阅读说明：本技术 以ccr5为靶点的覆盆子矢车菊素-3-o-半乳糖苷在制备抗hiv-1药物中的应用 (Application of raspberry cyanidin-3-O-galactoside with CCR5 as target spot in preparation of anti-HIV-1 drugs ) 是由 张清燕 李强 段晨晨 邓博文 刘真 桑锋 李�杰 钱洁玉 岳静宇 沈俊岭 李承乘 于 2021-07-28 设计创作，主要内容包括：本发明涉及以CCR5为靶点的覆盆子矢车菊素-3-O-半乳糖苷在制备抗HIV-1药物中的应用,可有效解决以CCR5为靶点拮抗受体的抗HIV-1中药天然小分子化合物,实现在制备抗HIV-1药物中的应用问题,该矢车菊素-3-O-半乳糖苷是从中药覆盆子筛选出的天然小分子化合物,具有抗HIV-1的活性,有效用于以CCR5为靶点制备抗HIV-1药物,本发明利用计算机辅助药物设计中的虚拟筛选技术,以CCR5为受体筛选出对CCR5有抑制作用的已知中药天然小分子化合物矢车菊素-3-O-半乳糖苷,以荧光素酶作为报告基因,确定其抗HIV活性,通过细胞毒性试验测试其肝毒性,并明确与CCR5的结合模式,作为CCR5抑制剂,是抗HIV药物上的创新。(The invention relates to an application of raspberry cyanidin-3-O-galactoside with CCR5 as a target spot in preparing an anti-HIV-1 medicament, which can effectively solve the application problem of an anti-HIV-1 Chinese medicinal natural small molecule compound with CCR5 as a target spot antagonistic receptor in preparing the anti-HIV-1 medicament, wherein the cyanidin-3-O-galactoside is a natural small molecule compound screened from the Chinese medicinal raspberry, has anti-HIV-1 activity, is effectively used for preparing the anti-HIV-1 medicament with CCR5 as the target spot, utilizes a virtual screening technology in computer-aided medicament design to screen a known Chinese medicinal natural small molecule compound cyanidin-3-O-galactoside with an inhibiting effect on CCR5 by taking CCR5 as a receptor, takes luciferase as a reporter gene to determine the anti-HIV activity, hepatotoxicity was tested by cytotoxicity assays and the binding pattern to CCR5 was well defined and is an innovation in anti-HIV drugs as a CCR5 inhibitor.)

1. Application of raspberry cyanidin-3-O-galactoside with CCR5 as target point in preparation of anti-HIV-1 drugs, wherein the raspberry cyanidin-3-O-galactoside is used as target pointThe glycoside has the formula of C₂₁H₂₁ClO₁₁The molecular structural formula is:

2. the use of a CCR 5-targeted raspberry procyanidin-3-O-galactoside in the preparation of an anti-HIV-1 drug according to claim 1, wherein the raspberry procyanidin-3-O-galactoside is screened from raspberry active ingredients.

3. The use of a CCR 5-targeted raspberry cyanidin-3-O-galactoside in the preparation of an anti-HIV-1 drug according to claim 2, wherein the raspberry cyanidin-3-O-galactoside is selected from raspberry by the method comprising:

consulting a traditional Chinese medicine system pharmacological database and an analysis platform, searching 25 CCR5 inhibitor active molecules, inducing 1300 theoretical inactive molecules Decoy molecules by DeceyFinder1.1 software, taking RCSB as a docking receptor, docking the active molecules and the Decoy molecules by Ledock and Vina, and enabling the binding energy to be less than or equal to-7 Kcal & mol^-1A positive result; SPSS20.0 is fitted with a receiver operation characteristic Curve to evaluate the sensitivity of two docking software, namely Ledock and Vina, the Area Under the Curve, namely Area Under Cutter (AUC), is taken as a judgment standard, and the docking result comprises four parameters: true positive TP, false positive FP, true negative TN and false negative FN; the calculation is disclosed as follows: the true positive rate TPR ═ Sensitivity ═ TP/(TP + FN); the false positive rate FPR is 1-specificity is 1-TN/(TN + FP); the detection result of the sensitivity of the docking software shows that Ledock scores higher in the test for CCR5 receptors, in order to more visually display the docking performance of Ledock software, the Ledock is used for docking positive drugs MVC and CCR5 again, the PYMOL1.0 visual docking result is compared with the reported combination of MVC-CCR5, and Ledock is used for virtually screening 29384 traditional Chinese medicine compounds in TCMSP;

ledock 29384 Chinese medicines for TCMSPThe method for virtually screening the compound comprises the following steps: mol2 format, importing file information of traditional Chinese medicine compounds into a notebook to establish ligands. txt files; ② boxes are arranged according to the MVC active binding site (Box position, X axis: 21.98, 41.407; Y axis: 4.105, 13.234; Z axis: 31.05, -14.489, grid size and spacing are set toAndremoving CCR5 water molecules, ligands, heteroatoms and the like by PYMOL1.0, carrying out hydrogenation and charging treatment, introducing a site coordinate into a binding site file, and inputting a docking instruction under a Linux system to screen a CCR5 inhibitor;

virtual screening to obtain binding energy less than or equal to-7 Kcal & mol^-1143 Chinese medicinal compounds, using Discovery Studio 2019 to check binding state, using MVC-CCR5 binding conformation as comparison, and using low energy value, no less than 10 residues identical to MVC-CCR5 binding conformation, less than 500 mass, and binding energy of-7.02 kcal & mol^-1The number of the same amino acid residues bound was 13, the molecular mass was 484.838, and the binding energy was-7.02 kcal.mol^-1Identified and determined by HPLC to be cyanidin-3-O-galactoside.

Technical Field

The invention relates to a medicine, in particular to an application of raspberry cyanidin-3-O-galactoside with CCR5 as a target spot in preparing an anti-HIV-1 medicine.

Background

AIDS is a human immunodeficiency disease caused by HIV-1 infection of AIDS virus, seriously threatens the health and life safety of people, and therefore, the treatment of AIDS arouses high attention of people. The C-C chemokine receptor 5(C-C chemokine type5, CCR5) contains 352 amino acids, is located on the cell membrane, belongs to a member of the G protein coupled receptor family, and plays an important role in the process of HIV invasion of target cells. When HIV virus invades target cells, the surface glycoprotein gp120 is combined with a target cell surface receptor CD4, a CCR5 binding site is exposed, and gp120 interacts with the binding site to further draw the space distance between the HIV virus and cell membranes. Subsequently, a series of conformational changes are generated on the virus surface glycoprotein gp41, so that the final fusion of the virus envelope and the cell membrane is realized, and the virus RNA enters the cell, so that the human is infected with AIDS. Therefore, inhibition of HIV binding to the CCR5 receptor is a key point in controlling HIV infection of target cells.

At present, although there are many drugs for treating AIDS, the curative effect and use are not satisfactory for various reasons, so the development and discovery of new anti-HIV-1 drugs is a technical problem that the industry hopes to solve. The traditional Chinese medicine has the technical advantages of treating various difficult and complicated diseases, and various anti-HIV-1 traditional Chinese medicine active ingredients are researched and virtually screened out by the medicine.

The virtual medicine screening technology is based on medicine design theory and selects some ideal compounds from great amount of compounds with the help of computer technology and professional application software. The combination condition of molecules and the active sites of receptors is simulated by a computer, and after a new lead compound is preliminarily screened out from the existing molecule library, the activity verification is carried out. The technology combines a computer-aided drug design technology and an activity experiment technology, and overcomes the defects of long period, large investment and the like in the traditional drug screening process.

At present, the CCR5 inhibitor only contains a western medicine Malavirus (Maraviroc, MVC), and has obvious clinical toxic and side effects, and the traditional Chinese medicine has obtained a certain clinical result in the treatment of AIDS. Compared with the simple HAART (highly effective anti-retroviral virus) treatment, the traditional Chinese medicine combination treatment has obvious treatment effect advantage on patients with immune reconstitution insufficiency and has low adverse reaction. In addition, the traditional Chinese medicine compound is proved to have anti-HIV effect, but the traditional Chinese medicine natural small molecule compound which takes CCR5 as a target point to inhibit HIV from entering target cells is not reported in a public way.

Disclosure of Invention

In view of the above situation, in order to overcome the defects of the prior art, the invention aims to provide an application of raspberry cyanidin-3-O-galactoside with CCR5 as a target point in preparation of an anti-HIV-1 medicament of an anti-HIV-1 Chinese medicinal natural small molecule compound capable of antagonizing CCR5 receptor, which can effectively solve the application problem of the anti-HIV-1 Chinese medicinal natural small molecule compound with CCR5 as a target point antagonistic receptor in preparation of the anti-HIV-1 medicament.

The technical scheme includes that the raspberry cyanidin-3-O-galactoside with CCR5 as a target point is applied to preparation of an anti-HIV-1 medicament, the cyanidin-3-O-galactoside is a natural small molecular compound screened from a traditional Chinese medicine raspberry, the molecular weight is 484.838, and the molecular formula is C₂₁H₂₁ClO₁₁The molecular structural formula is:

the screening method of the raspberry cyanidin-3-O-galactoside comprises the steps of consulting a Traditional Chinese Medicine system pharmacological Database and an Analysis Platform (TCMSP, Traditional Chinese Medicine Systems pharmacological Database and Analysis Platform), searching for 25 CCR5 inhibitors (active molecules), inducing 1300 theoretical inactive molecules (Decoy molecules) by DeceyFinder1.1 software, using CCR5(PDB ID: 4MBS) in RCSB (http:// www.rcsb.org) as a docking receptor, docking the active molecules and the Decoy molecules by Ledock and Vina to enable the binding energy to be less than or equal to-7 Kcal mol^-1A positive result; the SPSS20.0 is fit to a Receiver operating characteristic Curve (ROC) to evaluate the sensitivity of two docking software, namely, Ledock and Vina, and the Area Under the Curve (AUC) is used as a judgment criterion, and the docking result includes four parameters: true Positive (TP), False Positive (FP), True Negative (TN) and False Negative (FN); the calculation is disclosed as follows: true Positive Rate (TPR) ═ Sensitivity ═ TP/(TP + FN); false Positive Rate (FPR) ═ 1-specificity ═ 1-TN/(TN + FP). Docking software sensitivity measurements showed that Ledock scored higher in the test for the CCR5 receptor, as shown in figure 2; in order to more visually display the docking performance of Ledock software, the positive drugs MVC and CCR5 are docked again by utilizing Ledock, the PYMOL1.0 visualizes the docking result, and the reported combination of MVC-CCR5 is used as a comparison; the original combination state is coincided with the original combination state through Ledock re-butt jointThe degree is extremely high, so that the Ledock is utilized to virtually screen the TCMSP database;

the method for virtually screening 29384 traditional Chinese medicine compounds in TCMSP by Ledock comprises the following specific steps: the method comprises the following steps of firstly, downloading compounds in TCMSP data (https:// tcmspw.com). Mol2 format, importing the file information of the traditional Chinese medicine compounds into a notebook to establish ligands.txt files; ② the Box (Box) position (X-axis: 21.98, 41.407; Y-axis: 4.105, 13.234; Z-axis: 31.05, -14.489) is set according to the MVC active binding site, the grid size and spacing are set asAndremoving CCR5 water molecules, ligands, heteroatoms and the like by PYMOL1.0, carrying out hydrogenation and charging treatment, introducing a site coordinate into a binding site file, and inputting a docking instruction under a Linux system to screen a CCR5 inhibitor;

virtual screening to obtain binding energy less than or equal to-7 Kcal & mol^-1143 Chinese medicinal compounds with binding state checked by using Discovery Studio 2019, MVC-CCR5 binding conformation as comparison, cyanidin-3-O-galactoside with low energy value, same residues as MVC-CCR5 binding conformation not less than 10, mass less than 500 and CAS number as limitation condition, and binding energy of-7.02 kcal & mol^-113 same amino acid residues, 484.838 molecular weight, 27214-71-7 CAS number (login number), and C molecular formula₂₁H₂₁ClO₁₁The molecular structural formula is:

the cyanidin-3-O-galactoside has anti-HIV-1 activity, is effectively used for preparing anti-HIV-1 medicines by taking CCR5 as a target spot, and realizes the application of the cyanidin-3-O-galactoside in preparing the anti-HIV-1 medicines.

The invention relates to an anti-HIV-1 traditional Chinese medicine natural small molecule compound capable of antagonizing CCR5 receptor, which is characterized in that a virtual screening technology in computer-aided drug design is utilized, CCR5 is used as a receptor to screen a known traditional Chinese medicine natural small molecule compound cyanidin-3-O-galactoside having an inhibiting effect on CCR5, luciferase is used as a reporter gene to determine the anti-HIV activity of the compound, the hepatotoxicity of the compound is tested through a cytotoxicity test, and a binding mode with CCR5 is determined, so that the molecular structure of the cyanidin-3-O-galactoside can be used as a CCR5 inhibitor, becomes a technical basis for designing a new anti-HIV drug, has wide development potential, is an innovation on anti-HIV drugs, and has good economic and social benefits.

Drawings

FIG. 1 is a molecular structural formula diagram of the small molecule compound cyanidin-3-O-galactoside of the invention.

FIG. 2 is a graph of Ledock and Vina receiver operating characteristics of the small molecule compound, cyanidin-3-O-galactoside of the present invention.

FIG. 3 is a gradient dilution virus fluorescence image of the small molecule compound cyanidin-3-O-galactoside of the invention.

FIG. 4 is a diagram of the gradient dilution virus CCK-8OD value of the small molecule compound cyanidin-3-O-galactoside of the invention.

FIG. 5 is a graph of the expression levels of IC50 luciferase from MAGI-CCR5 cells after treatment with a small molecule compound, cyanidin-3-O-galactoside of the present invention.

FIG. 6 is a graph showing the cytotoxicity of MAGI-CCR5 after treatment with cyanidin-3-O-galactoside, a small molecule compound of the invention.

FIG. 7 is a graph showing the cytotoxicity of LO2 after treatment with cyanidin-3-O-galactoside, a small molecule compound of the invention.

FIG. 8 is the nuclear magnetic H spectrum of small molecule compound cyanidin-3-O-galactoside.

Detailed Description

The following detailed description of the embodiments of the present invention refers to the accompanying drawings.

The invention relates to an application of cyanidin-3-O-galactoside with CCR5 as a target spot in preparation of an anti-HIV-1 medicament, wherein the cyanidin-3-O-galactoside is screened from raspberry, and the screening method comprises the following steps:

review of the pharmaceutical literature for 25 CCR5 inhibitors (active molecules, Actives), DeceyFinder1.1 software induced 1300 theoretical inactive molecules (Decoy molecules), RCSB (http:// www.rcsb.org) CCR5(PDB ID: 4MBS) as docking acceptor, Ledock and Vina docked active and Decoy molecules to bind with a binding energy of ≦ 7Kcal mol^-1A positive result; the SPSS20.0 is fit to a Receiver operating characteristic Curve (ROC) to evaluate the sensitivity of two docking software, namely, Ledock and Vina, and the Area Under the Curve (AUC) is used as a judgment criterion, and the docking result includes four parameters: true Positive (TP), False Positive (FP), True Negative (TN) and False Negative (FN); the calculation is disclosed as follows: true Positive Rate (TPR) ═ Sensitivity ═ TP/(TP + FN); false Positive Rate (FPR) ═ 1-specificity ═ 1-TN/(TN + FP). Docking software sensitivity measurements showed that Ledock scored higher in the test for the CCR5 receptor, as shown in figure 2; in order to more visually display the docking performance of Ledock software, the positive drugs MVC and CCR5 are docked again by utilizing Ledock, the PYMOL1.0 visualizes the docking result, and the reported combination of MVC-CCR5 is used as a comparison; the coincidence degree of the TCMSP database and the original combination state is extremely high through Ledock re-docking, so that the TCMSP database is virtually screened by utilizing Ledock;

the method for virtually screening 29384 traditional Chinese medicine compounds in TCMSP by Ledock comprises the following specific steps: the method comprises the following steps of firstly, downloading compounds in TCMSP data (https:// tcmspw.com). Mol2 format, importing the file information of the traditional Chinese medicine compounds into a notebook to establish ligands.txt files; ② the Box (Box) position (X-axis: 21.98, 41.407; Y-axis: 4.105, 13.234; Z-axis: 31.05, -14.489) is set according to the MVC active binding site, the grid size and spacing are set asAndremoving CCR5 water molecules, ligands, heteroatoms and the like by PYMOL1.0, carrying out hydrogenation and charging treatment, introducing a site coordinate into a binding site file, and inputting a docking instruction under a Linux system to screen a CCR5 inhibitor;

virtual screening to obtain binding energy less than or equal to-7 Kcal & mol^-1143 Chinese medicinal compounds are obtained by checking binding state with Discovery Studio 2019, MVC-CCR5 binding conformation as comparison, and small molecule compounds with low energy value, no less than 10 residues identical to that in MVC-CCR5 binding conformation, less than 500 mass and CAS number as limiting conditions, and have binding energy of-7.02 kcal & mol as identified and determined by HPLC^-1The number of the same amino acid residues is 13, the molecular weight is 484.838, the CAS number (accession number) is 27214-71-7, the name is cyanidin-3-O-galactoside, and the molecular formula is C₂₁H₂₁ClO₁₁The molecular structural formula is:

the cyanidin-3-O-galactoside is a natural small molecular compound of a raspberry as a traditional Chinese medicine, has activity of resisting HIV-1, is effectively used for preparing an anti-HIV-1 medicine by taking CCR5 as a target spot, realizes the application of the cyanidin-3-O-galactoside in preparing the anti-HIV-1 medicine, and has very good effect through detection and experiments, and the related data are as follows:

firstly, screening natural small molecular compounds of traditional Chinese medicines:

consult Chinese medicine literature library to find out 25 CCR5 inhibitors (active molecules, Actives), DeceyFinder1.1 software induces 1300 theoretical inactive molecules (Decoy molecules), CCR5(PDB ID: 4MBS) in RCSB (http:// www.rcsb.org) is used as docking receptor, Ledock and Vina dock active molecules and Decoy molecules so as to bind with the binding energy less than or equal to-7 Kcal mol^-1The result is positive. SPSS20.0 is fitted with a Receiver operating characteristic Curve (ROC) to evaluate the sensitivity of two docking software, namely Ledock and Vina, the Area Under the Curve (AUC) is used as a judgment standard, and the docking result comprises four parameters: true Positive (TP), False Positive (FP), True Negative (TN) and False Negative (FN). The calculation is disclosed as follows: true Positive Rate (TPR) ═ Sensitivity ═ TP/(TP + FN); false Positive Rate (FPR) ═ 1-specificity ═ 1-TN/(TN + FP). Docking software sensitivity measurements showed that Ledock scored higher in the test for the CCR5 receptor as shown in figure 2. In order to more visually display the docking performance of Ledock software, the positive drugs of MVC and CCR5 are docked again by utilizing Ledock, and the docking result is visualized by PYMOL1.0, wherein the reported combination of MVC-CCR5 is used as a comparison. The coincidence degree of the TCMSP database with the original combination state is extremely high through Ledock re-docking, so that the TCMSP database is virtually screened by utilizing Ledock.

The method for virtually screening 29384 traditional Chinese medicine compounds in TCMSP by Ledock comprises the following specific steps: the method comprises the following steps of firstly, downloading compounds in TCMSP data (https:// tcmspw.com). Mol2 format, importing the file information of the traditional Chinese medicine compounds into a notebook to establish ligands.txt files; ② the Box (Box) position (X-axis: 21.98, 41.407; Y-axis: 4.105, 13.234; Z-axis: 31.05, -14.489) is set according to the MVC active binding site, the grid size and spacing are set asAndremoving CCR5 water molecules, ligands, heteroatoms and the like by PYMOL1.0, and carrying out hydrogenation and charge treatment. And (3) importing the site coordinates into a binding site file, and inputting a docking instruction under a Linux system to screen a CCR5 inhibitor.

Virtual screening to obtain binding energy less than or equal to-7 Kcal & mol^-1143 Chinese medicinal compounds. Using Discovery Studio 2019 to look at the binding status, MVC-CCR5 binding conformation as a comparison gave cyanidin-3-O-galactoside (SJ) with low energy value, no less than 10 identical residues, mass less than 500 and with CAS number as the limiting conditions. SJ binding energy of-7.02 kcal.mol^-1The number of the same amino acid residues is 13, the molecular weight is 484.838, and the CAS number is 27214-71-7;the raspberry is from raspberry, and is identified by HPLC liquid chromatography, wherein the chromatographic column comprises: c18 column, 4.6X 250mm X5 μm; mobile phase methanol: 0.3% phosphoric acid water 28: 72, wavelength λ 520nm, flow rate 1mL/min, 35 ℃, sample dissolved in 50% methanol, peak result:

	retention time (minutes)	Area (microvolt/sec)	% area
				1	6.120	15934	0.47
2	10.075	3363449	99.53

And (3) analysis results:

detecting items	Quality standard	Results
			Traits	Dark brown solid, dissolved in methanol, acid water	Conform to
Purity of	Not less than 98.0% (area normalization method)	99.53％(520nm)
			Mass spectrometry	Should conform to its structure	Conform to
Nuclear magnetism	Should conform to its structure	Conform to

See fig. 8 for a spectral diagram.

Second, Activity test

1 materials of the experiment

Experimental cells and plasmids:

293T cell strain, MAGI-CCR5 cell strain and LO2 cell strain are preserved in a virus laboratory of AIDS research center of the first subsidiary hospital of university of medicine in south river; DH-5. alpha. E.coli was purchased from Shanghai Biopsis; the recombinant plasmids JRFL and PLAI were gifted by Shanghai Pasteur institute of Chinese academy.

Experimental drugs:

lip2000 transfection reagent (batch: 1526341) was purchased from Invitrogen, usa; plasmid extraction kits (cat # 740410.5) were purchased from Macherey-Nagel, Germany; geneticin (batch: 10788700) was purchased from Gibco, USA; puromycin (cat # P8833) was purchased from Sigma, USA; hygromycin B solution (cat # 10843555001) was purchased from Roche, USA; CCK-8 kit (batch: K10186133EF5E) was purchased from APEXBIO, USA; fetal bovine serum (batch: RB35935) was purchased from Synechol; cell lysates (batch # 0000381551), luciferase reporter gene assay kit (batch # 0000151502) were purchased from PROMEGA, usa; PBS buffer (batch No. 20191221), DMEM high-sugar medium (batch No. 20200110), Opti-MEM serum-free medium (batch No. 2152784), and 0.25% trypsin-EDTA digest (batch No. 20191211) were purchased from Beijing Soilebo; the traditional Chinese medicine natural small molecular compound cyanidin-3-O-galactoside is obtained from Szechwan Egyptian Biotech limited company.

An experimental instrument:

Thermo3111 CO₂cell culture chamber (USA), THZ-92B gas bath constant temperature oscillator (Shanghai Bonews), DL-CJ-2ND ultra clean bench (Harbin east Union), Olympus CKX41-A32PH inverted fluorescence phase contrast microscope (Japan), QL-861 vortex instrument (Linbel instrument), HFsafe1200 biosafety cabinet (Shanghai Lianshi), 3K3D desktop high speed refrigerated centrifuge (Sigma in Germany), NanoVue Plus nucleic acid protein tester (Biochrom USA), SpectraMaxi3 multifunctional enzyme reader (USA molecular instrument), SpectraMaxL chemiluminescence plate reader (USA molecular instrument).

2. Pseudovirus preparation and infection capacity detection experiment:

(1) double plasmid transformed DH5 alpha colibacillus

Plasmid PLAI (containing HIV envelope protein gene) JRFL (containing HIV skeleton gene) 4 uL each, respectively adding into 40 uL DH5 alpha Escherichia coli centrifuge tube, mixing, ice bath for 45min, water bath at 42 deg.C for 90s, and ice bath for 2 min. Adding into 700 μ L non-resistant LB liquid culture medium, mixing, 200r min^-1Culturing at 37 deg.C for 40min, transferring to resistant LB agar medium plate containing ampicillin, sterilizing, coating with glass rod, inverting the plate, and culturing at 37 deg.C overnight to obtain Escherichia coli containing recombinant plasmid.

(2) Plasmid extraction and concentration detection

Selecting single Escherichia coli containing recombinant plasmid, respectively mixing in a shake tube containing 4mL resistance LB liquid culture medium, at 37 deg.C for 250r min^-1Pouring antibiotics after 6hIn a sexual LB liquid culture medium, the temperature is 37 ℃, and the speed is 250 r.min^-1Overnight. Plasmid extraction is carried out strictly according to a plasmid extraction kit, and the concentration and the purity of the plasmid are measured on a nucleic acid detector.

(3) Double plasmid transfection 293T cell

293T cells in DMEM medium (10% FBS, diabody, high sugar) in CO₂Incubator (37 ℃, 5% CO content)₂) And (5) medium culture and normal liquid change and passage. Opti-MEM diluted plasmid (PLAI: JAFL ═ 3:1, total 8. mu.g) to 350. mu.L/well, shaken and mixed; the Opti-MEM diluted lip2000 transfection reagent 8. mu.L to 350. mu.L/well, the plasmid and transfection reagent solution were combined, shaken and mixed well, and left at room temperature for 20 min. 293T cells at a density of 1X 10⁶Transfection was performed in 6-well plates at a density of about 80%. Adding 700 mu L of combined solution of the plasmid and the transfection reagent into each hole; after 6h, the culture was continued for 48h by changing to DMEM complete medium. Collecting supernatant, subpackaging, and storing at-80 deg.C for use.

(4) DMEM medium for MAGI-CCR5 cells (10% FBS, diabody, high sugar, 0.2mg/mL geneticin, 0.1 mg. mL)^-1Hygromycin B, 1. mu.g.mL^-1Puromycin) in CO₂Incubator (37 ℃, 5% CO content)₂) And (5) medium culture and normal liquid change and passage. 1X 10⁴The density per well was seeded in 96-well plates at 100. mu.L per well. Discarding supernatant of a 96-well plate the next day, adding 100 mu L of pseudovirus according to a ratio of 1:1,1:2,1:4,1:8,1:16,1:32 and 1:64, taking a virus-free group as a blank control group, taking 4 multiple wells in each group, sucking and discarding cell supernatant of each well after culturing for 48 hours, adding 100 mu L/well of sterile PBS buffer solution for cleaning, placing in a shaking table for 5min, and discarding PBS supernatant solution; adding 35 μ L of assay Lysis Buffer into each well, and placing in a shaking table for 15 min; transferring 20 mu L of the solution after each hole is cracked to an L detection plate; preparing Luciferase Assay detection solution according to the specification, and putting the L detection plate into a SpectraMaxL chemiluminiscence plate reader to read data. Culturing a 96-well plate MAGI-CCR5 cell, abandoning the supernatant of the 96-well plate the next day, adding 100 mu L of pseudovirus according to the ratio of 1:1,1:2,1:4,1:8,1:16,1:32 and 1:64, taking a virus-free group as a blank control group, adding 20 mu L of CCK-8 reagent per well after 48 hours, incubating at 37 ℃ for 1 hour, and measuring the OD value by using an enzyme-linked immunosorbent assay.

3-cyanidin-3-O-galactoside pseudovirus activity detection experiment:

DMEM medium for MAGI-CCR5 cells (10% FBS, diabody, high sugar, 0.2mg/mL geneticin, 0.1 mg. mL)^-1Hygromycin B, 1. mu.g.mL^-1Puromycin) in CO₂Incubator (37 ℃, 5% CO content)₂) And (5) medium culture and normal liquid change and passage. 1X 10⁴The density per well was seeded in 96-well plates at 100. mu.L per well. Discarding supernatant of 96-well plate the next day, adding gradient diluted cyanidin-3-O-galactoside into 96-well plate, and diluting with gradient (V/V) of 200, 100, 50, 25, 12.5, 6.25, 3.125. mu. mol. L^-1Blank control group without Chinese medicine compound, each group has 3 multiple wells. Adding pseudovirus 100 μ L/well after 2h, continuing culturing for 48h, removing cell supernatant from each well, adding sterile PBS buffer solution 100 μ L/well, washing, and placing in shaking table for 5 min; discarding the PBS supernatant solution; adding 35 μ L of assay Lysis Buffer into each well, and placing in a shaking table for 15 min; transferring 20 mu L of the solution after each hole is cracked to an L detection plate; the Luciferase Assay was prepared according to the instructions, and the L Assay plate was placed in a SpectraMaxL chemiluminescent plate reader to read the data and the 50% inhibitory Concentration (50% inhibition Concentration, IC50) was calculated using GraphPad 8.0.

Detection experiment of cytotoxicity of 4-cyanidin-3-O-galactoside

(1) Detection of cytotoxicity of cyanidin-3-O-galactoside on MAGI-CCR5

DMEM medium for MAGI-CCR5 cells (10% FBS, diabody, high sugar, 0.2mg/mL geneticin, 0.1 mg. mL)^-1Hygromycin B, 1. mu.g.mL^-1Puromycin) in CO₂Incubator (37 ℃, 5% CO content)₂) And (5) medium culture and normal liquid change and passage. At 1 × 10⁴The density per well was seeded in 96-well plates at 100. mu.L per well. Discarding supernatant of 96-well plate, adding 100 μ L of certain diluted Chinese medicinal compound cyanidin-3-O-galactoside into 96-well plate, and diluting with gradient (V/V) of 200, 100, 50, 25, 12.5, 6.25, 3.125 μmol. L^-1Adding 3 multiple wells of blank control group without adding Chinese medicinal compounds, culturing for 48 hr, adding CCK-8 reagent 10 μ L into each well, incubating at 37 deg.C for 1 hr, and measuring with enzyme-labeling instrumentOD values were calculated as 50% toxicity concentrations (50% cytotoxin Concentration, CC50) using GraphPad 8.0.

(2) Detection of cytotoxicity of cyanidin-3-O-galactoside on LO2

LO2 cells were cultured in DMEM medium (10% FBS, diabody, high sugar) in a CO2 incubator (37 ℃ C., containing 5% CO2) and passaged normally. At 8X 10³The density per well was seeded in 96-well plates at 100. mu.L per well. Discarding supernatant of 96-well plate, adding 100 μ L of certain diluted Chinese medicinal compound cyanidin-3-O-galactoside into 96-well plate, and diluting with gradient (V/V) of 200, 100, 50, 25, 12.5, 6.25, 3.125 μmol. L^-1And 3 groups of 3 wells without adding the traditional Chinese medicine compound are cultured for 48 hours, 10 mu L of CCK-8 reagent is added into each well, the incubation is carried out for 1 hour at 37 ℃, an OD value is measured by an enzyme-linked immunosorbent assay, and the 50% toxicity Concentration (50% cytotoxin Concentration, CC50) is calculated by using GraphPad 8.0.

5. cyanidin-3-O-galactoside binds to CCR5 in the manner:

PYMOL1.0, Discovery Studio 2019 visually explores the interaction pattern of cyanidin-3-O-galactoside with the CCR5 receptor complex. Wherein the MVC-CCR5 conformation is used as a control, cyanidin-3-O-galactoside can be tightly combined with a hydrophobic pocket of CCR5, and main interaction forces comprise hydrogen bonds, van der Waals force, hydrophobic interaction and the like. The conformation of cyanidin-3-O-galactoside-CCR 5 is the same as that of MVC-CCR5, and the cyanidin-3-O-galactoside forms 4 hydrogen bonds with the key residues Thr105, Ser180, Tyr251 and Glu283, which indicates that the key residues play an extremely important role in the interaction of the two.

6. Statistical treatment

Performing normal distribution test on the obtained data by using SPSS20.0 software, and performing One-factor analysis of variance (One-Way ANOVA) on the data conforming to the normal distribution; data not conforming to normal distribution were transformed as mean ± standard deviationThe significance level was defined as 0.05.

7. Conclusion

The purity of the plasmids PLAI and JRFL is 1.845 and 1.836 respectively under the A260/A280 absorbance, and the plasmid can be used for transfection experiments. Luciferase expression can be detected 48h after the pseudovirus infects MAGI-CCR5 cells, luciferase expression after pseudovirus double-specific dilution is shown in figure 3, and the pseudovirus infection intensity is reduced along with the increase of the dilution factor. When the pseudovirus titer was too high, it was easy to inhibit cell growth (fig. 4), 1:8, the activity of the pseudovirus-infected cells is not significantly different from that of the blank control group at the beginning of dilution concentration (P)>0.05), the IC50 of the positive drug MVC in the pseudovirus model is 1.85 + -0.34 nmol.L^-1The pseudovirus model is proved to be reliable, so the pseudovirus concentration is diluted by 1:8 times to carry out experiments.

The luciferase expression level of the MAGI-CCR5 cell after cyanidin-3-O-galactoside treatment is reduced, which indicates that the pseudovirus can be antagonized to infect the MAGI-CCR5 cell; the IC50 of the strain is (28.35 +/-7.59) mu mol.L^-1(FIG. 5). cyanidin-3-O-galactoside has low toxicity to MAGI-CCR5 and LO2 cells, and has CC50 of (164.90 +/-42.95) mu mol.L on MAGI-CCR5 cells^-1(FIG. 6), CC50 for LO2 cells was (329.80. + -. 24.35) μmol. multidot.L^-1(FIG. 7).

Experiments show that the invention takes CCR5 as a target spot to screen known traditional Chinese medicine compounds in a TCMSP database, primarily screens cyanidin-3-O-galactoside which is a traditional Chinese medicine natural small molecule compound with potential anti-HIV activity through activity tests and cytotoxicity experiments, and defines the effective antiviral concentration range of the compound, the cyanidin-3-O-galactoside of the traditional Chinese medicine natural small molecule compound can be used as a lead antiviral compound, the antiviral activity of the compound is increased through next structural modification, early technical support is provided for deep research of the anti-HIV effect of the traditional Chinese medicine in the future, new application of the cyanidin-3-O-galactoside of the traditional Chinese medicine raspberry natural small molecule compound and the medicinal value and commercial value of the raspberry are exploited, and good economic and social benefits are achieved.