阅读说明：本技术 一种用于检测血管通透性的产品及其制备方法 (Product for detecting vascular permeability and preparation method thereof ) 是由 胡朝阳 于 2021-01-11 设计创作，主要内容包括：本发明属于分子生物学领域,具体涉及一种用于检测血管通透性的产品及其制备方法。具体技术方案为：利用ARPC4和/或TRAF2或基因制备的检测用产品。本发明使用分子生物学技术,筛选出一种与血管通透性相关度高的靶基因。本领域技术人员可以利用业内常规技术制备出系列可用于检测血管通透性的试剂、试纸等相同产品。利用本发明提供的靶基因及制备的检测用产品,可及时、快速、准确判断血管通透性是否发生改变。(The invention belongs to the field of molecular biology, and particularly relates to a product for detecting vascular permeability and a preparation method thereof. The specific technical scheme is as follows: a product for detection prepared from ARPC4 and/or TRAF2 or gene. The invention screens out a target gene with high correlation degree with vascular permeability by using a molecular biology technology. Those skilled in the art can prepare a series of reagents and test strips for detecting vascular permeability by using the conventional technology in the industry. The target gene and the prepared detection product provided by the invention can be used for timely, quickly and accurately judging whether the vascular permeability is changed.)

Use of the TRAF2 and/or ARPC4 genes in the preparation of a product for detecting vascular permeability.

2. Use according to claim 1, characterized in that: the product is one of a detection reagent, a detection test paper and a kit.

3. A product for detecting vascular permeability, characterized by: is prepared by using TRAF2 and/or ARPC4 gene.

4. Product for detecting vascular permeability according to claim 3, characterized in that: contains TRAF2 and/or ARPC4 gene binding sites.

5. Product for detecting vascular permeability according to claim 3, characterized in that: the product is detection test paper.

6. Product for detecting vascular permeability according to claim 5, characterized in that: the preparation method of the test paper comprises the following steps:

synthesizing ARPC4 and/or TRAF2 protein to obtain protein product, injecting the synthesized protein into mouse body, and synthesizing clone antibody;

performing ELISA experimental screening on the obtained cloned antibody, fixing an antibody on an ELISA base plate to serve as a reaction 1 antibody, and adding a human serum sample with diseases related to vascular permeability change; combining another antibody with immunofluorescent enzyme, adding the antibody serving as a second antibody into the reaction, detecting the fluorescence reaction, and selecting the antibody with the maximum fluorescence reaction;

preparation of colloidal gold marker: adding potassium carbonate solution into colloidal gold, mixing uniformly, adding the screened antibody, mixing uniformly, standing, adding confining liquid, mixing uniformly, standing, centrifuging after standing, removing supernatant, redissolving the precipitate with colloidal gold redissolution, and mixing fully to obtain a solution, namely a colloidal gold marker;

preparing the colloidal gold test paper: the glass fiber membrane was cut to the desired size and the sample pad was placed in the colloidal gold label for sufficient absorption. And then, uniformly drying the sample pad by using a blower to prepare a gold label pad, sequentially sticking the nitrocellulose membrane, the gold label pad, the sample pad and the absorbent paper, cutting the gold label pad into required sizes after the assembly is finished, and putting the gold label pad into a container.

7. A method of screening for a target gene for detecting capillary permeability, comprising: the method comprises the following steps:

(1) selecting a disease associated with vascular permeability changes;

(2) respectively carrying out platelet transcriptome sequencing on a patient suffering from the disease related to vascular permeability change and a normal person to obtain the platelet gene expression quantity of a disease sample and a normal sample;

(3) taking the normal sample gene expression amount as a reference, carrying out expression difference statistics on the genes of diseases related to vascular permeability change, and screening out the difference genes with the gene expression times of | Log2FC | > 2 and P _ value less than 0.05;

(4) carrying out KEGG database comparison on the screened differential genes to obtain a differential gene enrichment pathway;

(5) selecting metabolic pathways related to membrane transport regulation in the disease enrichment pathway, and selecting differential genes from the selected metabolic pathways; subsequently, the reaction is carried out as follows (6-1) to (8-1), or as follows (6-2) to (8-2);

(6-1) further screening characteristic genes having prognostic significance from the differential genes of step (5) by using protein interaction analysis and subject manipulation characteristic analysis and using the screened characteristic genes as preselected target genes;

(7-1) performing amplification verification on the preselected target genes, and calculating copy multiples in the disease sample and the normal sample;

(8-1) selecting genes with the largest expression difference multiple from the preselected target genes in the step (7-1), namely the genes which are required to be the target genes;

(6-2) selecting a gene common to the disease positively correlated with vascular permeability change and the disease negatively correlated with vascular permeability change from the differential genes of step (5) as a preselected target gene;

(7-2) expressing the preselected target genes in the diseases positively correlated to the vascular permeability change, in the diseases negatively correlated to the vascular permeability change and in a normal sample respectively, and counting the expression amount of each preselected target gene;

(8-2) selecting a preselected target gene, which has an expression higher than that in the normal sample in the disease positively correlated with vascular permeability change and an expression higher than that in the normal sample in the disease negatively correlated with vascular permeability change, as the desired target gene.

8. A method of screening a target gene for detecting capillary permeability according to claim 7, wherein: the disease related to vascular permeability change is atherosclerosis, and/or thrombosis susceptibility, and/or purpura.

9. A method of screening a target gene for detecting capillary permeability according to claim 8, wherein: the diseases related to vascular permeability change are thrombosis and purpura.

10. A method of screening a target gene for detecting capillary permeability according to claim 9, wherein: when the disease related to vascular permeability change is atherosclerosis, carrying out the steps (6-1) to (8-1); and/or, when the disease related to vascular permeability change is thrombosis and purpura, performing the steps (6-2) to (8-2).

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a product for detecting vascular permeability and a preparation method thereof.

Background

Blood vessels are an important component of the blood circulation system. Blood circulation is accomplished without blood vessel exchange of blood and tissue material in the tissue. Vascular permeability refers to the ability of a substance to pass through the walls of capillary blood vessels.

Vascular permeability is affected by the structure of the vessel wall. Typically, the vessel wall is capable of blocking the passage of macromolecules through the vessel wall. However, when a series of pathological processes such as inflammation, burn, shock, immune reaction, tumor metastasis and the like occur, the permeability of the vascular wall is changed. Therefore, the method can detect the vascular permeability timely, quickly and accurately, and has important significance for discovering certain diseases in advance and diagnosing certain diseases.

The existing method for detecting vascular permeability usually depends on the occurrence of inflammatory pathological process and the detection of urine protein, namely, depends on the 'after' index that vascular permeability is changed and even symptoms appear, and can not effectively make advance judgment on the change of vascular permeability in time.

Therefore, if a product which can quickly and accurately judge the vascular permeability change can be provided, the method has important scientific research value and practical significance.

Disclosure of Invention

The invention aims to provide a product for detecting vascular permeability and a preparation method thereof.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: use of the TRAF2 and/or ARPC4 genes in the preparation of a product for detecting vascular permeability.

Preferably, the product is one of a detection reagent, a detection test paper and a kit.

Correspondingly, the product for detecting the vascular permeability is prepared by using TRAF2 and/or ARPC4 genes.

Preferably, the protein contains TRAF2 and/or ARPC4 gene binding sites.

Preferably, the product is a test paper.

Preferably, the preparation of the test strip comprises the following steps:

synthesizing ARPC4 and/or TRAF2 protein to obtain protein product, injecting the synthesized protein into mouse body, and synthesizing clone antibody;

performing ELISA experimental screening on the obtained cloned antibody, fixing an antibody on an ELISA base plate to serve as a reaction 1 antibody, and adding a human serum sample with diseases related to vascular permeability change; combining another antibody with immunofluorescent enzyme, adding the antibody serving as a second antibody into the reaction, detecting the fluorescence reaction, and selecting the antibody with the maximum fluorescence reaction;

preparation of colloidal gold marker: adding potassium carbonate solution into colloidal gold, mixing uniformly, adding the screened antibody, mixing uniformly, standing, adding confining liquid, mixing uniformly, standing, centrifuging after standing, removing supernatant, redissolving the precipitate with colloidal gold redissolution, and mixing fully to obtain a solution, namely a colloidal gold marker;

preparing the colloidal gold test paper: the glass fiber membrane was cut to the desired size and the sample pad was placed in the colloidal gold label for sufficient absorption. And then, uniformly drying the sample pad by using a blower to prepare a gold label pad, sequentially sticking the nitrocellulose membrane, the gold label pad, the sample pad and the absorbent paper, cutting the gold label pad into required sizes after the assembly is finished, and putting the gold label pad into a container.

Accordingly, a method for screening a target gene for detecting capillary permeability, comprising the steps of:

(1) selecting a disease associated with vascular permeability changes;

(2) respectively carrying out platelet transcriptome sequencing on a patient suffering from the disease related to vascular permeability change and a normal person to obtain the platelet gene expression quantity of a disease sample and a normal sample;

(3) taking the normal sample gene expression amount as a reference, carrying out expression difference statistics on the genes of diseases related to vascular permeability change, and screening out the difference genes with the gene expression times of | Log2FC | > 2 and P _ value less than 0.05;

(4) carrying out KEGG database comparison on the screened differential genes to obtain a differential gene enrichment pathway;

(5) selecting metabolic pathways related to membrane transport regulation in the disease enrichment pathway, and selecting differential genes from the selected metabolic pathways; subsequently, the reaction is carried out as follows (6-1) to (8-1), or as follows (6-2) to (8-2);

(6-1) further screening characteristic genes having prognostic significance from the differential genes of step (5) by using protein interaction analysis and subject manipulation characteristic analysis and using the screened characteristic genes as preselected target genes;

(7-1) performing amplification verification on the preselected target genes, and calculating copy multiples in the disease sample and the normal sample;

(8-1) selecting genes with the largest expression difference multiple from the preselected target genes in the step (7-1), namely the genes which are required to be the target genes;

(6-2) selecting a gene common to the disease positively correlated with vascular permeability change and the disease negatively correlated with vascular permeability change from the differential genes of step (5) as a preselected target gene;

(7-2) expressing the preselected target genes in the diseases positively correlated to the vascular permeability change, in the diseases negatively correlated to the vascular permeability change and in a normal sample respectively, and counting the expression amount of each preselected target gene;

(8-2) selecting a preselected target gene, which has an expression higher than that in the normal sample in the disease positively correlated with vascular permeability change and an expression higher than that in the normal sample in the disease negatively correlated with vascular permeability change, as the desired target gene.

Preferably, the disease associated with vascular permeability changes is atherosclerosis, and/or thrombosis, and/or purpura.

Preferably, the diseases related to vascular permeability change are thrombosis and purpura.

Preferably, when the disease associated with vascular permeability changes is atherosclerosis, steps (6-1) to (8-1) are performed; and/or, when the disease related to vascular permeability change is thrombosis and purpura, performing the steps (6-2) to (8-2).

The invention has the following beneficial effects: the invention uses molecular biology technology to screen two target genes with high correlation degree with vascular permeability. A series of related products such as reagents and test paper which can be used for detecting vascular permeability can be prepared by the technical personnel in the field by utilizing the conventional technology in the industry. The target gene and the prepared detection product provided by the invention can be used for timely, quickly and accurately judging whether the vascular permeability is changed.

Drawings

FIG. 1 is a schematic diagram of an atherosclerotic disease differential gene enrichment pathway;

FIG. 2 is a qpcr gene copy number assay for the ARPC4 gene;

FIG. 3 is a schematic diagram of an enrichment pathway of a thrombosis-prone differential gene;

FIG. 4 is a schematic diagram of a purpura differential gene enrichment pathway;

FIG. 5 is a schematic diagram showing the results of expression levels of PLCG1, TRAF2 and LAD genes;

FIG. 6 is a diagram showing a structure of a colloidal gold test paper;

FIG. 7 shows the results of a negative test on vascular permeability test paper;

FIG. 8 shows the result of a positive test on the vascular permeability test paper.

Detailed Description

The present invention provides both target genes useful for detecting vascular permeability: ARPC4, TRAF 2. The gene can be further prepared into reagents, test paper, kits, detection agents and the like so as to detect the change condition of vascular permeability timely, quickly, conveniently and accurately.

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The first embodiment is as follows: screening of qualified target genes using atherosclerosis

1. Diseases closely related to vascular permeability changes are selected: atherosclerosis. Lipid metabolism disorder is the occurrence basis of atherosclerosis, and is gradually caused by fibrous tissue hyperplasia and calcium deposition along with accumulation of lipid and complex saccharides on the inner wall of a blood vessel, so that the artery wall is thickened and hardened, the blood vessel cavity is narrow, substance exchange of the blood vessel wall is influenced, and the permeability of the blood vessel is changed.

For the reasons stated above, this example selects atherosclerosis as the disease manifestation of altered capillary permeability.

2. Blood samples from patients with atherosclerosis and from normal individuals were subjected to thrombocyte transcriptome sequencing. Through case collection, 3 atherosclerosis samples are obtained in total, wherein the atherosclerosis samples comprise 1 male case and 2 female cases, and the age range is 45-77 years old. Meanwhile, 3 normal human samples, 2 male samples and 1 female sample are collected, and the age range is 40-80 years old. The expression levels of the platelet genes of the atherosclerosis and normal samples were obtained (58303 genes were examined in total).

3. Taking the normal sample gene expression amount as a control, carrying out expression difference statistics on the genes of atherosclerosis, and screening out the differential genes (3817 differential genes in total) with the gene expression times of | Log2FC | > 2 and P _ value < 0.05.

4. And (3) comparing the differential genes screened in the step (3) with a KEGG database to obtain an enrichment channel of the differential genes of the atherosclerotic diseases. 53 significantly different (P < 0.05) metabolic pathways were enriched (as shown in FIG. 1).

5. Among the 53 metabolic pathways shown in FIG. 1, Antigen processing and presentation (NF-signal pathway), Fluid shear stress and atherosclerosis pathway (Fluid shear stress and atherosclerosis) are involved in and regulate membrane transport and material exchange, information transfer of cells. The differential genes (94 in total) enriched in the above 3 metabolic pathways were selected.

6. Among the 94 differential genes selected in step 5, the first 20 genes with high average expression level (base Mean) were selected as preselected target genes. Then PPI protein interaction screening and analysis of characteristic curve of subject operation are carried out, and one-step screening is carried out to obtain 10 preselected target genes (HLA-E, ARPC1B, CDS2, CALM1, ARPC4, HLA-B, HCLS1, HLA-C, DNM3 and HLA-A).

7. Qpcr primers for the 10 preselected target genes selected in step 6 were designed using Primer Premier 6 software, as shown in table 1.

TABLE 1 qpcr primers for each preselected target Gene

8. The primer designed in step 7 is used for detecting the copy number of the qpcr gene, and the result is shown in table 2.

TABLE 2qpcr Gene copy number test results show

Gene	Disease 1	Disease 2	Disease 3	Normal 1	Normal 2	Normal 3
							HLA-E	2.05	1.56	3.49	1.17	0.94	0.890892
ARPC1B	1.42	0.70	1.75	1.02	1.10	0.876798
							CDS2	7.14	3.55	6.17	0.42	1.24	1.342618
CALM1	1.47	1.17	2.27	0.65	1.28	1.070749
							ARPC4	5.55	5.00	11.12	0.79	1.65	0.556974
HLA-B	0.94	1.12	2.10	0.73	1.00	1.272211
							HCLS1	7.72	2.82	6.00	0.93	0.88	1.188007
HLA-C	1.24	0.76	1.75	0.49	0.71	1.802528
							DNM3	1.01	0.69	1.50	1.30	0.742488	0.961776
HLA-A	1.87	1.95	5.19	0.68	1.221628	1.095917

The copy number of the ARPC4 gene is obtained to have the maximum difference multiple between the disease group and the normal group, and the target gene is selected (shown in figure 2).

Example two: screening target genes meeting requirements by using thrombosis susceptibility (YSZ) and thrombocytopenic purpura (ITP)

1. Two diseases closely related to vascular permeability changes were selected: thrombosis liability (YSZ) and thrombocytopenic purpura (ITP). Among them, the thrombosis liability syndrome is divided into hereditary thrombosis and acquired thrombosis, and the acquired thrombosis liability syndrome is manifested by changes in blood components (platelets, coagulation factors, anticoagulation factors, fibrinolysis and anti-fibrinolysis factors). Changes in vascular permeability can cause changes in blood composition and may lead to acquired thrombosis. Thrombocytopenic purpura is manifested by subcutaneous bleeding, thrombocytopenia in the blood, migration of plasma and other components of the blood into the subcutaneous tissue, and the like. Therefore, if the patient suffers from thrombocytopenic purpura, the permeability of the capillary vessels is often changed.

For the above reasons, this example selects acquired thrombosis liability and thrombocytopenic purpura as disease manifestations of altered capillary permeability. When the acquired thrombosis is suffered, the capillary permeability is weakened (namely the acquired thrombosis is inversely related to the change of the vascular permeability); in thrombocytopenic purpura, the permeability of the capillary vessels becomes stronger (i.e., thrombocytopenic purpura is positively correlated with vascular permeability change).

2. Blood samples of patients with acquired thrombosis, patients with thrombocytopenic purpura and normal persons were subjected to thrombocyte transcriptome sequencing. Comprises 20 patients with thrombosis, 51 patients with thrombocytopenic purpura, and 36 normal patients; among patients and normal persons, the age range of blood-supplying patients and normal persons is 14 to 94 years, and the male-female ratio is 1: 2. the expression levels of the platelet genes of the disease (YSZ, ITP) and the normal sample (normal) were obtained separately.

3. Taking the normal sample gene expression amount as a reference, respectively carrying out expression difference statistics on genes of thrombosis susceptibility (YSZ) and purpura (ITP), and screening out the difference genes with the gene expression times of | Log2FC | > 2 and P _ value < 0.05.

4. Comparing the differential genes screened from thrombosis and purpura with a KEGG database to obtain an enrichment pathway of the differential genes of the two diseases. The metabolic pathways with remarkable difference (P < 0.05) are enriched in 44 different genes in thrombosis easily (as shown in figure 3), and the metabolic pathways with remarkable difference are enriched in 33 different genes in purpura (as shown in figure 4).

5. In fig. 3, 44 metabolic pathways are shown, wherein ABC transport pathway, calcium signaling pathway, focal adhesion, MAPK signaling pathway and Rap1 signaling pathway are involved in and regulate membrane transport and material exchange, information transfer of cells. In the 33 metabolic pathways displayed in FIG. 4, the NF-. kappa.B signaling pathway is involved in membrane transport regulation. Selecting the differential genes from the metabolic pathways.

Three consensus genes (PLCG1, TRAF2 and LAD) were selected from these differential genes as preselected target genes.

6. The expression levels of the three genes in the two diseases and the expression levels of the three genes in the normal sample were subjected to T-test, and the results are shown in FIG. 5: in ITP, TRAF2 is expressed in higher than normal amounts; in YSZ, TRAF2 is expressed in lower than normal amounts. Thus TRAF2 is the desired gene of interest (target gene).

Example three: application of ARPC4 and TRAF2 genes in preparation of test paper for detecting capillary permeability

In the case of screening and determining ARPC4 and TRAF2 as target genes for detecting capillary permeability, one skilled in the art can prepare a series of products for detection, including but not limited to: a detection agent, a detection test paper, a kit and the like. The present embodiment provides a test strip that can be used for detection.

The preparation method of the test paper is shown in fig. 6, and specifically comprises the following steps:

1. the protein sequences of products corresponding to the ARPC4 and TRAF2 genes are obtained through NCBI website, and are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The synthesis of ARPC4 and TRAF2 proteins was performed to give protein products. The two synthesized proteins were injected into mice as a multi-antigen to synthesize a polyclonal antibody.

The obtained polyclonal antibody is screened by ELISA experiment. An antibody was immobilized on an ELISA plate as a reaction 1 antibody. Serum samples of atherosclerosis, multiple purpura and thrombosis were added. The other antibody was conjugated to immunofluorescent enzyme and added to the reaction as a secondary antibody. The fluorescence reaction was detected, and antibodies (1 antibody and 2 antibody) having the highest fluorescence reaction were selected.

2. Preparation of colloidal gold marker: adding 6 μ L potassium carbonate solution into 1mL colloidal gold, rapidly mixing, adding 25 μ L screened 1 antibody, mixing, and standing for 10 min. Add 10. mu.l of blocking solution, mix well and let stand for 10 minutes. After standing, the mixture was centrifuged at 9000rpm for 7 minutes, and the supernatant was discarded. Re-dissolving the precipitate to 1ml with colloidal gold re-solution, and mixing the solution with ultrasonic cleaning instrument to obtain colloidal gold labeled substance.

3. Preparing the colloidal gold test paper: cutting the glass fiber film into 5 × 3cm²The sample pad is placed in the colloidal gold label and allowed to absorb well. And then, uniformly drying the sample pad by using a blower to prepare the gold-labeled pad. As shown in fig. 6, the nitrocellulose membrane, the gold label pad (two layers), the sample pad, and the absorbent paper were sequentially attached, and after the assembly, the test paper strip with a width of 4mm was cut with scissors and placed in the card case.

The detection is carried out in two ways. Dot pattern: a small amount of the screened 1 antibody is absorbed by a capillary tube, the 1 antibody is lightly spotted on a detection line, and the spotted membrane is dried at room temperature. Drawing a line: the 1 antibody of 30. mu.L was pipetted by a pipette tip, then removed from the pipette tip, and the tip was used in conjunction with a ruler to scribe at the NC membrane test line position, as well as the 2 antibodies selected to scribe at the quality test line position.

4. And verifying the detection effect of the test paper. The prepared test paper is used for testing blood (20 each, half of men and women) of normal people, patients with acute nephritis, patients with atherosclerosis, patients with multiple purpura and patients with thrombosis. The results of the test strips for vascular permeability showing negative (unchanged permeability) are shown in FIG. 7, and the results of the test strips for vascular permeability showing positive (changed permeability) are shown in FIG. 8. The result shows that the test paper has 100 percent of detection accuracy on normal people and the detection accuracy on patients with vascular permeability change.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes, modifications, alterations, and substitutions which may be made by those skilled in the art without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Sequence listing

<110> Fenneng medical science and technology (Shanghai) Limited liability company

<120> a product for detecting vascular permeability and a preparation method thereof

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