阅读说明：本技术 哮喘生物标志物klrc1及其应用 (Asthma biomarker KLRC1 and application thereof ) 是由 杨永清 陈艳焦 王宇 徐玉东 尹磊淼 刘艳艳 于 2019-03-29 设计创作，主要内容包括：本发明属于哮喘诊断研究领域,具体提供了哮喘生物标志物KLRC1及其应用。本发明涉及疾病相关生物标志物及应用领域,首次公开了一种自然杀伤细胞受体KLRC1在哮喘患者中表达上调,提示检测KLRC1受体水平可以用于哮喘临床诊断、直接反映哮喘控制情况,以及KLRC1抗体运用于哮喘疾病防治的应用。KLRC1作为生物标志物用于哮喘的诊断、治疗方案选择、预后评估,客观、特异性高、灵敏度好。可克服原有哮喘检测大多为主观检测的局限。(The invention belongs to the field of asthma diagnosis research, and particularly provides an asthma biomarker KLRC1 and application thereof. The invention relates to disease-related biomarkers and the application field, and discloses the expression up-regulation of a natural killer cell receptor KLRC1 in an asthmatic patient for the first time, which prompts the detection of the KLRC1 receptor level to be used for clinical diagnosis of asthma and directly reflecting the asthma control condition, and the application of a KLRC1 antibody in the prevention and treatment of asthma diseases. KLRC1 is used as a biomarker for asthma diagnosis, treatment scheme selection and prognosis evaluation, and has objectivity, high specificity and good sensitivity. Can overcome the limitation that the prior asthma detection is mostly subjective detection.)

Use of KLRC1 for the preparation or screening of asthma detection reagents.

2. Use according to claim 1, characterized in that KLRC1 is used as biomarker.

3. The use according to claim 1, wherein the asthma detection reagent is used for the judgment of asthma, selection of a treatment regimen, and/or prognostic assessment.

4. The use according to claim 3, wherein the prognostic assessment of asthma refers to prognostic judgment of the course and/or outcome of asthma patients.

5. The use of claim 1, wherein the asthma detection reagent is a reagent that specifically recognizes KLRC 1.

6. Use according to claim 1, characterized in that the agent specifically recognizing KLRC1 is selected from an agent specifically recognizing KLRC1 gene or an agent specifically recognizing KLRC1 protein.

7. Use according to claim 6, wherein the agent specifically recognizing the KLRC1 gene is selected from any one or more of the following: (1) a primer for specifically amplifying KLRC1 gene or transcript, (2) a probe for specifically recognizing KLRC1 gene or transcript; the reagent specifically recognizing the KLRC1 protein is an antibody or a ligand of the KLRC1 protein.

8. The use of claim 7, wherein when the reagent specifically recognizing KLRC1 gene comprises a primer specifically amplifying KLRC1 gene or transcript, the sequence of the upstream primer of the primer specifically amplifying KLRC1 gene is as set forth in SEQ ID NO: 1 is shown in the specification; the downstream primer is shown as SEQ ID NO: 2, respectively.

9. Use of a reagent specifically recognizing KLRC1 for the preparation of a kit for the diagnosis of asthma.

10. An asthma detection kit, at least comprising an asthma detection reagent in the kit, wherein the asthma detection reagent is selected from a reagent specifically recognizing KLRC 1.

Technical Field

The invention belongs to the technical field of cell biology, and particularly relates to an asthma biomarker KLRC1 and application thereof.

Background

The diagnosis of asthma in the GINA guideline of 2018 is still based on the clinical symptoms and signs of the patient, combined with laboratory examination, imaging examination and respiratory function. The symptom signs are subjective indexes, which are not beneficial to disease diagnosis, the pulmonary function examination has certain contraindications (such as severe hypertension, coronary heart disease, pulmonary bullae and the like), and the bronchial excitation test has the risks of poor tolerance of patients, induction of bronchospasm and life threatening. There is a lack of direct objective diagnostic methods.

The current treatment regimen for asthma is mainly to control the symptoms of asthma, and the main methods are smooth muscle relaxation and airway hyperresponsiveness control. Asthma is recurrent despite regular inhalation of beta agonists and glucocorticoids, and each episode exacerbates pulmonary inflammation and further deterioration of pulmonary function.

KLRC1 is a 43kD type II transmembrane protein, expressed predominantly on NK cell membranes, belonging to the NK family of receptors (NKG2 family). From a protein structure perspective, KLRC1 belongs to the class C lectin-like receptor, also known as the killer lectin-like receptor (KLR); from a functional point of view, KLRC1 belongs to an inhibitory receptor capable of inhibiting NK cell killing. The prior literature reports that the receptor directly influences the activity of NK cells, and the KLRC1 antibody can be applied to a tumor treatment drug. At present, the relation between the receptor and asthma diseases is not researched at home and abroad, and the application of the KLRC1 antibody to asthma prevention and treatment is not reported.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide an asthma biomarker KLRC1 and application thereof.

In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:

in a first aspect of the invention, there is provided the use of KLRC1 for the preparation or screening of asthma detection reagents.

Further, KLRC1 was used as a biomarker.

The asthma detection reagent is used for judging asthma, selecting a treatment scheme and/or evaluating prognosis.

Prognostic assessment of asthma refers to the prognostic judgment of the course and/or outcome of an asthmatic patient.

Furthermore, the asthma detection reagent refers to a reagent for detecting the expression level of KLRC1 in a sample, and the prognosis is carried out according to the detection result so as to judge whether the patient has asthma, which treatment scheme is selected and/or the course and/or the fate of the patient.

In one embodiment, the prognostic determination includes determining the quality of life and/or asthma control of the patient.

Further, the higher the expression level of KLRC1, the lower the patient's quality of life score and the more severe the asthma. The lower the expression level of KLRC1, the higher the asthma quality of life score, the less asthmatic symptoms.

The higher the expression of KLRC1, the lower the asthma control test score and the more severe the asthma. The lower the expression level of KLRC1, the higher the asthma control test score and the lighter the asthma symptoms.

The expression level of KLRC1 may be the protein expression level of KLRC1, the transcription level of KLRC1 gene, or the gene expression level of KLRC 1.

The sample may be a whole blood RNA sample of the subject to be tested.

It should be noted that the asthma detection reagent and/or asthma prognosis reagent is not limited to be necessarily in a liquid form.

The Genbank accession number of KLRC1 gene is 3821.

In one embodiment, the agent specifically recognizing KLRC1 is selected from an agent specifically recognizing KLRC1 gene or an agent specifically recognizing KLRC1 protein.

The reagent specifically recognizing the KLRC1 gene is selected from any one or more of the following: (1) a primer for specifically amplifying KLRC1 gene or transcript, (2) a probe for specifically recognizing KLRC1 gene or transcript; the reagent specifically recognizing the KLRC1 protein is an antibody or a ligand of the KLRC1 protein.

The sequence of an upstream primer of the primer for specifically amplifying the KLRC1 gene is shown as SEQ ID NO: 1 is shown in the specification; the downstream primer is shown as SEQ ID NO: 2, respectively.

The antibody includes a monoclonal antibody or a polyclonal antibody.

In a second aspect of the invention, there is provided the use of an agent which specifically recognizes KLRC1 for the preparation of an asthma detection kit.

Further, KLRC1 was used as a biomarker.

The asthma detection reagent is used for judging asthma, selecting a treatment scheme and/or evaluating prognosis.

Prognostic assessment of asthma refers to the prognostic judgment of the course and/or outcome of an asthmatic patient.

Furthermore, the asthma detection reagent refers to a reagent for detecting the expression level of KLRC1 in a sample, and the prognosis is carried out according to the detection result so as to judge whether the patient has asthma, which treatment scheme is selected and/or the course and/or the fate of the patient.

In one embodiment, the prognostic determination includes determining the quality of life and/or asthma control of the patient.

Further, the higher the expression level of KLRC1, the lower the patient's quality of life score and the more severe the asthma. The lower the expression level of KLRC1, the higher the asthma quality of life score, the less asthmatic symptoms.

The higher the expression of KLRC1, the lower the asthma control test score and the more severe the asthma. The lower the expression level of KLRC1, the higher the asthma control test score and the lighter the asthma symptoms.

The expression level of KLRC1 may be the expression level of KLRC1 protein, the transcription level of KLRC1 gene, or the expression level of KLRC1 gene.

The sample may be a whole blood RNA sample of the subject to be tested.

It should be noted that the asthma detection reagent and/or asthma prognosis reagent is not limited to be necessarily in a liquid form.

In one embodiment, the agent specifically recognizing KLRC1 is selected from an agent specifically recognizing KLRC1 gene or an agent specifically recognizing KLRC1 protein.

The reagent specifically recognizing the KLRC1 gene is selected from any one or more of the following: (1) a primer for specifically amplifying KLRC1 gene or transcript, (2) a probe for specifically recognizing KLRC1 gene or transcript; the reagent specifically recognizing the KLRC1 protein is an antibody or a ligand of the KLRC1 protein.

The sequence of an upstream primer of the primer for specifically amplifying the KLRC1 gene is shown as SEQ ID NO: 1 is shown in the specification; the downstream primer is shown as SEQ ID NO: 2, respectively.

The antibody includes a monoclonal antibody or a polyclonal antibody.

The kit can be a real-time fluorescent quantitative PCR detection kit, and the basic principle of the kit is that a pair of specific primers of target polynucleotide is utilized, and in the PCR amplification reaction solution of the kit, the circular amplification of the target polynucleotide is realized through a fluorescent quantitative PCR amplification instrument, so that the aim of quickly and quantitatively detecting the polynucleotide is fulfilled.

In a third aspect, the invention provides an asthma detection kit, which at least comprises an asthma detection reagent selected from reagents specifically recognizing KLRC 1.

Further, KLRC1 was used as a biomarker.

The asthma detection reagent is used for judging asthma, selecting a treatment scheme and/or evaluating prognosis.

Prognostic assessment of asthma refers to the prognostic judgment of the course and/or outcome of an asthmatic patient.

Furthermore, the asthma detection reagent refers to a reagent for detecting the expression level of KLRC1 in a sample, and the prognosis is carried out according to the detection result so as to judge whether the patient has asthma, which treatment scheme is selected and/or the course and/or the fate of the patient.

In one embodiment, the prognostic determination includes determining the quality of life and/or asthma control of the patient.

Further, the higher the expression level of KLRC1, the lower the patient's quality of life score and the more severe the asthma. The lower the expression level of KLRC1, the higher the asthma quality of life score, the less asthmatic symptoms.

The higher the expression of KLRC1, the lower the asthma control test score and the more severe the asthma. The lower the expression level of KLRC1, the higher the asthma control test score and the lighter the asthma symptoms.

The expression level of KLRC1 may be the protein expression level of KLRC1, the transcription level of KLRC1 gene, or the gene expression level of KLRC 1.

The sample may be a whole blood RNA sample of the subject to be tested.

It should be noted that the asthma detection reagent and/or asthma prognosis reagent is not limited to be necessarily in a liquid form.

In one embodiment, the agent specifically recognizing KLRC1 is selected from an agent specifically recognizing KLRC1 gene or an agent specifically recognizing KLRC1 protein.

The reagent specifically recognizing the KLRC1 gene is selected from any one or more of the following: (1) a primer for specifically amplifying KLRC1 gene or transcript, (2) a probe for specifically recognizing KLRC1 gene or transcript; the reagent specifically recognizing the KLRC1 protein is an antibody or a ligand of the KLRC1 protein.

The sequence of an upstream primer of the primer for specifically amplifying the KLRC1 gene is shown as SEQ ID NO: 1 is shown in the specification; the downstream primer is shown as SEQ ID NO: 2, respectively.

The antibody includes a monoclonal antibody or a polyclonal antibody.

The kit can be a real-time fluorescent quantitative PCR detection kit, and the basic principle of the kit is that a pair of specific primers of target polynucleotide is utilized, and in the PCR amplification reaction solution of the kit, the circular amplification of the target polynucleotide is realized through a fluorescent quantitative PCR amplification instrument, so that the aim of quickly and quantitatively detecting the polynucleotide is fulfilled.

In a fourth aspect, the present invention provides a method for using the aforementioned kit, comprising the steps of:

(1) sample adding: respectively adding the sample genome cDNA and positive control or negative control into a PCR tube provided with a PCR reaction system to obtain the corresponding sample reaction tube, positive reaction tube or negative reaction tube, wherein the PCR reaction system contains the KLRC1 gene detection primer;

(2) and (3) PCR reaction: the reaction tube is arranged on a PCR instrument, and circulation parameters are set for carrying out PCR reaction;

(3) after the PCR reaction was completed, the results were analyzed.

The sample may be a whole blood RNA sample of the subject to be tested.

The present invention is not particularly limited to other components and final concentrations thereof in the PCR reaction system except for using the primer of the present invention, and one skilled in the art can establish the PCR reaction system based on the general components and concentrations thereof conventionally used in establishing the PCR system. The template (e.g., genomic DNA) for PCR amplification can also be extracted using methods conventional in the art.

Compared with the prior art, the invention has the following beneficial effects:

the invention relates to disease-related biomarkers and the application field, and discloses the expression up-regulation of a natural killer cell (NK) receptor KLRC1 in asthma models of asthmatics and mice for the first time, which prompts that the detection of the KLRC1 receptor level can be used for clinical diagnosis of asthma and directly reflecting the asthma control condition, and the application of a KLRC1 antibody in the prevention and treatment of asthma diseases. KLRC1 is used as a biomarker for asthma diagnosis, treatment scheme selection and prognosis evaluation, and has objectivity, high specificity and good sensitivity. Can overcome the limitation that the prior asthma detection is mostly subjective detection.

Drawings

FIG. 1 shows the expression levels of KLRC1 gene from whole blood RNA in normal and asthmatic groups (p < 0.05 for significant statistical significance).

FIG. 2 shows that the expression level of KLRC1 gene (FC > 2 or FC < 0.5) is statistically significant for whole blood RNA from asthmatics before and after treatment.

FIG. 3 shows the results of fitting ROC curves to the expression levels of KLRC1 in the normal and asthmatic groups.

FIG. 4 is a graph showing the relationship between KLRC1 expression level and asthma quality of life score of asthmatic patients.

FIG. 5 is a graph showing the expression level of KLRC1 as a function of the asthma control test score of asthmatic patients.

Detailed Description

KLRC1 is a 43kD type II transmembrane protein, expressed predominantly on NK cell membranes, belonging to the NK family of receptors (NKG2 family). From a protein structure perspective, KLRC1 belongs to the class C lectin-like receptor, also known as the killer lectin-like receptor (KLR); from a functional point of view, KLRC1 belongs to an inhibitory receptor capable of inhibiting NK cell killing.

The NKG2 molecule in KLRC1 can form a CD94-NKG2 heterodimer with a constant chain CD94 to specifically recognize human non-classical HLA-E molecules, and plays a role in inhibiting NK cell killing by utilizing an Immunoreceptor Tyrosine Inhibition Motif (ITIM) possessed by the NKG2 molecule. Evidence suggests that only one inhibitory receptor is generally expressed on the surface of each NK cell, and that KLRC1 receptor, which binds to non-classical HLA-E-class molecules, may play a critical role in NK cell activity.

The results of our study show that in clinical studies, the up-regulation of the expression of the KLRC1 receptor of asthma patients is verified by gene chip detection and RT-PCR. This receptor may provide a novel approach to the diagnosis and prevention of asthma.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Asthma detection kit and/or asthma prognosis judgment kit

The kit is based on the invention and adopts the real-time fluorescence PCR technologyFor detection, other conventional reagents required for PCR, such as: SYBR premix ex Taq, double distilled Water (ddH)₂O), a sample genome DNA extraction reagent, one or more of common PCR reaction reagents such as dNTPs, RT buffer, RNase, M-MLV-RTase and the like. Since the common PCR reagents can be purchased separately or configured by themselves through the market, the reagents can be assembled into the kit according to the actual needs of customers, and can be assembled into the kit for convenience.

The kit of the present invention may contain each set of primer pairs packaged independently, or may contain a prepared PCR detection mixture containing each set of primer pairs.

The PCR detection mixed solution can be prepared by self, and can also be obtained by directly adding primers into a general PCR detection mixed solution which is sold in the market and does not contain the primers. For example, the kit may further contain SYBR premix ex Taq (SYBR _ Premix) and double distilled Water (ddH)₂O). The PCR reaction system can be obtained by adding the primer of the invention and the DNA extract or cDNA of the sample to be detected.

Optionally, the kit may further comprise a positive control. The positive control is a cDNA sample containing KLRC1 gene expression.

Optionally, the kit may further comprise a negative control. The negative control may be a cDNA sample without KLRC1 gene expression.

Methods of use of the kit

The method comprises the following steps:

(1) sample adding: respectively adding the sample genome cDNA and positive control or negative control into a PCR tube provided with a PCR reaction system to obtain the corresponding sample reaction tube, positive reaction tube or negative reaction tube, wherein the PCR reaction system contains the KLRC1 gene detection primer;

(2) and (3) PCR reaction: the reaction tube is arranged on a PCR instrument, and circulation parameters are set for carrying out PCR reaction;

(3) after the PCR reaction was completed, the results were analyzed.

Optionally, in step (3), the conditions of the PCR reaction are set as: (a) 15S at 95 ℃; (b) 5S at 95 ℃; (c) 30S at 60 ℃; steps (b) - (c) were repeated 40 times.