a) DMR signals desensitized to 20nM arginine vasopressin (figure 1b) and antagonist signals (figure 1 c). The DMR signals of these probe molecules can be compared with DMR signals of subsequent samples to be tested, and used as a criterion for evaluating the activity results of the samples to be tested. At EC₅₀And IC₅₀Values were calculated using the values corresponding to the highest point of the data from the DMR signal response (fig. 1d), and EC of arginine vasopressin on HEK293 cell line₅₀The value was 4.88. + -. 1.72nM, IC₅₀The value was 1.13. + -. 0.24nM, and the IC50 value for SR49059 was 3.44. + -. 1.27 nM. These parameters serve as references for the concentration of agonist and antagonist added in subsequent experiments and also as references for the binding strength of the active molecule.

Example 2

Modeling of the V1a receptor on the CHO-V1a cell line

CHO-V1a cells were seeded in Epic optical biosensor 384 plates at approximately 1.5X 10 cells per well⁴After 24 hours of culture, the culture medium is aspirated, 30 μ L of incubated HBSS buffer solution is added, and the cell is placed on a signal detection position of an Epic instrument after standing for 1 hour until the cell state is stable. After establishing a baseline of 2min on an Epic instrument system, a gradient of V1a receptor agonist (e.g., arginine vasopressin, starting at a final concentration of 200nM, with a 1/2-fold stepwise decrease in concentration to 0.024nM for 14 concentration points) or antagonist (e.g., SR49059, starting at a final concentration of 2000nM, with a concentration of 1/2-fold stepwise decrease to 0.98pM for 12 concentration points) was added, 10. mu.L per well, and the cell response signal was monitored for 1h, as indicated in step S1, to obtain the DMR signal curve of the probe molecule. The baseline was re-established on the Epic instrumentation system, arginine vasopressin (10. mu.L) was added to each well at a final concentration of 50nM, and the cell response signal was monitored for 1h, as indicated by step S2, to obtain the desensitized DMR signal curve of the probe molecule.

The resulting signal response raw data were converted to data export using Imager Beta v3.7 software and the exported data was processed using Microsoft Excel 2010 and GraphPad Prism 6 software. After blank correction, the DMR signal for the agonist (figure 2a), DMR signal desensitized to 50nM arginine vasopressin (figure 2b) and antagonist signal for the antagonist (figure 2c) were obtained. D of these Probe moleculesThe MR signal can be compared with a DMR signal of a subsequent sample to be detected, and the comparison result is used as a judgment standard of an activity result of the sample to be detected. At EC₅₀And IC₅₀In the calculation of the values, the EC for arginine vasopressin on the CHO-V1a cell line was calculated using the values corresponding to the highest points of the data from the DMR signal response (FIG. 2d)₅₀The value was 2.01. + -. 0.58nM, IC₅₀The value was 8.67. + -. 1.68 nM. SR49059 IC₅₀The value was 281.19. + -. 91.54 nM. These parameters can be used as reference for the concentration of agonist and antagonist added to the cell in subsequent experiments, and also as reference for the binding strength of the active molecule.

Example 3 ligand screening of the V1a receptor

CHO-V1a cells were seeded in Epic optical biosensor 384 plates at approximately 1.5X 10 cells per well⁴After 24 hours of culture, the culture medium is aspirated, 30 μ L of incubated HBSS buffer solution is added, and the cell is placed on a signal detection position of an Epic instrument after standing for 1 hour until the cell state is stable. And establishing a base line for 2min on an Epic instrument system, adding calyx seu fructus physalis medicinal material fraction with the final concentration of 50 mug/mL, treating for 60min with 10 muL of each hole, and recording as S1 to obtain a DMR signal curve of the calyx seu fructus physalis medicinal material fraction. And (3) establishing a base line on an Epic instrument system again, adding arginine vasopressin (10 mu L) with the final concentration of 50nM into each hole, monitoring a cell response signal for 1h, obtaining the signal influence of the compound to be detected on the agonist arginine vasopressin, and recording as S2 to obtain a desensitization DMR signal curve of the calyx seu fructus physalis medicinal material fraction.

The resulting signal responses were converted to data leads using Imager Beta V3.7 software and the derived data were processed using Microsoft Excel 2010 and GraphPad Prism 6 software to generate the signal of the test compound on the CHO-V1a cell line (fig. 3a) and a signal influence plot for arginine vasopressin (fig. 3 b). The results show that fractions F3, F4, F5 and F19 caused a decrease in arginine vasopressin signaling in S2, possibly containing agonists or antagonists of V1 a.

The extraction and separation method of calyx seu fructus physalis is as follows. Pulverizing calyx Seu fructus Physalis, soaking in 70% ethanol water for 48 hr, ultrasonic extracting twice, and mixing extractive solutions. And (3) carrying out rotary evaporation and freeze-drying on the extracting solution, re-dissolving the extracting solution to 200 mg/mL by using 70% ethanol water, centrifuging to remove precipitates, passing the supernatant through an SPE (X1) column, and collecting the permeate and the eluent to obtain an SPE product solution. SPE product liquid is used as sample liquid and is prepared by liquid phase chromatographic separation. The chromatographic conditions are as follows: 85.85mg/mL calyx seu fructus physalis SPE (X1) product liquid; sample loading amount: 6mL of sample solution; preparing a column: RZA10_ C18_0.1(10 μm, 50X 240mm, 2016092202, 300 g); flow rate: 80 mL/min; balancing: methanol: 0.1% formic acid water ═ 5: 95, 3 BV; elution conditions: 0-5min, A/B, 5/95; 5-45min, A/B, 5/95-95/5; 45-55min, A/B, 95/5; and (3) detection: UV (254 nm), chromatogram is shown in FIG. 4. Receiving time: f1,0-4 min; f2,4-6 min; f3,6-10 min; f4, 10-12.5 min; f5,12.5-15 min; f6,15-17.5 min; f7,17.5-20 min; f8,20-22.5 min; f9,22.5-25 min; f10,25-27.5 min; f11,27.5-30 min; f12,30-32 min; f13, 32-34 min; f14,34-36 min; f15,36-38 min; f16,38-40 min; f17,40-42.5 min; f18,42.5-45 min; f19,45-50 min; f20,50-55 min.

Claims

1. A method for constructing a cell model of a label-free arginine vasopressin receptor and screening ligands is characterized in that:

1) the method comprises the steps that cells expressing V1a receptors endogenously or transfectally are used as a carrier, the cells are placed in an optical biosensor micropore plate for culture, the cells are placed at the signal detection position of an Epic instrument after the culture is finished, V1a receptor agonist or antagonist is added into the pores of the micropore plate, the real-time response signals are detected, the real-time response signals are obtained, the pharmacological characteristics of V1a receptor ligands are characterized and judged through the processing results of the real-time response signals, and therefore the construction of a label-free V1a receptor cell model is achieved;

2) the method comprises the steps of taking a cell which endogenously expresses or transfects and expresses a V1a receptor as a carrier, placing the cell in a micropore plate of an optical biosensor for culture, placing the cell at a signal detection position of an Epic instrument after the culture is finished, adding a sample to be detected into a hole of the micropore plate, detecting a real-time response signal to obtain the real-time response signal, representing and judging whether the sample to be detected is a ligand of a V1a receptor or not according to a processing result of the real-time response signal, and further representing the pharmacological property of the sample to be detected if the sample is the ligand of the V1a receptor, so that the cell without a marker V1a receptor is screened by the sample to be detected.

2. The method of construction and screening of claim 1, wherein: HEK293 with a V1a receptor or CHO-V1a with a V1a receptor is used as a carrier, a real-time response signal is obtained through an optical biosensor micropore plate, and a V1a receptor model is established through processing the real-time response signal;

the specific V1a receptor model is constructed by the following method:

C. removing the culture medium for culturing the cells in the microporous plate in the step B, adding HBSS buffer solution, and placing on an Epic instrument for balanced incubation until the cell state is stable (the change of the reflection wavelength signal detected by the instrument is +/-5 pm);

D. establishing a base line on a system of an Epic instrument, adding V1a receptor agonist or antagonist with different doses into different holes of a microplate as probe molecules, continuously monitoring cell response signals for designated time, and marking as step S1 to obtain a real-time response DMR signal curve of the probe molecules;

E. re-establishing a base line on the system of the Epic instrument, adding the same dose of V1a receptor agonist into different holes of the microplate, continuously monitoring the appointed time, and marking as a step S2 to obtain a real-time response DMR signal curve of the agonist;

F. mapping the probe molecule dosage in S1 to the DMR signal of S1 or the DMR response signal caused by the V1a receptor agonist in S2 (respectively as abscissa and ordinate), drawing a model dose-effect relationship curve, and calculating the pharmacological parameters of the agonist or antagonist on the V1a receptor (binding strength model EC)₅₀Value and model IC₅₀Value), the construction of the V1a receptor model on HEK293 or CHO-V1a cell lines was completed.

3. The method of construction and screening of claim 1, wherein: HEK293 with a V1a receptor or CHO-V1a with a V1a receptor is used as a carrier, a real-time response signal is obtained through an optical biosensor micropore plate, and a V1a receptor screening system is established through processing the real-time response signal; the specific V1a receptor screening system is constructed by the following method:

D. establishing a base line on a system of an Epic instrument, adding samples to be detected with different dosages into different holes of a microporous plate, continuously monitoring cell response signals until the specified time is reached, and recording the time as a step S1 to obtain a DMR signal curve of the samples to be detected;

E. re-establishing the baseline on the Epic instrument system, adding the V1a receptor agonist with the same required concentration into different holes of the microplate, continuing to monitor the solution for the designated time, and marking the solution as step S2, wherein the required concentration is the model EC of the agonist on the V1a receptor₅₀value-EC₁₀₀A value;

G. if the sample to be detected is an agonist or antagonist of the V1a receptor, drawing a dose-effect relationship curve according to the corresponding relationship (respectively serving as an abscissa and an ordinate) between the dosage of the sample to be detected in S1 and the DMR signal of S1 or the DMR response signal caused by the V1a receptor agonist in S2, and calculating the pharmacological parameter (the binding strength EC) of the sample to be detected on the V1a receptor (the binding strength EC)₅₀Value sum IC₅₀Value), completing the screening and binding strength determination of the tested sample on the V1a receptor model.

4. The method of construction and screening of claim 1, wherein: HEK293 with a V1a receptor or CHO-V1a with a V1a receptor is used as a carrier, a real-time response signal is obtained through an optical biosensor microplate, and a V1a receptor agonist screening system is established through processing the real-time response signal; the specific V1a receptor agonist screening system is constructed by the following method:

D. establishing a base line on a system of an Epic instrument, adding V1a receptor antagonists with different final concentrations into different holes of a microplate, and continuously monitoring cell response signals until a designated time, which is marked as step S1;

E. re-establishing the base line on the system of the Epic instrument, adding the sample to be detected into the hole of the micropore plate, continuing to monitor the sample until the specified time is recorded as step S2, wherein the required concentration is EC of the sample to be detected on the cell₅₀value-EC₁₀₀Obtaining a DMR signal curve of the sample to be detected;

F. superposing the horizontal and vertical coordinates of the DMR signal curve of the sample to be detected and the DMR signal curve of the sample without the antagonist, and if the DMR signal curve of the sample to be detected is positioned below the DMR signal curve of the sample without the antagonist, indicating that the sample to be detected is possible to be an agonist of a V1a receptor; if coincident or above, this indicates that it is not an agonist of the V1a receptor.

G. If the sample to be tested is an agonist of the V1a receptor, the dosage of the antagonist in S1 corresponds to the corresponding relation between the DMR response signals caused by the sample to be tested in S2 (respectively as the abscissa and the ordinateCoordinate), plotting dose-effect relationship curve, calculating pharmacological parameter (binding strength IC) of tested sample on V1a receptor₅₀Value), completing the screening of the agonist of the tested sample on the V1a receptor model.

5. The method of construction and screening according to any one of claims 2 to 4, wherein: the V1a receptor agonist used is one or both of the following: arginine vasopressin, lysine vasopressin; the V1a receptor antagonist used is one or two or three of SR49059, Conivaptan (Conivaptan) and Relcovaptan (Relcovaptan); in the model construction method, the V1a receptor agonist used in the step S1 has a concentration of 0.0001-100000 nM; in the model construction method, the V1a receptor antagonist concentration used in step S1 is 0.0001-100000 nM.

6. The method of construction and screening according to any one of claims 2 or 3, wherein: the V1a receptor agonist concentration used in step S2 is the EC obtained on that cell₅₀value-EC₁₀₀Values, where the concentration of arginine vasopressin used was 4-200 nM.

7. The method of construction and screening according to any one of claims 2 to 4, wherein: the processing time of step S1 is 0-300 minutes, and the processing time of step S2 is 3-300 minutes.

8. The method of claim 1, wherein the V1a receptor or the signal transduction pathway thereof is screened for agonists or antagonists.

9. The method of claim 1, wherein the binding strength of a ligand for the V1a receptor to the V1a receptor is detected.