CN112433053A

CN112433053A - Application of Tetraspin-14 protein detection reagent in preparation of lung cancer screening kit

Info

Publication number: CN112433053A
Application number: CN202011311801.1A
Authority: CN
Inventors: 李丽; 陈红英; 陈菲; 包春娟
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2020-11-20
Filing date: 2020-11-20
Publication date: 2021-03-02

Abstract

The invention relates to the field of in-vitro diagnostic reagents, in particular to an application of a Tetrasmanin-14 protein detection reagent in preparation of a lung cancer screening kit. The invention discovers for the first time that the level of Tetrasmanin-14 protein in plasma exosome of lung cancer patient is obviously higher than that of healthy people. According to the invention, the reagent for detecting the Tetraspin-14 protein is used for preparing the lung cancer screening kit, so that the effective screening of the lung cancer can be realized.

Description

Application of Tetraspin-14 protein detection reagent in preparation of lung cancer screening kit

Technical Field

The invention relates to the field of in-vitro diagnostic reagents, in particular to an application of a Tetrasmanin-14 protein detection reagent in preparation of a lung cancer screening kit.

Background

Lung cancer is one of the most common malignant tumors in the world, the morbidity and mortality of the lung cancer are on the rising trend year by year, the morbidity is at the top of the world at present, and the health and the life of human beings are seriously threatened.

The lung cancer is a disease good in occult, clinical symptoms are often shown only when the disease develops to the advanced stage, 70-80% of lung cancer patients are already at the middle and advanced stages when the lung cancer symptoms are diagnosed, cancer cells are diffused, the best curing time is missed, and the five-year survival rate is low. For early-stage lung cancer patients, the survival rate and the survival quality of the patients can be greatly improved by 5 years and more through timely treatment. Early diagnosis of lung cancer and effective screening are therefore of paramount importance.

The screening of the lung cancer refers to that the conventional physical examination is carried out on people without lung cancer related symptoms, and the lung cancer is found in time before the symptoms appear. If the lung cancer molecular marker in the plasma can be found, the molecular marker has important significance for prompting a clinician to take relevant treatment measures or decisions for a patient at an early stage.

The Tetraspanin-14 protein (Uniprot code: Q8NG11) is responsible for regulating ADAM 10-mediated GP6 and CDH 5.

No prior art related to lung cancer of Tetraspan-14 protein is found at present.

Disclosure of Invention

The invention aims to provide a novel lung cancer marker and application of a detection reagent of the marker in preparation of a lung cancer screening kit.

The technical scheme of the invention comprises the following steps:

application of a reagent for detecting Tetraspin-14 protein in preparation of a lung cancer screening kit.

As the application, the reagent for detecting the Tetraspin-14 protein is a reagent for enzyme-linked immunosorbent assay.

As the application, the reagent for detecting the Tetraspin-14 protein is a western blot reagent.

As mentioned above, the reagent for detecting the Tetraspin-14 protein is a reagent for a protein chip detection method.

As the aforementioned use, the reagent for detecting the Tetraspin-14 protein is a reagent for detecting the Tetraspin-14 protein in human plasma exosomes.

A lung cancer screening kit comprises a reagent for detecting Tetrasmanin-14 protein.

As the kit, the reagent for detecting the Tetraspin-14 protein is a reagent for enzyme-linked immunosorbent assay.

As the kit, the reagent for detecting the Tetraspin-14 protein is a western blot reagent.

As the kit, the reagent for detecting the Tetraspin-14 protein is a reagent for a protein chip detection method.

As the kit, the reagent for detecting the Tetraspin-14 protein is a reagent for detecting the Tetraspin-14 protein in human plasma exosome.

The key point of the invention is that the content of the Tetrasmanin-14 protein in human plasma exosomes is determined to be obviously related to the risk of suffering from lung cancer, so that the risk of suffering from lung cancer can be judged by detecting the content of the Tetrasmanin-14 protein in the human plasma exosomes, various means disclosed in the prior art can be adopted as a means for specifically detecting the Tetrasmanin-14 protein in the human plasma exosomes, and an enzyme-linked immunoassay method (protein chip) is specifically adopted for detection in the embodiment of the invention, but the method is not limited to the means, and any method capable of detecting the content of the Tetrasmanin-14 protein can be used for lung cancer screening.

The invention provides a new lung cancer screening marker and a new lung cancer screening kit, which can realize effective screening of lung cancer; and the plasma exosome can be used as a detection sample, so that the harm to a patient is low. The invention has good application prospect.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The foregoing aspects of the present invention are explained in further detail below with reference to specific embodiments. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1: comparison of Tetraspan-14 protein levels in lung cancer patients (LC), healthy control (NC) plasma exosomes.

FIG. 2: lung cancer patients (LC) were analyzed by ROC with healthy controls (NC).

Detailed Description

Example 1 relationship between Tetrasmanin-14 protein in plasma exosomes and Lung cancer

First, clinical data

40 lung cancer patients and 40 healthy controls were selected, and the basic information was as follows:

basic information	Patients with lung cancer	Healthy controls
			Number of people	40	40
Age (age)	58.8±11.5	50.1±14.3
			Proportion of male	33(82.5％)	17(42.5％)

Second, detection principle

The method is the same as the basic enzyme-linked immunosorbent assay (ELISA) detection method.

A Tetraspanin-14 protein antibody (VVNTQCGYDVR amino acid sequence for specifically recognizing the Tetraspanin-14 protein) is fixed on the protein chip, and after the protein chip is incubated with plasma exosome protein, if the Tetraspanin-14 protein exists in the exosome protein, the Tetraspanin-14 protein can be bound; washing to remove unbound antibodies and other proteins, binding the fluorescent-labeled Tetraspin-14 protein antibody to the Tetraspin-14 protein for detection, and reading a signal by a fluorescence scanner, wherein the strength of the signal is in positive correlation with the affinity and the quantity of the antibody.

SEQ ID NO.1：VVNTQCGYDVR

Third, method

1. The preparation method of the plasma exosome protein comprises the following steps:

the sample was removed from-80 ℃ and centrifuged at 12000g for 15 minutes at 4 ℃ and the supernatant was transferred to a new centrifuge tube and filtered through a 0.22. mu.M microporous membrane, and then the exosomes were isolated using PTM-EVs kit manufactured by PTM corporation according to the instructions. Adding Urea with a final concentration of 8M and protease inhibitor for ultrasonic cleavage, and using a BCA kit for protein concentration determination.

2. Detection of Tetraspin-14 protein in plasma exosomes

The reagents used in this section were as follows:

the method comprises the following specific steps:

1) rewarming: taking out the chip from a refrigerator at-80 deg.C, putting in a refrigerator at 4 deg.C for rewarming for half an hour, and then putting in room temperature for rewarming for 15 min;

2) and (3) sealing: fixing 14 blocks in the rewarming chip, adding sealing liquid into each block after fixing, placing on a side swing bed, and sealing at room temperature for 3 hr;

3) incubation of plasma exosome protein samples: after the sealing is finished, pouring the sealing liquid completely, then quickly adding the prepared plasma exosome protein incubation liquid, wherein the sample loading volume is 200 mu L, and incubating overnight at 4 ℃ by laterally swinging a shaker at 20rpm (diluting the plasma exosome protein and the incubation liquid in a ratio of 1: 50 to obtain the plasma exosome protein incubation liquid);

4) cleaning: taking out the chip and the chip fence together, sucking out the sample, then quickly adding PBST with the same volume, and circulating for several times to ensure that no cross contamination exists among plasma exosome protein samples when the chip fence is detached. After the chip fence is removed, the chip is placed in a chip cleaning box with cleaning solution, and is cleaned for 3 times (10 min each time) by a horizontal shaking table at room temperature of 80 rpm;

5) and (3) secondary antibody incubation: transferring the chip into an incubation box added with 3mL of secondary antibody incubation liquid, laterally swinging a shaker at 40rpm, keeping out of the sun, and keeping at room temperature for 1 hr;

6) cleaning: the chip was removed (note that the upper surface of the chip was not touched or scratched), and placed in a chip washing cassette containing a washing solution, and washed 3 times 10min each time, on a horizontal shaker at room temperature and 80 rpm. After completion with ddH₂O cleaning for 2 times, 10min each time;

7) drying;

8) scanning: scanning by using a crystal core LuxScan 10K microarray chip scanner;

9) data extraction: opening the corresponding GAL file (recording the position of protein in the chip), aligning the chip image and each array of the GAL file integrally, pressing an automatic alignment button, extracting data and storing.

Fourthly, the result

The mean expression level of the Tetraspin-14 protein in plasma exosomes of lung cancer patients was 1.57 (protein-to-quantitative ratio), and the mean expression level of the healthy control Tetraspin-14 protein was 0.61. The lung cancer group was statistically significant compared to healthy controls (p <0.01) (fig. 1). The lung cancer group and the healthy control have the ROC analysis result of 97.5 percent of specificity and 17.5 percent of sensitivity (figure 2); the Tetraspanin-14 protein in the plasma exosome can be shown to specifically distinguish lung cancer from healthy control.

The results show that the level difference of the Tetraspin-14 protein in the plasma exosomes of the lung cancer patients and the non-lung cancer patients is obvious, and the aim of screening the lung cancer can be fulfilled by detecting the level of the Tetraspin-14 protein in the plasma exosomes.

Example 2 composition of the detection kit of the invention and methods of use thereof

Kit composition

Detection kit (14 persons):

the reagent for preparing the plasma exosome protein needs to be self-prepared, and can be a commercial plasma exosome protein extraction kit.

Second, kit using method

The same as in the third section of example 1.

The kit can screen the risk of lung cancer of a human to be detected by detecting the level of the Tetrasmanin-14 protein in the plasma exosome: if the level of Tetraspan-14 protein is high (relative to healthy people), the risk of lung cancer is high, and if the level of Tetraspan-14 protein is low, the risk of lung cancer is low. The method can be used for the auxiliary diagnosis of clinical lung cancer, provides effective basis for patients to take relevant treatment measures or decisions, and has good clinical application prospect.

SEQUENCE LISTING

<110> Sichuan university Hospital in western China

Application of <120> Tetraspin-14 protein detection reagent in preparation of lung cancer screening kit

<130> GYKH1094-2020P0111925CC20JS038

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 11

<212> PRT

<213> Artificial sequence

<400> 1

Val Val Asn Thr Gln Cys Gly Tyr Asp Val Arg

1 5 10

Claims

1. Application of a reagent for detecting Tetraspin-14 protein in preparation of a lung cancer screening kit.

2. The use according to claim 1, wherein the reagent for detecting the Tetraspanin-14 protein is a reagent for enzyme-linked immunosorbent assay.

3. The use according to claim 1, wherein the reagent for detecting the Tetraspanin-14 protein is a western blot reagent.

4. The use according to claim 1, wherein the reagent for detecting the Tetraspanin-14 protein is a reagent for a protein chip detection method.

5. The use according to any one of claims 1 to 4, wherein the reagent for detecting the Tetraspin-14 protein is a reagent for detecting the Tetraspin-14 protein in human plasma exosomes.

6. A lung cancer screening kit is characterized by comprising a reagent for detecting Tetrasmanin-14 protein.

7. The kit according to claim 6, wherein the reagent for detecting the Tetraspanin-14 protein is a reagent for enzyme-linked immunosorbent assay.

8. The kit of claim 6, wherein the reagent for detecting the Tetraspanin-14 protein is a western blot reagent.

9. The kit according to claim 6, wherein the reagent for detecting the Tetraspanin-14 protein is a reagent for a protein chip detection method.

10. The kit according to any one of claims 6 to 9, wherein the reagent for detecting the Tetraspanin-14 protein is a reagent for detecting the Tetraspanin-14 protein in human plasma exosomes.