CN114636822A - HIV latent infection marker and application thereof - Google Patents

HIV latent infection marker and application thereof Download PDF

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CN114636822A
CN114636822A CN202011482869.6A CN202011482869A CN114636822A CN 114636822 A CN114636822 A CN 114636822A CN 202011482869 A CN202011482869 A CN 202011482869A CN 114636822 A CN114636822 A CN 114636822A
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lamp2
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张丽军
卢洪洲
尹林
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SHANGHAI PUBLIC HEALTH CLINICAL CENTER
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Abstract

The invention belongs to the technical field of biomedicine, relates to an HIV latent infection marker and application thereof, and particularly relates to target markers LAMP2, CD36 and CD47 for treatment and diagnosis of HIV latent infection, in particular to application of LAMP2, CD36 and CD47 proteins as HIV latent infection markers in preparation of HIV latent infection diagnosis and differential diagnosis reagents for LAMP2, CD36 and CD47 and novel anti-HIV latent infection medicines. In the invention, WB is used for detecting HIV latent infection patients in PBMC; detecting LAMP2, CD36 and CD47 in the plasma of HIV latently infected patients by using an ELISA technology; detecting LAMP2, CD36 and CD47 in resting CD4+ T cells of HIV latently infected patients by using a flow cytometry technology; the novel anti-HIV latent infection medicine takes LAMP2, CD36 and CD47 as medicine targets.

Description

HIV latent infection marker and application thereof
Technical Field
The invention belongs to the technical field of biomedicine, relates to an HIV latent infection marker and application thereof, and particularly relates to target markers LAMP2, CD36 and CD47 for treatment and diagnosis of HIV latent infection, in particular to application of LAMP2, CD36 and CD47 proteins as HIV latent infection markers in preparation of HIV latent infection diagnosis and differential diagnosis reagents for LAMP2, CD36 and CD47 and novel anti-HIV latent infection medicines.
Background
It is reported that aids is still a serious infectious disease worldwide, and although highly effective antiretroviral therapy (HAART) can effectively control the replication of viruses, the existence of virus reservoir causes the incomplete elimination of HIV, which becomes the biggest obstacle to the radical treatment of aids, so the research of virus reservoir becomes the focus and difficulty of the aids research at present.
Resting memory CD4+T is the largest diseaseA toxic reservoir, which has a low decay rate despite a low number of these cells; the gene silencing produced by the integration of the viral genome makes it difficult for the immune system to recognize and the integrated virus does not replicate and thus evades the attack of HAART drugs. Therefore, by exploring some latent characteristic proteins of HIV and deeply researching the latent infection mechanism of HIV, a new drug target can be provided for HIV elimination, and a new idea is provided for AIDS cure.
The consensus in the industry, the search of disease-specific proteins or proteins with abnormal expression as biomarkers has important significance for the diagnosis of clinical diseases; especially, the research on the physiological functions is a great application of combining proteomics with clinical treatment; comparing the change of HIV latent cell protein by using a marking method (iTRAQ marking or isotope marking) by applying a mass spectrum technology, and performing up-regulation or down-regulation, and then verifying and researching functions of differential protein in vitro (cell level) and in vivo (clinical samples), thereby providing a new biomarker for clinical diagnosis and treatment; and through deep molecular mechanism research, a new HIV latent infection treatment target is searched, and a new HIV latent infection treatment medicine is developed.
Based on the current state of the prior art, the inventors of the present application intend to provide a new HIV latent infection marker, and particularly relate to the use of LAMP2, CD36 and CD47 proteins as HIV latent infection markers and the use thereof, especially the use of LAMP2, CD36 and CD47 proteins as HIV latent infection markers in the preparation of reagents for diagnosing and identifying HIV latent infection aiming at LAMP2, CD36 and CD47, and new anti-HIV latent infection drugs.
Among the references relevant to the present invention are,
1.Eisele E,Siliciano RF:Redefining the viral reservoirs that prevent HIV-1eradication.Immunity 2012,37(3):377-388.
2.Paavilainen VO,Bertling E,Falck S,Lappalainen P:Regulation of cytoskeletal dynamics by actin-monomer-bindingproteins.Trends CellBiol2004,14(7):386-394.
3.Carlier MF,PernierJ,Montaville P,Shekhar S,Kuhn S:Control of polarized assemblyofactin filaments in cell motility.CellMolLifeSci2015,72(16):3051-3067.
4.Carlier MF,PernierJ,Avvaru BS:Control ofactin filament dynamics at barbed ends byWH2 domains:from cappingto permissive and processive assembly.Cytoskeleton(Hoboken)2013,70(10):540-549.
5.Matarrese P,Malorni W:Human immunodeficiencyvirus(HIV)-1proteins and cytoskeleton:partners inviral lifeand hostcell death.CellDeath Differ2005,12Suppl 1:932-941.
6.LevyJA:HIVpathogenesis:25yearsofprogressand persistentchallenges.Aids2009,23(2):147-160.7.Jacob T,Van den Broeke C,van Troys M,Waterschoot D,Ampe C,Favoreel HW:Alphaherpesviral US3 kinase induces cofilin dephosphorylation to reorganize the actin cytoskeleton.J Virol 2013,87(7):4121-4126.。
disclosure of Invention
The invention aims to provide HIV latent infection markers and application thereof based on the current state of the prior art, and particularly relates to target markers LAMP2, CD36 and CD47 for treatment and diagnosis of HIV latent infection and application thereof.
The invention takes HIV latently infected U1 cells as a research model to obtain an HIV latently infected marker and a new target for treating HIV latently infected, and particularly relates to LAMP2, CD36 and CD47 proteins as HIV latently infected markers; the invention also provides application of the LAMP2, CD36 and CD47 proteins in preparation of diagnostic reagents for latent HIV infection and targeted drugs for latent HIV infection.
The invention utilizes an HIV latent cell line U1 and a contrast U937 cell line thereof, and proteome research of analyzing plasma membranes of U1 cells and U937 cells through an iTRAQ-labeled proteomics technology shows that LAMP2, CD36 and CD47 are low in expression in HIV latent infected cells U1, and the LAMP2, CD36 and CD47 proteins can be used as HIV latent infection markers.
The expression of CD36 and CD47 is detected by an immunoblotting (WB) method by taking U1 and U937 cells and J-Lat and Jurkat cells as models, and the results show that the protein expression of CD36 and CD47 is down-regulated in two latent cells;
collection therapyEDTA anticoagulation of AIDS patients and healthy volunteers for more than 6 months, separation of resting CD4+T cells, followed by WB for detection of resting CD4+The expression of LAMP2 in T cells shows that the expression of LAMP2 is down-regulated in resting cells; the cell model proves that LAMP2, CD36 and CD47 can be used as HIV latent infection markers.
The target markers LAMP2, CD36 and CD47 are detected by WB or flow cytometry, and the method comprises the following steps:
(1) WB sampling technology for detecting LAMP2, CD36 and CD47 in blood cells
Blood is taken, PBMCs are purified, and the WB technology is adopted to detect LAMP2, CD36 and CD47 in the PBMCs;
(2) detection of LAMP2, CD36 and CD47 in plasma by ELISA technique
Blood was taken, plasma was separated and LAMP2, CD36 and CD47 in the plasma were detected using ELISA techniques.
(3) Detection of LAMP2, CD36 and CD47 in resting CD4+ T cells by flow cytometry
Blood was taken and LAMP2, CD36 and CD47 in resting CD4+ T cells were detected by flow cytometry.
Preferably, in the present invention, WB is used to detect LAMP2, CD36 and CD47 in PBMCs.
Preferably, the present invention uses ELISA technology to detect LAMP2, CD36 and CD47 in plasma.
Preferably, the present invention uses flow cytometry to detect LAMP2, CD36 and CD47 in blood.
The experimental results of the present invention show that,
1) studies using iTRAQ-based proteomics techniques showed that LAMP2, CD36 and CD47 were down-regulated in protein expression levels in HIV latent cells;
2) the down-regulation of protein expression of CD36 and CD47 in HIV latent cells is verified by J-Lat and U1 cell models;
3) resting CD4 in HIV latency+LAMP2 protein expression was down-regulated in T cells.
The invention is proved by experiments to obtain a novel HIV latent infection marker: LAMP2, CD36 and CD47, and the LAMP2, CD36 and CD47 proteins can be used as HIV latent infection markers for preparing HIV latent infection diagnosis and differential diagnosis reagents for LAMP2, CD36 and CD47 and new anti-HIV latent infection targeted therapeutic drugs.
In the present invention, the abbreviations used are as shown in Table 1,
TABLE 1 list of abbreviations
Acronyms English name Name of Chinese
HIV human immunodeficiency virus AIDS virus
LC-MS Liquid chromatography-Mass spectrometry Liquid chromatography tandem mass spectrometry
LAMP2 Lysosomal Associated Membrane Protein 2 Lysosomal associated membrane protein 2
WB Western blot Immunoblotting
ELISA Enzyme linked immunosorbent assay Enzyme linked immunosorbent assay
。
Drawings
Figure 1, 110 different proteins and their heatmaps.
FIG. 2, WB detects protein expression of CD36, CD47 and LAMP2, wherein,
expression of CD36, CD47, CD4 and ITGA6 in U1 and U937 cells, and J-Lat and Jurkat cells; B. protein expression of LAMP2 in resting CD4+ T cells.
Detailed Description
1. The materials and equipment used in the experiments of the present invention are as follows,
1.1 cell lines
1) U937 cells: human histiocyte lymphoma cells purchased from Shanghai cell Bank of Chinese academy of sciences.
2) U1 cells: HIV latently infects the cell model, the mother cell is U937 cell, presented by the university of Zandong, Jiangshik and Lulu laboratories.
3) Jurkat cell line: human peripheral blood leukemia T cells, offered as a gift by professor's laboratory of Xujian Qing, the public health clinic center of Shanghai city. J-Lat cell line: the Jurkat cell line was derived from HIV latent cells and was offered by professor's laboratory, Xujian, national institutes of public health and clinical center, Shanghai.
1.2 reagents and consumables
Figure BDA0002837164130000041
Figure BDA0002837164130000051
1.4 Experimental instrumentation
Figure BDA0002837164130000052
Example 1 cell culture and sample preparation
1 cell recovery
1) U1, U937, J-Lat and Jurkat cells taken out of a liquid nitrogen tank or a refrigerator at-80 ℃ are put into water at 37 ℃ and quickly shaken back and forth to melt the cells as soon as possible.
2) The cryopreserved tube was transferred to a biosafety cabinet, the cell suspension was transferred to a 15mL centrifuge tube, 5mL of 1640 complete medium was added, 1000rpm was used, and centrifugation was performed for 5 min.
3) The supernatant was discarded and the pellet was resuspended in 1mL 1640 complete medium.
4) Cell count and cell density adjusted to 5X 105one/mL, which was transferred to a T25 cell culture flask (5-6 mL/flask).
5) Screwing the cover of the culture flask, ventilating air, placing in a 37 deg.C incubator with 5% CO2And (5) culturing.
2 passage of cells
1) The suspension cell culture in logarithmic growth phase was transferred to a 15mL centrifuge tube in a biosafety cabinet, centrifuged at 1000rpm for 5 min.
2) The supernatant was discarded, washed once with 3mL of 1 XPBS, 1000rpm and centrifuged for 5 min.
3) Cell count, density adjusted to 5X 105one/mL, which was transferred to a T25 cell culture flask (5-6 mL/flask).
4) Screwing the cover of the culture flask, ventilating with air, placing in an incubator at 37 deg.C and 5% CO2Culturing for 2-3 days and passing once.
3 cell cryopreservation
1) The suspension cell culture was transferred to a 15mL centrifuge tube, centrifuged at 1000rpm for 5 min.
2) Wash once with 1 XPBS.
3) Resuspending the pellet with cell freezing medium to a concentration of about 1-2X 106Cell suspension was transferred to cryopreservation tubes, 1 mL/tube, and cell name, cryopreservation date and cell number were recorded.
4) And (3) placing the freezing tube in a programmed cooling box, placing the freezing tube in a refrigerator at the temperature of-80 ℃, and transferring the freezing tube into a liquid nitrogen tank or a sample box after 24 hours.
4 plasma Membrane protein extraction
As a concrete procedure, refer to the Plasma Membrane Protein Extraction Kit (ab65400, abcam corporation)
1) Expansion of suspension cells to 5-8X 108500g, centrifuge for 5 min.
2) Wash with 1 XPBS and discard the supernatant.
3) The pellet was resuspended in 1mL of precooled Homogenize Buffer Mix, placed on ice, and ground 50 times with a glass homogenizer until the cells were completely disrupted.
4) The homogenate was transferred to a 1.5mL centrifuge tube at 4 ℃ for 10min at 700 g.
5) The supernatant was transferred to a new 1.5mL centrifuge tube at 4 ℃ for 30min at 10,000 g.
6) The precipitation is full membrane protein, including cytoplasmic membrane protein and organelle membrane protein, and part of the full membrane protein is reserved.
7) Resuspend the pellet in 300. mu.L of supernatant from the kit, add 200. mu.L of subnatant from the kit, mix, label A, incubate for 5min on ice.
8) A new 1.5mL centrifuge tube was added with 200. mu.L of supernatant and 200. mu.L of subnatant, and mixed well, labeled B.
9) Centrifuge A, B tubes, 1000g, 10 min.
10) The upper layer in tube A was transferred to a new 1.5mL centrifuge tube, labeled C.
11) Mix the lower layer of tube A with the upper layer of tube 100 μ L B, 1000g, 10 min.
l2) collect the upper layer into C tube, then mix the lower layer of 100. mu. L B tube uniformly, 1000g, 10 min.
13) The upper layer was collected with 5 volumes of ddH2Dilution with O and incubation on ice for 5 min.
14) Removing supernatant at 4 deg.C for 10min, precipitating to obtain plasma membrane protein, and freezing the plasma membrane protein and whole membrane protein in-80 deg.C refrigerator for later use.
5 protein assay
1) The bidirectional lysate is used to lyse the protein.
2) Protein concentration was measured by the Broadford method.
3)20 μ g, 100 μ g of protein were dispensed and stored in a-80 ℃ freezer for later use.
Example 2 iTRAQ-based proteomic experiments
1. Reductive alkylation and enzymatic hydrolysis of plasma membrane proteins
1) The assay was performed using 6 of the 8-labeled iTRAQ markers, with 3 replicates for each sample. 100ug of plasma membrane protein from U1 and U937 cells, respectively, were transferred to a 1.5mL centrifuge tube, labeled, and brought to a volume of 30. mu.L.
2) Add reducing agent 120. mu.L into each tube, mix by vortexing, and centrifuge instantaneously. Repeat once for thorough mixing. Wrapping the centrifuge tube with sealing film, and water bath at 37 deg.C for 1 hr.
3) Add 120. mu.L of reducing agent per tube, vortex and mix well, centrifuge instantaneously, repeat once. Standing at room temperature for 10-15min, and keeping away from light.
4) The new ultrafiltration tube was labeled and the sample was transferred to the ultrafiltration tube at 4 ℃ for 40min at 12,000 rpm. If the sample is not completely centrifuged down, the time is suitably extended.
5) 50 μ L of EAB1 was added to each tube at 4 ℃ and 12,000rpm, and centrifuged for 30min to remove all liquid from the filter.
6) The solution at the bottom of the ultrafiltration tube was discarded, 150. mu.L buffer2 was added to each tube, centrifuged at 12,000rpm at 4 ℃ for 40 min.
7) 150 μ L of EAB1 was added to each tube, centrifuged at 12,000rpm at 4 ℃ for 40min, and the waste solution at the bottom of the ultrafiltration tube was discarded.
8) Repeat step 7 twice.
9) The filter membrane of the ultrafiltration tube was removed, 1mL of chromatography pure water was added to each tube, the ultrafiltration tube was repeatedly rinsed, and the rinse was discarded.
10) Filling the filter membrane of the ultrafiltration tube, adding 100 mu L of the enzymolysis solution, sealing the ultrafiltration tube by using a sealing membrane, and carrying out water bath at 37 ℃ for 15-16 h.
2. Peptide fragment collection and labeling reaction after enzymolysis
1) Taking out the protein sample after enzymolysis, centrifuging at room temperature of 12,000rpm for 10min, and collecting the digested peptide fragment at the bottom of the ultrafiltration tube.
2) 50 μ L of EAB2 was added to the ultrafiltration tube at 12,000rpm and centrifuged for 10 min. Repeating the steps once to fully collect peptide fragments.
3) Transferring the peptide fragment to a new centrifugal tube with the volume of 1.5mL, marking, sealing with a sealing film, perforating the sealing film with a perforator, and freeze-drying in a freeze dryer.
4) Samples were removed and 100. mu. of LTEAB2 was added and vortexed thoroughly.
5) The iTRAQ reagent was removed from the refrigerator, equilibrated to room temperature, and centrifuged instantaneously.
6) Add 280. mu.L of isopropanol to each labeling reagent, vortex and mix, centrifuge instantaneously, repeat once.
7) And adding the marking reagent into the peptide fragment solution, marking, uniformly mixing by vortex, performing instantaneous centrifugation, and standing at room temperature for 2 hours. U1 cell labeling: 114. 115, 116, U937 cell markers 117, 118, 119.
8) After the reaction, 400. mu.L of purified chromatographic water was added thereto, and the mixture was left at room temperature for 30 min.
9) Mixing the marked samples, uniformly mixing by vortex, centrifuging to the bottom of the tube, subpackaging in a plurality of 1.5mL centrifuge tubes, sealing the tube openings by using sealing films, perforating by using a puncher, and freeze-drying by using a freeze dryer. Stored at-80 ℃ for later use.
3.2D-LC-MS/MS analysis
1) Strong cation exchange chromatographic separation
a) The lyophilized sample was reconstituted with 120. mu.L of mobile phase A, vortexed, mixed, centrifuged at 12,000rpm for 10min at room temperature, and 110ul of the solution was aspirated into a sample vial. Elution was performed by an Agilent 1100 instrument using a strong cation exchange (SCX) chromatography column (2.1 mm. times.150 mm, C18,5 μm, Agilent Technologies).
b) The polypeptide was eluted by gradient elution with 2% acetonitrile (pH10) and 98% acetonitrile (pH10) as eluents at a flow rate of 0.3mL/min for 1h, which was divided into 10 fractions, which were then lyophilized.
2) Reverse chromatography-TripleTOF analysis
a) The fractions were redissolved in Nano-RPLC Buffer A (containing 0.1% formic acid and 5% acetonitrile) first by dissolving inLine Nano-RPLC liquid chromatography on Eksigent nanolC-UltraTM2D system, pre-column with C18 (100. mu. m.times.3 cm, C18,3 μm,
Figure BDA0002837164130000081
) Desalting is carried out.
b) The fractions were then automatically passed through a C18 reverse phase chromatography column (C18,3 μm,350 μm. times.0.5 mm,
Figure BDA0002837164130000082
) And carrying out gradient elution by using 0.1% formic acid and 2% acetonitrile as a mobile phase A and 0.1% formic acid and 98% acetonitrile as a mobile phase B at the flow rate of 0.3mL/min for 70 min.
c) The mass spectrometry adopts Triple TOF 5600(AB SCIEX) in combination with a nanoliter spray III ion source, the spray voltage reaches 2.5kV, the air curtain pressure reaches 30PSI, the atomization pressure is 5PSI, and the obtained ions have wide range, high sensitivity and resolution, short time and high efficiency.
4 plasma membrane protein qualitative and quantitative
1) Database search and protein identification
Data processing was performed using Protein Pilot Software v.5.0(AB SCIEX, USA) Software, a database of which was derived from transcriptome sequencing data. When the protein is qualitative, MS/MS data of an experiment and theoretical mass spectrum data obtained by simulating a database are matched, so that the identification result of the protein is obtained. Because of the presence of biological variations, we performed FDR (error rate) estimation by PSPEP software.
2) Differential protein screening
1) In each experiment, 3 groups of authentic proteins were screened, and then differential proteins were screened for authentic proteins.
Screening criteria: a) authentic proteins: FDR is less than 1 percent, and the number of peptide fragments is more than or equal to 1.
b) Differential protein: fold difference >1.5 or <0.67, p < 0.05.
2) The Weinn graph shows the condition of the differential protein screened by each repeated group, and the specific steps are as follows:
a) opening the website http:// bioinformatics. psb. element. be/webtools/Venn/;
b) putting the differential protein screened in the front into a txt file;
c) clicking 'select file' in the upload file area, and uploading txt file;
d) click "Submit"; and obtaining the Wien picture and copying the picture.
Example 3 validation of CD36 and CD47 differential proteins WB
1 protein electrophoresis
1) Adding equal volume of 2 Xloading buffer solution into 20 μ g of protein, mixing by vortex, performing metal bath at 100 deg.C for 10min for protein denaturation, at 4 deg.C, 12000rpm, and centrifuging for 10 min.
2) And (4) placing the solidified gel glass plate in an electrophoresis tank, adding an electrode buffer solution, and paying attention to leakage detection. The comb was carefully removed and the protein sample was carefully added using a pipette gun, with one pre-stained protein Marker on each side of the sample as a reference.
And (3) connecting an electrophoresis device, keeping the constant pressure of the concentrated gel at 50V for 40min, separating the gel at 120V when a pre-dyed marker appears, finishing gel running when bromophenol blue approaches the bottom of the separation gel, and closing the electrophoresis device.
2-turn film
1) Cutting PVDF film into pieces with the same size as the glue, soaking in methanol for 10s, ddH2O rinsing, and soaking in membrane transfer buffer.
2) The gel was peeled off and also soaked in the membrane transfer buffer, waiting for 15 min.
3) Appropriate amount of membrane transfer buffer solution was added to the flat-bottom tray, and 2 translucent membrane pads and 2 porous pads were soaked inside.
4) Manufacturing a film transfer unit: black plates, membrane pads, porous pads, gels, PVDF membranes, porous pads, membrane pads, white plates.
5) And transferring the film transfer unit into a film transfer groove, wherein the black plate corresponds to the black groove. And filling the film transfer tank with a film transfer buffer solution, and connecting a power supply. 200V,300mA, and 1h for film rotation.
3 immunoblotting
1) And (3) after the membrane transfer is finished, transferring the pre-dyed Marker and the protein to a PVDF membrane, and sealing the sealing liquid at the room temperature for 2 hours.
2) Primary antibody was diluted with blocking solution, applied to PVDF membrane, carefully not to generate air bubbles, and incubated at room temperature for 2h or overnight in a refrigerator at 4 ℃.
Na+/K+-ATPase, prohibitin, β -actin antibodies at a dilution ratio of 1:5000, 1:2000, and 1:1000, respectively.
3) The membrane was washed 3 times 10 min/time with 0.05% TBS-T.
4) The secondary antibody was diluted with blocking solution at a dilution ratio of 1:2000, applied to PVDF membrane, with care taken not to generate air bubbles, and incubated at room temperature for 1 h.
5) The membrane was washed 3 times with 0.05% TBS-T for 10 min/time.
4 chemiluminescence
And uniformly mixing the chemiluminescent substrates A and B1:1, adding the mixture onto a PVDF membrane, placing the PVDF membrane into an instrument for exposure for 3-10 min, and storing corresponding pictures.
Experimental example 4LAMP2 on resting CD4 of HIV-latent patients+Expression in T cells
1 volunteers into groups
The clinical trial scheme was reviewed by the ethical committee of the public health clinical center in Shanghai to obtain written informed consent of volunteers.
1) 5 healthy adult male subjects (no hepatitis B, hepatitis C, syphilis and HIV infection, normal liver and kidney function and other laboratory tests) are added, and 100mL of EDTA anticoagulation blood is collected.
2) 5 HIV infectors are used as patients, the patients receive antiviral treatment for more than 6 months, the virus load after treatment is less than 50copies/mL, and no other combined infection such as viral hepatitis, syphilis, tuberculosis and the like exists, and no other complication exists.
2 resting CD4 in clinical specimens (HIV-latently patients)+Isolation of T cells
1) Using CD4+T cell extraction kit (Milteyi biotec, 130096533)
a) Centrifuging at 200g for 5min, and using 10 times7Add 40. mu.L buffer to each cell.
b) Adding CD4+ T cell anti-biotin antibody mixture for every 107mu.L of each cell was added.
c) Mixing, and incubating at 4 deg.C for 10 min.
d) By 10 each7 Add 30. mu.L buffer to each cell.
e) Adding CD4+T cell MicroBead mix, 10 per batch710 μ L of each cell was added.
f) Mixing, and incubating at 4 deg.C for 10min to obtain cell suspension.
g) The volume is enlarged by 3 times because the MS column is easy to block due to excessive cell density, and the cells which pass through the MS column and are subjected to negative screening are CD4+T cells
2) Resting CD4+T cells
a) Centrifuging at 200g for 5min, and using 10 times7An individual CD4+T cells were loaded with 40. mu.L of buffer.
b) Adding CD25 anti-biotin antibody, CD69 anti-biotin antibody, HLA-DR anti-biotin antibody every 107Adding 5 mu L of each cell;
c-g) the above steps to obtain the negative selection cell, namely resting CD4+T cells.
And reserving a part of cells for flow identification, centrifuging the rest part of cells, removing supernatant, and freezing in a refrigerator at the temperature of-80 ℃ for later use.
Detection of resting CD4 with flow antibody+Yield and typing of T cells
1) At least 5X 10 samples are required for each sample to be tested5The cells were centrifuged at 600g for 5min at room temperature, and the supernatant was discarded.
2) Wash once with staining buffer (PBS containing 1% BSA) and discard the supernatant.
3) Antibodies that need to be stained in multiple colors are formulated together. CD3-ECD, CD4-V450, CD25-PC7, CD69-APC-CY7 and HLA-DR-APC are in one group, and CD3-ECD, CD4-V450, CD45RA-PC7, CD27-PE and CCR7-APC are in the other group.
4) Fluorescent antibodies were added to the samples, diluted with staining buffer to give a final volume of 40 μ L per sample, and mixed well.
4) To adjust for the complement, one single stain was made for each dye, and the final volume of each sample was 40 μ L.
5) And dyeing for 20min at room temperature in dark.
6) Add 800. mu.L of staining buffer, mix well, 600g, centrifuge for 5min, discard the supernatant.
7) The washing was repeated.
8) Adding 1% paraformaldehyde, and fixing at 4 deg.C for 15 min. 600g, centrifuge for 5min, and discard the supernatant.
9) The pellet was resuspended in 100. mu.L of staining buffer and stored at 4 ℃ for future use.
Note that: in order to extend the life of the antibody, the antibody application needs to be performed on ice.
In the embodiment of the invention, the obtained data are analyzed by SPSS 22.0 statistical software, the experiment is repeated for 2-3 times, and the statistical difference is that P is less than 0.05.
The experimental results of the invention show that: CD36, CD47 and LAMP2 down-regulated protein expression in the HIV latent cell line U1 (compared to the control cell line U937); WB results from U1, U937, J-Lat and Jurkat cell lines showed that the protein content of CD36 and CD47 in HIV-latent cells (U1, J-Lat) was much lower than in their control cells (U937, Jurkat); resting CD4 of HIV patient+Protein expression of LAMP2 in T cells was significantly lower than in healthy volunteers.
The experimental results show that the CD36, the CD47 and the LAMP2 can be used as markers of HIV latent infection, and the CD36, the CD47 and the LAMP2 can be used for preparing targeted drugs for treating HIV latent infection and diagnostic kits for HIV latent infection.

Claims (8)

  1. A marker for latent HIV infection, wherein the marker is LAMP2, CD36, and CD47 protein.
  2. 2. Use of the HIV latent infection marker of claim 1 in the preparation of a reagent for diagnosis and differential diagnosis of HIV latent infection by LAMP2, CD36 and CD47, and a novel anti-HIV latent infection drug.
  3. 3. Use of the HIV latent infection marker of claim 1 in the preparation of a medicament for the treatment of HIV latent infection against LAMP2, CD36, and CD 47.
  4. 4. The use according to claim 2, characterized in that the markers LAMP2, CD36 and CD4 are target markers for HIV latent infection diagnosis and differential diagnosis reagents, which are detected by WB or flow cytometry, comprising the following steps:
    (1) WB sampling technology for detecting LAMP2, CD36 and CD47 in blood cells
    Blood is taken, PBMCs are purified, and the WB technology is adopted to detect LAMP2, CD36 and CD47 in the PBMCs;
    (2) detection of LAMP2, CD36 and CD47 in plasma by ELISA technique
    Blood is taken, plasma is separated, and LAMP2, CD36 and CD47 in the plasma are detected by adopting an ELISA technology;
    (3) detection of LAMP2, CD36 and CD47 in resting CD4+ T cells by flow cytometry
    Blood was taken and LAMP2, CD36 and CD47 in resting CD4+ T cells were detected by flow cytometry.
  5. 5. The use of claim 4, wherein WB is used to detect LAMP2, CD36 and CD47 in PBMCs.
  6. 6. Use according to claim 4, wherein in said method the plasma is assayed for LAMP2, CD36 and CD47 using ELISA techniques.
  7. 7. Use according to claim 4, wherein the method is used to detect LAMP2, CD36 and CD47 in blood using flow cytometry.
  8. 8. The use of claim 3, wherein the therapeutic agent targets LAMP2, CD36 and CD 47.
CN202011482869.6A 2020-12-15 2020-12-15 HIV latent infection marker and application thereof Pending CN114636822A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106176753A (en) * 2016-07-15 2016-12-07 南方医科大学 RVX 208 is as the application of HIV 1 latent infection inversion agent
US20170322141A1 (en) * 2015-01-19 2017-11-09 Sri International Measuring HIV Reservoirs with Optical Scanning

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170322141A1 (en) * 2015-01-19 2017-11-09 Sri International Measuring HIV Reservoirs with Optical Scanning
CN106176753A (en) * 2016-07-15 2016-12-07 南方医科大学 RVX 208 is as the application of HIV 1 latent infection inversion agent

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