Method for detecting campylobacter jejuni cell swelling toxin CdtB in food

Abstract

The invention discloses a method for detecting campylobacter jejuni cell swelling toxin CdtB in food, and provides application of a campylobacter jejuni cell swelling lethal toxin CdtB monoclonal antibody to detection of campylobacter jejuni in food for the first time. The invention aims to establish the detection kit and the detection method based on the campylobacter jejuni cell swelling toxin CdtB monoclonal antibody, and the detection kit and the detection method have relatively strong specificity and sensitivity. The CdtB monoclonal antibody prepared by the invention can be combined with CdtB protein secreted by campylobacter jejuni in food, so that the detection of the campylobacter jejuni is realized. The detection speed is high, semi-quantitative detection can be achieved, the detection sensitivity is high, and the lowest CdtB mass of 0.01 microgram can be detected.

Description

Technical Field

[0001] This invention belongs to the fields of monoclonal antibody technology and microbial detection technology, specifically relating to a method for detecting Campylobacter jejuni cell swelling toxin CdtB in food. Background Technology

[0002] Campylobacter jejuni is an important foodborne zoonotic pathogen worldwide. Infection can cause gastrointestinal diseases such as diarrhea and bloody stools, and in more serious cases, it can lead to autoimmune diseases such as Guillain-Barré syndrome. Its pathogenic mechanism is not yet clear.

[0003] The virulence factors identified in Campylobacter jejuni include flagellin, adhesion-associated proteins, type VI secretion system, and cytotoxic edematoxin. CDT, an important exocrine protein, is a bacterial genotoxin that interferes with eukaryotic cell cycle progression by damaging cellular DNA, leading to G2 / M arrest and apoptosis. The cytotoxic edematoxin of Campylobacter jejuni is composed of three genes: cdtA, cdtB, and cdtC. CdtB, as the functional active unit of the whole toxin, possesses toxic effects. As an exotoxin, it is difficult to extract, and currently there is no detection method to detect the presence of CdtB protein residues in the food supply chain. Summary of the Invention

[0004] Objective of the Invention: Addressing the shortcomings of existing technologies, this invention provides the application of a monoclonal antibody against Campylobacter jejuni cell-induced edema toxin CdtB protein in the preparation of reagents for detecting CdtB in samples from various food industry chains. The purpose of this invention is to establish a detection kit and method based on a monoclonal antibody against Campylobacter jejuni cell-induced edema toxin CdtB, exhibiting high specificity and sensitivity, suitable for immunological detection. This invention utilizes a specific monoclonal antibody to detect residual CdtB protein in treated food samples, achieving a detection method currently lacking for CdtB in target samples from the food industry chain.

[0005] This invention also provides an application of a method for detecting Campylobacter jejuni cell swelling toxin CdtB in food industry samples based on this monoclonal antibody, providing a foundation for the detection of Campylobacter jejuni.

[0006] Technical solution: In order to achieve the above objectives, the monoclonal antibody against Campylobacter jejuni cell swelling lethal toxin CdtB protein described in this invention is used in the preparation of reagents for detecting Campylobacter jejuni cell swelling lethal toxin CdtB in different food samples. The Campylobacter jejuni CdtB protein monoclonal antibody is a monoclonal antibody CdtB monoclonal antibody secreted by monoclonal hybridoma cells 1F5.

[0007] The monoclonal antibody against Campylobacter jejuni cell swelling lethal toxin CdtB protein is mouse IgG2b subtype.

[0008] The monoclonal antibody against Campylobacter jejuni cell swelling lethal toxin CdtB protein specifically binds to Campylobacter jejuni CdtB protein; the amino acid sequence of Campylobacter jejuni CdtB protein is shown in SEQ ID NO.1.

[0009] Furthermore, the Campylobacter jejuni cell swelling toxin CdtB is Campylobacter jejuni cell swelling toxin CdtB from a sample in the fresh food production and processing industry chain.

[0010] Furthermore, the Campylobacter jejuni cell swelling toxin CdtB is Campylobacter jejuni cell swelling toxin CdtB from fresh poultry meat production and processing industry chain samples.

[0011] The kit for detecting Campylobacter jejuni infection and CdtB protein residues according to the present invention includes a monoclonal antibody against Campylobacter jejuni cell swelling lethal toxin CdtB protein, CdtB standard protein, blocking solution, HRP-labeled secondary antibody, chromogenic solution, and other components of the antibody kit.

[0012] The present invention provides a method for detecting Campylobacter jejuni cell swelling toxin CdtB based on the aforementioned Campylobacter jejuni CdtB protein monoclonal antibody, comprising the following steps:

[0013] Western blotting was used for detection: protein samples were added and transferred after electrophoresis, followed by blocking; after washing the membrane, monoclonal antibody against Campylobacter jejuni cell swelling lethal toxin CdtB protein was added and incubated overnight at 4°C; the antibody was recovered the next day, and after washing the membrane, HRP-labeled goat anti-mouse antibody was added and incubated at room temperature; after washing the membrane, the results were observed by exposure using an imaging system.

[0014] Furthermore, the method for detecting Campylobacter jejuni cell swelling toxin CdtB can be an in vitro detection method.

[0015] This process involves obtaining samples from the food industry chain, processing and testing them to determine if Campylobacter jejuni infection is present in the food samples, and extracting residual CdtB protein from positive samples for Western blotting detection.

[0016] Furthermore, the processing method for the food industry chain samples is as follows: take fresh food samples, slightly crush them, place them on ice, add lysis working solution, grind and crush them, place them in an ice bath for full lysis, centrifuge to collect the supernatant, add sample loading buffer, and after metal bath, the protein samples obtained are used for Western blot experiments.

[0017] Preferably, the detection method is Western blotting; the detection method includes the obtained food sample and the Campylobacter jejuni CdtB protein monoclonal antibody.

[0018] Preferably, the protein included in the detection line detection method is the CdtB protein naturally secreted by Campylobacter vacuoles and expressed in prokaryotes.

[0019] This invention enables the detection of CdtB secretion in samples infected with Campylobacter jejuni by processing samples from the food industry chain, as well as the detection of CdtB secretion in samples from different industry chains.

[0020] This invention provides a monoclonal antibody against Campylobacter jejuni cell swelling lethal toxin CdtB protein. The monoclonal antibody against Campylobacter jejuni cell swelling lethal toxin CdtB protein is derived from hybridoma cells 1F5 of CdtB in the literature: Preparation of monoclonal antibodies against prokaryotic expression of Campylobacter jejuni cdtB and cdtC and their products.

[0021] In this invention, the monoclonal antibody subtype determination shows that the Campylobacter jejuni cell swelling lethal toxin CdtB protein monoclonal antibody is mouse IgG2b subtype. The Campylobacter jejuni cell swelling lethal toxin CdtB protein monoclonal antibody specifically binds to Campylobacter jejuni CdtB protein. The Campylobacter jejuni CdtB protein is selected from the amino acid sequence of the Campylobacter jejuni CdtB region from amino acid position 19 to 268 as shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2.

[0022] In this invention, the Campylobacter jejuni CdtB protein, as a reactant, can specifically react with the Campylobacter jejuni cell-induced lethal toxin CdtB protein monoclonal antibody, indicating that the Campylobacter jejuni cell-induced lethal toxin CdtB protein monoclonal antibody can specifically recognize the specific antigenic epitope of Campylobacter jejuni CdtB protein. Therefore, providing the Campylobacter jejuni cell-induced lethal toxin CdtB protein monoclonal antibody provides a basis for the application of detecting Campylobacter jejuni CdtB protein.

[0023] This invention provides an application of a method for detecting Campylobacter jejuni cell swelling toxin CdtB in food samples based on this monoclonal antibody.

[0024] In this invention, the preferred immunological detection method is Western blotting. This invention does not impose any special restrictions on the method of use; any Western blotting detection procedure well-known in the art can be employed.

[0025] In this invention, when the detection method is preferably Western blotting, the detection method includes the obtained food industry sample and the Campylobacter jejuni CdtB protein monoclonal antibody. This invention does not impose any special limitations on other components of the detection method; reagents well-known in the art can be used, such as blocking buffer, washing buffer, antibody diluent, and chromogenic solution.

[0026] In this invention, Western blotting was used to verify the specificity of the monoclonal antibody against Campylobacter jejuni cell-induced lethal toxin CdtB protein. The results showed that the monoclonal antibody against Campylobacter jejuni cell-induced lethal toxin CdtB protein could specifically recognize Campylobacter jejuni CdtB protein. Therefore, the monoclonal antibody against Campylobacter jejuni cell-induced lethal toxin CdtB protein can detect CdtB protein by processing protein samples obtained from food industry chain samples, thereby achieving the detection purpose.

[0027] In this invention, the protein included in the detection limit detection method is the CdtB protein naturally secreted by Campylobacter jejuni and expressed in prokaryotes.

[0028] This invention provides a method for detecting Campylobacter jejuni in food industry chain samples. In this invention, the naturally secreted CdtB protein sample obtained from the food industry chain sample is obtained.

[0029] In this invention, a monoclonal antibody against the edematous lethal toxin CdtB protein of Campylobacter jejuni specifically binds to CdtB, followed by HRP-labeled secondary antibody and chromogenic reaction, thus achieving visualized protein detection. The antibody and detection method of this invention were used to detect Campylobacter jejuni in vitro with a CFU level of 10-1. 9 This detection method can only detect CdtB protein expressed by Campylobacter jejuni when the concentration of CdtB protein purified from prokaryotic expression reaches 0.01 μg. The detection limit of this method for CdtB protein obtained from different samples has been clarified, thus providing a method for detecting Campylobacter jejuni in samples from the food industry chain, and for detecting the presence of Campylobacter jejuni infection and CdtB protein residues.

[0030] This invention employs a specific method for processing samples from the food industry chain. After extracting the protein, CdtB protein can be detected, achieving in vitro detection. Currently, there is no other method to detect residual CdtB protein in food industry chain samples.

[0031] The specific tissue sample processing method of this invention is as follows: protein extraction, transfer to a membrane, blocking with 5% BSA at room temperature for two hours, diluting the Campylobacter jejuni cell swelling lethal toxin CdtB protein monoclonal antibody with 5% BSA at a ratio of 1:1000, incubating overnight at 4°C with shaking, washing with PBST five times with shaking for 5 minutes each time, diluting HRP-labeled goat anti-mouse secondary antibody with 5% BSA at a ratio of 1:5000, incubating at room temperature with shaking for 1 hour, washing with PBST seven times with shaking for 5 minutes each time, selecting a chromogenic solution, uniformly mixing chromogenic solutions A and B in a 1:1 ratio, incubating the NC membrane in the dark for 1 minute, and then photographing and storing. This invention detected Campylobacter jejuni reaching a CFU of 10 in vitro. 9 The detection method can detect CdtB protein expressed by Campylobacter jejuni, and the detection method can detect the presence of CdtB protein when the amount of CdtB protein purified by prokaryotic expression reaches 0.01 μg.

[0032] This invention is the first to achieve specific detection of the active subunit CdtB of Campylobacter jejuni toxin CDT.

[0033] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0034] This invention proposes the application of a monoclonal antibody against Campylobacter jejuni cell swelling lethal toxin CdtB protein in the preparation of reagents for detecting Campylobacter jejuni cell swelling lethal toxin CdtB in samples from the food industry chain. This method enables the detection of Campylobacter jejuni cell swelling toxin CdtB in samples from various food industry chains. The invention offers several advantages: rapid detection speed (results within 20 hours of sample acquisition); semi-quantitative detection capability (allowing for semi-quantitative analysis of the analyte); high sensitivity (detecting as low as 0.01 μg of CdtB); and wide applicability (different samples can be detected using this method after simple pretreatment). Attached Figure Description

[0035] Figure 1 Western blot analysis of CdtB monoclonal antibody.

[0036] M: 180kDaPrestainedProtein Marker; 1: BL21(DE3)(pET-CdtB)whole protein; 2: BL21(DE3)(pET)whole protein;

[0037] Figure 2 This study describes a Western blot analysis based on the detection limit of Campylobacter jejuni CFU.

[0038] M: 180kDa PrestainedProtein Marker; 1: CFU=101 2: CFU = 10 2 3:CFU=10 3 ;

[0039] 4:CFU=10 4 ;5:CFU=10 5 6:CFU=10 6 7: CFU = 10 7 ;8:CFU=10 8 ;9:CFU=10 9 ;

[0040] Figure 3 This study describes a Western blot analysis based on the concentration gradient of purified CdtB protein to detect the limit of protein concentration.

[0041] M: 180kDaPrestainedProtein Marker; 1: 10μg r-CdtB; 2: 5μg r-CdtB; 3: 2.5μgr-CdtB; 4: 1μg r-CdtB; 5: 0.5μg r-CdtB; 6: 0.25μg r-CdtB; 7: 0.1μg r-CdtB; 8: 0.05μg r-CdtB; 9: 0.01μg r-CdtB; 10: 0.005μg r-CdtB; 11: 0.001μg r-CdtB;

[0042] Figure 4 Western blot analysis of proteins extracted from samples from the food industry chain.

[0043] M: 180kDaPrestainedProtein Marker; 1: T-4-5; 2: S51-3-5; 3: S52-3-3; Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0045] The materials and their sources used in the specific embodiments of this invention are as follows:

[0046] 1. Strains and plasmids

[0047] The NCTC11168 and pGEX-6p-1 vectors were provided by Yangzhou University (Preparation of monoclonal antibodies against prokaryotic expression of Campylobacter jejuni cdtB and cdtC and their products, Yangzhou University, 2013), and pET-30a was purchased from Dalian Takara Bio Co., Ltd.

[0048] 2. Main reagents

[0049] Restriction endonucleases BamHI and XhoI, Primestar max DNAPolymerase, and gel extraction kits were purchased from Takara Bio Inc. (Dalian). One-step ligase C115, protein marker, and 5× Loading Buffer were purchased from Novizan Biotechnology Co., Ltd. Bacterial genome extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd. Isopropyl β-D-thiogalactoside (IPTG), kanamycin (Kan), and LB medium were purchased from Sangon Biotech (Shanghai) Co., Ltd. HRP-goat anti-mouse IgG, DMSO, and mouse monoclonal antibody subclass kits were purchased from Sigma-Aldrich. All other reagents were domestically produced analytical grade.

[0050] 3. Main instruments and equipment

[0051] PCR instrument (BIO-RAD), gel imaging analysis system (BIO-RAD), water bath (Shanghai Jinghong Equipment Co., Ltd.), centrifuge (Eppendorf), Bio-Rad semi-Dry transfer cell (BIO-RAD), inverted fluorescence microscope and inverted microscope (Leica), CO2 incubator (Thermo), microplate reader (BIO-TEK), benchtop refrigerated centrifuge (Eppendorf), SDS-PAGE electrophoresis system and transfer equipment (BIO-RAD); ELISA strips and cell culture plates (JETBIOFIL).

[0052] Example 1

[0053] The monoclonal antibody against Campylobacter jejuni cell-induced swelling toxin CdtB protein in this embodiment was obtained from the secretion of hybridoma cells 1F5, as reported in existing literature, and was provided by Yangzhou University. For details, please refer to the following papers: Preparation of Monoclonal Antibodies for Prokaryotic Expression of Campylobacter jejuni cdtB and cdtC and Their Products, Yangzhou University, 2013; or: Research Progress on Campylobacter jejuni Cell-Induced Swelling Toxin, Chinese Journal of Zoonoses, 2012, 28(10):1048-1050. Its Western blot analysis is as follows: Figure 1 As shown.

[0054] Example 2

[0055] Western Blot detection method detection line

[0056] Resuscitate Campylobacter jejuni ATCC81-176, stored at -70℃, and grow on CCDA agar plates for 20 h. Then, subculture the plates completely onto MH blood agar plates and incubate at 42℃ under microaerophilic conditions for 18 h. Harvest the bacteria and inoculate into 10 mL of MH medium, starting at an OD concentration of [missing value]. 600The culture medium was incubated at 0.1°C, 42°C, and 150 rpm under microaerophilic conditions until the bacterial count in the liquid culture medium reached 1 × 10⁻⁶ cells / mL. 9 CFU / mL. Take 1 mL of bacterial suspension and perform serial dilutions to harvest bacterial cells of different concentrations. Centrifuge at 12000 rpm for 1 min at 4℃ to harvest the bacterial precipitate and culture medium supernatant. Sonicate the bacterial precipitate under ice bath conditions and centrifuge at 8000 rpm for 10 min at 4℃ to obtain the lysis supernatant. Take 40 μL of the supernatant and add 10 μL of loading buffer. Incubate at 100℃ for 10 min in a metal bath and temporarily store at -20℃ for Western blot experiments.

[0057] The CdtB protein of Campylobacter jejuni expressed in prokaryotic cells was used as the original sample at a concentration of 1.1 mg / mL. It was then serially diluted at concentrations of 0.5 mg / mL, 0.25 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, and 0.01 mg / mL to obtain protein samples. 40 μL of the diluted protein was added to 10 μL of loading buffer and incubated in a metal bath at 100 °C for 10 min. The samples were then temporarily stored at -20 °C for Western blot experiments.

[0058] The specific Western blot experimental procedure was as follows: protein was extracted, transferred to a membrane, and blocked at room temperature with 5% BSA for two hours. The monoclonal antibody against Campylobacter jejuni cell swelling lethal toxin CdtB protein was diluted with 5% BSA at a ratio of 1:1000. After overnight incubation at 4°C with shaking, the membrane was washed with PBST five times for 5 minutes each time. The HRP-labeled goat anti-mouse secondary antibody was diluted with 5% BSA at a ratio of 1:5000. The membrane was incubated at room temperature with shaking for 1 hour. After washing with PBST seven times for 5 minutes each time, the chromogenic solution A and B were mixed evenly in a 1:1 ratio. The NC membrane was then incubated in the dark for 1 minute before photographing and storing the image.

[0059] The results showed that Campylobacter jejuni reached a CFU level of 10 in vitro culture conditions. 9 This detection method can detect CdtB protein expressed by Campylobacter jejuni. Figure 2 The detection method can detect the presence of CdtB protein when the amount of CdtB protein purified from prokaryotic expression reaches 0.01 μg. Figure 3 ).

[0060] Example 3

[0061] Take fresh poultry meat samples from the production and processing chain, gently crush them using sterile scissors and forceps, and place them in a 1.5 mL centrifuge tube on ice. Add 500 μL of lysis working buffer, homogenize in a homogenizer, and incubate on ice for complete lysis. Cycle at 12000 rpm for 10 min at 4℃, collect 40 μL of the supernatant, add 10 μL of loading buffer, and incubate at 100℃ for 10 min. Store the resulting protein sample at -20℃ for Western blot experiments.

[0062] Example 4

[0063] Detection method of monoclonal antibody against Campylobacter jejuni cell edema lethal toxin CdtB protein in central kitchen food production and processing chain

[0064] Add 15 μL of the protein sample prepared in Example 3 to a prepared 12% SDS-PAGE gel and electrophoresis at 120V for 90 min. After electrophoresis, transfer is performed for 8 min. After transfer, the NC membrane is placed in 5% BSA and blocked at room temperature for 2 h. Discard the blocking solution, wash the membrane 3 times with PBST, add CdtB monoclonal antibody diluted 1:1000 with 5% BSA and incubate overnight at 4°C. The next day, recover the antibody, wash the membrane 5 times with PBST for 5 min each time, add HRP-labeled goat anti-mouse antibody diluted 1:5000 with 5% BSA and incubate at room temperature for 1 h, wash the membrane 7 times with PBST, and observe the results using an imaging system. Figure 4 The presence of CdtB protein was detected in fresh poultry meat processing samples from Campylobacter jejuni isolate T4-2-5, indicating that Campylobacter jejuni in infected samples secretes CdtB protein.

[0065] This invention obtains a monoclonal antibody against Campylobacter jejuni cell-lethal swelling toxin CdtB, determines its titer, and applies it. First, a Western blotting detection method based on this monoclonal antibody is established, and the detection limit of this method is provided, demonstrating excellent specificity and sensitivity. This detection method is applied to process samples from the food industry chain to obtain residual protein samples in food. The method is simple and easy to implement, providing a new approach for the detection of microbial toxins. The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of this invention. It should be noted that those skilled in the art can make several improvements and modifications to this invention without departing from the principles of this invention, and these improvements and modifications also fall within the scope of protection of the claims of this invention.

Claims

1. The monoclonal antibody against Campylobacter jejuni cell swelling lethal toxin CdtB protein is used in the preparation of reagents for detecting Campylobacter jejuni cell swelling lethal toxin CdtB in samples from different food industry chains. The monoclonal antibody against Campylobacter jejuni cell swelling lethal toxin CdtB protein is a CdtB monoclonal antibody.

2. The application according to claim 1, characterized in that, The CdtB monoclonal antibody was obtained from hybridoma cells 1F5.

3. The application according to claim 1, characterized in that, The monoclonal antibody against the CdtB protein, a swollen lethal toxin of Campylobacter jejuni, is mouse IgG2b subtype.

4. The application according to claim 1, characterized in that, The monoclonal antibody against Campylobacter jejuni cell swelling lethal toxin CdtB protein specifically binds to Campylobacter jejuni CdtB protein; the amino acid sequence of Campylobacter jejuni CdtB protein is shown in SEQ ID NO.

1.

5. The application according to claim 1, characterized in that, The Campylobacter jejuni cell swelling toxin CdtB is a Campylobacter jejuni cell swelling toxin CdtB from a sample in the food production and processing industry chain.

6. The application according to claim 1, characterized in that, The Campylobacter jejuni cell swelling toxin CdtB is a Campylobacter jejuni cell swelling toxin CdtB from a fresh poultry meat production and processing industry chain sample.

7. A kit for detecting Campylobacter jejuni infection and CdtB protein residue, characterized in that, It includes a monoclonal antibody against Campylobacter jejuni cell swelling lethal toxin CdtB protein, CdtB standard protein, blocking solution, HRP-labeled secondary antibody, and chromogenic solution.

8. A method for detecting Campylobacter jejuni cell swelling toxin CdtB in food industry samples based on the Campylobacter jejuni CdtB protein monoclonal antibody according to claim 1, characterized in that, Includes the following steps: Western blotting was used for detection: protein samples were added and transferred after electrophoresis, followed by blocking; after washing the membrane, monoclonal antibody against Campylobacter jejuni cell swelling lethal toxin CdtB protein was added and incubated overnight at low temperature; the antibody was recovered the next day, and after washing the membrane, HRP-labeled goat anti-mouse antibody was added and incubated at room temperature; after washing the membrane, the results were observed by exposure using an imaging system.

9. The method according to claim 8, characterized in that, Samples were obtained from the food industry chain, and residual CdtB protein was extracted from infected positive samples for Western blotting detection.

10. The method according to claim 8, characterized in that, The processing method for the food industry chain samples is as follows: take fresh food industry chain samples, slightly crush them, place them on ice, add lysis working solution, grind and crush them, place them in an ice bath for full lysis, centrifuge them, take the supernatant, add sample loading buffer, and after metal bath, the protein samples obtained are used for Western blot experiments.

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