An immunogen, a method for detecting cronobacter based on specific proteins and application

Abstract

The application discloses an immunogen, a method for detecting Cronobacter based on specific proteins, and application, and the amino acid sequence of the immunogen is SEQ ID NO. 1. The application obtains specific proteins that can represent Cronobacter based on screening and verification, and the specific proteins are recombinantly expressed through a prokaryotic system and used as immunogens to prepare Cronobacter monoclonal antibodies based on a hybridoma technology. A colloidal gold rapid detection method for Cronobacter is established by using the prepared monoclonal antibodies. The detection method solves the problems of high detection cost, limited detection environment and complex operation in the current Cronobacter detection method, the test strip can rapidly and simply detect Cronobacter and be applied to actual samples, and the rapid detection of Cronobacter has important significance.

Description

Technical Field

[0001] This invention belongs to the field of bacterial detection technology, and in particular to an immunogen, a method for detecting Cronobacterium based on specific proteins, and its application. Background Technology

[0002] Cronobacter is a Gram-negative, facultative anaerobic foodborne pathogen that can multiply in the intestines of humans and animals. Its contamination with reconstituted infant formula has led to widespread concern regarding infant meningitis, sepsis, and enterocolitis. Cronobacter can also cause bacteremia and osteomyelitis in the elderly and immunocompromised adults. *Cronobacter sakazakii* and *Cronobacter malonaticus* within the genus are highly pathogenic, while other bacteria in the genus are clinically insignificant. The national food safety standard GB 4789.40-2016, "Microbiological Examination of Food - Examination of Cronobacter sakazakii," stipulates that *Cronobacter* must not be detected in infant formula, milk and dairy products, or their raw materials.

[0003] Traditional physicochemical methods are cumbersome, prone to errors and omissions, and insensitive, failing to achieve accurate quantitative results and meet the requirements for rapid detection. While various methods based on molecular biology principles, such as PCR, multiplex PCR, quantitative real-time PCR, and loop-mediated isothermal amplification, have overcome the sensitivity and time limitations of traditional culture methods and effectively solved the problem of missed bacterial detection, they generally suffer from limitations in the detection environment and high detection costs.

[0004] Colloidal gold chromatography, due to its simplicity and speed, has been developed into various diagnostic kits and is widely used for detecting bacteria and viruses. Based on immunological principles, antibodies are labeled onto colloidal gold, and paired antibodies are immobilized to a matrix. The color reaction induced by antigen-antibody binding on the colloidal gold is then used to determine the result.

[0005] A search revealed no patent publications related to this invention's patent application. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an immunogen, a method for detecting Cronobacter based on specific proteins, and its application.

[0007] The technical solution adopted by this invention to solve its technical problem is:

[0008] An immunogen for preparing a monoclonal antibody that can specifically bind to Cronobacter, wherein the amino acid sequence of the immunogen is SEQ ID NO.1.

[0009] The preparation steps of the immunogen described above are as follows:

[0010] Based on the screening and combination of specific fragments in the long-chain fatty acid outer membrane transporter protein and outer membrane protein X, which are specific genes of Cronobacter genus, the final sequence obtained is the specific gene sequence of Cronobacter genus; a vector is constructed and the target protein is recombinantly expressed through a prokaryotic system, and the target protein is purified by affinity chromatography to obtain the final sequence.

[0011] The amino acid sequence of the long-chain fatty acid outer membrane transporter protein is SEQ ID NO.2, and the amino acid sequence of the outer membrane protein X protein is SEQ ID NO.3.

[0012] Monoclonal antibodies were prepared using the immunogen described above.

[0013] The application of monoclonal antibodies as described above in the preparation of detection products for detecting or assisting in the detection of Cronobacter.

[0014] Test strips containing monoclonal antibodies as described above.

[0015] The application of the test strips described above in the detection of Cronobacter pylori when the purpose is not for the diagnosis or treatment of disease.

[0016] A method for detecting Cronobacter using a specific protein based on an immunogen as described above, wherein the method is not intended for the diagnosis or treatment of a disease, and the method utilizes a monoclonal antibody that can specifically bind to Cronobacter for detection, wherein the monoclonal antibody is an immunogen expressed by a target protein in a recombinant vector constructed by screening and combining specific fragments of long-chain fatty acid outer membrane transporter protein and outer membrane protein X protein in Cronobacter.

[0017] Furthermore, the method is based on the double-antibody sandwich method and combined with colloidal gold technology. It uses test strips to detect Cronobacter. The control line of the test strip is coated with goat anti-mouse secondary antibody. The paired antibody combination of the prepared monoclonal antibody is used as the gold-labeled capture antibody on the gold-labeled pad and the detection antibody coated on the detection line, respectively.

[0018] Furthermore, the required colloidal gold solution was prepared using the trisodium citrate reduction method;

[0019] Alternatively, the test strip consists of a sample pad, a nitrocellulose membrane strip, and an absorbent pad;

[0020] Alternatively, the bacterial solution to be tested can be dropped onto the test strip, and the Cronobacterium can be detected based on the color development of the test strip;

[0021] Alternatively, select goat anti-mouse secondary antibody with a detection antibody concentration of 1.0 mg / mL and diluted 8 times.

[0022] A test strip for detecting Cronobacterium using the immunogen described above, the preparation steps of which are as follows:

[0023] Step 1: Based on the screening and combination of specific fragments from the long-chain fatty acid outer membrane transporter protein and outer membrane protein X, which are specific genes in Cronobacter genus, a recombinant vector was constructed. The target protein expressed was an immunogen. Mouse monoclonal antibodies were prepared using the hybridoma principle. The amino acid sequence of the long-chain fatty acid outer membrane transporter protein is SEQ ID NO.2, and the amino acid sequence of the outer membrane protein X is SEQ ID NO.3.

[0024] Step 2: Monoclonal antibodies are labeled onto colloidal gold solution to obtain colloidal gold probes;

[0025] Step 3: Assemble the colloidal gold test strip to prepare the test strip for detecting Cronobacter.

[0026] The advantages and positive effects of this invention are as follows:

[0027] 1. This invention can highly specifically detect Cronobacter spp. strains in food, while failing to identify common foodborne pathogens such as Escherichia coli, Salmonella enteritidis, Listeria monocytogenes, Staphylococcus aureus, and Pseudomonas aeruginosa. This invention solves the problems of high detection costs, limited detection environments, and complex operations in current Cronobacter detection methods; the test strip of this invention can quickly and easily detect Cronobacter and be applied to actual samples.

[0028] 2. This invention is based on screening and verification to obtain proteins specific to the genus *Cronobacter*. These specific proteins are recombinantly expressed using a prokaryotic system and used as immunogens to prepare *Cronobacter* monoclonal antibodies using hybridoma technology. The immunogen sequence is SEQ ID NO.1. Using the prepared monoclonal antibodies, a rapid colloidal gold detection method for *Cronobacter* is established. This invention solves the problems of high detection cost, limited detection environment, and complex operation in current *Cronobacter* detection methods. The test strip of this invention can rapidly and easily detect *Cronobacter* and can be applied to actual samples, which is of great significance for the rapid detection of *Cronobacter*.

[0029] 3. This invention prepares monoclonal antibodies based on immunological detection principles and combines them with colloidal gold technology for the specific detection of Cronobacter. It is applicable to various settings and offers the advantage of ease of operation. It utilizes colloidal gold technology to detect Cronobacter sakazakii and most other strains within the Cronobacter genus.

[0030] 4. The test strip of this invention is an immunochromatographic test strip prepared using mouse monoclonal antibody against a specific protein of *Cronobacter* genus combined with colloidal gold technology. The color intensity of the detection line is positively correlated with the concentration of *Cronobacter* bacterial solution in the sample, and the results can be observed visually within 2 minutes. This invention can detect *Cronobacter sakazakii* and most strains within the *Cronobacter* genus, with a detection limit of 10⁻⁶ for *Cronobacter* bacterial solutions. 6 The cfu / mL concentration has been successfully applied to actual sample testing. The test strip of this invention features short testing time, simple operation, and good specificity. Attached Figure Description

[0031] Figure 1 This is an electrophoresis diagram of recombinant protein expression in this invention; wherein, M is the protein molecular weight standard; 1 is the pre-induction bacterial cell; 2 is the post-induction bacterial cell; 3 is the post-induction bacterial cell supernatant; and 4 is the post-induction bacterial cell precipitate.

[0032] Figure 2 This is a diagram showing the purification of the recombinant protein in this invention; wherein, the purified β protein is detected by SDS-PAGE. M. Protein molecular weight standard; 1. Pre-induction bacterial cells; 2. Post-induction bacterial cells; 3. Post-induction bacterial cell supernatant; 4. Post-induction precipitate; 5. Protein elution buffer; 6-8. Purified β protein;

[0033] Figure 3 This is an electron microscope image of colloidal gold in this invention;

[0034] Figure 4 This is a schematic diagram of the assembly of the test strip in this invention;

[0035] Figure 5 This is a diagram showing the optimization of conditions for the test strip in this invention; wherein, (a) the effect of monoclonal antibody dosage on antibody-modified AuNPs; and (b) the effect of potassium carbonate addition on antibody-modified AuNPs.

[0036] Figure 6 This is a graph showing the detection limit of the test strip in this invention;

[0037] Figure 7 This is a diagram illustrating the specificity determination of the test strips in this invention; wherein, in (a) 1. *Cronobacter sakazakii* ATCC29544; 2. *Escherichia coli*; 3. *Staphylococcus aureus*; 4. *Salmonella enteritidis*; 5. *Listeria monocytogenes*; 6. *Pseudomonas aeruginosa*. In (b) 1. *Cronobacter malondialdehyde* TJCIQsak10607; 2. *Cronobacter zurich* JKY3874; 3. *Cronobacter moginsica* ATCC513294; ​​4. *Cronobacter dublinum* BJCIQ4061; 5. *Cronobacter univarsica* TJCIQsak90308;

[0038] Figure 8This is a diagram showing the actual sample testing of the test strip in this invention. Detailed Implementation

[0039] The embodiments of the present invention are described in detail below. It should be noted that these embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0040] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0041] An immunogen for preparing a monoclonal antibody that can specifically bind to Cronobacter, wherein the amino acid sequence of the immunogen is SEQ ID NO.1.

[0042] The preparation steps of the immunogen described above are as follows:

[0043] Based on the screening and combination of specific fragments in the long-chain fatty acid outer membrane transporter protein and outer membrane protein X, which are specific genes of Cronobacter genus, the final sequence obtained is the specific gene sequence of Cronobacter genus; a vector is constructed and the target protein is recombinantly expressed through a prokaryotic system, and the target protein is purified by affinity chromatography to obtain the final sequence.

[0044] The amino acid sequence of the long-chain fatty acid outer membrane transporter protein is SEQ ID NO.2, and the amino acid sequence of the outer membrane protein X protein is SEQ ID NO.3.

[0045] Monoclonal antibodies were prepared using the immunogen described above.

[0046] The application of monoclonal antibodies as described above in the preparation of detection products for detecting or assisting in the detection of Cronobacter.

[0047] Test strips containing monoclonal antibodies as described above.

[0048] The application of the test strips described above in the detection of Cronobacter pylori when the purpose is not for the diagnosis or treatment of disease.

[0049] A method for detecting Cronobacter using a specific protein based on an immunogen as described above, wherein the method is not intended for the diagnosis or treatment of a disease, and the method utilizes a monoclonal antibody that can specifically bind to Cronobacter for detection. The monoclonal antibody is an immunogen expressed by a recombinant vector constructed by screening and combining a specific gene fragment of a long-chain fatty acid outer membrane transporter protein in Cronobacter and a specific fragment of outer membrane protein X.

[0050] Preferably, the method is based on the double-antibody sandwich method and combined with colloidal gold technology, using test strips to detect Cronobacter. The control line of the test strip is coated with goat anti-mouse secondary antibody. The paired antibody combination of the prepared monoclonal antibody is used as the gold-labeled capture antibody on the gold-labeled pad and the detection antibody coated on the detection line, respectively.

[0051] Preferably, the required colloidal gold solution is prepared by the reduction method of trisodium citrate;

[0052] Alternatively, the test strip consists of a sample pad, a nitrocellulose membrane strip, and an absorbent pad;

[0053] Alternatively, the bacterial solution to be tested can be dropped onto the test strip, and the Cronobacterium can be detected based on the color development of the test strip;

[0054] Alternatively, select goat anti-mouse secondary antibody with a detection antibody concentration of 1.0 mg / mL and diluted 8 times.

[0055] A test strip for detecting Cronobacterium using the immunogen described above, the preparation steps of which are as follows:

[0056] Step 1: Based on the screening and combination of specific fragments from the long-chain fatty acid outer membrane transporter protein and outer membrane protein X, which are specific genes in Cronobacter genus, a recombinant vector was constructed. The target protein expressed was an immunogen. Mouse monoclonal antibodies were prepared using the hybridoma principle. The amino acid sequence of the long-chain fatty acid outer membrane transporter protein is SEQ ID NO.2, and the amino acid sequence of the outer membrane protein X is SEQ ID NO.3.

[0057] Step 2: Monoclonal antibodies are labeled onto colloidal gold solution to obtain colloidal gold probes;

[0058] Step 3: Assemble the colloidal gold test strip to prepare the test strip for detecting Cronobacter.

[0059] Monoclonal antibodies were labeled onto colloidal gold solution to obtain colloidal gold probes.

[0060] Based on the principle of the double-antibody sandwich method, the optimal antibody pairing combination is used as the gold-labeled antibody and the detection antibody, respectively.

[0061] The colloidal gold test strip was assembled, thus preparing the test strip for detecting Cronobacter.

[0062] Specifically, the relevant preparation and detection examples are as follows:

[0063] A recombinant vector was constructed based on the final sequence obtained by screening and combining specific fragments from the long-chain fatty acid outer membrane transporter protein and outer membrane protein X of Cronobacter. The target protein expressed is an immunogen. Mouse monoclonal antibodies were prepared using the hybridoma principle. The amino acid sequence of the immunogen is SEQ ID NO.1.

[0064] Example 1: Preparation method of test strip for detecting Cronobacter pylori

[0065] Follow these steps:

[0066] Step 1: A recombinant vector was constructed based on the final sequence obtained by screening and combining specific fragments from the long-chain fatty acid outer membrane transporter protein and outer membrane protein X of Cronobacter spp. The target protein expressed was an immunogen. Mouse monoclonal antibodies were prepared using the hybridoma principle. The amino acid sequence of the immunogen is SEQ ID NO.1.

[0067] SEQ ID NO.1:

[0068] MetLysLysIleAlaTyrAlaArgAlaLysCysLeuSerAlaLeuIleGluArgTyrAlaAlaCysValLeuAlaGlyAspLeuProGlnValSerAlaGlySerIleIleAlaGlyGlyAlaValA laGlyThrLeuProGlyLeuValAlaThrValThrGlyGlnSerGlyLysIleLeuGlyValIleGlyAspProGlnThrAlaSerPheThrTyrThrAlaGlnLeuGlyAlaGluLysAspArgThr AlaAlaGlyGlyIleGluAspGlyValTyrAsnSerAspThrGlnGlyLysAsnGlnTyrIleAlaHisLeuLysTyrGlyIleThrAlaGlyAspGluTrpGlyGlyProAlaTyrArgPheGlyT rpAsnAlaLeuAsnAspTrpAlaGlyIleLeuTyrGluSerIleTyrGlyValProGluSerIleTyrGlyValProGlunGlnArgValGlyValGlyTyrSerIleSerIleProAspLysAlaGlnAlaThrAsnGlyValAsp

[0069] Step 2: Monoclonal antibodies are labeled onto colloidal gold solution to obtain colloidal gold probes.

[0070] Step 3: Assemble the colloidal gold test strip, thus preparing the test strip for detecting Cronobacter.

[0071] Preferably, the specific implementation method of step one can be as follows:

[0072] The final sequence fragments obtained by screening and combining specific fragments from the long-chain fatty acid outer membrane transporter protein and outer membrane protein X, which are specific genes of Cronobacter, are encoded into corresponding nucleotide sequences. The final sequence is the specific gene sequence of Cronobacter. Recombinant expression of the target protein is then performed using a prokaryotic system. The constructed recombinant plasmid is transformed chemically and cultured. IPTG is used to induce recombinant protein expression under the following conditions: 0.1 mmol / L IPTG, 30℃ for 5 h. Figure 1 The image shows the results of SDS-PAGE electrophoresis. The induced target protein was clearly expressed, with a newly added band at 20 kDa. Affinity chromatography was used to purify the target protein, with metal chelate chromatography being the most widely used and offering high purity in a single step. Details are as follows:

[0073] a. Sonicate the solvent used in the purification process to remove air bubbles.

[0074] b. Add an appropriate amount of Ni-NTAHis Bind to the chromatography column, wash away any residual ethanol with plenty of sterile water, rinse with the prepared Binding Buffer, and equilibrate the chromatography column.

[0075] c. Mix the high-concentration urea protein denaturing solution and the binding solution in equal volumes, and circulate them onto the column at 4°C using a peristaltic pump for two to three hours until they are fully bound to the resin at a speed of 6 rpm.

[0076] d. After thorough mixing, connect the prepared imidazole eluents of different concentrations to the peristaltic pump and collect them in portions in the EP tube.

[0077] e. After washing away excess proteins, elute the target protein with a higher concentration of imidazole elution buffer and collect the proteins separately.

[0078] f. After elution, remove the resin suspension from the chromatography tube, add 20% ethanol, and store in a refrigerator for long-term preservation. Figure 2 As shown, after purification, a single target protein band was observed with high purity and concentration, and a molecular weight of approximately 20 kDa. The target protein was used as an immunogen to immunize mice to prepare Cronobacter monoclonal antibodies. High-titer mouse spleen cells were selected and fused with myeloma cells. Through further culture, screening, and cloning, hybridoma cell lines secreting specific antibodies were obtained. The specific methods are as follows:

[0079] (1) Preparation of SP2 / 0 myeloma cells

[0080] Myeloma cells can proliferate continuously and be passaged indefinitely in vitro. Therefore, by taking advantage of this characteristic, they can be fused with spleen cells that secrete specific antibodies to obtain a large number of monoclonal antibodies with good specificity and high titer.

[0081] Cell resuscitation: SP2 / 0 cells were removed from the liquid nitrogen tank and quickly placed in 42°C warm water to thaw. The cells were gently centrifuged, the supernatant was aspirated, and the cells were resuspended in growth medium and transferred to a culture flask for incubation in a CO2 incubator. Once the cells were in good growth condition and had completely covered the bottom of the culture flask, they were passaged for expansion culture in preparation for cell fusion.

[0082] (2) Anatomy and spleen handling

[0083] Five to seven days before fusion, mice with high titers were selected for sprint immunization. Solvents and equipment required for the experiment were sterilized and prepared for use.

[0084] a. Mice were euthanized by cervical dislocation after blood was collected from their eyeballs, and disinfected for 5 minutes. PEG 4000 and DMEM culture medium were then incubated at 37°C for later use.

[0085] b. Secure the disinfected mouse to a sterile dissection table;

[0086] c. Use clean scissors and tweezers to cut open the skin layer to expose the peritoneum, cut open the peritoneum to expose the abdominal cavity; remove the spleen and transfer it to a disposable petri dish containing 5 ml of DMEM culture medium.

[0087] d. Repeat the washing of the spleen, place it in a cell sieve, and gently grind it with a grinder until the cells pass through the sieve and enter the culture medium. Do not over-grind. Gently mix with a pipette, then transfer to a centrifuge tube and let stand for 10 minutes to allow excess tissue to settle.

[0088] (3) Treatment of spleen cells and SP2 / 0 myeloma cells:

[0089] a. Add 2 mL of fetal bovine serum to a 15 mL centrifuge tube;

[0090] b. After the excess tissue has settled, slowly add the spleen cell suspension to a centrifuge tube containing serum. The cell layer and serum layer are clearly separated. This method can filter mycoplasma from the suspension.

[0091] c. Centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend in 10 mL of cell culture medium, and mix well;

[0092] d. Centrifuge at 1000 rpm for 5 min, discard the supernatant, slowly add 1 mL of erythrocyte lysis buffer, mix well, add 2 mL of fetal bovine serum to stop the reaction, take 8 mL of culture medium, mix well, centrifuge, resuspend, mix well and wait for counting; select myeloma cells in the logarithmic growth phase, blow the tumor cells off the cell culture flask, wash twice, resuspend in an appropriate volume of DMEM culture medium, mix well and wait for counting;

[0093] (4) Cell counting

[0094] 10 μL of each type of cell was aspirated into an EP tube containing 40 μL of DMEM culture medium, diluted five times, and then added to a hemocytometer for observation and counting under a microscope.

[0095] (5) Cell fusion

[0096] After cell counting, spleen cells and tumor cells were mixed at a ratio of 10:1, thoroughly mixed, and centrifuged twice. Residual liquid was blotted away with sterile filter paper, and the bottom of the centrifuge tube was gently tapped to loosen the cell clumps. The fusion temperature was strictly controlled at 37℃. The preheated fusion agent PEG was slowly added dropwise to the cell clumps at the bottom of the tube while gently stirring. After the fusion agent had fully reacted, 15 mL of culture medium was slowly added to terminate the fusion.

[0097] After fusion, centrifuge and discard the supernatant. Gently resuspend the cells in HAT medium and add them to cell culture plates, maintaining a cell count of 2-3 × 10⁶ cells per well. 5 Approximately 250 μL / well.

[0098] On the second day after fusion, observe whether the cells are contaminated. On the third and fourth days, add 50 μL of HT medium every other day, using a gentle touch to avoid blowing away the cell colonies. When the cell colonies in the wells have grown to 1 / 4 of the wells, perform the first cell screening.

[0099] (6) Indirect ELISA screening of positive wells

[0100] The purified specific protein was coated onto an ELISA plate at a concentration of 1 μg / mL. The supernatant culture medium from the observable cell colonies in the wells was used as the primary antibody for incubation. Wells with an OD value of 1.0 or higher were considered positive.

[0101] First screening: Select wells with higher OD values. After repeating the measurement three times, change the medium in each well. Once the cells have grown to a confluence of cells in a 96-well plate, transfer them to a 24-well cell culture plate for further culture.

[0102] Second screening: Select cells with good growth and re-analyze the cell supernatant using an indirect competitive ELISA method to screen for cells with high titers and good specificity. Change the medium and continue to expand the culture. After multiple passages, replace the medium with DMEM complete culture medium for further culture.

[0103] (7) Cell Cloning

[0104] Hybridoma cells after fusion are unstable. In order to remove the influence of multiple hybridoma cell lines, cell cloning must be performed to screen for hybridoma cell lines with strong growth capacity and stable antibody secretion.

[0105] Limiting dilution cell cloning steps:

[0106] a. Select wells with high cell titers, resuspend the cells, count them, dilute them to a concentration of about three cells per well, and seed them into 96-well plates.

[0107] b. Add an appropriate amount of feeder cells to each well to promote the growth of hybridoma cells.

[0108] c. After cloning, observe cell growth and check for contamination. When the cell colonies are large, add HT medium.

[0109] d. When the cells have covered 1 / 10 of the bottom of the culture wells, perform indirect ELISA again. Select cell colonies with high positive values ​​for at least three cloning processes, selecting wells with a single cell colony for cloning each time. This will ultimately yield a cell line that stably secretes antibodies against a single antigenic epitope. Inoculate the expanded hybridoma cells into the peritoneal cavity of mice, collect a large amount of ascites fluid, and purify the antibody.

[0110] The specificity of the monoclonal antibodies was verified by indirect ELISA: the purified antibodies were used to verify the specificity of 5 strains of Cronobacter spp. and 5 other foodborne pathogens outside the spp. The results are shown in Table 1. The two antibodies showed good specificity against strains of Cronobacter spp.

[0111] Table 1. Results of ELISA detection of Cronobacter Specificity.

[0112]

[0113]

[0114] Preferably, the specific implementation method of step two of the present invention can be: synthesizing 20nm colloidal gold, such as... Figure 3 As shown, the optimal combination of paired antibodies is selected, and the monoclonal antibody is labeled on the colloidal gold solution to obtain the colloidal gold probe, which is the capture antibody, and the detection line is coated with the detection antibody.

[0115] The specific method is as follows: This experiment uses the trisodium citrate method to prepare colloidal gold. 100 mL of ultrapure water and the rotor are added together to a clean round-bottom flask. The solution is heated to 100°C and kept boiling for 3 minutes. 1 mL is accurately removed using a pipette, and 1 mL of 1% (w / w) chloroauric acid is added. The mixture is stirred and heated until fully mixed and boiled. To prepare 20 nm colloidal gold particles, 2.25 mL of 1% (w / w) trisodium citrate solution is added. When the solution boils, the trisodium citrate solution is quickly added while maintaining high-speed stirring. A vigorous redox reaction occurs at high temperature, and the solution color changes from light yellow to a stable wine-red and remains stable. The solution is then cooled to room temperature at the original stirring speed and stored at 4°C in the dark for later use. Under alkaline conditions, because the colloidal gold particles have a negative charge, they can undergo non-covalent electrostatic adsorption with the positively charged groups in the target protein, thus stably adhering to the target protein without significantly affecting its biological activity. Antigens or antibodies attached to the surface of colloidal gold can guide colloidal gold microparticles into cells and immobilize antibodies on a solid-phase support. Because of the high electron density of gold particles, these labels accumulate at a certain concentration (i.e., 10⁻⁶) at the antigen-antibody reaction site. 7 pcs / mm 2 When the antibody is applied, visible red dots will appear. Add the antibody to be labeled at the optimal labeling amount of 10 μg to the colloidal gold, adjust the pH to approximately 8 with K2CO3, and then slowly add appropriate amounts of BSA and PEG20000 to block and stabilize the gold-labeled antibody. Centrifuge at 14000 g / min for 30 min at 4℃, remove the supernatant; resuspend the precipitate in the gold-labeled working solution to obtain the labeled antibody, which can be stored at 4℃ for later use.

[0116] Preferably, the specific implementation method of step three of the present invention can be as follows: cut a suitable gold-labeled pad, soak it in the treatment solution for 15 minutes, vacuum dry it for 24 hours, and then place it in a sealed and dry environment for use. Assemble the absorbent pad, gold-labeled pad, nitrocellulose membrane, and PVC board, as follows. Figure 4 As shown. Nitrocellulose membrane processing method: Use a membrane scribing instrument to scribble the goat anti-mouse secondary antibody and the above-mentioned detection antibody onto the nitrocellulose membrane, respectively, to serve as the test line and control line of the test strip, spaced approximately 5 mm apart. After vacuum drying, use. Cut the assembled test strip into 6 mm wide strips and store in a sealed, dry environment. When testing the sample, drop the sample directly onto the sample pad and let it stand for 5 minutes to determine the result. If red bands appear at both the test line and control line, the result is positive; if a red band appears only at the control line, the result is negative; if there is no band at the control line or a band at the T line, the test strip is invalid.

[0117] The first optimization of the detection conditions for the test strip of this invention: In order to improve the stability of the modified gold nanoparticles and achieve optimal detection performance, the pH value and antibody modification amount were optimized, such as... Figure 5 As shown. In this invention, antibodies are mainly modified onto the surface of AuNPs via electrostatic adsorption. Adjusting the pH to near the isoelectric point of the antibody facilitates antibody adsorption onto the AuNPs surface. In this experiment, the pH value of the system was optimized by adding different amounts of K2CO3. The highest absorbance value at OD520 nm of AuNPs was observed when the amount of K2CO3 added was 7 μL, indicating that the amount of K2CO3 added was suitable and beneficial to antibody modification. The amount of antibody affects the labeling effect. Too little antibody will produce more free AuNPs, while too much antibody may cause non-specific adsorption or result in antibody residue that cannot fully exert its function. The highest OD520 nm value was observed when the amount of antibody modified was 10 μg. When the amount of K2CO3 added was 7 μL and the amount of antibody was 10 μg, the AuNPs-mAb was the most stable, and therefore, this was selected as the most suitable modification condition.

[0118] The second optimization of the detection conditions for the test strip of this invention is as follows: the detection limit of the test strip is determined by observing the color change. If the control line shows a distinct color and the test line does not change color, the result is negative. If both the test line and the control line show color changes, the result is positive. If the control line does not show color, the test is invalid. Since the intensity of the control line color has a significant impact on the test results, the concentration of the detection antibody and the dilution factor of the goat anti-mouse secondary antibody have been optimized to make the test strip results more accurate, as shown in Table 2. If the concentration of the detection antibody is the same, increasing the dilution factor of the goat anti-mouse secondary antibody will cause the control line to become lighter in color. Conversely, assuming a consistent dilution factor for the goat anti-mouse secondary antibody, increasing the concentration of the detection antibody will cause the test line to become darker. A large dilution factor for the secondary antibody will result in a colorless control line, making it difficult to accurately interpret the results. Excessively high concentrations of the secondary antibody and detection antibody at the control and test lines will cause them to bind with most of the antibody in the solution, resulting in excessively dark colors and reducing the sensitivity of the detection method. However, when the detection antibody concentration is 1.0 mg / mL and the goat anti-mouse secondary antibody is diluted 8 times, the colors of the control and test lines on the test strip are appropriate and uniform. Therefore, a detection antibody concentration of 1.0 mg / mL and a dilution of 8 times with the goat anti-mouse secondary antibody was chosen for subsequent experiments.

[0119] Table 2 Optimization of antibody coating amount and goat anti-mouse secondary antibody dilution factor.

[0120]

[0121] Note: C represents the control line, T represents the test line, "+" indicates color, and "-" indicates no color.

[0122] Example 2: Determination of the detection limit of the test strip

[0123] Cronobacter sakazakii was inoculated into freshly prepared LB liquid medium and incubated overnight at 37°C. Bacterial counts were performed using the plate count method, followed by 10-fold dilutions to 10⁻⁶. 8 10 7 10 6 10 5 10 4 10 3 cfu / mL, add samples diluted to different concentrations onto the test strip.

[0124] As the concentration of Cronobacter sakazakii bacterial solution increases, the color of the test line gradually darkens, such as... Figure 6 As shown, when the bacterial concentration is 10... 6 At cfu / mL, the detection line showed a clear color reaction, confirming that the detection limit of the Cronobacter clonal antibody colloidal gold immunochromatographic test strip of this invention is 10. 6 cfu / mL.

[0125] Example 3: Determination of the specificity of the test strip

[0126] Common foodborne pathogens and the standard strain ATCC29544 of Cronobacter sakazakii were selected for specificity verification; five strains from different species within the Cronobacter genus were selected for genus coverage determination.

[0127] The above bacterial solutions were added dropwise onto the test strips, and the results are as follows: Figure 7 As shown, it can be seen that, except for *Cronobacter sakazakii*, which can be detected by the test strip, the other common pathogens were not detected, indicating that the invention can specifically identify *Cronobacter*. In the verification of the detection of different species within the *Cronobacter* genus, the test strip showed obvious color reactions, confirming that the invention has good coverage for the *Cronobacter* genus, and most strains of the *Cronobacter* genus can be detected.

[0128] Example 4: Testing of actual samples with test strips. Milk powder was selected as the sample to be tested. 25g of milk powder was weighed and placed in 225mL of LB liquid culture medium. Different concentration gradients of Cronobacter sakazakii bacterial suspension (2.2×10⁻⁶) were added. 0 2.2×10 1 2.2×10 2 and 2.2×10 3 Inoculate the sample with cfu / mL and incubate at 37°C for 0–7 hours. Analyze the sample solution to determine the sensitivity of the test strip in actual sample detection. Analyze the enriched sample every 1 hour to determine the sensitivity of the test strip in actual sample detection. Figure 8 As shown, after 8 hours of enrichment, the T line showed a more obvious color, indicating that the detection limit for Cronobacter sakazakii in the tested milk powder was 2.2 × 10⁻⁶.1 cfu / mL.

[0129] The sequences used in this invention are as follows:

[0130] The amino acid sequence of the immunogen is SEQ ID NO.1:

[0131] MetLysLysIleAlaTyrAlaArgAlaLysCysLeuSerAlaLeuIleGluArgTyrAlaAlaCysValLeuAlaGlyAspLeuProGlnValSerAlaGlySerIleIleAlaGlyGlyAlaValA laGlyThrLeuProGlyLeuValAlaThrValThrGlyGlnSerGlyLysIleLeuGlyValIleGlyAspProGlnThrAlaSerPheThrTyrThrAlaGlnLeuGlyAlaGluLysAspArgThr AlaAlaGlyGlyIleGluAspGlyValTyrAsnSerAspThrGlnGlyLysAsnGlnTyrIleAlaHisLeuLysTyrGlyIleThrAlaGlyAspGluTrpGlyGlyProAlaTyrArgPheGlyT rpAsnAlaLeuAsnAspTrpAlaGlyIleLeuTyrGluSerIleTyrGlyValProGluSerIleTyrGlyValProGlunGlnArgValGlyValGlyTyrSerIleSerIleProAspLysAlaGlnAlaThrAsnGlyValAsp

[0132] The amino acid sequence of the long-chain fatty acid outer membrane transporter protein is SEQ ID NO.2:

[0133]

[0134] The amino acid sequence of outer membrane protein X is SEQ ID NO.3:

[0135] MetLysLysIleAlaCysLeuSerAlaLeuAlaCysValLeuAlaValSerAlaGlySerAlaValAlaGlyThrAlaThrValThrGlyGlyTyrAlaGlnSerAspAlaGlnGlyValMetAsnL ysMetAsnGlyPheAsnLeuLysTyrArgTyrGluPheAspAspThrAsnProLeuGlyValIleGlySerPheThrTyrThrGluLysAspArgThrGluAspGlyValTyrGlyLysAsnGlnTyr TyrGlyIleThrAlaGlyProAlaTyrArgLeuAsnAspTrpAlaSerIleTyrGlyValValGlyValGlyTyrAspLysAlaGlnAlaThrAsnGlyValAspLysAlaAspThrSerAspTyrG lyPheSerTyrGlyAlaGlyLeuGlnPheAsnProValGlnAspValAlaLeuAspPheSerTyrGluGlnSerArgValArgAsnThrAspIleGlyThrTrpIleAlaGlyValGlyTyrArgPhe

[0136] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. An immunogen for preparing monoclonal antibodies that specifically bind to Cronobacter, characterized in that: The amino acid sequence of the immunogen is SEQ ID NO.

1.

2. The method for preparing the immunogen as described in claim 1, characterized in that: The preparation steps are as follows: Based on the screening and combination of specific fragments in the long-chain fatty acid outer membrane transporter protein and outer membrane protein X, which are specific genes of Cronobacter genus, the final sequence obtained is the specific gene sequence of Cronobacter genus; a vector is constructed and the target protein is recombinantly expressed through a prokaryotic system, and the target protein is purified by affinity chromatography to obtain the final sequence. The amino acid sequence of the long-chain fatty acid outer membrane transporter protein is SEQ ID NO.2, and the amino acid sequence of the outer membrane protein X protein is SEQ ID NO.3.

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