CN106771194B - A method of detection pathogenic microorganism - Google Patents

Abstract

The invention belongs to the field of nanobiological technology detection, and specifically refers to a method for detecting pathogenic microorganisms based on displaying polypeptide ligands and enzyme-like nanometer materials on the surface of phages. Use the multifunctional biological nanoprobe to incubate with the target pathogenic microorganism to be detected, and then realize its detection under the chromogenic system; among them, the multifunctional biological nanoprobe is screened from the landscape phage library by using the biopanning technology The phage monoclonal that specifically binds to the pathogenic microorganism is further isolated and purified to obtain the protein at the corresponding site of the phage displaying the specific polypeptide; then the phage capsid protein displaying the specific polypeptide is assembled on the imitation enzyme nanomaterial. The probe of the invention can not only specifically recognize and bind target microorganisms, but also can realize signal amplification due to its imitation enzyme activity, thereby realizing the immunocolorimetric quantitative detection of Vibrio parahaemolyticus. The method is simple in operation, low in cost, high in specificity and sensitivity, and provides an effective way for sensitive detection of pathogenic microorganisms.

Description

A method for detecting pathogenic microorganisms

technical field

The invention belongs to the field of nano-biotechnology detection, and specifically refers to a method for detecting pathogenic microorganisms based on assembling a multifunctional biological nano-probe based on displaying polypeptide ligands on the surface of phages and imitating enzyme nano-materials.

Background technique

Pathogenic microorganisms refer to organisms that can directly or indirectly cause damage to humans and animals. They are of various types and are very common in humans, threatening almost everyone. Foodborne diseases caused by foodborne pathogenic microorganisms are currently the most common threat to human beings and society. Vibrio parahaemolyticus (Vibrio parahaemolyticus) is one of the most prevalent pathogens in the public health care system worldwide, and it is also the most important microorganism that contaminates common food and aquatic products. Foodborne diseases caused by such pathogenic microorganisms, such as food poisoning, gastroenteritis, sepsis, toxic shock syndrome and other fatal diseases, are currently a crucial problem for the medical system at home and abroad.

Traditional methods for detecting pathogenic microorganisms mainly rely on specific microbiology, biochemistry and molecular biology techniques, such as plate culture and PCR. Although these traditional methods have good sensitivity and specificity, the time required for detection is relatively long (generally 3-7 days), so they are not suitable for on-site rapid detection. At present, researchers have developed a variety of rapid detection methods with high sensitivity and specificity, which can be mainly divided into methods based on immunology and methods based on biosensors. The characteristics and difficulties of these two methods are the selection of antibodies that can specifically recognize and bind to target microorganisms. However, antibody molecules have disadvantages such as complex preparation, high cost, and poor stability, which greatly limit their wide application in the biological field. Therefore, it is inevitable to develop targeting-specific ligands with wide practicability, low cost, reproducibility, strong stability and good specificity. Specific peptides have the advantages of high stability, small molecular weight, high specificity, and easy synthesis, and have become new targeting reagents. Phage display technology can display exogenous random polypeptides on the surface of phage capsid proteins, and the constructed phage display library can obtain target-specific polypeptide ligands through high-throughput screening.

Nanozyme is a nanomaterial with enzyme-like activity, and its essence is a chemical catalyst. Nanozymes have the advantages of low cost, high stability, large surface area, and strong catalytic activity. At present, the study of nanozymes has become a hot topic in related fields in recent years. Transition metal oxide nanozymes are the most widely studied nanozymes. Because of their unique physical and chemical properties, nanozymes have broad application prospects in the fields of catalysis, biomedicine, detection, and environmental protection.

In summary, there is an urgent need for a polypeptide ligand that can specifically bind to target microorganisms to achieve specific recognition and binding of target microorganisms. At the same time, there is an urgent need for a nanozyme with excellent enzyme-like activity to achieve signal amplification and effectively combine the two. , to achieve sensitive, specific and rapid detection of pathogenic microorganisms.

Contents of the invention

The purpose of the present invention is to provide a method for preparing multifunctional biological nanoprobes to detect pathogenic microorganisms based on displaying polypeptide ligands on the surface of phages and imitating enzyme nanomaterials.

To achieve the above object, the technical solution adopted in the present invention is:

A method for detecting pathogenic microorganisms, which uses a multifunctional biological nanoprobe to incubate with a target pathogenic microorganism to be detected, and then realizes its detection under a chromogenic system; wherein, the multifunctional biological nanoprobe adopts the main coat of a phage The shell protein pVIII is used as a template to synthesize enzyme-like nanomaterials; then, the phage monoclonal that specifically binds to pathogenic microorganisms is screened from the landscape phage library using biopanning technology, and then further isolated and purified to obtain the main capsid of the phage displaying specific polypeptides protein; then the phage capsid protein displaying specific polypeptides is assembled on the imitation enzyme nanomaterial. Thus constructing a multifunctional biological nanoprobe. The probe can not only specifically recognize and bind target microorganisms, but also can achieve signal amplification due to its imitation enzyme activity, thereby realizing the quantitative detection of target pathogenic microorganisms.

The preparation of the multifunctional biological nanoprobe is as follows: the enzyme-like nanomaterial manganese dioxide is synthesized by the protein template method; then the phage monoclonal specifically binding to Vibrio parahaemolyticus is screened out from the landscape phage library by using the biopanning technique , and then further purified to obtain the phage capsid protein pVIII (fusion pVIII) displaying specific polypeptides, and then the fusion pVIII was assembled on the surface of manganese dioxide material to construct a multifunctional biological nanoprobe for detecting Vibrio parahaemolyticus. The probe can not only specifically recognize and bind target microorganisms, but also can achieve signal amplification due to its imitation enzyme activity, thereby realizing the immunocolorimetric quantitative detection of Vibrio parahaemolyticus. Wherein, the manganese dioxide imitation enzyme nanometer material is MnO ₂ nano sheet (MnO ₂ NSs) with imitation peroxidase activity.

The construction of the multifunctional biological nanoprobe is as follows. First, tert-butyl carbamate (BOC) is used to protect the N-terminus of fusion pVIII, which has binding activity with Vibrio parahaemolyticus, and then its C-terminus is treated with 1-ethyl-3 -(3-Dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinimide (NHS) was activated to assemble fusion pVIII on the surface of pVIII protein residues on the surface of manganese dioxide imitation enzyme nanosheets Then add trifluoroacetic acid to remove BOC protection of fusion pVIII, so that its N-terminus can restore the activity of specific binding to Vibrio parahaemolyticus.

The polypeptide ligands displayed on the surface of the phage target the pathogenic microorganism Vibrio parahaemolyticus, and are panned from the landscape phage f8/9 library using biopanning technology. The specificity test successfully obtained a phage monoclonal that can specifically recognize Vibrio parahaemolyticus. The phages were further amplified in large quantities and the phage fusion pVIII displaying specific polypeptides were separated and purified by saturated phenol.

The multifunctional biological nanoprobe is constructed by interacting the C-terminus of fusion pVIII with the N-terminus of the surface protein residue of the manganese dioxide material through an amide bond.

During the construction process of the multifunctional biological nanoprobe, the molar ratio of fusion pVIII to manganese dioxide material is 1000:1.

For the detection of Vibrio parahaemolyticus, first fix fusion pVIII in a 96-well plate, block with BSA for 1 hour, wash with PBS buffer, and then incubate the Vibrio parahaemolyticus statically, then add the above-mentioned multifunctional manganese dioxide nanoprobe for incubation, and the incubation conditions Incubate at 37°C for 1 hour, wash with PBS buffer, and add chromogenic solution to react; the fixed amount and fixed concentration of fusion pVIII in the 96-well plate are 100 μl and 40 μg/ml. The chromogenic system was 140 μl of acetic acid-sodium acetate buffer (10 mM, pH 4), 20 μl of 10 mM H ₂ O ₂ and 20 μl of 6 mM tetramethylbenzidine (TMB).

The beneficial effects of the present invention are:

The present invention adopts biological panning technology to obtain phage polypeptide ligands that can specifically recognize and bind Vibrio parahaemolyticus from phage libraries by panning; It can catalyze the peroxidase substrate to produce a clear color reaction to achieve signal amplification. In the present invention, phage polypeptide ligands are assembled on the surface of manganese dioxide nanomaterials to construct a multifunctional nanoprobe that can specifically recognize and bind Vibrio parahaemolyticus, and can also realize signal amplification, realizing the sensitive detection of Vibrio parahaemolyticus. Specific detection has very important practical significance. specifically is:

1. The present invention adopts the bio-panning technology to pan the phage library to obtain the high-affinity and high-specificity phage nonapeptide ligand VQTVQIGSD of Vibrio parahaemolyticus. The polypeptide ligand has high stability, small molecular weight, high specificity and easy synthesis.

2. The present invention adopts BOC protection and EDC/NHS activation to assemble Vibrio parahaemolyticus-specific binding phage fusionpVIII on the surface of MnO ₂ NSs to construct a novel multifunctional nanoprobe. The probe can not only specifically recognize the target microorganism Vibrio parahaemolyticus, but also realize signal amplification due to its imitation peroxidase activity.

3. The detection limit of Vibrio parahaemolyticus in the method of the present invention is 15 cfu/mL, which is lower than the detection limit of other methods reported so far. The proposed method has the advantages of wide detection range, good selectivity, low price, and is applicable to the measurement of real marine samples, opening up a promising detection method in the fields of environmental chemistry, biotechnology, biotechnology, catalysis industry, and biological evaluation .

Description of drawings

Figure 1 is a schematic diagram of the detection of Vibrio parahaemolyticus provided by the embodiment of the present invention, including the construction process of the multifunctional nanoprobe: BOC protection of fusion-pVIII (A), EDC/NHS activation of the C-terminal of fusion-pVIII, and MnO ₂ Assembly of NSs, removal of BOC protection at the N-terminal of fusion-pVIII (B), and detection of Vibrio parahaemolyticus (C).

Fig. 2 is the recovery rate of three rounds of biopanning provided by the embodiment of the present invention.

Fig. 3 is the test results of monoclonal specificity of three selected phages provided in the embodiment of the present invention.

Fig. 4 is the working curve for detecting Vibrio parahaemolyticus provided by the embodiment of the present invention.

Fig. 5 is the specific test result for detecting Vibrio parahaemolyticus provided by the embodiment of the present invention.

Detailed ways

The detection method involved in the present invention will be described in detail below through examples, but it does not constitute a limitation to the present invention.

The invention adopts the biological panning technology to screen out the phage monoclonal specifically combined with Vibrio parahaemolyticus from the landscape phage library, and isolates and purifies the main capsid protein pVIII (fusionpVIII) of the phage displaying the specific polypeptide. A multifunctional biological nanoprobe was constructed by interacting the C-terminal of fusion pVIII with the N-terminal of the surface of manganese dioxide material with excellent peroxidase imitation through amide bond.

Example 1

1) Preparation of peptide ligands displayed on the surface of phage

Using the food-borne pathogen Vibrio parahaemolyticus as the target, the landscape phage f8/9 library was panned using biopanning technology, and through the specificity test, a phage monoclonal capable of specifically recognizing Vibrio parahaemolyticus was successfully obtained . The phages were further amplified in large quantities and the phage fusion pVIII displaying specific polypeptides were separated and purified by saturated phenol.

First, V. parahaemolyticus was inoculated into a 96-well plate and allowed to dry overnight. Mix 10 μl of f8/9 landscape phage library (about 10 ¹³ phages/ml) with 45 μl of library blocking solution, let stand at room temperature for 1 hour, and then aspirate the phage library that cannot bind to target cells after incubation. Add 100 μl of washing buffer (containing 0.5% BSA, 0.1% Tween-20) to the Vibrio parahaemolyticus cells and incubate at room temperature for 10 min, discard the buffer, and then wash again, repeating this process for 6-8 times to remove cells Bacteriophages that are not firmly bound to surfaces. Aspirate off the washing buffer, add elution buffer (containing 1mg/ml BSA, 0.1mg/ml phenol red, 0.2M glycine) to Vibrio parahaemolyticus cells, let stand at room temperature for 10min, and elute the cells bound to parahaemolyticus Phages on the surface of Vibrio cells. Transfer the eluate to a 1.5ml centrifuge tube containing 19 μl of neutralizing solution (containing 1M Tris, pH 9.1), oscillate to make it evenly mixed, the color of the solution changes rapidly from yellow to wine red, and measure the titer of the obtained phage, Total phage output from the first round of panning labeled V. parahaemolyticus cells.

The phage obtained in the first round were then amplified, purified and titered for the next round of phage panning. The recovery rate of phages is used to monitor each round of panning process, and the calculation method of the recovery rate is: recovery rate = total amount of input phages/ total amount of output phages × 100%. The response rates of the three rounds of panning are shown in Figure 2.

It can be seen from Figure 2 that after three rounds of biopanning, the phages capable of binding to Vibrio parahaemolyticus cells were effectively enriched. Single clones were randomly selected on the titer plate for PCR identification. According to the identification results, the PCR amplified fragments were sent to the company for sequencing and the sequencing results were analyzed. The results are shown in Table 1. According to the analysis results, the corresponding E. coli colonies displaying the complete exogenous random nonapeptide were activated, amplified, and the corresponding phages were purified from them. The concentration of the purified phages was measured and stored in a refrigerator at 4°C for later use.

Finally, a specificity test is performed. Inoculate Vibrio parahaemolyticus in the experimental group and Edwardsiella in the control group, Escherichia coli Trans 5α, Proteus vulgaris, Bacillus cereus, Staphylococcus aureus and Vibrio anguillarum in different wells of the 96-well plate, and dry overnight. On the next day, 50 μl of phage (10 ⁷ TU) was added to each well, and incubated at room temperature for 1 hour. Then use a method similar to the biopanning process to obtain the number of cell surface phages and intracellular phages on each cell through steps such as washing, elution, lysis, and titer determination, and calculate the phage recovery rate corresponding to each cell According to the recovery rate, the binding specificity of the phage to the target cells was determined, and the results are shown in Figure 3.

It can be seen from Figure 3 that the sequence of the polypeptide ligand displayed on the surface of the Vibrio parahaemolyticus phage is VQTVQIGSD, which can bind to Vibrio parahaemolyticus with high affinity and specificity.

Table 1 shows the polypeptide sequences displayed by the selected phage monoclonals provided in the examples of the present invention.

Then, replace the above-mentioned targets with other pathogenic bacteria, and then use bio-panning technology to pan from the landscape phage f8/9 library, and pass the specificity test to obtain a phage monoclonal that can specifically recognize the corresponding target. The phage is further amplified in large quantities, and the protein at the corresponding site of the phage displaying a specific polypeptide is separated and purified by saturated phenol.

2) The preparation of the above manganese dioxide imitation enzyme nanosheets (containing protein residues) can refer to the literature (Han, L., Shao, C., Liang, B., Liu, A., 2016. Genetically Engineered Phage-Templated MnO ₂ Nanowires: Synthesis and Their Application in Electrochemical Glucose Biosensor Operated at Neutral pH Condition.ACS Appl.Mater.Inter.8(22),13768-13776.), replace the M13 phage reported in the literature with the M13 phage coat protein, then Synthesize manganese dioxide-like enzyme nanosheets (MnO ₂ NSs), the specific method is as follows:

Add 1 mg of M13 phage major capsid protein (pVIII) to 10 ml of MnAc ₂ aqueous solution (1 mM), and stir at room temperature for 1 h. The pH was then adjusted to 10 with NaOH solution. Stirring was continued at room temperature for 6h. Finally, the reaction system is filtered through a 0.22 μm filter membrane to remove unreacted MnAc ₂ and NaOH, and MnO ₂ NSs can be obtained.

3) Probe construction:

Firstly, BOC was used to protect the N-terminus of fusion pVIII, which has the characteristic of binding to Vibrio parahaemolyticus, and then its C-terminus was activated with EDC/NHS, and assembled according to fusion pVIII/MnO ₂ NSs = 1000 (molar ratio) (4°C static overnight), fusion pVIII assembled on the surface of MnO ₂ NSs (containing protein residues) through amide bonds. Then, fusion pVIII was deprotected by BOC by adding trifluoroacetic acid, so that its N-terminus could restore the activity of specifically binding to Vibrio parahaemolyticus, so that the MnO ₂ NSs@fusion-pVIII functional nanoprobe could be obtained. As shown in Figure 1C.

Example 2

Detection of Vibrio parahaemolyticus:

Using the peroxidase-like activity of MnO ₂ NSs obtained above and the specific binding performance of fusion pVIII to Vibrio parahaemolyticus, a label-free enzyme-like nanoprobe based on the specific phage pVIII fusion protein was constructed to detect Vibrio parahaemolyticus method.

Detection: 100 μl of 40 μg/ml fusion pVIII was added to a 96-well plate, and fixed overnight at 4°C. Wash with PBS buffer (10 mM, pH 7.4) 3 times to remove unfixed fusion pVIII. Block with 10mg/ml BSA solution for 1h, wash twice with PBS buffer (10mM, pH 7.4), add target microorganism Vibrio parahaemolyticus, incubate at 37°C for 2h, then add 100μl 0.5mg/ml multifunctional nano The probe was incubated at 37°C for 1 h, washed three times with PBS buffer (10 mM, pH 7.4), and then added 140 μl acetic acid-sodium acetate buffer (10 mM, pH 4), 20 μl 10 mM H ₂ O ₂ and 20 μl 6 mM TMB, react at room temperature for 30min. Finally, the absorbance value at 650 nm was measured.

The method established according to the present invention can realize the sensitive and specific detection of Vibrio parahaemolyticus, as shown in Figure 4, its detection range is 0-10 ⁴ cfu/ml, and the detection limit is 15 cfu/mL, which is lower than other methods reported so far detection limit. At the same time, after a specificity test, it was found that the detection method can realize the selective detection of Vibrio parahaemolyticus, as shown in FIG. 5 .

In summary, the present invention obtains specific phage polypeptide ligands of Vibrio parahaemolyticus through biopanning technology, and assembles them on the surface of manganese dioxide nanomaterials with good imitation peroxidase activity to obtain a Novel multifunctional nanoprobes. Using this probe, an immunocolorimetric method for the detection of Vibrio parahaemolyticus was developed with a detection limit as low as 15 cfu/mL, which is lower than that of other methods reported so far. The proposed method has the advantages of wide detection range, good selectivity, low price, and is applicable to the detection of real marine samples, opening up a promising detection method in the fields of environmental chemistry, biotechnology, biotechnology, catalytic industry and biological evaluation. method.

Above-mentioned record is the detailed description that the present invention's preferred specific embodiment and the embodiment that provide in conjunction with accompanying drawing are described in detail, but the present invention is not limited to above-mentioned embodiment and embodiment, also is not limited to nano imitation enzyme, any biological enzyme (such as spicy pepper Root peroxidase, etc.) and any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are all included in the protection scope of the present invention Inside.

SEQUENCE LISTING

<110> Qingdao University

<120> A method for detecting pathogenic microorganisms

<130>

<160> 1

<170> PatentIn version 3.1

<210> 1

<211> 9

<212> PRT

<213> Vibrio parahaemolyticus

<220>

<221> PROPEP

<222> (1)..(9)

<223>

<400> 1

Val Gln Thr Val Gln Ile Gly Ser Asp

1 5

Claims (4)

1. a kind of method of detection pathogenic microorganism, it is characterised in that：Utilize multifunctional bio nano-probe and target to be detected Pathogenic microorganism is incubated, and then be can be realized under color development system and is detected to it；Wherein, multifunctional bio nano-probe is The bacteriophage monoclonal specifically bound with pathogenic microorganism is filtered out from landscape phage library using biopanning technique, It further splits, purify the bacteriophage major capsid protein for obtaining and thering is specific polypeptide to express from purifying displaying；Then show There is the phage capsid protein of specific polypeptide to be assembled in imitative enzyme nano material；

The multifunctional bio nano-probe is to be filtered out and secondary haemolysis from landscape phage library using biopanning technique The bacteriophage monoclonal of vibrios specific binding, further purifying, which obtains displaying, has the phage protein of specific polypeptide main Capsid protein pVIII（fusion pVIII）, then by fusion pVIII be assembled in protein template method synthesis imitative enzyme nanometer material Material surface is to build the multifunctional bio nano-probe of detection vibrio parahaemolytious.

2. the method for detection pathogenic microorganism as described in claim 1, it is characterised in that：The multifunctional bio nano-probe Structure first with t-butyl carbamate（BOC）Protection fusion pVIII's is active with being combined with vibrio parahaemolytious N-terminal, then by its C-terminal 1- ethyls -3-（3- dimethyl aminopropyls）Carbodiimides（EDC）/ n-hydroxysuccinimide (NHS) it is activated, fusion pVIII is assembled in imitative enzyme nano material（Surface p VIII protein residues）Afterwards, trifluoro is added Fusion pVIII are gone BOC to protect by acetic acid so that its N-terminal restores the activity with vibrio parahaemolytious specific binding, you can.

3. the method for detection pathogenic microorganism as described in claim 2, it is characterised in that：The multifunctional bio nano-probe It is that the N-terminal of the C-terminal of fusion pVIII and the pVIII protein residues of imitative enzyme nano-material surface is interacted by amido bond What structure obtained.

4. by claim 1,2 or the method for 3 any one of them detection pathogenic microorganism, it is characterised in that：It is described multi-functional Fusion pVIII and the molar ratio of imitative enzyme nano material are 1000 in the building process of biological nano probe:1.