Method for enriching O: 3 type enterocolitis yersinia by Gp17 magnetic beads

Abstract

The invention relates to the technical field of microbiological detection, in particular to a method for enriching O: 3 type enterocolitis yersinia by Gp17 magnetic beads, which comprises the following steps: step 1, expressing and purifying Gp17 protein through a prokaryotic expression system; step 2, coupling the purified Gp17 protein in the step 1 with carboxyl modified magnetic nanoparticles to prepare Gp17 functionalized magnetic beads; step 3, mixing the Gp17 functionalized magnetic beads prepared in the step 2 with a sample to be detected, and enriching O: 3 type enterocolitis yersinia through magnetic field separation; 4, the enriched O: 3 type enterocolitis yersinia is cultured or subjected to molecular biology detection.The method for enriching the O: 3 type enterocolitis yersinia through the Gp17 magnetic beads has the advantages that compared with existing immunomagnetic beads and Gp17 functional magnetic beads, the specificity is better, enrichment of specific serotypes can be achieved, and the method is suitable for large-scale popularization and application. And the Gp17 protein can be massively produced in vitro by utilizing a prokaryotic expression technology and is easy to obtain, the cost is reduced, and the difference between batches is small.

Description

Technical Field

[0001] The invention relates to the technical field of microbial detection, and in particular to a method for enriching O:3 type Yersinia enterocolitica with Gp17 magnetic beads. Background Art

[0002] Foodborne diseases are a type of disease that cannot be ignored in the field of public health worldwide, and pathogen contamination is one of the most important factors leading to foodborne disease outbreaks. The World Health Organization reports that about 1.7 million people die from bacterial-related diseases each year. In recent years, with the increase in demand for refrigerated foods and the development of cold chain transportation, the risks posed by foodborne psychrophilic bacteria cannot be ignored. Psychrophilic bacteria can grow slowly at low temperatures and produce toxins, leading to a decline in food quality. There are many pathogenic bacteria and conditional pathogens among psychrophilic bacteria, which may be potentially pathogenic to people with low immunity and easily cause food poisoning. Common psychrophilic bacteria include Yersinia, Listeria monocytogenes, and Pseudomonas. Among them, Yersinia enterocolitica, as a zoonotic intestinal pathogen and foodborne pathogen, can cause various gastrointestinal syndromes and is one of the four major causes of human gastrointestinal diseases. In severe cases, it can lead to death.

[0003] Yersinia enterocolitica is 0.5-0.8μm wide and 1-3μm long. It does not form spores, has no capsule, and has flagella. It can grow at 0-45℃, with an optimal growth temperature of 25-32℃. When it proliferates under refrigerated conditions (4-8℃), it will produce a heat-resistant toxin. Due to the high heterogeneity of the bacteria, there are more than 70 serotypes classified based on the O antigen on the surface of the bacteria. Among them, the main serotypes that are harmful to humans include O:3, O:9, O:8, O:5,27, etc. At present, O:3 and O:9 are mainly prevalent in my country and Europe, and O:8 is mainly prevalent in the United States. The bacteria has strong adaptability and exists in large quantities in the environment. It can survive and reproduce in fresh food or refrigerated environments, causing certain epidemiological risks and posing a threat to food safety. The main source of human infection with Yersinia is animal food, raw or undercooked pork, as well as contaminated dairy products, plants and seafood. It is one of the three most common hazards associated with eating pork in human clinical cases and one of the two main hazards identified in pork carcasses. Since Yersinia enterocolitica remains in an uncultivable but viable state in natural samples and can grow and reproduce under refrigerated conditions at 4°C, eating contaminated meat poses a potential health risk to consumers. Food safety issues caused by Yersinia enterocolitica have always been a matter of great concern in the fields of food safety and public health at home and abroad. In view of the hazards of this bacterium to food safety and human health, it is required to be able to separate and detect Yersinia enterocolitica in food or other samples. However, the existing "gold standard" and molecular biology rapid detection methods generally have problems such as expensive detection equipment, insufficient sensitivity and complex sample pretreatment, which makes it difficult to effectively monitor potentially contaminated food samples. In addition, the matrix components in food are complex, and Yersinia enterocolitica is usually contaminated at low concentrations, with low content and difficult to detect, requiring samples to be pre-enriched and cultured for a long time.

[0004] The commonly used enrichment method for this bacterium is cold enrichment, but this method has a long culture cycle of 2-3 weeks, which may cause the growth of other psychrophilic competing bacteria such as Pseudomonas and other species of Yersinia, and cannot meet the requirements of inspection timeliness, especially when foodborne diseases break out. Hot enrichment only takes 2-3 days, but the detection rate is lower than that of cold enrichment. In addition, magnetic nanoparticles can be used to quickly separate foodborne pathogens in samples through the action of magnetic fields, significantly shortening the enrichment time. Based on magnetic separation, magnetic beads can be combined with metal-organic frameworks, antibodies, aptamers or functional proteins to selectively capture and enrich target bacteria in a short time, thereby significantly improving the sensitivity of the detection method and improving the detection rate and accuracy in actual samples. The commonly used immunomagnetic separation (IMS) technology currently is to label anti-Yersinia enterocolitica antibodies such as IgG and OmpF on magnetic beads, combine them with pathogenic bacteria through antigen-antibody reaction, and combine them with PCR technology. It has high sensitivity, but has disadvantages such as low specificity and reaction with non-target bacteria. In addition, the antibody preparation process is complicated and requires multiple immunizations in animals. There are large batch differences, which is not conducive to the protection of animal welfare.

[0005] Therefore, in view of the above situation, it is urgent to develop a method for enriching O:3 type Yersinia enterocolitica using Gp17 magnetic beads to overcome the shortcomings in current practical applications. Summary of the invention

[0006] The object of the present invention is to provide a method for enriching O:3 type Yersinia enterocolitica using Gp17 magnetic beads to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for enriching O:3 type Yersinia enterocolitica using Gp17 magnetic beads comprises the following steps:

[0009] Step 1, preparing Gp17 protein: expressing and purifying Gp17 protein through a prokaryotic expression system, wherein the Gp17 protein is derived from a short-tailed bacteriophage and has the ability to specifically bind to O:3 type Yersinia enterocolitica;

[0010] Step 2, preparing Gp17 functionalized magnetic beads: coupling the Gp17 protein purified in step 1 with carboxyl-modified magnetic nanoparticles to prepare Gp17 functionalized magnetic beads;

[0011] Step 3, enriching Yersinia enterocolitica type O:3: mixing the Gp17 functionalized magnetic beads prepared in step 2 with the sample to be tested, and separating by magnetic field to enrich Yersinia enterocolitica type O:3;

[0012] Step 4, detection: culture or molecular biological detection of the enriched O:3 type Yersinia enterocolitica.

[0013] As a further solution of the present invention: in step 1, the method for preparing the Gp17 protein comprises the following steps:

[0014] S1. Determine the primary, secondary and tertiary structures of Gp17 protein by bioinformatics analysis;

[0015] S2, constructing a recombinant expression vector containing the Gp17 gene, and transforming it into a prokaryotic expression host for expression;

[0016] S3. Purify Gp17 protein by affinity chromatography.

[0017] As a further solution of the present invention: in step S2, the prokaryotic expression host is Escherichia coli BL21 (DE3), the recombinant expression vector is pET32a (+), and the N-terminus of the Gp17 protein carries a His tag.

[0018] As a further solution of the present invention: In step 2, the method for preparing the Gp17 functionalized magnetic beads comprises the following steps:

[0019] A1, activating the carboxyl-modified magnetic nanoparticles with an activator;

[0020] A2, mixing the activated magnetic nanoparticles with the Gp17 protein in a coupling buffer, and coupling at room temperature with shaking for 2 hours;

[0021] A3. Remove the uncoupled Gp17 protein by magnetic separation, and block the unreacted active sites on the surface of the magnetic beads with blocking solution.

[0022] As a further solution of the present invention: in step 3, the sample to be tested includes a food sample, an environmental sample or a clinical sample, and the food sample is pork, seafood or dairy products.

[0023] As a further solution of the present invention: in step 3, the enrichment rate of the Gp17 functionalized magnetic beads for O:3 type Yersinia enterocolitica is above 80%.

[0024] As a further solution of the present invention: in step 3, the Gp17 functionalized magnetic beads are capable of enriching type O:3 Yersinia enterocolitica from a sample with a concentration of 1 CFU / mL.

[0025] As a further solution of the present invention: in step 4, the detection method includes plate culture method, PCR method or fluorescence microscope observation method.

[0026] As a further solution of the present invention: in step 2, the Gp17 protein of the Gp17 functionalized magnetic beads can specifically bind to the O antigen polysaccharide of O:3 type Yersinia enterocolitica, and does not bind to the surface antigens of O:8 type, O:5 type and O:9 type Yersinia enterocolitica and other common foodborne pathogens.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention is based on the specific recognition ability of Gp17 for O:3 type Yersinia enterocolitica, expresses and purifies Gp17, combines its N-terminus with carboxyl-modified magnetic nanoparticles, prepares Gp17 functionalized magnetic beads that can specifically bind to O:3 type Yersinia enterocolitica, utilizes the separation effect of the magnetic field, quickly and accurately enriches the target bacteria, and combines with improved Y culture medium to detect O:3 type Yersinia enterocolitica. Compared with existing immunomagnetic beads, Gp17 functionalized magnetic beads have better specificity, can achieve the enrichment of specific serotypes, and Gp17 protein can be mass-produced in vitro using prokaryotic expression technology, is easy to obtain, reduces costs, and has small batch differences. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of bioinformatics analysis in an embodiment of the present invention;

[0030] Among them, a is the hydrophilicity / hydrophobicity analysis of Gp17 by Prot Scale, b is the signal peptide prediction of Gp17 by SignalP 6.0, c is the signal peptide and transmembrane region prediction of Gp17 by Phobius, d is the transmembrane region prediction of Gp17 by TMHMM 2.0, e is the transmembrane region prediction of Gp17 by DeepTMHMM, f is the secondary structure analysis of Gp17 by Psipred, g is the secondary structure analysis of Gp17 by PHYRE, h is the domain analysis of Gp17 by UniProt, i is the domain analysis of Gp17 by InterPro, j is the homology modeling of 3-143aa and 537-644aa of Gp17 by SWISS-MODEL, k is the de novo modeling of Gp17 by I-TASSER, and l is the molecular docking of Gp17-OPS.

[0031] Figure 2 This is a schematic diagram of the construction and recombination of the prokaryotic expression vector in the embodiment of the present invention.

[0032] Figure 3 Schematic diagram of soluble expression of Gp17 in an embodiment of the present invention.

[0033] Figure 4 Schematic diagram of the SDS-PAGE results of Gp17 nickel column purification in the embodiment of the present invention.

[0034] Figure 5 This is a schematic diagram of the ELISA test of the affinity of Gp17 protein to O:3 type Yersinia enterocolitica in an example of the present invention.

[0035] Figure 6 This is a schematic diagram of fluorescence microscopy imaging of the Gp17-bacteria complex in an embodiment of the present invention;

[0036] Among them, a is the superposition field (Gp17+ bacteria), b is the fluorescence field (Gp17), and c is the bright field (bacteria).

[0037] Figure 7 This is a schematic diagram of the specificity of Gp17 protein for O:3 type Yersinia enterocolitica tested by ELISA in an example of the present invention.

[0038] Figure 8 This is a schematic diagram of verifying the enrichment rate of Gp17 functionalized magnetic beads for O:3 type Yersinia enterocolitica in an embodiment of the present invention;

[0039] Among them, a is the original bacterial solution without adding magnetic beads, b is the supernatant after adding original carboxyl magnetic beads, c is the supernatant after adding Gp17 functionalized magnetic beads, and d is the supernatant after adding Gp17 functionalized magnetic beads-bacteria.

[0040] Fig. 9 This is a schematic diagram of SEM characterization of the capture of O:3 type Yersinia enterocolitica by Gp17 functionalized magnetic beads in an embodiment of the present invention;

[0041] Among them, a is O:3 type Yersinia enterocolitica, b is Gp17 functionalized magnetic beads, c is Gp17 functionalized magnetic beads-O:3 type Yersinia enterocolitica complex, and d is a mixture of original carboxyl magnetic beads and O:3 type Yersinia enterocolitica.

[0042] Fig.10 This is a schematic diagram of verifying the specificity of Gp17 functionalized magnetic beads to O:3 type Yersinia enterocolitica in an embodiment of the present invention;

[0043] Among them, a is the mixed original bacterial solution without adding magnetic beads, b is the supernatant of the suspension after adding Gp17 functionalized magnetic beads, c is the enrichment and re-cultivation after adding Gp17 functionalized magnetic beads, and d is the single colony serotype PCR result.

[0044] Fig.11 Schematic diagram of PCR results of different concentrations of O:3 type Yersinia enterocolitica in the examples of the present invention.

[0045] Fig.12 This is a schematic diagram of the capture rate of O:3 type Yersinia enterocolitica by Gp17 functionalized magnetic beads in an embodiment of the present invention;

[0046] Where a is 10 without adding magnetic beads. 2 CFU / mL original bacterial solution, b is the supernatant after adding original carboxyl magnetic beads, c is the supernatant after adding Gp17 functionalized magnetic beads, and d is the supernatant after adding Gp17 functionalized magnetic beads-bacteria.

[0047] Fig.13 Schematic diagram of the sensitivity of Gp17 functionalized magnetic beads to O:3 type Yersinia enterocolitica at low concentrations in an embodiment of the present invention;

[0048] Where a is 10 without adding magnetic beads. 1 CFU / mL original bacterial solution, b is the supernatant after adding original carboxyl magnetic beads, c is the supernatant after adding Gp17 functionalized magnetic beads, and d is the supernatant after adding Gp17 functionalized magnetic beads-bacteria.

[0049] Fig.14 Schematic diagram of the sensitivity of Gp17 functionalized magnetic beads to O:3 type Yersinia enterocolitica at extremely low concentrations in an embodiment of the present invention;

[0050] Where a is 10 without adding magnetic beads. 0 CFU / mL original bacterial solution, b is the supernatant after adding original carboxyl magnetic beads, c is the supernatant after adding Gp17 functionalized magnetic beads, and d is the supernatant after adding Gp17 functionalized magnetic beads-bacteria.

[0051] Fig.15 This is a schematic diagram of the detection of spiked pork in an embodiment of the present invention;

[0052] Where a is 10 1 CFU / mL spiked pork sample solution, b is the supernatant after adding Gp17 functionalized magnetic beads, c is the culture after adding Gp17 functionalized magnetic beads for enrichment, d is 10 0 CFU / mL spiked pork sample stock solution, e is the supernatant after adding Gp17 functionalized magnetic beads, and f is the culture after enrichment with Gp17 functionalized magnetic beads.

[0053] Fig.16 This is a schematic diagram of spiked seafood detection in an embodiment of the present invention;

[0054] Where a is 10 1 CFU / mL spiked seafood sample stock solution, b is the supernatant after adding Gp17 functionalized magnetic beads, c is the culture after adding Gp17 functionalized magnetic beads for enrichment, d is 10 0 CFU / mL spiked seafood sample stock solution, e is the supernatant after adding Gp17 functionalized magnetic beads, and f is the culture after enrichment with Gp17 functionalized magnetic beads.

[0055] Fig.17 Schematic diagram of enrichment of target bacteria by Gp17 functionalized magnetic beads in an embodiment of the present invention. DETAILED DESCRIPTION

[0056] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0057] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0058] See also Fig.17 The present invention provides a method for enriching O:3 type Yersinia enterocolitica with Gp17 magnetic beads, comprising the following steps:

[0059] Step 1, preparing Gp17 protein: expressing and purifying Gp17 protein through a prokaryotic expression system, wherein the Gp17 protein is derived from short-tailed bacteriophage φYeO3-12 and has the ability to specifically bind to O:3 type Yersinia enterocolitica;

[0060] Step 2, preparing Gp17 functionalized magnetic beads: coupling the Gp17 protein purified in step 1 with carboxyl-modified magnetic nanoparticles to prepare Gp17 functionalized magnetic beads;

[0061] Step 3, enriching Yersinia enterocolitica type O:3: mixing the Gp17 functionalized magnetic beads prepared in step 2 with the sample to be tested, and separating by magnetic field to enrich Yersinia enterocolitica type O:3;

[0062] Step 4, detection: culture or molecular biological detection of the enriched O:3 type Yersinia enterocolitica.

[0063] In one embodiment of the present invention, see Figure 1 In step 1, the method for preparing the Gp17 protein comprises the following steps:

[0064] S1. Determine the primary, secondary and tertiary structures of Gp17 protein by bioinformatics analysis;

[0065] S2, constructing a recombinant expression vector containing the Gp17 gene, and transforming it into a prokaryotic expression host for expression;

[0066] S3. Purify Gp17 protein by affinity chromatography.

[0067] In step S2, the prokaryotic expression host is Escherichia coli BL21 (DE3), the recombinant expression vector is pET32a (+), and the N-terminus of the Gp17 protein carries a His tag.

[0068] In one embodiment of the present invention, see Figure 1-Figure 4 In step 2, the method for preparing the Gp17 functionalized magnetic beads comprises the following steps:

[0069] A1, activating the carboxyl-modified magnetic nanoparticles with an activator;

[0070] A2, mixing the activated magnetic nanoparticles with the Gp17 protein in a coupling buffer, and coupling at room temperature with shaking for 2 hours;

[0071] A3. Remove the uncoupled Gp17 protein by magnetic separation, and block the unreacted active sites on the surface of the magnetic beads with blocking solution.

[0072] In one embodiment of the present invention, see Figure 5-Figure 16 In step 3, the sample to be tested includes a food sample, an environmental sample or a clinical sample, and the food sample is pork, seafood or dairy products.

[0073] In step 3, the enrichment rate of the Gp17 functionalized magnetic beads for type O:3 Yersinia enterocolitica is greater than 80%.

[0074] In step 3, the Gp17 functionalized magnetic beads are capable of enriching Yersinia enterocolitica type O:3 from a sample with a concentration of 1 CFU / mL.

[0075] In step 4, the detection method includes plate culture method, PCR method or fluorescence microscope observation method.

[0076] In one embodiment of the present invention, in step 2, the Gp17 protein of the Gp17 functionalized magnetic beads can specifically bind to the O antigen polysaccharide of Yersinia enterocolitica type O:3, and does not bind to the surface antigens of other serotypes of Yersinia enterocolitica such as type O:8, type O:5 and type O:9 and other common foodborne pathogens.

[0077] In the process of enriching Yersinia enterocolitica type O:3 with Gp17 magnetic beads, the specific experimental method is as follows:

[0078] 1. Prepare Gp17 protein through bioinformatics analysis, vector construction and expression purification;

[0079] 1.1 Bioinformatics Analysis

[0080] The biological information of Gp17, such as primary structure, secondary structure, and tertiary structure, was systematically and comprehensively analyzed using relevant servers and software to better perform subsequent prokaryotic expression and predict and verify the protein's ability to bind to the target. Analysis of its physical and chemical properties using Prot Param showed that the molecular weight of Gp17 was 69.4 kDa, the theoretical isoelectric point was 6.02, it was slightly acidic, hydrophilic, stable, and could be expressed in prokaryotes; analysis of the hydrophilicity / hydrophobicity, signal peptide, and transmembrane region of Gp17 using websites and servers such as Prot Scale, SignalP 6.0, Phobius, TMHMM 2.0, and DeepTMHMM showed that Gp17 was a hydrophilic protein without a signal peptide or transmembrane region, and was an extracellular protein, so there was no need to remove or modify the amino acid sequence (such as Figure 1 ae in (shown in );

[0081] The prediction of protein secondary structure and domain by Psipred, PHYRE, UniProt, InterPro and other websites and servers showed that Gp17 contains 199 α-helices (Hh), accounting for 30.85%, and has a relatively stable structure. The 539-645aa segment is the C-terminus, which is the intramolecular chaperone autoprocessing domain (ICA), belongs to the peptidase family S74, can catalyze trimerization-dependent autoprotein hydrolysis, has a single domain, and can be expressed as a whole (e.g. Figure 1 The tertiary structure was predicted by SWISS-MODEL homology modeling, and it was found that Gp17 had a low match with the existing models in the database, and there was no template protein with high homology. Only partial tertiary structure models could be established, among which the model composed of 3-143aa was established on the N-terminal template of the phage T7 tail fiber protein gp17, and the model composed of 537-644aa was established on the ICA domain template of the subtype 2 MyRF of the myelin regulatory factor (as shown in Figure 1). Figure 1 The I-TASSER ab initio method successfully predicted and constructed the three-dimensional structure of Gp17 and visualized it, with a C-score of -0.63 (as shown in j in Figure 2). Figure 1 The tertiary structure predicted by I-TASSER was used for molecular docking with the target OPS. The binding energy between OPS and Gp17 was -6.1 kcal / mol, indicating that they can be stably bound, providing a certain theoretical basis for subsequent experiments (such as Figure 1 1 in the figure).

[0082] 1.2 Vector construction

[0083] Enhanced green fluorescent protein (EGFP) was added to the N-terminus of Gp17 protein (sequence number NC_001271.1), and the two ends of the EGFP+Gp17 gene were treated with restriction endonucleases NcoI and XhoI, and then connected to the cloning vector TOP10 to construct a recombinant cloning plasmid, and the rare codons were optimized, and then sent to GenScript Biotech to synthesize the target gene. The plasmid was transformed into E. coli. DH5α for culture, and double enzyme digestion was performed after sequencing and alignment were correct. The target fragment was connected to the prokaryotic expression vector pET32a (+) to construct a recombinant expression plasmid, retaining the trxA solubilizing tag and His tag (such as Figure 2 The plasmid was transformed into Escherichia coli BL21 (DE3), double enzyme digestion verification, sequencing, and seed preservation.

[0084] 1.3 Expression and purification

[0085] Take 50 μL of E. coli BL21 (DE3) containing Gp17 protein and inoculate it into 5 mL of LB medium containing 100 μg / mL ampicillin, and culture it at 37°C and 180 rpm overnight as the activated seed liquid. Take several tubes of 5 mL of LB medium containing ampicillin, inoculate each with 50 μL of seed liquid (1:100 inoculation), and culture at 37°C and 180 rpm until OD 600 The concentration of IPTG was 0.6, and different concentrations of IPTG were added. The cells were cultured at 37°C for 8h and 16°C for 20h to induce expression. The uninduced bacterial solution was used as a negative control. After ultrasonic disruption, the supernatant and precipitate components were subjected to SDS-PAGE gel electrophoresis and stained. The target band size was predicted to be about 113.5kDa, including about 70kDa of Gp17, 26.9kDa of fluorescent tag EGFP, 11.8kDa of solubility tag trxA, and His tag. Through experimental optimization, the soluble expression conditions of Gp17 protein were 0.5mM, cultured at 16°C for 16h or 37°C for 8h (such as Figure 3 The Gp17 protein was expanded and cultured, induced to express at 0.5 mM and 16°C, and then ultrasonically broken, centrifuged and the supernatant was collected and purified by nickel gravity column affinity chromatography (as shown in Figure 4 shown).

[0086] 2. Verify the affinity and specificity of Gp17 protein to Yersinia enterocolitica type O:3;

[0087] 2.1 ELISA to verify the affinity of Gp17 protein to Yersinia enterocolitica type O:3

[0088] O:3 type enterocolitica Yersinia was cultured overnight to the logarithmic phase, washed with sterile 1×PBS and resuspended, 100 μL of bacterial solution was added to each well of a 96-well plate, coated overnight at 25°C, and washed 3 times with PBST. 100 μL of 2% BSA was added to each well, blocked at 37°C for 1 hour, and washed 3 times with PBST. The purified Gp17 protein was gradiently diluted (200, 150, 125, 100, 75, 50, 30, 20, 10, 5, 2.5, 1 μg / mL), 100 μL was added to each well, the negative control was 1×PBS, each gradient was repeated 2 wells, incubated at room temperature for 1 hour, and washed 3 times with PBST. 100 μL of anti-His monoclonal antibody-HRP (1:5000) was added to each well, incubated at 37°C for 1 hour, and washed 3 times with PBST. Add 100 μL of colorimetric reagent to each well, incubate at 37°C for 10 min, add 50 μL of stop solution to each well, and read the OD 450 The negative control average was calculated, and the equilibrium dissociation constant (Kd) value was calculated after subtracting the negative control value from the experimental group value, that is, the concentration required to achieve half of the maximum binding at equilibrium. The results showed that the equilibrium dissociation constant (Kd) value was 44.38±5.34μg / mL, that is, 88.76μg / mL of Gp17 protein had the highest affinity for O:3 type Yersinia enterocolitica, such as Figure 5 shown.

[0089] 2.2 Confocal characterization of Gp17 protein binding to Yersinia enterocolitica type O:3

[0090] Take appropriate amount of fresh culture to 10 9 CFU / mL of O:3 type Yersinia enterocolitica was washed and resuspended in sterile 1× PBS, and then mixed and incubated with an appropriate amount of Gp17 protein. The Gp17-bacteria complex was imaged by fluorescence microscopy using a spectral laser confocal microscope FV-1000. Figure 6 As shown, the results showed that Gp17 protein could bind well to the surface of O:3 type Yersinia enterocolitica and was not affected by the N-terminal EGFP fluorescent tag.

[0091] 2.3 ELISA to verify the specificity of Gp17 protein to Yersinia enterocolitica type O:3

[0092] Yersinia enterocolitica O:3, O:8, O:5,27, O:5, O:6,30, Listeria monocytogenes, Vibrio parahaemolyticus and Escherichia coli grown to logarithmic phase were coated on 96-well ELISA plates. Blocked with 2% BSA. 90 μg / mL of Gp17 protein was added to each well for incubation. The negative control was PBS. Each group was repeated 2 wells. Anti-His monoclonal antibody-HRP (1:5000) was added to each well. Color development, termination, and OD reading 450The mean value of negative control was calculated, and the SD was calculated from 3 replicates after subtracting the negative control from the experimental group value. Dunnett's multiple comparison test was performed between O:3 Yersinia enterocolitica and other strains. The results showed that Gp17 protein could specifically bind to O:3 Yersinia enterocolitica, but had no specific binding to other serotypes of Yersinia enterocolitica and other common foodborne pathogens. The difference was extremely significant, such as Figure 7 shown.

[0093] 3. Detect the enrichment effect of Gp17 functionalized magnetic beads on Yersinia enterocolitica type O:3;

[0094] 3.1 Enrichment rate of Yersinia enterocolitica type O:3 by Gp17 functionalized magnetic beads

[0095] Prepare Gp17 functionalized magnetic beads. Take an appropriate amount of carboxyl magnetic beads, wash them with reaction buffer, add activator, and activate them by shaking at room temperature for 15 minutes. Wash the activated carboxyl magnetic beads with coupling solution, add an appropriate amount of Gp17 protein, and couple them by shaking at room temperature for 2 hours. After magnetic separation, take the supernatant to calculate the coupling rate. Add blocking solution to the magnetic bead complex and shake at room temperature for 30 minutes. Wash the magnetic beads with preservation solution and adjust the volume to the working concentration, and store at 4°C.

[0096] Take 1 mg of the prepared Gp17 functionalized magnetic beads and 1 mg of the blocked original carboxyl magnetic beads and add them to 10 3 CFU / mL O:3 type Yersinia enterocolitica suspension was incubated at room temperature for 30 minutes with shaking, and the supernatant and magnetic bead-bacteria complex were obtained after magnetic separation. The original bacterial solution without magnetic beads, the supernatant after adding the original carboxyl magnetic beads, the supernatant after adding Gp17 functionalized magnetic beads, and the Gp17 functionalized magnetic bead-bacteria complex were plated for culture.

[0097] The results showed that the enrichment rate of O:3 Yersinia enterocolitica by Gp17 functionalized magnetic beads at this concentration could reach 87.2%, and the original carboxyl magnetic beads had a small amount of binding with O:3 Yersinia enterocolitica. The supernatant basically did not contain O:3 Yersinia enterocolitica, indicating that Gp17 functionalized magnetic beads could efficiently identify O:3 Yersinia enterocolitica; after the Gp17 functionalized magnetic beads-bacteria complex was cultured, a large number of O:3 Yersinia enterocolitica appeared on the plate, indicating that Gp17 functionalized magnetic beads had no toxicity to O:3 Yersinia enterocolitica, and the O:3 Yersinia enterocolitica in the Gp17 functionalized magnetic beads-bacteria complex was a live bacterium that could be cultured and identified on the plate, such as Figure 8 shown.

[0098] 3.2 SEM Characterization of Yersinia enterocolitica Type O:3 Captured by Gp17-functionalized Magnetic Beads

[0099] The O:3 type Yersinia enterocolitica, Gp17 functionalized magnetic beads, and Gp17 functionalized magnetic beads-O:3 type Yersinia enterocolitica complex were fixed on the cell slides, and the negative control was a mixture of the original carboxyl magnetic beads and O:3 type Yersinia enterocolitica. After dehydration, freeze drying and gold spraying, SEM scanning electron microscopy was performed. The width of Yersinia enterocolitica is 0.5-0.8μm, the length is 1-3μm (such as Fig. 9 The diameter of the magnetic beads is 2 μm (as shown in a in Fig. 9 b), Gp17-functionalized magnetic beads-O:3-type Yersinia enterocolitica complex (as shown in Fig. 9 c), negative control as Fig. 9 As shown in d, the original carboxyl magnetic beads have a small amount of binding to O:3 type Yersinia enterocolitica, the Gp17 functionalized magnetic beads can bind to the surface of O:3 type Yersinia enterocolitica, and a single magnetic bead can bind to multiple target bacteria.

[0100] 4. Verify the specificity of Gp17-functionalized magnetic beads for Yersinia enterocolitica type O:3;

[0101] Take 10 3 CFU / mL of O:3, O:8, O:5,27, O:5, O:6,30 types of Yersinia enterocolitica, as well as Listeria monocytogenes, Vibrio parahaemolyticus, and Escherichia coli were mixed in equal proportions. Take 1 mg of the prepared Gp17 functionalized magnetic beads, add it to the mixed bacterial suspension, incubate it at room temperature with shaking for 30 minutes, and obtain the supernatant and magnetic bead-bacteria complex after magnetic separation, and spread them on plates for culture. Pick 4-6 single colonies on the magnetic bead complex plate for PCR verification of serotype. The results showed that the number of colonies on the supernatant plate was less than that on the original bacterial solution plate, and there were a large number of colonies with consistent morphology on the magnetic bead complex plate, and the PCR results of multiple single colonies picked were all O:3 type Yersinia enterocolitica, indicating that Gp17 functionalized magnetic beads can accurately identify and enrich O:3 type Yersinia enterocolitica. Fig.10 As shown, it is consistent with the ELISA results.

[0102] 5. Verify the sensitivity of Gp17-functionalized magnetic beads in capturing Yersinia enterocolitica type O:3;

[0103] The O:3 type Yersinia enterocolitica was diluted in series and PCR was performed at each concentration. 1 mg of the prepared Gp17 functionalized magnetic beads and 1 mg of the blocked original carboxyl magnetic beads were added to 10 2 , 10 1 , 10 0The suspension of Yersinia enterocolitica type O:3 at a CFU / mL was incubated at room temperature with shaking for 30 minutes. After magnetic separation, the supernatant and Gp17 functionalized magnetic beads-bacteria complex were obtained and plate-spread cultured respectively.

[0104] The results showed that conventional PCR could detect 10 5 CFU / mL O:3 type Yersinia enterocolitica (such as Fig.11 In contrast, Gp17 functionalized magnetic beads can enrich and detect as low as 10 0 CFU / mL of O:3 type Yersinia enterocolitica. 2 CFU / mL, only a small amount of strains appeared in the supernatant plate after adding Gp17 functionalized magnetic beads, and the capture rate of Gp17 functionalized magnetic beads for O:3 type Yersinia enterocolitica was still more than 80% (e.g. Fig.12 As shown). 1 , 10 0 CFU / mL, no strains appeared in the supernatant plate after adding Gp17 functionalized magnetic beads, indicating that all strains were captured, that is, Gp17 functionalized magnetic beads can still enrich and detect O:3 type enterocolitica Yersinia (such as Fig.13 and Fig.14 shown).

[0105] 6. Testing of spiked samples;

[0106] Take 1g of fresh pork and seafood respectively, add 9mL of sterile 1×PBS and grind into homogenate, irradiate with ultraviolet light for 30min to sterilize, centrifuge at 10000rpm for 5min, take the supernatant, and filter with 0.22μm filter membrane to sterilize. Add O:3 type enterocolitica to the filtrate to a final concentration of 10 1 , 10 0 CFU / mL is the spiked sample. Take 1 mg of the prepared Gp17 functionalized magnetic beads and add them to the spiked sample. Incubate at room temperature for 30 min with shaking. After magnetic separation, the supernatant and magnetic bead-bacteria complex are obtained and plated for culture.

[0107] The results show that in 10 1 CFU / mL of spiked pork (such as Figure 5 shown) and seafood (as Fig.16There are a small number of strains in the sample (as shown in the figure), and no strains appear in the supernatant after adding Gp17 functionalized magnetic beads, indicating that the strains in the sample are all captured by Gp17 functionalized magnetic beads, and Gp17 functionalized magnetic beads still have good bacterial capture ability in real samples; more strains appear in the plate after enrichment and re-culture with Gp17 functionalized magnetic beads, indicating that Gp17 functionalized magnetic beads have no toxicity to O:3 type Yersinia enterocolitica, and the bacteria in the Gp17 functionalized magnetic beads-bacteria complex are live bacteria and can be cultured and identified on plates; the number of bacteria in the enriched and re-cultured plates is significantly greater than that in the original bacterial solution, indicating that Gp17 functionalized magnetic beads have the ability to enrich bacteria from low-abundance samples.

[0108] Therefore, based on the specific recognition ability of Gp17 protein for O:3 type Yersinia enterocolitica, the present application expressed and purified Gp17, evaluated its affinity and specificity by ELISA, and performed confocal characterization, and found that Gp17 had high affinity and good specificity for O:3 type Yersinia enterocolitica; Gp17 was coupled with carboxyl-modified magnetic nanoparticles with a diameter of 2 μm to prepare Gp17 functionalized magnetic beads, and the separation effect of the magnetic field was used to quickly and accurately enrich the target bacteria, with an enrichment rate of up to 87.2%, good specificity and high sensitivity, and could reach 10 0 CFU / mL, fresh pork and seafood were spiked to 10 1 , 10 0 CFU / mL, and found that Gp17 functionalized magnetic beads can enrich O:3 type Yersinia enterocolitica from low-abundance samples. Compared with existing immunomagnetic beads, Gp17 functionalized magnetic beads have higher sensitivity and better specificity, can achieve the enrichment of specific serotypes, have low-abundance enrichment capabilities, and Gp17 protein can be mass-produced in vitro using prokaryotic expression technology, which is easy to obtain, reduces costs, and has small batch-to-batch differences.

[0109] It should be noted that in the present invention, it should be understood that although this specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that those skilled in the art can understand.

Claims

1. A method for enriching Yersinia enterocolitica type O:3 using Gp17 magnetic beads, characterized in that: The following steps are involved: Step 1, preparing Gp17 protein: expressing and purifying Gp17 protein through a prokaryotic expression system, wherein the Gp17 protein is derived from a short-tailed bacteriophage and has the ability to specifically bind to O:3 type Yersinia enterocolitica; Step 2, preparing Gp17 functionalized magnetic beads: coupling the Gp17 protein purified in step 1 with carboxyl-modified magnetic nanoparticles to prepare Gp17 functionalized magnetic beads; Step 3, enriching Yersinia enterocolitica type O:3: mixing the Gp17 functionalized magnetic beads prepared in step 2 with the sample to be tested, and separating by magnetic field to enrich Yersinia enterocolitica type O:3; Step 4, detection: culture or molecular biological detection of the enriched O:3 type Yersinia enterocolitica.

2. The method for enriching Yersinia enterocolitica type O:3 using Gp17 magnetic beads according to claim 1, characterized in that: In step 1, the method for preparing the Gp17 protein comprises the following steps: S1. Determine the primary, secondary and tertiary structures of Gp17 protein by bioinformatics analysis; S2, constructing a recombinant expression vector containing the Gp17 gene, and transforming it into a prokaryotic expression host for expression; S3. Purify Gp17 protein by affinity chromatography.

3. The method for enriching Yersinia enterocolitica type O:3 using Gp17 magnetic beads according to claim 2, characterized in that: In step S2, the prokaryotic expression host is Escherichia coli BL21, the recombinant expression vector is pET32a, and the N-terminus of the Gp17 protein carries a His tag.

4. The method for enriching Yersinia enterocolitica type O:3 using Gp17 magnetic beads according to claim 1, characterized in that: In step 2, the method for preparing the Gp17 functionalized magnetic beads comprises the following steps: A1, activating the carboxyl-modified magnetic nanoparticles with an activator; A2, mixing the activated magnetic nanoparticles with the Gp17 protein in a coupling buffer, and coupling at room temperature with shaking for 2 hours; A3. Remove the uncoupled Gp17 protein by magnetic separation, and block the unreacted active sites on the surface of the magnetic beads with blocking solution.

5. The method for enriching Yersinia enterocolitica type O:3 using Gp17 magnetic beads according to claim 1, characterized in that: In step 3, the sample to be tested includes a food sample, an environmental sample or a clinical sample, and the food sample is pork, seafood or dairy products.

6. The method for enriching Yersinia enterocolitica type O:3 using Gp17 magnetic beads according to claim 1, characterized in that: In step 3, the enrichment rate of the Gp17 functionalized magnetic beads for type O:3 Yersinia enterocolitica is greater than 80%.

7. The method for enriching Yersinia enterocolitica type O:3 using Gp17 magnetic beads according to claim 1, characterized in that: In step 3, the Gp17 functionalized magnetic beads are capable of enriching Yersinia enterocolitica type O:3 from a sample with a concentration of 1 CFU / mL.

8. The method for enriching Yersinia enterocolitica type O:3 using Gp17 magnetic beads according to claim 1, characterized in that: In step 4, the detection method includes plate culture method, PCR method or fluorescence microscope observation method.

9. The method for enriching Yersinia enterocolitica type O:3 using Gp17 magnetic beads according to claim 4, characterized in that: In step 2, the Gp17 protein of the Gp17 functionalized magnetic beads can specifically bind to the O antigen polysaccharide of Yersinia enterocolitica type O:3, and does not bind to the surface antigens of Yersinia enterocolitica type O:8, O:5 and O:9 and other common foodborne pathogens.

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