Application of KCTD12 protein in diagnosis of azithromycin resistance of child patient suffering from mycoplasma pneumonia

Abstract

The invention provides application of KCTD12 protein in diagnosis of azithromycin resistance of child patients suffering from mycoplasma pneumonia, and relates to the technical field of biology. The research of the inventor finds that the protein expression of the serum exosome KCTD12 of a child with mycoplasma pneumonia having response to azithromycin treatment is up-regulated compared with that of a child with mycoplasma pneumonia having no response to azithromycin treatment. Therefore, the serum exosome KCTD12 protein can be used as a marker of the azithromycin resistance of a child patient suffering from mycoplasma pneumonia, and is used for evaluating the azithromycin resistance of the child patient.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of KCTD12 protein in diagnosing azithromycin resistance in children with mycoplasma pneumonia. Background Art

[0002] Mycoplasma pneumoniae (M. pneumoniae) is one of the most common pathogens of community-acquired pneumonia (CAP) in children. Mycoplasma pneumoniae pneumonia (MPP) in children accounts for 10%-40% of CAP in children and is a major concern of clinical pediatricians. Although MPP is usually a benign, self-limiting disease, it can also develop into severe, life-threatening pneumonia in pediatric cases. Refractory mycoplasma pneumoniae pneumonia (RMPP) is defined as the continued fever and worsening of clinical symptoms and imaging manifestations in children after taking macrolide antibiotics for 1 week or more. The disease progresses rapidly in children with RMPP, and a large area of ​​the lungs may be affected, moderate to large pleural effusions, pleural thickening, lung abscesses, pneumothorax, etc. may occur in a short period of time. In severe cases, it may lead to atelectasis, acute respiratory distress syndrome (ARDS) and even systemic inflammatory response syndrome, which can often affect multiple extrapulmonary organs and even endanger life. In recent years, the incidence of RMPP has increased, seriously endangering the life and quality of life of children, and posing new challenges to clinicians. Therefore, studying the regulatory mechanism of RMPP formation has important social and economic significance.

[0003] Currently, macrolides, such as azithromycin, are the first-line drugs for the treatment of MPP. It has been reported that the mechanism of azithromycin resistance in M. pneumoniae pneumonia is related to functional mutations in 23S rRNA, but this does not fully explain the principle of azithromycin resistance. The biological mechanism of azithromycin resistance in M. pneumoniae pneumonia in children remains largely unknown and requires further study.

[0004] In recent years, the problem of azithromycin resistance has become increasingly serious. The lack of effective clinical biomarkers to predict treatment response has made treatment selection challenging. At present, azithromycin resistance is determined based on genetic testing. As an in vitro test, it is difficult to judge the situation in the host. According to current research, genetic resistance does not fully reflect the efficacy of the drug. Therefore, it is urgent to find biomarkers of host resistance to azithromycin to determine the in vivo situation of azithromycin resistance in Mycoplasma pneumoniae pneumonia.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The first object of the present invention is to provide a reagent for detecting KCTD12 protein for use in the preparation of a product for diagnosing azithromycin resistance in children with mycoplasma pneumonia, so as to solve the above technical problems.

[0007] The second object of the present invention is to provide a product for diagnosing azithromycin resistance in children with mycoplasma pneumonia.

[0008] In order to achieve the above objectives, the following technical solutions are adopted:

[0009] In a first aspect, the present invention provides the use of a reagent for detecting KCTD12 protein in the preparation of a product for diagnosing azithromycin resistance in children with mycoplasma pneumonia.

[0010] As a further technical solution, the KCTD12 protein is the KCTD12 protein in serum exosomes.

[0011] As a further technical solution, the expression of serum exosomal KCTD12 protein was upregulated in children with Mycoplasma pneumonia who responded to azithromycin compared with children with Mycoplasma pneumonia who did not respond to azithromycin.

[0012] As a further technical solution, the product includes a kit.

[0013] In a second aspect, the present invention provides a product for diagnosing azithromycin resistance in children with mycoplasma pneumonia, wherein the product is used to detect the expression level of KCTD12 protein in serum exosomes.

[0014] As a further technical solution, the product includes a marker for identifying the KCTD12 protein.

[0015] As a further technical solution, the marker includes an antibody that binds to the KCTD12 protein.

[0016] As a further technical solution, the product also includes reagents for extracting serum exosomes.

[0017] As a further technical solution, the test sample of the product includes blood.

[0018] As a further technical solution, the product is a reagent or a kit.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The inventors have found that compared with children with mycoplasma pneumonia who did not respond to azithromycin treatment, the expression of serum exosomal KCTD12 protein in children with mycoplasma pneumonia who responded to azithromycin treatment was upregulated. Therefore, serum exosomal KCTD12 protein can be used as a marker of azithromycin resistance in children with mycoplasma pneumonia and can be used to evaluate the resistance of children to azithromycin. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 Proteomic analysis of serum exosomes in children with Mycoplasma pneumoniae pneumonia. A is a schematic diagram of the proteomic analysis process of serum exosomes. B is the morphology of isolated exosomes under electron microscopy. C is the size of isolated exosomes analyzed using nanoparticle tracking analysis. D is PCA analysis showing the expression of exosomal proteins between azithromycin-sensitive and resistant patients.

[0023] Figure 2 Identification of differentially expressed proteins in serum exosomes of children with Mycoplasma pneumoniae pneumonia. A shows differentially expressed proteins in responders compared with non-responders before azithromycin treatment, B shows the expression and aggregation of differentially expressed proteins in responders compared with non-responders before azithromycin treatment, C shows differentially expressed proteins in responders compared with non-responders after treatment with azithromycin, and D shows the expression and aggregation of differentially expressed proteins in responders compared with non-responders after azithromycin treatment.

[0024] Figure 3 To evaluate KCTD12 protein as a biomarker for azithromycin resistance in Mycoplasma pneumoniae;

[0025] Figure 4 Standard curve for KCTD12 protein assay by ELISA. DETAILED DESCRIPTION

[0026] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0027] In a first aspect, the present invention provides the use of a reagent for detecting KCTD12 protein in the preparation of a product for diagnosing azithromycin resistance in children with mycoplasma pneumonia.

[0028] The inventors have found that compared with children with mycoplasma pneumonia who do not respond to azithromycin, the expression of serum exosomal KCTD12 protein in children with mycoplasma pneumonia who respond to azithromycin is upregulated. Therefore, serum exosomal KCTD12 protein can be used as a marker of azithromycin resistance in children with mycoplasma pneumonia and can be used to evaluate the resistance of children to azithromycin.

[0029] In some optional embodiments, the product comprises a kit.

[0030] In a second aspect, the present invention provides a product for diagnosing azithromycin resistance in children with mycoplasma pneumonia, wherein the product is used to detect the expression level of KCTD12 protein in serum exosomes.

[0031] The product provided by the present invention detects the expression level of KCTD12 protein in the serum exosomes of children with mycoplasma pneumonia, thereby diagnosing azithromycin resistance in children with mycoplasma pneumonia.

[0032] In some optional embodiments, the product includes a marker that recognizes the KCTD12 protein.

[0033] The identification and quantification of KCTD12 protein are achieved through markers.

[0034] In some alternative embodiments, the marker comprises an antibody that binds to the KCTD12 protein.

[0035] In some optional embodiments, the product further comprises a reagent for extracting serum exosomes.

[0036] By extracting exosomes from blood or serum, KCTD12 protein in exosomes can be further detected.

[0037] In some optional embodiments, the test sample of the product includes blood.

[0038] In some optional embodiments, the product is a reagent or a kit.

[0039] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0040] Example 1

[0041] The experimental process is as follows Figure 1 As shown in Figure 1, exosomes were isolated and proteomic analysis was performed on serum samples from 20 children with Mycoplasma pneumoniae pneumonia. The expression level of KCTD12 was identified by LC-MS / MS, and its potential as a biomarker was verified using PRM.

[0042] 1. Sample collection: Collect serum samples from patients with Mycoplasma pneumoniae.

[0043] Twenty children with Mycoplasma pneumoniae pneumonia who received a single dose of azithromycin were randomly enrolled in this study. Responders (R) and non-responders (NR) were defined by evaluating the response of respiratory and inflammatory symptoms to treatment. Serum samples were collected from peripheral blood of each patient before and after azithromycin treatment, and the patients were divided into responders and non-responders based on the response of respiratory and inflammatory symptoms to treatment.

[0044] 2. Exosome isolation: Exosomes were isolated from serum using a total exosome isolation kit.

[0045] First, the collected serum was centrifuged at 10,000 g for 30 minutes at 4°C to remove cell debris. After collecting free serum, exosomes were isolated using the total exosome isolation kit (4478360, Thermo Fisher Scientific). In brief, serum was added to the total exosome isolation reagent (volume ratio: 5:1). After vortexing, the mixture was incubated at 4°C for 30 minutes and then centrifuged at 10,000 g for 10 minutes. The exosomes contained in the precipitate were collected and resuspended in 1X PBS. Nanoparticle tracking analysis (NTA) was performed using ZetaViewPMX 110 (produced by Particle Metrix, Meerbusch, Germany) to measure the particle size and concentration of isolated exosomes. First, the isolated exosome samples were appropriately diluted with 1X PBS buffer, and then the NTA measurements were recorded using the ZetaView system ( Figure 1C in ). For transmission electron microscopy (TEM) characterization, the exosome-containing particles were first fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, followed by secondary fixation with 2% osmium tetroxide. After dehydration, the particles were embedded in propylene oxide (provided by Electron microscopy sciences, Inc.) for subsequent ultramicrotome preparation. Finally, a transmission electron microscope (model Jeol Electric 1400, manufactured by Jeol Ltd.) equipped with a digital camera was used to examine the morphology and structure of the particles ( Figure 1 B).

[0046] 3. Proteomic analysis: Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to analyze exosome proteins, and PCA analysis was performed on the main components of exosome proteins. The results are as follows Figure 1 As shown in D, the differences in the expression levels of exosome proteins in the peripheral blood of responders and non-responders were screened, and 27 differentially expressed proteins (DEPs) were successfully identified in responders, including 13 upregulated and 14 downregulated proteins (Table 1).

[0047] The specific analysis method is as follows:

[0048] The generated MS / MS data were processed using the Maxquant search engine (v.1.5.2.8). Tandem mass spectra were linked to the human Uniprot database with a reverse bait database to search for human proteins. The mass tolerance for precursor ions was set to 20 ppm in the first search and 5 ppm in the main search, and the mass tolerance for fragment ions was set to 0.02 Da. The carbamidomethyl group on Cys was assigned as a fixed modification and acetylation modification. Oxidation on Met was assigned as a variable modification. The FDR was adjusted to <1% and the minimum score for modified peptides was set to >40.

[0049] The R package DESeq2< was used to set Student's t test and log2 fold change (log2FC) ≥ 1.5, and p value 0.05 as the cutoff value for DEPs search, and the R package ggplot was used to generate the relevant volcano and heat maps ( Figure 2 ).

[0050] Figure 2 A in the figure shows the proteins differentially expressed in responders compared with non-responders before azithromycin treatment; Figure 2 Panel B shows the expression and aggregation of differentially expressed proteins in responders compared with nonresponders before azithromycin treatment; Figure 2 C in the figure shows the proteins differentially expressed in responders compared with non-responders after treatment with azithromycin; Figure 2Panel D shows the expression and aggregation of differentially expressed proteins in responders compared to non-responders after azithromycin treatment.

[0051] Table 1 27 differentially expressed proteins in exosomes of responders and non-responders before azithromycin treatment

[0052]

[0053]

[0054] 4. Biomarker quantification: Parallel reaction monitoring (PRM) was used to quantify the differentially expressed proteins, and it was found that potassium channel tetramerization domain 12 (KCTD12) was significantly upregulated before azithromycin administration, while transferrin (TF), lactotransferrin (LTF) and myeloperoxidase (MPO) were downregulated. Therefore, KCTD12 was finally identified as a candidate marker for azithromycin resistance.

[0055] Example 2

[0056] The expression levels of serum exosomal KCTD12 protein in 30 MPP patients were analyzed by ELISA, and the sensitivity and specificity in diagnosing azithromycin resistance were evaluated by ROC curve.

[0057] First, the BTB / POZ domain-containing protein KCTD12 (KCTD12) ELISA kit (MBS7201044, Mybiosource) was used to detect different concentrations of KCTD12 protein according to the manufacturer's instructions to obtain a standard curve of KCTD12 protein (e.g. Figure 4 The ELISA kit was then used to determine the expression level of KCTD12 protein in serum exosomes of 30 MPP patients (15 non-responders and 15 responders). Before AZT (azithromycin) treatment, KCTD12 levels in responders were upregulated compared with non-responders ( Figure 3 Next, receiver operating characteristic (ROC) analysis was performed to evaluate the sensitivity and specificity of KCTD12 in predicting azithromycin resistance. The cutoff value of KCTD12 was 65.9 ng / ml, with a sensitivity of 80%, a specificity of 80%, and an area under the curve (AUC) value of 0.8137 ( Figure 3 (right picture in the figure).

[0058] Therefore, the present invention uses serum exosomes and proteomics technology to screen out biomarkers for non-responders to azithromycin, selects KCTD12 as a candidate biomarker, and determines the expression level of KCTD12 by ELISA. Combined with the receiver operating characteristic (ROC) curve analysis, the CUTOFF value is determined, which can be used to judge the patient's resistance to azithromycin.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of reagents for detecting KCTD12 protein in the preparation of products for diagnosing azithromycin resistance in children with mycoplasma pneumonia.

2. The use according to claim 1, characterized in that: The KCTD12 protein is the KCTD12 protein in serum exosomes.

3. The use according to claim 1, characterized in that: Compared with children with mycoplasma pneumonia who did not respond to azithromycin, the expression of serum exosomal KCTD12 protein was upregulated in children with mycoplasma pneumonia who responded to azithromycin.

4. The use according to claim 1, characterized in that: The products include kits.

5. A product for diagnosing azithromycin resistance in children with mycoplasma pneumonia, characterized in that: The product is used to detect the expression level of KCTD12 protein in serum exosomes.

6. The product according to claim 5, characterized in that The product includes a marker that recognizes the KCTD12 protein.

7. The product according to claim 6, characterized in that The marker includes an antibody that binds to the KCTD12 protein.

8. The product according to claim 5, characterized in that The product also includes reagents for serum exosome extraction.

9. The product according to claim 5, characterized in that The test sample of the product includes blood.

10. The product according to claim 5, characterized in that The product is a reagent or a kit.

Images