METHOD FOR SIMULTANEOUSLY DETECTING EXOSOME MEMBRANE PROTEIN AND MRNA

Abstract

A method for simultaneously detecting an exosome membrane protein and mRNA is provided. The method can perform simultaneous detection on exosomes separated and purified from the same sample by labeling an exosome membrane protein with a fluorescent antibody and labeling a target gene mRNA with a molecular beacon, wherein labeling the exosome with the molecular beacon is specifically performed by means of an in-situ exosome capture well plate or chip, and each well or chip in the in-situ exosome capture well plate includes a fluorescein-labeled molecular beacon; and the molecular beacon is a specific DNA probe for detecting the target gene mRNA. The present invention utilizes an in-situ exosome capture well plate or chip technology to detect a biomarker gene mRNA contained in an exosome of a biological sample.

Claims

1. A method for simultaneously detecting exosome membrane protein and mRNA, wherein the method can perform simultaneous detection on exosomes separated and purified from the same sample by labeling an exosome membrane protein with a fluorescent antibody and labeling a target gene mRNA with a molecular beacon; wherein labeling the exosome with the molecular beacon is specifically performed by means of an in-situ exosome capture well plate or chip, and each well or chip in the in-situ exosome capture well plate comprises a fluorescein-labeled molecular beacon; and the molecular beacon is a specific DNA probe for detecting the target gene mRNA.

2. The method for simultaneously detecting exosome membrane protein and mRNA of claim 1, wherein a 5′ stem and loop on the specific DNA probe are completely complementary to the target gene; and a 3′ stem is partially complementary to the 5′ stem; a 5′ terminal and a 3′ terminal are respectively modified by a fluorophore and a quencher group, and partial bases on the loop are modified by locked nucleic acid.

3. The method for simultaneously detecting exosome membrane protein and mRNA of claim 1, wherein the specific DNA probe is self-designed and modified for synthesis according to a target gene sequence; and the fluorescent antibody for labeling the exosome membrane protein is a fluorescein-labeled monoclonal antibody.

4. The method for simultaneously detecting exosome membrane protein and mRNA of claim 1, wherein the specific DNA probe is coated with a cationic lipid composite nanoparticle.

5. (canceled)

6. The method for simultaneously detecting exosome membrane protein and mRNA of claim 1, wherein the sample comprises: cell culture supernatants, isolated laboratory animal plasma, serum, isolated human plasma, serum, urine, and other body fluids or excrement samples.

7. The method for simultaneously detecting exosome membrane protein and mRNA of claim 1, wherein the sample is from a source of a living cell, an animal, human body fluid or an excrement sample.

8-10. (canceled)

11. The method for simultaneously detecting exosome membrane protein and mRNA of claim 2, wherein the specific DNA probe is coated with a cationic lipid composite nanoparticle.

12. The method for simultaneously detecting exosome membrane protein and mRNA of claim 3, wherein the specific DNA probe is coated with a cationic lipid composite nanoparticle.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 is a schematic diagram showing the detection principle of an exosome membrane protein PDL1 and mRNA thereof as an example;

[0024] FIG. 2 shows expression of a membrane protein PDL1 and mRNA of Example 2 in an exosome of a lung cell line;

[0025] FIG. 3 shows expression of the membrane protein PDL1 and mRNA of Example 2 in an exosome of a lung tumor;

[0026] FIG. 4 shows expression of the membrane protein GP73 and mRNA of Example 2 in an exosome of a liver cell line;

[0027] FIG. 5 shows expression of a membrane protein GP73 and mRNA of Example 2 in an exosome of a liver tumor;

DESCRIPTION OF THE EMBODIMENTS

[0028] The present invention is further described in combination with embodiments below.

Example 1: Design of a Specific Molecular Beacon (Targets PDL1 and GP73 were Set as Examples)

[0029] It is crucial to design a specific molecular beacon for detecting a target gene for an exosome capture well plate or chip to detect specific nucleic acid. For this purpose, the Applicant designed a molecular beacon having a special stem-loop structure in combination with the features of a target gene; a 5′ stem and loop were completely complementary to the target gene; a 3′ stem was partially complementary to the 5′ stem; a 5′ terminal and 3′ terminal were respectively modified by a fluorophore and a quencher group; and partial bases on the loop were modified by locked nucleic acid; the specific PDL1 molecular beacon had a specific sequence as shown in Table 1; the sequence shown in SEQ ID No. 1 had the base modification mode: base 1 was modified by 6FAM; bases 10, 13, 16, 19, 22, 25 and 28 were modified by LNA, and base 36 was modified by BHQ1; the sequence shown in SEQ ID No. 2 had the base modification mode: base 1 was modified by 6FAM; bases 10, 13, 16, 19, 22, 25 and 28 were modified by LNA, and base 35 was modified by BHQ1; the sequence shown in SEQ ID No. 3 had the base modification mode: base 1 was modified by 6FAM; bases 10, 13, 16, 19, 22, and 25 were modified by LNA, and base 34 was modified by BHQ1; the sequence shown in SEQ ID No. 4 had the base modification mode: base 1 was modified by 6FAM; bases 10, 13, 16, 19, 22, 25 and 28 were modified by LNA, and base 35 was modified by BHQ1; the sequence shown in SEQ ID No. 5 had the base modification mode: base 1 was modified by 6FAM; bases 10, 13, 16, 19, 22, 25 and 28 were modified by LNA, and base 35 was modified by BHQ1; and the sequence shown in SEQ ID No. 6 had the base modification mode: base 1 was modified by 6FAM; bases 10, 13, 16, 19, 22, 25 and 28 were modified by LNA, and base 36 was modified by BHQ1. For this purpose, the Applicant designed a molecular beacon having a special stem-loop structure in combination with the features of a target gene; a 5′ stem and loop were completely complementary to the target gene; a 3′ stem was partially complementary to the 5′ stem; a 5′ terminal and 3′ terminal were respectively modified by a fluorophore and a quencher group; and partial bases on the loop were modified by locked nucleic acid; the specific PDL1 molecular beacon had a specific sequence as shown in Table 1; the sequence shown in SEQ ID No. 1 had the base modification mode: base 1 was modified by 6FAM; bases 10, 13, 16, 19, 22, 25 and 28 were modified by LNA, and base 36 was modified by BHQ1; the sequence shown in SEQ ID No. 3 had the base modification mode: base 1 was modified by 6FAM; bases 10, 13, 16, 19, 22, 25 and 28 were modified by LNA, and base 35 was modified by BHQ1; the sequence shown in SEQ ID No. 3 had the base modification mode: base 1 was modified by 6FAM; bases 10, 13, 16, 19, 22, and 25 were modified by LNA, and base 40 was modified by BHQ1; the sequence shown in SEQ ID No. 5 had the base modification mode: base 1 was modified by 6FAM; bases 10, 13, 16, 19, 22, 25 and 28 were modified by LNA, and base 35 was modified by BHQ1; the sequence shown in SEQ ID No. 5 has the base modification mode: base 1 was modified by 6FAM; bases 10, 13, 16, 19, 22, 25 and 28 are modified by LNA, and base 35 was modified by BHQ1; the sequence shown in SEQ ID No. 6 has the base modification mode: base 1 was modified by 6FAM; bases 10, 13, 16, 19, 22, 25 and 28 were modified by LNA, and base 36 was modified by BHQ1; and the sequence shown in SEQ ID No. 7 has the base modification mode: base 1 was modified by 6FAM; bases 10, 13, 16, 19, 22, 25 and 28 were modified by LNA, and base 36 was modified by BHQ1.

[0030] The specific molecular beacon designed by the present invention improves the binding specificity of a molecular beacon to a target gene to the maximum extent, and reduces the background fluorescence intensity of the reaction. After the molecular beacon was synthesized, to prove its binding specificity to the corresponding target gene and optimum working temperature, we designed the following Table 3, thus choosing the optimum molecular beacon based on the maximum Signal to Noise Ratio (SNR), and its working temperature.

TABLE-US-00001 TABLE 1 PDL1 probe sequence Sequence (5′-3′, “+” represented that LNA Modifying No. modified base) group 1 5′ 6FAM/ 6FAM-CGCGATCGG+AGG+ATG+TGC+CAG+AGG+TAG+TTGATCGCG- BHQ1/ BHQ1 3′ (as shown in SEQ ID No. 1) LNA 2 5′ 6FAM/ 6FAM-CGCGATCGC+TAT+GGT+GGT+GCC+GAC+TAC+AGATCGCG- BHQ1/ BHQ1 3′ (as shown in SEQ ID No. 2) LNA 3 5′ 6FAM/ 6FAM-CGCGATCTG+GTG+CCG+ACT+ACA+AGC+GAAGATCGCG- BHQ1/ BHQ1 3′ (as shown in SEQ ID No. 3) LNA 4 5′ 6FAM/ 6FAM-CGCGATCTG+GTG+CCG+ACT+ACA+AGC+GAA+TGATCGCG- BHQ1/ BHQ1 3′ (as shown in SEQ ID No. 4) LNA 5 5′ 6FAM/ 6FAM-CGCGATCGG+AGG+ATG+TGC+CAG+AGG+TAG+TGATCGCG- BHQ1/ BHQ1 3′ (as shown in SEQ ID No. 5) LNA 6 5′ 6FAM/ 6FAM-CGCGATCGC+TAT+GGT+GGT+GCC+GAC+TAC+AAGATCGCG- BHQ1/ BHQ1 3′ (as shown in SEQ ID No. 6) LNA

TABLE-US-00002 TABLE 2 GP73 probe sequence Sequence (5′-3′, “+” represented that LNA modified Modifying No. base) group 1 5′ 6FAM/ 6FAM-CGCGATCGG+CGG+CGA+CTT+CAT+GCT+GCG+AGATCGCG- BHQ1/ BHQ1 3′ (as shown in SEQ ID No. 7) LNA 2 5′ 6FAM/ 6FAM-CGCGATCGA+CTT+CAT+GCT+GCG+ACG+CCC+GTT+TGATCGCG- BHQ1/ BHQ1 3′ (as shown in SEQ ID No. 8) LNA 3 5′ 6FAM/ 6FAM-CGCGATCCG+CCC+TGC+GGA+CCC+TGC+CTT+CGATCGCG- BHQ1/ BHQ1 3′ (SEQ ID No. 9) LNA 4 5′ 6FAM/ 6FAM-CGCGATCCC+AGG+GCT+GCT+TGC+TTG+TCT+GTC+TCAGATCGCG- BHQ1/ BHQ1 3′ (as shown in SEQ ID No. 10) LNA 5 5′ 6FAM/ 6FAM-CGCGATCTG+CCA+GGG+CTG+CTT+GCT+TGT+CTG+TGATCGCG- BHQ1/ BHQ1 3′ (as shown in SEQ ID No. 11) LNA 6 5′ 6FAM/ 6FAM-CGCGATCGC+GAC+GCC+CGT+TTC+CCA+AGC+CGATCGCG- BHQ1/ BHQ1 3′ (as shown in SEQ ID No. 12) LNA 7 5′ 6FAM/ 6FAM-CGCGATCGC+TGC+GAC+GCC+CGT+TTC+CCA+AGGATCGCG- BHQ1/ BHQ1 3′ (as shown in SEQ ID No. 13) LNA

TABLE-US-00003 TABLE 3 37° C. 42° C. 50° C. 55° C. Pure Tem- Mole- Template + Tem- Molecular Template + Tem- Molecular Template + Tem- Molecular Template + water* plate cular Molecular plate beacon Molecular plate beacon Molecular plate beacon Molecular beacon beacon beacon beacon beacon Body An An An An An An An An An An An An fluid** exosome exosome exosome exosome exosome exosome exosome exosome exosome exosome exosome exosome capture capture capture capture capture capture capture capture capture capture capture capture well well well well well well well well well well well well plate plate plate plate plate plate plate plate plate plate plate plate or chip or chip or chip or chip or chip or chip or chip or chip or chip or chip or chip or chip + a + a + a + a + a + a + a + a + a + a + a + a control test test control test test control test test control test test sample sample sample sample sample sample sample sample sample sample sample 2 1 2 1 2 1 2 1 *A fluorescent reader was used to read the fluorescence intensity. **A TIRF microscope was used to detect the fluorescence intensity.

Example 2: Detection Test (Targets PDL1 and GP73 were Respectively Set as Examples)

[0031] I. Exosome Isolation

[0032] 1. 200 ul sample was taken (a cell culture supernatant, an isolated laboratory animal plasma, serum, isolated human plasma, serum, urine, and other body fluid or excrement sample), and 12000×g were centrifuged for 30 min at room temperature to remove cells and fragments;

[0033] 2. supernatant was transferred to a new EP tube, and a 100 ul exosome precipitate reagent was added;

[0034] 3. the above materials were mixed evenly for incubation for 30 min at 4° C.;

[0035] 4. 10,000×g were centrifuged for 30 min at room temperature;

[0036] 5. supernatant was removed by suction, and then 100 ul 1×PBS was taken to resuspend the precipitate rich in the exosome, standing at 4° C. for further use.

[0037] II. Purification of an Exosome Chromatographic Column

[0038] 1. Chromatographic column balancing: 100 ul equilibrium liquid was added and 9000×g were centrifuged for 1 min;

[0039] 2. sample loading: 100 ul resuspending solution was put on a column; and 9000×g were centrifuged for 1 min;

[0040] 3. eluting: a 50 ul eluent was added, and 9000×g were centrifuged for 1 min.

[0041] III. Detection of an Exosome Capture Well Plate

[0042] 1. The well plate or chip (each well in the well plate or chip can be coated with multiple fluorescein-labeled molecular beacons shown in Table 1 or Table 2, and the molecular beacon was coated with composite cationic lipid nanoparticles); and then the purified exosome eluent was added to sample wells;

[0043] 2. negative and positive controls (negative and positive controls were a nematode gene segment and a target gene fragment respectively coated with nanoparticles) were added to the subsequent sample wells;

[0044] 3. a PDL1 or GP73 fluorescent antibody was added according to a volume ratio of 1:1000;

[0045] 4. the above materials were incubated for 1 h at 42° C.;

[0046] 5. The well plate was washed by 1×PBS for three times, and an TIRF microscope was used to collect fluorescence images;

[0047] 6. DXimageV1 software was used to analyze the images to configure a cut-off value automatically, and interpret the result of the sample to be tested automatically.

[0048] IV. Test Results

[0049] 60 cases from a conventional normal human hepatic cell line HL-7702, a hepatoma carcinoma cell line HepG2, a normal lung cell line HLF-1 and a lung carcinoma cell line A549, and plasma samples of a patient suffering benign and malignant liver and lung tumors were configured respectively; and the results were shown in FIGS. 2, 3, 4, and 5; after the exosome capture well plate or chip was imaged under a TIRF microscope, the total fluorescence intensity of the exosome PDL1 membrane protein and mRNA from the lung carcinoma cell line A549 and malignant lung tumor was higher than that of the normal lung cell line HLF-1 and benign lung tumor. The total fluorescence intensity of the exosome GP73 membrane protein and mRNA from the hepatoma carcinoma cell line HepG2 and malignant liver tumor was higher than that of the normal lung cell line HL-7702 and benign liver tumor, indicating that the technology can simultaneously detect the target membrane protein and mRNA of the exosome.