Marker for salmonella infection and application thereof

Abstract

The invention discloses a salmonella infection marker and application thereof. By constructing a salmonella challenge model and combining a proteomics technology, it is successfully identified that ZNF143 can be used as a potential protein marker of salmonella infection, so that a more direct, rapid and accurate salmonella detection approach is provided, and a solid theoretical foundation is laid for developing a novel, efficient and accurate salmonella detection technology.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a marker of salmonella infection and application thereof. Background Art

[0002] As the most common bacterial pathogen endangering the poultry industry, Salmonella can not only reduce egg production and hatching rate through vertical infection, increase the possibility of egg contamination, but also directly cause a large number of poultry deaths, seriously restricting the development of China's poultry industry. In addition, salmonellosis, a zoonotic disease caused by Salmonella, poses a severe challenge to public health and safety. Severe cases of infection can induce sepsis, leading to high mortality.

[0003] At present, in poultry farming, vaccination is an effective means of preventing and treating bacterial diseases such as Salmonella, but it is easy for the body and strains to develop drug resistance, and the long-term irrational use of antibiotics has led to a widening of the drug resistance spectrum, thus leading to the spread of multi-drug resistant Salmonella. There is no clear conclusion on whether the residues of vaccines in the body are harmful to humans and the environment, so relying solely on vaccines and other drugs for prevention and treatment is not a sustainable and feasible method.

[0004] Given the severity and widespread nature of Salmonella infection, research on the infection mechanism, disease progression, and host resistance of specific diseases such as avian Salmonella has been widely conducted worldwide. However, due to the lack of comprehensive and systematic phenotypic evaluation indicators and evaluation systems for disease resistance and health status, the progress of related research has been relatively slow. In production practice, the detection of Salmonella mainly relies on the traditional serum plate agglutination test. Although this method has shown certain practicality in processing large-scale samples, its detection range and accuracy have certain limitations, and it is difficult to meet the needs of high-precision and high-reliability detection. Therefore, in order to obtain more accurate test results, the serum plate agglutination test is usually combined with other advanced detection technologies, such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) technology, for comprehensive application and cross-validation in the production process.

[0005] Due to the lack of comprehensive and systematic phenotypic evaluation indicators and evaluation systems for disease resistance and health status, the progress of related research is relatively slow. In view of the shortcomings of existing detection methods in efficiency and accuracy, it is urgent to develop an innovative detection technology that aims to efficiently indicate the infection status of Salmonella through a specific protein marker. This technology is expected to provide a more direct, rapid and accurate way to detect Salmonella, thereby significantly improving detection efficiency and diagnostic accuracy. Specifically, the research will focus on screening and identifying protein markers that can specifically identify Salmonella infection, and then developing a detection system based on this marker. This system is not only required to have high sensitivity and specificity, but also needs to meet the needs of simple operation, high cost-effectiveness and applicability to various testing environments. Summary of the invention

[0006] The purpose of the present invention is to provide a marker of Salmonella infection and application thereof.

[0007] To achieve the purpose of the present invention, in a first aspect, the present invention provides a marker for Salmonella infection, wherein the marker is ZNF143 protein.

[0008] For example, the amino acid sequence of the chicken ZNF143 protein has a reference sequence number of XM_040701211.2 in NCBI.

[0009] In a second aspect, the present invention provides ZNF143 protein as a marker for Salmonella infection and use thereof in the preparation of a product for detecting Salmonella infection.

[0010] In a third aspect, the present invention provides the use of a substance for detecting a marker, namely, ZNF143 protein, in the preparation of a reagent or a kit for detecting Salmonella infection, diagnosing Salmonellosis or evaluating therapeutic efficacy.

[0011] The substance includes a substance for detecting the marker at the gene or protein level. For example, the substance is an antibody that specifically binds to the marker, or the substance is a primer for detecting the expression amount of the coding gene corresponding to the marker.

[0012] In the present invention, the Salmonella ( Salmonella ) including Salmonella Enteritidis ( Salmonella e nteritidis).

[0013] In the present invention, the animal infected by Salmonella is poultry, such as chicken.

[0014] In the present invention, the expression level of ZNF143 in animals infected with Salmonella is reduced (lower than that in animals not infected with Salmonella).

[0015] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention constructs a Salmonella challenge model and combines proteomics technology to successfully identify ZNF143 as a potential protein marker for Salmonella infection, laying a solid theoretical foundation for the development of new, efficient and accurate Salmonella detection technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 In the preferred embodiment of the present invention, the Salmonella Enteritidis challenge model was successfully constructed, and there were differences in immune phenotypes between the control group and the challenge group.

[0017] Figure 2 In a preferred embodiment of the present invention, a significantly differentially expressed protein ZNF143 was screened out based on the PBMC proteome, which can indicate Salmonella infection.

[0018] Figure 3 In a preferred embodiment of the present invention, there is a significant difference in the expression level of ZNF143 between the control group and the challenge group in the immune tissue transcriptome.

[0019] DETAILED DESCRIPTION

[0020] Existing detection methods, such as serum plate agglutination test, have limitations in detection range and accuracy, and are difficult to meet the detection requirements of high accuracy and high reliability. By screening and identifying ZNF143 protein as a new protein marker, the present invention provides a more direct, rapid and accurate way to detect Salmonella.

[0021] The present invention adopts the following technical solution: The present invention selects "Guangming No. 2" white-feathered broiler chicken (hereinafter referred to as Guangming white chicken) and Salmonella enteritidis 50335 ( SalmonellaEnteritidis 50335) strain was used as the experimental material to construct a Salmonella enteritidis challenge model. By using liquid chromatography tandem mass spectrometry (LC-MS / MS) technology, the proteins of peripheral blood mononuclear cells (PBMCs) of each sample were deeply measured and comprehensively analyzed, and the protein closely related to Salmonella infection, zinc finger protein 143 (ZNF143), was successfully screened out. ZNF143 is a C2H2-type transcription factor composed of 7 zinc finger structures, which is widely expressed in different tissues and cells of most vertebrates. Related studies have shown that the expression of ZNF143 in cancer tissues and embryonic development is higher than that in normal tissues. It can play an important role in cell activities such as cell cycle, cell proliferation and division by participating in the activation of coding and non-coding genes.

[0022] The main improvements of the present invention are as follows: (I) By constructing a Salmonella Enteritidis attack model, the Salmonella infection environment is simulated to ensure the authenticity and reliability of the experimental conditions.

[0023] (ii) Liquid chromatography tandem mass spectrometry (LC-MS / MS) is a highly sensitive, high-resolution protein analysis technology that can perform in-depth measurement and comprehensive analysis of proteins in complex biological samples.

[0024] (III) By comparing the protein expression profiles of peripheral blood mononuclear cells (PBMCs) in the infection group and the control group, a protein closely related to Salmonella infection, zinc finger protein 143 (ZNF143), was screened out. As a C2H2-type transcription factor, ZNF143 may play an important regulatory role in the process of Salmonella infection, and changes in its expression level can reflect the infection status.

[0025] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0026] Example 1 Development of markers for Salmonella infection 1. Experimental Methods 1. Experimental animals and sample preparation The experimental group consisted of 261 Guangdong white chickens, which were transferred to two sterile isolation cabins at 1 day old to serve as the challenge group and the control group. 30 μL of blood was collected from the sub-wing vein for Salmonella infection detection to confirm that there were no individuals infected with Salmonella. The challenge group was given artificial oral administration of Salmonella Enteritidis CMCC(B) 50335 (SE) for 3 consecutive days starting at 6 days old (8.5×108 CFU / mL, 0.5mL / mouse), the control group was simultaneously gavaged with the same dose of PBS, and blood was collected from the jugular vein and samples were collected 24 hours after the last challenge (9 days old). All individual cecal tonsil samples were sequenced by RNA-Seq. 20 individuals were randomly selected from the challenge group and the control group, and 2mL of blood was taken to separate PBMC cells from the whole blood by Ficoll density gradient centrifugation. The remaining whole blood was placed in a coagulant tube to separate serum for the determination of inflammatory factors and antibody levels in the serum. Cecal tissue was collected from all individuals for cecal bacterial load determination.

[0027] 2. Analysis of differential protein in PBMC proteome (1) PBMC protein extraction The samples were taken out from -80°C, and 4 volumes of lysis buffer (1% SDS, 1% protease inhibitor (purchased from Merck Millipore)) were added to each group of samples, and ultrasonic lysis was performed. Centrifugation was performed at 4°C, 12000g for 10 min, and the supernatant was transferred to a new centrifuge tube, and the protein concentration was determined using a BCA kit.

[0028] (2) Protein digestion with pancreatic enzymes Take an equal amount of protein from each sample for enzymatic hydrolysis, adjust the volume to the same with lysis buffer, add 1 volume of pre-cooled acetone, vortex and mix, then add 4 volumes of pre-cooled acetone, and precipitate at -20℃ for 2h. Centrifuge at 4500g for 5min, discard the supernatant, and wash the precipitate with pre-cooled acetone 2-3 times. After drying the precipitate, add TEAB with a final concentration of 200mM, ultrasonically break up the precipitate, add trypsin (purchased from Promega) at a ratio of 1:50 (protease: protein, m / m), and enzymatically hydrolyze overnight. Add dithiothreitol (DTT) to a final concentration of 5mM, and reduce at 56℃ for 30min. Then add iodoacetamide (IAA) to a final concentration of 11 mM, and incubate at room temperature in the dark for 15min.

[0029] (3) Liquid chromatography-mass spectrometry analysis The peptides were dissolved in liquid chromatography mobile phase A and separated using the Easy-nLC1000 ultra-high performance liquid phase system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an acetonitrile-water solution containing 0.1% formic acid. The liquid phase gradient was set as follows: 0-14min, 6%-24%B; 14-16min, 24%-35%B; 16-18min, 35%-80%B; 18-20min, 80%B, and the flow rate was maintained at 500nl / min. After the peptides were separated by the ultra-high performance liquid phase system, they were injected into the Capillary ion source for ionization and then into the timsTOF Pro mass spectrometer for data acquisition. The ion source voltage was set to 1.75kV, and the peptide parent ion and its secondary fragments were detected and analyzed using TOF. The data acquisition mode used was data-independent parallel accumulation serial fragmentation (dia-PASEF) mode. The primary mass spectrometry scan range was set to 300-500 m / z (MS / MS scan range). After one primary mass spectrum was acquired, 20 (Number of PASEF scan) PASEF (MS / MS mode) acquisitions were performed. The secondary mass spectrometry scan was in the range of 400-850 m / z, with every 7 m / z as a window.

[0030] (4) Screening of differentially expressed proteins related to disease resistance After common functional annotations were performed on the identified proteins, one-way analysis of variance (ANOVA) was performed on the four groups to calculate the significant P-values ​​of multiple groups, and the differential proteins between multiple groups were screened for analysis based on the P-value; the fold change (FC) and T-test significant P-value between the two groups were calculated based on the quantitative results, and the differences were screened according to the set thresholds, and statistical graphs related to the difference analysis were drawn. The functional classification statistical analysis of the differential proteins between the two groups was performed, including GO secondary classification, subcellular localization classification, COG / KOG classification and KEGG pathway enrichment analysis, to screen candidate proteins related to anti-Salmonella infection.

[0031] 2. Test results 1. The Salmonella Enteritidis challenge model was successfully established, and there were differences in immune phenotypes between the control group and the challenge group ( Figure 1 ). After the construction of the Salmonella enteritidis challenge model was completed, the cecal bacterial load of the challenge group was measured to ensure that Salmonella could be detected. The serum inflammatory factors (IFN-γ, IL-6 and IL-8) and the IgA antibody content in the serum of the challenge group and the control group were detected and analyzed. The results showed that the concentrations of IFN-γ and IL-6 increased significantly after the challenge, while the concentration of IgA decreased significantly. The above results indicate that the model was successfully constructed.

[0032] 2. Based on the PBMC proteome, a significantly differentially expressed protein, ZNF143, was screened out, which can indicate Salmonella infection ( Figure 2 ). By analyzing the PBMC protein groups of the Enteritidis Salmonella challenge group and the control group, the significantly different protein ZNF143 was screened out based on the screening criterion of P-value < 0.05. In order to verify the potential value of this protein in the prediction of Salmonella infection, the ROC (receiver operating characteristic) curve analysis method was further used. The ROC curve analysis results of ZNF143 showed that its AUC (area under the curve) value was as high as 0.902, which was not only much higher than the random prediction level (AUC = 0.5), but also significantly higher than the average level of general diagnostic or prediction models, indicating that this protein has extremely high accuracy and reliability in predicting Salmonella infection.

[0033] 3. There was a significant difference in the expression of ZNF143 between the control group and the challenge group in the immune tissue transcriptome ( Figure 3 Based on the transcriptome data of cecal tonsils of 261 individuals, the relative expression level (FPKM) of the gene encoding ZNF143 protein in the challenge group and the control group was analyzed. The results showed that compared with the control group, ZNF143 The expression level was significantly reduced, which verified the PBMC proteome results and confirmed that the expression level of ZNF143 protein can effectively predict Salmonella infection in poultry.

[0034] Existing detection methods, such as serum plate agglutination test, have limitations in detection range and accuracy, and are difficult to meet the detection requirements of high accuracy and high reliability. By screening and identifying ZNF143 protein as a new protein marker, the present invention provides a more direct, rapid and accurate way to detect Salmonella.

[0035] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A marker of Salmonella infection, characterized in that The marker is ZNF143 protein; The amino acid sequence of the chicken ZNF143 protein is referenced in NCBI as XM_040701211.

2.

2. Application of ZNF143 protein as a marker of Salmonella infection in the preparation of products for detecting Salmonella infection; The amino acid sequence of the chicken ZNF143 protein is referenced in NCBI as XM_040701211.

2.

3. Use of a substance for detecting the marker of claim 1 in the preparation of a reagent or kit for detecting Salmonella infection, diagnosing salmonellosis or evaluating therapeutic efficacy.

4. The use according to claim 3, characterized in that: The substances include substances for detecting the markers at the gene or protein level.

5. The use according to claim 4, characterized in that: The substance is an antibody that specifically binds to the marker, or the substance is a primer for detecting the expression level of a coding gene corresponding to the marker.

6. The use according to any one of claims 2 to 5, characterized in that: The Salmonella includes Salmonella Enteritidis.

7. The use according to any one of claims 2 to 5, characterized in that: The animals infected with Salmonella are poultry; Preferably, the animal is a chicken.

8. The use according to any one of claims 2 to 5, characterized in that: ZNF143 expression was reduced in animals infected with Salmonella.

Images