Cronobacter bacteriophage tail silk protein and application thereof in bacterial detection

Abstract

The invention provides a cronobacter bacteriophage tail silk protein TFP08 and an application of the cronobacter bacteriophage tail silk protein TFP08 in cronobacter detection. The amino acid sequence and the nucleotide sequence of the tail silk protein are respectively shown as SEQ ID NO.1 and SEQ ID NO.2; the electrochemical biosensor based on the tail silk protein can specifically recognize cronobacter of different species, is good in stability, and can be stored at room temperature for 7 days, and the detection signal is reduced within 7%; the electrochemical biosensor constructed by using the tail silk protein can complete the detection of target bacteria within 30 minutes, and shows a good linear relationship in a range of 102-107 CFU / mL, and the detection limit is 23 CFU / mL; the method can be used for on-site detection and rapid screening of cronobacter, and can provide an excellent identification element and a powerful technical support platform for cronobacter detection in the fields of food safety, environment detection, clinical diagnosis and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology and microbial detection technology, and in particular to a Cronobacter phage tail fiber protein and its application in bacterial detection. Background Art

[0002] Cronobacter is an important foodborne pathogen that can cause neonatal meningitis, necrotizing colitis and bacteremia, with a mortality rate of up to 40%-80%. The main infection channel of Cronobacter is infant formula milk powder, which has caused many large-scale milk powder recalls. Cronobacter has been officially confirmed as a Class A pathogen by FAO-WHO, and is also a pathogen that is currently under the control of dairy manufacturers. my country's current national standards also clearly stipulate that Cronobacter must not be detected in formula foods for infants aged 0-6 months. Therefore, rapid and accurate detection of Cronobacter is an important means to prevent related food safety incidents.

[0003] The traditional colony counting method is still used as the "gold standard" for detecting Cronobacter, but its detection cycle is long, usually requiring 3-4 days. Molecular biological technology-based real-time fluorescence quantitative PCR, loop-mediated constant temperature nucleic acid amplification, rolling circle amplification and constant temperature amplification technologies have effectively improved the sensitivity of Cronobacter detection and shortened the detection time, but in order to reduce the interference of the sample matrix, complex and cumbersome pre-treatment steps are required; based on immunological technology, enzyme-linked immunosorbent, immunomagnetic beads, colloidal gold test strips, immunosensors, etc., have high sensitivity and strong specificity, especially immunomagnetic separation technology can quickly selectively capture and separate enriched target bacteria from complex matrices, but immunological technology relies on antibody recognition, traditional antibody preparation is complex, production cycle is long, and cost is high, resulting in the application of immunological technology being limited. In addition, new antibody substitutes such as nucleic acid aptamers are developing rapidly, but the recognition performance of aptamers is highly dependent on their folding structure and conformation, and this conformation is very easy to change in complex matrices, affecting its recognition affinity and specificity. Therefore, it is urgent to explore new recognition elements to break through the bottleneck of Cronobacter detection technology.

[0004] Bacteriophages are viruses that can specifically recognize and infect host bacteria. In recent years, novel detection methods using phages as recognition elements have received widespread attention due to their high specificity, accuracy, and reduced assay time. Compared with antibodies, phages are easier to produce and tolerate extreme pH, temperature, and organic solvents. However, the complex interaction mechanism of the intact phage structure limits the development of new phage recognition elements. Phage tail fiber protein is an important structural protein in the phage tail, which is mainly responsible for the specific recognition and adsorption of phages and host bacteria. At present, there are no reports on the detection of Cronobacter using phage tail fiber protein. Summary of the invention

[0005] In response to the above problems, the present invention successfully obtained the Cronobacter phage tail fiber protein TFP08 through gene recombination and prokaryotic expression, and used it as the recognition molecule to construct an electrochemical biosensor for Cronobacter, which can be applied to on-site detection and rapid screening of Cronobacter in food, environment and clinical samples.

[0006] Specifically, the present invention adopts the following technical solutions:

[0007] First, the present invention provides a Cronobacter phage tail fiber protein, whose amino acid sequence is shown in SEQ ID No. 1, and whose nucleotide sequence is shown in SEQ ID No. 2. The applicant named the tail fiber protein TFP08.

[0008] Secondly, the present application provides the use of tail fiber protein with an amino acid sequence as shown in SEQ ID No. 1 in detecting Cronobacter for non-medical purposes, such as detecting Cronobacter in the fields of food, environment, clinical diagnosis, etc.

[0009] Specifically, the above application refers to: using the above-mentioned tail fiber protein TFP08 to prepare an electrochemical biosensor, and then using differential pulse voltammetry based on the sensor to detect the content of Cronobacter in the sample, and substituting the peak current signal measured by the electrochemical workstation into the regression equation to obtain the content of Cronobacter in the sample; the above regression equation is A=1.57-0.085lgC, where A is the peak current signal and C is the concentration of Cronobacter.

[0010] The preparation method of the electrochemical biosensor is as follows: 1 ml AuNPs was mixed with 1 ml GO (1 mg / mL), 40 μL nafion was added, and ultrasonic stirring was performed to obtain AuNPs@GO; 5 μL AuNPs@GO was fixed on the surface of the gold electrode to obtain the AuNPs@GO / gold electrode; the AuNPs@GO / gold electrode was then immersed in an ethanol solution containing 4 mM MUA overnight, and then activated with a mixture of EDC (0.4 M) and NHS (0.1 M); the electrode was then washed with deionized water; 5 ul of 5 mg / mL tail fiber protein TFP08 was dropped on the top of the electrode for incubation to obtain the TFP 08 / AuNPs@GO / gold electrode; the TFP 08 / AuNPs@GO / gold electrode was then washed with deionized water, and then the electrode was immersed in a 100 μL 10 mM Ni 2+ The electrochemical biosensor is obtained.

[0011] The present application utilizes the recognition and adsorption of specific bacterial host receptors by the bacteriophage tail fiber protein receptor binding protein during the recognition process of the bacteriophage. The protein is a trimer rich in β structures, which are folded and entangled in a β helical structure, further improving the stability, and is not easily affected by enzymes, temperature and pH, and has specificity and stability. Compared with the prior art, the beneficial effects of the present invention are:

[0012] 1. The Cronobacter phage tail fiber protein TFP08 provided by the present invention has a wide recognition range and has good recognition ability for both standard strains and clinical isolated strains.

[0013] 2. The Cronobacter phage tail fiber protein TFP08 provided by the present invention has strong specificity and strong recognition ability for Cronobacter, but has no recognition ability for non-target bacteria Escherichia coli (ATCC 25922, ATCC 36150), Staphylococcus aureus (CMCC26001) and Salmonella (ATCC 13076).

[0014] 3. The electrochemical biosensor prepared based on the Cronobacter phage tail fiber protein can be effectively used for on-site detection and rapid screening of Cronobacter. It has high specificity and does not require pre-treatment of the matrix. The detection time is only about 30 minutes, which can shorten the existing detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of protein detection;

[0016] (A) Gel electrophoresis of TFP08; 1: recombinant plasmid His-ORF08-pET28a, 2: double enzyme digestion map of recombinant plasmid, M: DNA molecular mass standard; (B) is the verification result of the transformation process; (C) SDS-PAGE map of TFP08; M: protein molecular mass standard. 1:

[0017] 1: precipitated protein. 2: supernatant protein. 3: purified supernatant protein; (D) Western blot analysis of TFP08; M: protein molecular weight standard, 1: purified TFP08.

[0018] Figure 2 This is the result of ELISA method to determine the binding ability of tail fiber protein TFP08 to host bacteria.

[0019] Figure 3 The biofilm interferometry technique was used to determine the molecular interaction of tail fiber protein TFP08 with LPS on the surface of host bacteria.

[0020] Figure 4 The construction and measurement results of the electrochemical biosensor.

[0021] Figure 5 The results of the specificity and storage stability test of TFP08 electrochemical sensor.

[0022] Figure 6 The linear range and sensitivity test results of TFP08 electrochemical sensor.

[0023] Figure 7 Schematic diagram of the preparation of electrochemical biosensor based on bacteriophage tail fiber protein TFP08. DETAILED DESCRIPTION

[0024] The raw materials, strains, and reagents used in the examples were obtained from conventional commercial sources unless otherwise specified.

[0025] GO and AuNPs were purchased from Nanjing Xianfeng Nanomaterials Technology Co., Ltd.;

[0026] Nafion was purchased from Beijing Zhongke Nong'an Technology Co., Ltd.;

[0027] Cronobacter b2, b29, and b30 are all preserved in Jiangsu Academy of Agricultural Sciences. The strains are conventional strains, as disclosed in the document “Foods 2024, 13, 871.”;

[0028] Staphylococcus aureus (CMCC 26001), Escherichia coli (ATCC 25922, ATCC 36150) and Salmonella (ATCC 13076) were standard strains purchased from the Chinese Culture Collection and the American Type Culture Collection.

[0029] Escherichia coli top10 strain was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0030] LB liquid medium (1 L): add 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride to 950 mL of water. Adjust the pH to 7.0 with 1 M NaOH and make up to 1 L; sterilize by high pressure at 121°C for 20 min and store at 4°C.

[0031] Kanamycin resistance LB medium: Add kanamycin to LB liquid medium at a final concentration of 50 μg / mL.

[0032] Kanamycin resistance plate: Add kanamycin with a final concentration of 50 μg / mL and agar with a final concentration of 1.2% to LB liquid culture medium.

[0033] Anti-His tag mouse monoclonal antibody and HRP-labeled goat anti-mouse IgG (H+L) antibody were purchased from Biotechnology Company.

[0034] Example 1 Expression of tail fiber protein

[0035] The recombinant plasmid HisORF08-pET28a(+) (GenScript Biotech) was mixed with 100 μL of E. coli BL21(DE3)pLysS (GenScript Biotech). The solution was placed in an ice bath for 10 minutes, incubated in a 42°C water bath for 90 seconds, and then transferred to an ice bath for 3 minutes. Next, 1 ml of LB liquid medium was added, and the mixture was incubated at 37°C for 45 minutes while shaking at 180 rpm to recover the cells. 60 μL of the culture was added to a kanamycin-resistant plate, and the plate was inverted and incubated overnight at 37°C.

[0036] Inoculate a single colony containing the recombinant plasmid into 5 ml of kanamycin resistant LB medium and incubate overnight at 37°C. Transfer 2 ml of the above culture into 200 ml of kanamycin resistant LB medium and incubate at 37°C with continuous shaking at 180 rpm. When OD 600 When the value reaches about 0.6, transfer to an ice bath for 10 minutes, then add isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.1mM, and after induction and expression in a shaker at 16°C for 16 hours, collect the bacterial cells by centrifugation (10000rpm, 10 minutes, 4°C) and wash three times with Tris-HCl (50mM, pH=7.2); resuspend the bacteria in 20mL Tris-HCl (50mM, pH=7.2), and sonicate the cells. Centrifuge the cells (12000, 15 minutes, 4°C) to separate the supernatant and the precipitate. The supernatant and precipitate samples are used for SDS-PAGE to verify the expression, and the detection results are as follows Figure 1 As shown in C, lane 1 is the precipitation detection result, and lane 2 is the supernatant detection result.

[0037] The synthesized gene was linked to the enzyme to construct a recombinant plasmid, which was then transfected into the expression strain Escherichia coli top10 strain. Single clones were picked on a kanamycin-resistant plate for NcoI / BamHI double enzyme digestion verification. The results were as follows: Figure 1 As shown in A, the gene fragment of the tail fiber protein TFP is about 2600 bp in size. The recombinant plasmid was then transformed into the E. coli expression strain BL21 (DE3). The T7 promoter universal primer was used to verify the transformed BL21 (DE3). The test results are shown in Figure 1 As shown in B, the gene fragment size is also about 2600 bp, indicating that the recombinant plasmid was successfully transferred to the E. coli expression strain BL21 (DE3).

[0038] The constructed expression vector was inoculated into kanamycin-resistant LB liquid medium at a 1% inoculum, and the expression was induced at 16°C and 210rpm for 20h. The medium was placed in an ice box and ultrasonically broken (300W, working 3s, rest 4s, 10min) for 3 cycles; after the ultrasonication, the medium was centrifuged at 12000rpm and 4°C for 10min, the supernatant and precipitate were separated, and the protein expression was identified by SDS-PAGE electrophoresis (140V, 60min). The test results are as follows: Figure 1 As shown in C, the target protein is mainly present in the supernatant, indicating that the target protein TFP08 is a soluble protein, which is conducive to subsequent experiments.

[0039] The protein TFP08 was then added to a protein purification column containing 5 mL of HisPurTM nickel-NTA resin, incubated at 4°C for 30 min, and allowed to flow through the resin bed. The protein was purified using 25mM, 50mM, 100mM, 250mM and 500mM imidazole, respectively, and the eluate was collected. The concentration of the eluate was determined by SDS-PAGE, and the target protein was concentrated using a 30KDa ultrafiltration tube at 4000g for 10 min. The purified protein was obtained, which was a soluble protein. The applicant named it tail fiber protein TFP08, and its amino acid sequence and nucleotide sequence are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The purified TFP08 was analyzed using western blotting, and the results are as follows Figure 1 As shown in D, there is a target band near 90 kDa, and the concentration of the tail fiber protein was finally measured using a BCA kit and was 5 mg / mL.

[0040] Example 2: Determination of the binding ability of tail fiber protein TFP08 to host bacteria using ELISA method

[0041] Cronobacter (b2) and Staphylococcus aureus (CMCC 26001) were cultured in LB liquid medium overnight, centrifuged at 5000rpm for 3min, washed three times with sterile water, and finally resuspended with sterile PBS. Add b2 strain, CMCC 26001 strain and blank sample (PBST buffer containing 5% mass concentration of skim milk powder) to 96-well plate, 100μL per well, incubated at 4℃ overnight. Wash four times with 0.05% (mass concentration, the same below) PBST (phosphate Tween-20 buffer), add 250μL of 5% mPBS to each well, and block at 37℃ for 2h. Wash three times with PBST, add 100μL of TFP08 (10μg / mL) to each well, and incubate at 37℃ for 1h. After 1 hour, wash three times with PBST, add 100 μL of anti-His tag mouse monoclonal antibody to each well (antibody: PBST diluted at a volume ratio of 1:3000) and incubate at 4°C overnight. After overnight, wash three times with PBST, add 100 μL of HRP-labeled goat anti-mouse antibody IgG (H+L) and incubate at 37°C for 1 hour. Add 10 μL of citrate buffer (CPBS), 10 μL of 3,3',5,5'-tetramethylbenzidine TMB colorimetric solution and 25 μL of H2O2 (0.65%) and incubate for 15 minutes. Finally, add 50 μL of H2SO4 (2M) to terminate the reaction. Measure OD using an enzyme reader 450 .

[0042] Test results such as Figure 2 As shown, it can be seen that the affinity of tail fiber protein to host bacteria b2 is much higher than that of blank sample mPBS and non-host bacteria 26001.

[0043] Example 3: Using biofilm interferometry to analyze the binding ability of tail fiber protein TFP08 to host bacterial receptor LPS

[0044] Existing studies believe that the receptor of Cronobacter may be on the lipopolysaccharide (LPS) on its surface. Therefore, this example first used an LPS extraction kit (purchased from Beijing Solebow Technology Co., Ltd.) to extract the LPS of Cronobacter (b2), and then used the Octet Red96 biomembrane interferometer to analyze the affinity between TFP08 and LPS. 200 μM TFP was loaded onto the APS biosensor for 600 s, and the binding of LPS to the immobilized TFP was measured. The process used association for 340 s and then dissociation for 300 s (the specific steps were performed according to the sensor manual). The results are as follows. Figure 3 As shown, it can be seen that the tail fiber protein TFP08 has a strong binding ability to LPS on the surface of host bacteria b2.

[0045] Example 4 Construction of electrochemical biosensor based on TFP08

[0046] 1 ml of gold nanoparticles (AuNPs) was mixed with 1 ml of monolayer graphene oxide (GO, 1 mg / mL), 40 μL of nafion was added, and ultrasonic stirring was performed for 30 minutes to obtain AuNPs@GO; 5 μL of AuNPs@GO was fixed on the surface of the gold electrode (dried at room temperature and the mixture was deposited on the surface of the gold electrode) to obtain AuNPs@GO / gold electrode; then the AuNPs@GO / gold electrode was immersed in an ethanol solution containing 4 mM 11-mercaptoundecanoic acid (MUA) MUA overnight (using the thiol functional group of MUA to combine with AuNPs), and then activated with a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 0.4 M) and N-hydroxysulfosuccinimide sodium salt (NHS, 0.1 M) for 30 minutes, and then the electrode was washed three times with deionized water to remove EDC and NHS; then 5 ul of 5 mg / mL The TFP08 solution was dropped on the top of the electrode with a pipette and incubated for 60 minutes. TFP08 was fixed and modified on the electrode (the amide bond between the two was covalently bonded), and a TFP08 / AuNPs@GO / gold electrode was obtained. After the incubation period, the TFP08 / AuNPs@GO / gold electrode was washed with deionized water to remove unbound TFP08. Finally, the electrode was immersed in 100 μL 10 mM Ni 2+ The His tag on TFP 08 was connected to obtain an electrochemical biosensor. The preparation flow chart is shown in Figure 7 The sensor preparation method is a conventional method in the art, as disclosed in the document "Talanta, 2024, 270, 125561."

[0047] The electrochemical biosensor uses Ni 2+ Generate a current signal, TFP08 captures bacteria and GO amplifies the current signal, and differential pulse voltammetry (DPV) can be used to detect Cronobacter b2. This detection method is a conventional method in the art, as disclosed in the document "Microchimica Acta, 2024, 191: 550". The specific detection steps of this embodiment are as follows: 5 μL of Cronobacter is dripped onto the surface of the electrochemical biosensor electrode based on TFP08 prepared in this embodiment, incubated for 30 minutes, and then washed with PBS to remove bacteria that are not bound, using differential pulse voltammetry (DPV), and the measurement is performed on a CHI 830C electrochemical station. Electrochemical measurements all use a three-electrode system-working electrode, platinum counter electrode and silver / silver chloride reference electrode. DPV measurements are performed in a PBS solution with a pH value of 7.4. The measurement potential range is set to -600 to -200mV, the amplitude is 100mV, and the pulse period is 0.1 second.

[0048] Test results such as Figure 4As shown in the figure, the bare gold electrode does not show an oxidation peak (red line) in the PBS solution, and then TFP08 and Ni 2+ After binding to the gold electrode surface, an oxidation peak appeared (gold electrode-TFPs-Ni 2+ ), indicating that TFP08 and Ni2+ were successfully combined with the gold electrode surface. After AuNPs@GO was combined with TFP08, an oxidation peak also appeared (gold electrode-AuNPs@GO-TFPs-Ni 2+ ), and the current signal is significantly enhanced, indicating that AuNPs@GO can amplify the current signal. After adding Cronobacter (b2) to c (gold electrode-AuNPs@GO-TFPs-Ni 2+ -b2), the current signal decreased significantly, indicating that the target bacteria were successfully captured by TFP08 and the electrochemical biosensor was successfully constructed.

[0049] Example 5 Specificity and storage stability of TFP08 electrochemical sensor

[0050] In order to investigate the specificity of the detection method, this example uses the TFP08 electrochemical sensor prepared in Example 4 to simultaneously detect the bacterial count of 10 8 CFU / mL of target bacteria (Cronobacter b2, b29, b30) and non-target bacteria Escherichia coli (ATCC 25922, ATCC 36150), Staphylococcus aureus (CMCC 26001), and Salmonella (ATCC 13076) were tested.

[0051] Test results such as Figure 5 As shown in A, compared with the blank control, the current signal of the TFP08 electrochemical sensor significantly decreased after capturing Cronobacter sakazakii (b29), Cronobacter malonaticus (b30), and Cronobacter dublinensis (b2), while the current signals of non-target bacteria, such as Escherichia coli, Salmonella, and Staphylococcus aureus, were basically the same as those of the blank control. This shows that TFP08 has good specificity.

[0052] In addition, this example also evaluates the storage stability of the electrochemical biosensor TFP08. Figure 5 As shown in B, AuNPs@GO, TFP08 and Ni 2+ After layer-by-layer assembly, the samples were stored at room temperature for 7 days. The current signal was measured using DPV. This shows that the TFP08 electrochemical sensor has good stability at room temperature.

[0053] Example 6 Linear range and sensitivity of TFP08 electrochemical sensor

[0054] In order to evaluate the linear range sensitivity of the TFP08 electrochemical sensor detection method prepared in Example 4, Cronobacter (b2) was diluted in a gradient manner (10 2 ~10 7 CFU / mL), this example uses TFP08 electrochemical biosensor to detect DPV of Cronobacter at different concentrations.

[0055] The results are as follows Figure 6 As shown in A, as the concentration of Cronobacter increased from 2.3×10 1 The CFU / mL increased to 3.7×10 7 CFU / mL, Ni 2+ The peak current gradually decreased, and the concentration of Cronobacter was inversely proportional to the current, indicating that Cronobacter could hinder the generation of current. 2 -10 7 The CFU / mL range showed a good linear relationship. 2 =0.9963. The limit of detection (LOD) was 23 CFU / mL. Figure 6 As shown in B, the regression equation is A=1.57-0.085lgC, where A is the peak current signal and C is the concentration of Cronobacter. This shows that the TFP08 electrochemical sensor has a wide linear range and high sensitivity, which can meet the needs of Cronobacter detection.

[0056] Example 7 Detection of Cronobacter in infant formula and leafy vegetables using TFP08 electrochemical sensor

[0057] Preparation of infant formula samples: Dissolve with sterile deionized water according to the instructions and set aside;

[0058] Preparation of fresh lettuce samples: The purchased fresh lettuce is first washed with distilled water, then cut into small pieces, and sterilized under ultraviolet light for 30 minutes on both sides, and set aside.

[0059] In this embodiment, the sample in the quantitative test of Cronobacter was diluted and tested with reference to the national food safety standard of China (GB 4789.40-2024): 10 mL and 10 g skim milk powder or lettuce were mixed with 90 mL sterile deionized water. Different concentrations of Cronobacter were added to the prepared samples, and then the plate count method (GB 4789.2-2022 "National Food Safety Standard Food Microbiology Test Total Colony Count Determination" and TFP08 electrochemical biosensor were used for experiments, and the results of the plate count method were used as a control.

[0060] Table 1 Determination of Cronobacter in different infant formula and lettuce by TFP08 electrochemical biosensor

[0061]

[0062] The test results are shown in Table 1. The recovery rates of Cronobacter were between 99.23% and 102.46%, and the RSDs were all less than 1.17%, indicating that this method can be used for the detection of Cronobacter in complex foods.

[0063] Example 8 Detection of Cronobacter in Farm Wastewater Samples and Bovine Serum by TFP08 Electrochemical Sensor

[0064] The farm wastewater and bovine serum were filtered through a 0.22 μm filter membrane, respectively, and then 2 mL of the sample was mixed with 18.0 mL of sterile water for later use.

[0065] Different concentrations of Cronobacter were added to the prepared samples, and then the plate count method and the TFP08 electrochemical biosensor prepared in Example 4 were used for detection, and the results of the plate count method were used as a control. The results are shown in Table 2.

[0066] Table 2 Determination of Cronobacter in farm wastewater and bovine serum by TFP08 electrochemical biosensor

[0067]

[0068] As can be seen from Table 2, the electrochemical sensor prepared using TFP08 can be used for the detection of Cronobacter in environmental and clinical samples.

Claims

1. A Cronobacter phage tail fiber protein, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. The gene encoding the tail fiber protein as claimed in claim 1, wherein the nucleotide sequence thereof is shown in SEQ ID NO.

2.

3. Use of the tail fiber protein as claimed in claim 1 in detecting Cronobacter.

4. The use according to claim 3, characterized in that: The tail fiber protein is used to prepare an electrochemical biosensor to detect Cronobacter in the environment or food.

5. The use according to claim 4, characterized in that: Based on the electrochemical biosensor, the content of Cronobacter in the sample is detected by differential pulse voltammetry, and the measured peak current signal is substituted into the regression equation to obtain the content of Cronobacter in the sample; the regression equation is: A=1.57-0.085lgC, where A is the peak current signal and C is the concentration of Cronobacter.

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