CN114539362B - A botulinum toxin-specific substrate peptide, detection kit and detection method - Google Patents

Abstract

The invention relates to the technical field of analysis and detection, in particular to a botulinum toxin-specific substrate peptide, a detection kit and a detection method. The present invention provides botulinum toxin-specific substrate peptides, wherein the substrate peptides are one or more of the polypeptides whose amino acid sequences are shown in SEQ ID NO.1-4. The specific, sensitive and accurate identification of botulinum toxin and its serotype can be realized by using the specific substrate peptide in combination with the serotype-specific target peptide segment generated by botulinum toxin cleavage. The present invention also provides a detection kit and a detection method based on the specific substrate peptide. Using the kit and the method, the botulinum toxin and its serotype can be identified rapidly, high-throughput, sensitively and accurately. Toxin detection provides technical support.

Description

A botulinum toxin-specific substrate peptide, detection kit and detection method

technical field

The invention relates to the technical field of analysis and detection, in particular to a botulinum toxin-specific substrate peptide, a detection kit and a detection method.

Background technique

Botulinum toxin (botulinum neurotoxin) is a natural protein toxin known to be the most toxic to humans produced by Clostridium botulinum, with a median lethal dose of 0.1-1.0ng/kg. Botulinum toxin is divided into seven serotypes: A, B, C, D, E, F, and G. Among them, A, B, E, and F are toxic to humans, C and D can cause animal poisoning, and G rarely seen. The two types of botulinum toxin A and B are widely used clinically in many fields such as neurology, plastic surgery, and rehabilitation. Clostridium botulinum poisoning can cause foodborne outbreaks. The identification and quality control of botulinum toxin serotypes play a very important role in the fields of medical treatment and epidemic prevention and control.

At present, the mouse animal experiment is still the national standard method for the detection of botulinum toxin, but this method is cumbersome to operate, has a large coefficient of variation of results, a long test period, a large demand for animals, and a high cost. Therefore, it is necessary to find an alternative method for animal experiments. Research is mainly carried out from the aspects of immunology, cytology, mass spectrometry and so on. Currently, only the botulinum toxin type A ELISA kit is the only commercially available botulinum toxin detection kit. Therefore, it is of great significance to establish a rapid and sensitive identification and typing method of botulinum toxin.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a botulinum toxin-specific substrate peptide, a detection kit and a detection method.

The main idea of the present invention is as follows: the active (toxic) botulinum toxin can be used as a specific site for enzymatic cleavage of the peptide segment. Substrate peptides specific for botulinum toxin of serotypes B, E, and F. The peptide will form two new type-specific target peptides after being specifically cleaved by active botulinum toxin in the sample. Mass spectrometry detection of these two target peptides can determine whether there is botulinum toxin in the sample and determine the serotype.

Specifically, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a botulinum toxin-specific substrate peptide, wherein the substrate peptide is one or more of the polypeptides whose amino acid sequences are shown in SEQ ID NO. 1-4, wherein SEQ ID NO. 1 In the sequence shown, Xaa represents norleucine, and in the sequence shown in SEQ ID NO. 3, Xaa represents homoarginine.

Among the above-mentioned substrate peptides, the substrate peptide whose amino acid sequence is shown in SEQ ID NO.1 is a botulinum toxin type A specific substrate peptide, which can be specifically and efficiently cleaved by botulinum toxin type A and produce two characteristic The product peptide is not cleaved by botulinum toxin types B, E and F.

The substrate peptide whose amino acid sequence is shown in SEQ ID NO.2 is a botulinum toxin type B-specific substrate peptide, which can be specifically and efficiently cleaved by botulinum toxin type B to produce two characteristic product peptides, and does not Cleaved by botulinum toxin types A, E and F.

The substrate peptide whose amino acid sequence is shown in SEQ ID NO.3 is an E-type botulinum toxin-specific substrate peptide, which can be specifically and efficiently cleaved by E-type botulinum toxin and produce two characteristic product peptides without Cleaved by botulinum toxin types A, B and F.

The substrate peptide whose amino acid sequence is shown in SEQ ID NO.4 is an F-type botulinum toxin-specific substrate peptide, which can be specifically and efficiently cleaved by F-type botulinum toxin to produce two characteristic product peptides, and does not Cut by botulinum toxin types A, B, and E.

The N-terminus and/or C-terminus of the above-mentioned substrate peptides may further comprise modifications of acetyl and amino groups.

In some embodiments of the present invention, the botulinum toxin-specific substrate peptide provided by the present invention is the following polypeptide:

Type A: Acetyl-RGSNKKPIDAGNQRATRXLGGR-NH ₂ ; wherein, X represents norleucine;

Type B: LSELDDRAADLQAGASQFESSAAKLKRKYWWKNLK;

Type E: WWWAKLGQEIDRTNRQKDXIMAKADSNKR-NH ₂ ; wherein, X represents homoarginine;

Type F: TSNRRLQQTQAQVDEVVDIRMVNVDKVLERDQKLSELDDRADAL.

Among them, the mass-to-charge ratio m/z of the A-type specific substrate peptide is 2406±1, the mass-to-charge ratio of the B-type specific substrate peptide is 4025±1, and the mass-to-charge ratio of the E-type specific substrate peptide is 4025±1. The ratio m/z was 3614±1, and the mass-to-charge ratio m/z of the F-type specific substrate peptide was 5111±1.

The identification of serotypes A, B, E and F of botulinum toxin using the above four substrate peptides combined with the characteristic target peptides produced by cleavage has high specificity and sensitivity.

In a second aspect, the present invention provides a biological material related to the above-mentioned botulinum toxin-specific substrate peptide, wherein the biological material is any one of the following (1)-(4):

(1) a nucleic acid molecule encoding the botulinum toxin-specific substrate peptide;

(2) an expression cassette comprising the nucleic acid molecule described in (1);

(3) a vector comprising the nucleic acid molecule described in (1);

(4) A host cell comprising the nucleic acid molecule described in (1), or the expression cassette described in (2), or the vector described in (3).

Based on the amino acid sequence and codon rules of the botulinum toxin-specific substrate peptide provided above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the botulinum toxin-specific substrate peptide. Degeneracy, the nucleotide sequence of the nucleic acid molecule encoding the botulinum toxin-specific substrate peptide is not unique, but all nucleic acid molecules capable of encoding polypeptides having the above amino acid sequences are within the scope of the present invention.

In the above (2), the expression cassette may consist of a promoter and a gene encoding the botulinum toxin-specific substrate peptide. Depending on the expression requirements and the different upstream and downstream sequences of the expression cassette, the expression cassette may also contain a terminator, or contain other transcriptional and translational regulatory elements such as enhancers.

In the above (3), the vector may be a plasmid vector, a bacteriophage, a virus, or the like, wherein the plasmid vector includes a replicating vector and a non-replicating vector, and the replicating vector includes an expression vector and a cloning vector.

In the above (4), the host cell is a microbial cell or an animal cell, the microbial cell includes but not limited to Escherichia coli, and the animal cell can be an animal cell commonly used for protein expression.

In a third aspect, the present invention provides a characteristic polypeptide group for detecting botulinum toxin, wherein the characteristic polypeptide group is the polypeptide group whose amino acid sequence is shown in SEQ ID NO. The group of polypeptides shown in SEQ ID NO.9-10 and/or the group of polypeptides shown in SEQ ID NO.11-12, wherein, in the sequence shown in SEQ ID NO.6, Xaa represents norleucine acid, Xaa in the sequence shown in SEQ ID NO. 9 represents homoarginine.

The characteristic peptide group is a peptide group with a mass-to-charge ratio m/z of 1427±1 and 999±1, a peptide group with a mass-to-charge ratio m/z of 1760±1 and 2283±1, and a mass-to-charge ratio m/ Groups of peptides with z of 2501±1 and 1133±1 and/or groups of peptides with m/z ratios of 3784±1 and 1346±1, respectively.

The above-mentioned characteristic polypeptide group consists of two, four, six or eight characteristic polypeptides.

The characteristic polypeptide is the product peptide produced after the botulinum toxin-specific substrate peptide is cleaved by its corresponding serotype botulinum toxin (types A, B, E, and F). Among them, the peptide group whose amino acid sequence is shown in SEQ ID NO.5-6 (mass-to-charge ratio m/z is 1427±1 and 999±1, respectively) is a substrate peptide whose amino acid sequence is shown in SEQ ID NO.1 It is produced by cleavage of botulinum toxin type A; the polypeptide group whose amino acid sequence is shown in SEQ ID NO. The substrate peptide shown in 2 is produced by cleavage of botulinum toxin type B; the peptide group whose amino acid sequence is shown in SEQ ID NO. The substrate peptide whose amino acid sequence is shown in SEQ ID NO.3 is produced by cleavage of E-type botulinum toxin; the polypeptide group whose amino acid sequence is shown in SEQ ID NO.11-12 (mass-to-charge ratio m/z is 3784±1 respectively) and 1346±1) are produced by the cleavage of F-type botulinum toxin from the substrate peptide whose amino acid sequence is shown in SEQ ID NO.4.

In some embodiments of the present invention, the characteristic polypeptides provided by the present invention are the following polypeptides:

Type A characteristic polypeptides: Acetyl-RGSNKKPIDAGNQ and RATRXLGGR-NH ₂ ; wherein, X represents norleucine;

Type B characteristic polypeptides: LSELDDRAADLQAGASQ and FESSAAKLKRKYWWKNLK;

E-type characteristic polypeptides: WWWAKLGQEIDRTNRQKDX and IMAKADSNKR-NH ₂ , where X represents homoarginine;

F-type characteristic polypeptides:

TSNRRLQQTQAQVDEVVDIRMVNVDKVLERDQ and KLSELDDRADAL.

In a fourth aspect, the present invention provides any one of the following applications (1) to (3) of the botulinum toxin-specific substrate peptide or the biological material or the characteristic polypeptide group for detecting botulinum toxin:

(1) Application in the preparation of products for the detection of botulinum toxin;

(2) Application in the preparation of products for the identification of botulinum toxin serotypes A, B, E and F;

(3) Application in the detection of botulinum toxin for non-disease diagnosis purposes.

In the above (1), the detection of botulinum toxin is preferably to detect whether the sample to be tested contains botulinum toxin, especially whether it contains botulinum toxin A, B, E and/or F serotype. Wherein, the botulinum toxin-specific substrate peptide can be prepared as the reaction substrate of the detection reagent.

In the above (2), the identification of botulinum toxin serotypes A, B, E and F is preferably to identify which of the serotypes A, B, E, and F the botulinum toxin contained in the sample to be tested is. Wherein, the botulinum toxin-specific substrate peptide can be prepared as the reaction substrate of the detection reagent.

The above-mentioned sample to be tested can be a sample that has been determined to contain or is suspected to contain botulinum toxin, and the botulinum toxin serotype contained in the sample can be identified by using the substrate peptide and detection method provided by the present invention, or it can be uncertain whether it contains botulinum toxin or not. A toxin sample can be determined whether it contains botulinum toxin and the botulinum toxin serotype it contains by using the substrate peptide and the detection method provided by the present invention.

The above-mentioned samples to be tested include but are not limited to human or animal body fluids (such as urine or blood), vomitus or excrement, food, cosmetics, medicines, environmental samples (such as water or soil), etc.

The above products include detection reagents or kits.

In a fifth aspect, the present invention provides a botulinum toxin detection kit, the kit comprises the following (1) and/or (2):

(1) the botulinum toxin-specific substrate peptide;

(2) The characteristic peptide group for the detection of botulinum toxin.

In order to facilitate the processing and detection of the sample to be tested, the kit further includes a sample enrichment reagent and/or a reaction buffer;

Wherein, the sample enrichment reagent comprises streptavidin-biotin magnetic beads coupled antibodies corresponding to botulinum toxin serotypes;

The reaction buffer contained 0.05-0.1 M Hepes, 20-30 mM DTT and 15-25 μM ZnCl ₂ .

The above-mentioned sample enrichment reagent can enrich the botulinum toxin in the sample to be tested by magnetic beads, which is convenient for the detection of botulinum toxin.

The above reaction buffer is used as a reaction for botulinum toxin cleavage of its corresponding specific substrate peptide.

In addition, the kit may further comprise one or more selected from PBST, BSA, α-hydrogen-4-hydroxycinnamic acid, and standard calibration solution for mass spectrometers.

The above kit detects the sample to be tested based on mass spectrometry (preferably matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS)) technology, and uses the sample to be tested to carry out enzymatic cleavage of the botulinum toxin-specific substrate peptide to obtain Enzymatic cleavage product; perform mass spectrometry analysis on the enzymatic cleavage product, and determine whether the sample to be tested contains botulinum toxin and the serum it contains botulinum toxin according to the amino acid sequence of the enzyme cleavage product or the mass-to-charge ratio m/z of the mass spectrum peak type.

Specifically, if the enzyme cleavage product contains a polypeptide whose amino acid sequence is shown in SEQ ID NO. The sample contains botulinum toxin serotype A;

If the digested product contains a polypeptide whose amino acid sequence is shown in SEQ ID NO.7-8, or the mass spectrum peak contains characteristic peaks with mass-to-charge ratio m/z of 1760±1 and 2283±1, the sample to be tested contains Botulinum toxin serotype B;

If the digested product contains a polypeptide whose amino acid sequence is shown in SEQ ID NO. 9-10, or the mass spectrum peak contains characteristic peaks with mass-to-charge ratio m/z of 2501±1 and 1133±1, the sample to be tested contains botulinum toxin serotype E;

If the digested product contains a polypeptide whose amino acid sequence is shown in SEQ ID NO.11-12, or the mass spectrum peak contains characteristic peaks with mass-to-charge ratio m/z of 3784±1 and 1346±1, the sample to be tested contains F serotype botulinum toxin;

Wherein, in the sequence shown in SEQ ID NO.6, Xaa represents norleucine, and in the sequence shown in SEQ ID NO.9, Xaa represents homoarginine.

In a sixth aspect, the present invention provides a non-disease diagnosis purpose botulinum toxin detection method, the method comprising:

The botulinum toxin-specific substrate peptide is subjected to enzymatic cleavage treatment with the sample to be tested to obtain an enzymatic cleavage product;

The enzyme cleavage product is subjected to mass spectrometry analysis, and at least one of the following results is obtained according to the amino acid sequence of the enzyme cleavage product or the mass-to-charge ratio m/z of the mass spectrometry peak:

(1) Whether the sample to be tested contains botulinum toxin;

(2) The serotype of botulinum toxin contained in the sample to be tested.

Among them, the method for judging the result according to the mass-to-charge ratio m/z of the mass spectrum peak is:

If the digested product contains a polypeptide whose amino acid sequence is shown in SEQ ID NO.5-6, or the mass spectrum peak contains characteristic peaks with mass-to-charge ratio m/z of 1427±1 and 999±1, the sample to be tested contains Serotype A botulinum toxin;

If the digested product contains a polypeptide whose amino acid sequence is shown in SEQ ID NO.7-8, or the mass spectrum peak contains characteristic peaks with mass-to-charge ratio m/z of 1760±1 and 2283±1, the sample to be tested contains Botulinum toxin serotype B;

If the digested product contains a polypeptide whose amino acid sequence is shown in SEQ ID NO.9-10, or the mass spectrum peak contains characteristic peaks with mass-to-charge ratio m/z of 2501±1 and 1133±1, the sample to be tested contains botulinum toxin serotype E;

If the digested product contains a polypeptide whose amino acid sequence is shown in SEQ ID NO.11-12, or the mass spectrum peak contains characteristic peaks with mass-to-charge ratio m/z of 3784±1 and 1346±1, the sample to be tested contains F serotype botulinum toxin;

Wherein, in the sequence shown in SEQ ID NO.6, Xaa represents norleucine, and in the sequence shown in SEQ ID NO.9, Xaa represents homoarginine.

Preferably, the method includes:

Mix the sample to be tested with the PBST solution (the preferred mixing ratio is: the volume ratio of the sample to be tested to the PBST solution is 1:10-20) to obtain the first mixed solution;

Mixing the first mixed solution with the sample enrichment reagent (the preferred mixing ratio is: the volume ratio of the sample enrichment reagent to the sample to be tested is 2-3:1) to obtain a second mixed solution;

After incubating the second mixed solution at 15-30°C for 30min-2h, the magnetic bead precipitation is collected to obtain the first magnetic bead precipitation (after the magnetic bead precipitation is collected, the step of washing the magnetic beads is preferably also included, and the washing can be performed first. using PBST, then water);

Mixing the first magnetic bead precipitation with BSA, incubating at 25-40° C. (preferably the incubation time is 0.5-2 h), collecting the magnetic bead precipitation to obtain the second magnetic bead precipitation;

Mix the second magnetic bead precipitation with the reaction buffer and the botulinum toxin-specific substrate peptide (the preferred mixing ratio is: the volume ratio of the sample to be tested to the botulinum toxin-specific substrate peptide is 1:10-15 , the volume ratio of the reaction buffer to the sample to be tested is 1.5-2:1), react at 30-40°C for 30min-4h;

After the reaction is completed, the reaction supernatant is collected for mass spectrometry analysis (preferably, the reaction supernatant is covered with α-hydro-4-hydroxycinnamic acid and then subjected to mass spectrometry).

Wherein, the sample enrichment reagent comprises streptavidin-biotin magnetic bead conjugated antibody;

The reaction buffer contained 0.05-0.1 M Hepes, 20-30 mM DTT and 15-25 μM ZnCl ₂ .

The beneficial effects of the present invention are: the present invention combines biochemical methods with mass spectrometry technology, and provides four specific substrates for the identification and analysis of all botulinum toxin types A, B, E and F with human toxicity. By combining these four specific substrate peptides with the serotype-specific target peptides produced by specific cleavage, more specific and sensitive identification of botulinum toxin and its serotype can be achieved.

The present invention also provides a detection kit and a detection method containing a specific substrate peptide. Using the kit and method, the botulinum toxin and its serotype can be identified rapidly, with high throughput, sensitivity and accuracy. The advantages are as follows. :

(1) The detection method of the present invention can complete the detection of four active botulinum toxins A, B, E, and F in 96 samples that are toxic to humans within 1-6 hours. The detection sensitivity is comparable to that of animal experiments, and the detection time is long. Much smaller than the current gold standard animal experiments for botulinum toxin detection (at least 3 rounds of experiments, each round of experiments at least 3 days, 96 samples need at least 1 month to complete the four serotypes of botulinum toxin detection, and requires a large number of mice);

(2) The sample consumption of the detection method of the present invention is only 10 μl, which is far lower than the sample consumption of one animal experiment (2 mice, at least 100 μl of sample);

(3) The detection method of the present invention can directly determine whether the botulinum toxin contained in the sample is active, but the only imported commercial kit at present, the ELISA kit for the detection of botulinum toxin type A, cannot determine the detected botulinum toxin type A. whether the botulinum toxin is active;

(4) Using the kit of the present invention for 18 samples for the international assessment task, 6 of them were identified within 5 hours. Compared with the ELISA and animal experiments conducted at the same time, the sensitivity and specificity were better than ELISA kit, the detection time is much shorter than the 96-hour round of animal experiments, and the minimum detection sample concentration reaches 0.5 ng/mL.

The detection kit and detection method provided by the invention can truly realize the characteristics of accuracy, high throughput, rapidity, easy use and economy, and are suitable for botulinum toxin production quality control, clinical diagnosis, disease monitoring, epidemiological investigation, public health Emergency treatment and other related fields provide new technical support for botulinum toxin detection, and provide solutions for botulinum toxin detection methods and detection reagents that lack independent intellectual property rights in my country, and botulinum toxin detection still relies on animal experiments.

Description of drawings

Fig. 1 is the specificity detection result of the substrate peptide in Example 1 of the present invention.

Figure 2 shows the mass spectrometry detection results of products obtained by cleaving substrate peptides of botulinum toxin type B at different concentrations in Example 3 of the present invention.

Fig. 3 is the optimization result of incubation time after adding streptavidin magnetic beads coupled with biotinylated monoclonal antibody in Example 3 of the present invention.

FIG. 4 is the mass spectrogram result of the positive sample detected in Example 4 of the present invention.

Detailed ways

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention.

Biotin and streptomycin-coupled magnetic beads used in the following examples were purchased from Thermo Fisher; reagents such as Hepes, DTT, ZnCl ₂ , and ultrapure water were purchased from Sigma; standard calibration solutions for mass spectrometers were purchased from From Bruker Corporation.

Example 1 Determination and synthesis of botulinum toxin serotype-specific substrate peptides

In the present invention, through screening and optimization, four substrate peptides specific for botulinum toxin A, B, E, and F serotypes are obtained respectively, and the amino acid sequences of these four substrate peptides are respectively as SEQ ID NO.1-4 said.

The above four substrate peptides are synthesized (synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.), and the synthesized substrate peptides are as follows:

Type A: Acetyl-RGSNKKPIDAGNQRATRXLGGR-NH ₂ ; wherein, X represents norleucine;

Type B: LSELDDRAADLQAGASQFESSAAKLKRKYWWKNLK;

Type E: WWWAKLGQEIDRTNRQKDXIMAKADSNKR-NH ₂ ; wherein, X represents homoarginine;

Type F: TSNRRLQQTQAQVDEVVDIRMVNVDKVLERDQKLSELDDRADAL.

After the above-mentioned four substrate peptides are specifically cleaved by their corresponding serotypes of botulinum toxin, the mass-to-charge ratios of the generated target peptides are as follows:

Type A, m/z 1427±1,999±1; Type B, m/z 1760±1, 2283±1; Type E, m/z 2501±1, 1133±1; Type F, m/z 3784± 1,1346±1.

For samples known to contain botulinum toxin of two serotypes A and B, the four specific substrate peptides A, B, E and F obtained from the above screening were simultaneously detected (refer to the detection method in Example 3). In the optimized detection method, four specific substrate peptides A, B, E and F were added to the samples of each serotype at the same time).

The results are shown in Figure 1. The above four specific substrate peptides can accurately identify the corresponding botulinum toxin serotype in the sample, and each specific substrate peptide can only be produced by the cleavage of its corresponding serotype botulinum toxin. Characteristic peptide with 100% specificity.

Example 2 Development of botulinum toxin detection kit

This embodiment provides a botulinum toxin detection kit, which includes reagent 1, reagent 2, reagent 3, reagent 4, reagent 5, reagent 6, and reagent 7;

Among them, Reagent 1 is PBST solution; Reagent 2 is sample enrichment reagent, Reagent 3 is 1-2 mg/mL BSA solution; Reagent 4 is reaction buffer; Reagent 5 is four substrate peptides synthesized in Example 1 (sequences such as SEQ ID NO.1-4); reagent 6 is a saturated solution of α-hydrogen-4-hydroxycinnamic acid (CHCA); reagent 7 is a standard calibration solution of mass spectrometer.

The composition of the above reaction buffer is as follows: 0.05M Hepes, 25mM DTT, 20μM _ZnCl2 , prepared in ultrapure water.

The above-mentioned sample enrichment reagent is used to enrich the trace amount of botulinum toxin in the sample, and it contains streptavidin-biotin magnetic bead conjugated antibody. The preparation method is as follows:

(1) Monoclonal antibody biotinylation

After 10 mg of biotin was equilibrated to room temperature, 224 μL of ultrapure water was added to obtain a 10 mM biotin stock solution; the botulinum toxin A, B, E, F serotype-specific botulinum toxin monoclonal antibodies were prepared according to the The mass is formulated into a solution with a concentration of 1-4 mg/mL; the volume of the biotin stock solution added is 60-80 V/3 (μL), where V is the antibody volume (mL). Break off the tail of the protein purification column gel column, unscrew the cap, place it in a 1.5 ml centrifuge tube, remove the storage solution by centrifugation, and suck off the excess liquid at the bottom. Change to a new collection tube and add 30-130 μL of sample. After the sample is absorbed, centrifuge to collect the product.

(2) Streptavidin Magnetic Bead-Conjugated Biotinylated Monoclonal Antibody

Take 100 μL of magnetic beads and put them in an eight-row tube, put them on a magnetic stand for 2 min, and discard the supernatant; add 100 μL of PBST, pipette evenly, and let stand on a magnetic stand for 1-2 min to remove the supernatant , add 100 μL PBST, resuspend the magnetic beads, and transfer to a 2 mL EP tube. According to the ratio of 2-15 μg antibody to 50-100 μL magnetic beads, add the biotinylated antibody solution obtained in step (1) to the resuspended magnetic beads, couple at room temperature for 1 h, and place them on a magnetic stand for 2 min, discard the supernatant, add 100 μL PBST for washing, and resuspend to obtain the sample enrichment reagent.

Example 3 Establishment of botulinum toxin detection method

1. Determination of sample dilution and detection limit

The botulinum toxin type B sample toxin concentrations were 300 ng/mL, 150 ng/mL, and 30 ng/mL, respectively, diluted 1:1, 1:4, and 1:20 by volume with PBST, and then 20 μL of streptavidin was added. The biotinylated monoclonal antibody was coupled to the magnetic beads and incubated at room temperature for 1 h. After subsequent washing, blocking and enzymatic cleavage reactions, mass spectrometry detection was performed. The optimal dilution factor was determined based on the peak response value of the enzyme digestion mass spectrometer.

The results are shown in Figure 2, and the peak response value of the enzyme digestion mass spectrum decreases with the increase of the dilution ratio. When the maximum dilution factor is 1:20, the peak of the enzyme-cleavage mass spectrum can still be detected, that is, the detection line of botulinum toxin type B can reach 1.5 ng/mL.

(2) Optimization of incubation time

The above samples were diluted with PBST according to an appropriate ratio, and after adding streptavidin magnetic beads coupled with biotinylated monoclonal antibodies, incubated at room temperature for 30 min, 1 h and 2 h, respectively. After subsequent washing, blocking and enzymatic cleavage reaction, it was detected on the machine. The optimal incubation time was determined based on the peak response value of the enzyme digestion mass spectrometer.

The results are shown in Figure 3, and the peak response value of the enzyme digestion mass spectrum increased gradually with the prolongation of the incubation time. The peak response value of the enzyme digestion mass spectrometer after incubation for 2h has a small increase. Considering the overall analysis time and the composition of different samples, the incubation time is selected to be 30min-1h.

(3) Optimization of enzyme digestion reaction time

The above samples were diluted with PBST according to an appropriate ratio, and after streptavidin magnetic beads coupled with biotinylated monoclonal antibodies were added, they were incubated at room temperature for a period of time. Followed by washing, blocking, enzyme digestion reaction. Take 30min, 1h, 2h, 4h and 6h of the enzyme digestion reaction supernatant, respectively, and test on the machine. The optimal reaction time was determined based on the peak response value of the enzyme digestion mass spectrum.

The results showed that the response value of the enzyme digestion mass spectrum peak gradually increased with the prolongation of the reaction time. When the botulinum toxin in the sample was in the high concentration range, the corresponding enzyme digestion mass spectrum peak could be detected within 30 minutes, and gradually increased with the prolongation of time. When the botulinum toxin in the sample is in the low concentration range, the corresponding enzyme-cleavage mass spectrometry peak can be detected within 4 hours, and the response value of the mass spectrometer peak can be slightly improved by extending the reaction time to 6 hours.

Therefore, considering the concentration of botulinum toxin in the sample and the overall analysis time, the supernatant was taken for detection after 30 minutes of reaction, and the reaction was stopped if botulinum toxin was detected. If no botulinum toxin was detected, continue the reaction to 4h and take the supernatant for detection.

The mass spectrometer used for data acquisition was Microflex LRF from Bruker. Data acquisition was performed in reflection mode with a mass range of 900–5500 Da, 18.00 kV for Ion source 1, 15.20 kV for Ion source 2, 8.70 kV for the lens, and 8.70 kV for the reflector. (Reflector) is 19.00 kV and the laser frequency is 60 Hz. The mass axis calibration of mass spectrometry was carried out using Bruker peptide calibrator, and the average deviation of molecular mass after calibration was less than 200 ppm.

Based on the above optimization results, the present embodiment provides a method for detecting botulinum toxin. The method utilizes the detection kit of Example 2 for detection, including the following steps:

Add 200 μL of Reagent 1 to a 2 mL EP tube, then add 10 μL of the sample to be tested, pipette evenly; add 20 μL of Reagent 2, seal with parafilm, and incubate at room temperature for 1 h; Place on the magnetic stand for 2 min, discard the supernatant; add 200 μL of reagent 1, pipette evenly, place on the magnetic stand for 2 min, discard the supernatant; add 230 μL sterilized water, pipette evenly Place on the magnetic rack for 2 min, discard the supernatant; add 20 μL of reagent 3, incubate at 37°C for 1 h, place the eight-row tube on the magnetic rack for 2 min, discard the supernatant; add 18 μL of reagent 4 , 2 μL of reagent 5, mix by pipetting, and react at 37°C for 30 min~4 h; after the reaction, place the eight-row tube on the magnetic stand for 2 min, take 1 μL of the reaction supernatant to spot the target, cover the solution of reagent 6, and put it on the Mass spectrometry analysis, the mass spectrometer was calibrated with reagent 7, and whether the sample contained botulinum toxin was determined according to whether the type-specific corresponding residue peak was detected, and which serotype of meat was determined according to the mass-to-charge ratio of the type-specific residue peak. Poisonous toxins.

Example 4 Evaluation of the actual detection effect of the test kit

Using the detection kit in Example 2 of the present invention and the detection method in Example 3, 18 samples of the international assessment task were tested. The sample was diluted 1:20, the reaction time was 30min-4h, and the mass spectrometry detection mode was reflection mode.

The results showed (Figure 4) that 6 of the positive samples were accurately and qualitatively identified within 5 hours, of which 4 samples contained botulinum toxin type A, 1 sample contained botulinum toxin type B, and 1 sample contained both A and B. serotype of botulinum toxin. Compared with the animal experiments conducted at the same time, the detection time is much shorter than the 96-hour round of the animal experiments, and the minimum concentration of the detected samples is 10ng/mL. The method has better specificity than animal experiments.

The detection method of the invention has the advantages of short analysis time, high accuracy, easy operation, low cost, etc. The method is a practical detection technology for all serotypes of botulinum toxin that is toxic to humans, and solves the problem of the lack of meat at present. The bottleneck problem of rapid toxin detection kits provides key technical support for the diagnosis of Clostridium botulinum infection and botulinum toxin-related diseases, epidemic prevention and control, and rapid detection of botulinum toxin in related products.

Although the present invention has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

sequence listing

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<212> PRT

<213> Artificial Sequence

<400> 2

Leu Ser Glu Leu Asp Asp Arg Ala Ala Asp Leu Gln Ala Gly Ala Ser

1 5 10 15

Gln Phe Glu Ser Ser Ala Ala Lys Leu Lys Arg Lys Tyr Trp Trp Lys

20 25 30

Asn Leu Lys

35

<210> 3

<211> 29

<212> PRT

<213> Artificial Sequence

<400> 3

Trp Trp Trp Ala Lys Leu Gly Gln Glu Ile Asp Arg Thr Asn Arg Gln

1 5 10 15

Lys Asp Xaa Ile Met Ala Lys Ala Asp Ser Asn Lys Arg

20 25

<210> 4

<211> 44

<212> PRT

<213> Artificial Sequence

<400> 4

Thr Ser Asn Arg Arg Leu Gln Gln Thr Gln Ala Gln Val Asp Glu Val

1 5 10 15

Val Asp Ile Arg Met Val Asn Val Asp Lys Val Leu Glu Arg Asp Gln

20 25 30

Lys Leu Ser Glu Leu Asp Asp Arg Ala Asp Ala Leu

35 40

<210> 5

<211> 13

<212> PRT

<213> Artificial Sequence

<400> 5

Arg Gly Ser Asn Lys Lys Pro Ile Asp Ala Gly Asn Gln

1 5 10

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 6

Arg Ala Thr Arg Xaa Leu Gly Gly Arg

1 5

<210> 7

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 7

Leu Ser Glu Leu Asp Asp Arg Ala Ala Asp Leu Gln Ala Gly Ala Ser

1 5 10 15

Gln

<210> 8

<211> 18

<212> PRT

<213> Artificial Sequence

<400> 8

Phe Glu Ser Ser Ala Ala Lys Leu Lys Arg Lys Tyr Trp Trp Lys Asn

1 5 10 15

Leu Lys

<210> 9

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 9

Trp Trp Trp Ala Lys Leu Gly Gln Glu Ile Asp Arg Thr Asn Arg Gln

1 5 10 15

Lys Asp Xaa

<210> 10

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 10

Ile Met Ala Lys Ala Asp Ser Asn Lys Arg

1 5 10

<210> 11

<211> 32

<212> PRT

<213> Artificial Sequence

<400> 11

Thr Ser Asn Arg Arg Leu Gln Gln Thr Gln Ala Gln Val Asp Glu Val

1 5 10 15

Val Asp Ile Arg Met Val Asn Val Asp Lys Val Leu Glu Arg Asp Gln

20 25 30

<210> 12

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 12

Lys Leu Ser Glu Leu Asp Asp Arg Ala Asp Ala Leu

1 5 10

Claims (20)

1. A botulinum toxin type A specific substrate peptide, wherein the substrate peptide is a polypeptide having an amino acid sequence shown in SEQ ID No.1, and Xaa represents norleucine in the sequence shown in SEQ ID No. 1.
2. 2. A biomaterial, characterized in that it is any one of the following (1) to (4):

   (1) a nucleic acid molecule encoding a botulinum toxin type A specific substrate peptide of claim 1;

   (2) an expression cassette comprising the nucleic acid molecule of (1);

   (3) a vector comprising the nucleic acid molecule of (1);

   (4) a host cell comprising the nucleic acid molecule of (1), or comprising the expression cassette of (2), or comprising the vector of (3).
3. 3. A characteristic polypeptide group for detecting botulinum toxin type A, which is characterized by consisting of polypeptides with amino acid sequences shown as SEQ ID No.5-6, wherein Xaa represents norleucine in the sequence shown as SEQ ID No. 6.
4. 4. Any one of the following (1) - (2) for the botulinum toxin type A specific substrate peptide according to claim 1, the biological material according to claim 2, or the set of polypeptides characteristic of botulinum toxin type A according to claim 3:

   (1) the application of the product for detecting the botulinum toxin type A;

   (2) use in the detection of botulinum toxin type A for non-disease diagnostic purposes.
5. 5. A botulinum toxin type A detection kit, characterized in that the kit comprises the following (1) and (2):

   (1) a botulinum toxin type a specific substrate peptide of claim 1;

   (2) the set of polypeptides for detecting botulinum toxin type A according to claim 3.
6. 6. The botulinum toxin detection kit of claim 5, wherein the kit further comprises a sample enrichment reagent and/or a reaction buffer;

   the sample enrichment reagent comprises streptavidin-biotin magnetic bead coupled antibody;

   the reaction buffer contains 0.05-0.1M Hepes, 20-30mM DTT and 15-25. mu.M ZnCl ₂ 。
7. 7. The botulinum toxin detection kit of claim 5 or 6, wherein the kit further comprises one or more selected from the group consisting of PBST, BSA, alpha-hydro-4-hydroxycinnamic acid, mass spectrometer standard calibration solutions.
8. 8. A method for detecting botulinum toxin type a for non-disease diagnostic purposes, the method comprising:

   carrying out enzyme digestion treatment on the botulinum toxin specific substrate peptide according to claim 1 by using a sample to be tested to obtain an enzyme digestion product;

   and (3) carrying out mass spectrometry on the enzyme digestion product, and judging whether the sample to be detected contains the botulinum toxin A or not according to the amino acid sequence of the enzyme digestion product or the mass-to-charge ratio m/z of a mass spectrum peak.
9. 9. The method for detecting botulinum toxin type A for non-disease diagnosis according to claim 8, wherein the sample to be tested contains botulinum toxin type A if the enzyme-cleaved product contains a polypeptide having an amino acid sequence as shown in SEQ ID nos. 5-6 or the mass spectrum peak contains characteristic peaks with mass-to-charge ratios m/z of 1427 ± 1 and 999 ± 1.
10. 10. The method according to claim 8 or 9, characterized in that the method comprises:

    mixing a sample to be detected with a PBST solution to obtain a first mixed solution;

    mixing the first mixed solution with a sample enrichment reagent to obtain a second mixed solution;

    incubating the second mixed solution at 15-30 ℃ for 30min-2h, and collecting magnetic bead precipitate to obtain first magnetic bead precipitate;

    mixing the first magnetic bead precipitate with BSA (bovine serum albumin), incubating at 25-40 ℃, and collecting the magnetic bead precipitate to obtain a second magnetic bead precipitate;

    mixing the second magnetic bead precipitate with a reaction buffer and the botulinum toxin type A specific substrate peptide according to claim 1, and reacting at 30-40 ℃ for 30min-4 h;

    after the reaction is finished, collecting reaction supernatant for mass spectrometry;

    wherein the sample enrichment reagent comprises streptavidin-biotin magnetic bead coupled antibody;

    the reaction buffer contains 0.05-0.1M Hepes, 20-30mM DTT and 15-25. mu.M ZnCl ₂ 。
11. The substrate peptide group specific to botulinum toxin types 11, A, B, E and F is characterized in that the substrate peptide group comprises polypeptides with amino acid sequences shown in SEQ ID NO.1-4, wherein Xaa in the sequence shown in SEQ ID NO.1 represents norleucine and Xaa in the sequence shown in SEQ ID NO.3 represents homoarginine.
12. 12. A biomaterial, characterized in that the biomaterial is any one of the following (1) to (4):

    (1) a nucleic acid molecule encoding A, B, E and a botulinum toxin type F specific substrate pepset of claim 11;

    (2) an expression cassette comprising the nucleic acid molecule of (1);

    (3) a vector comprising the nucleic acid molecule of (1);

    (4) a host cell comprising the nucleic acid molecule of (1), or comprising the expression cassette of (2), or comprising the vector of (3).
13. 13. A characteristic polypeptide group for detecting A, B, E and botulinum toxin F, wherein the characteristic polypeptide group consists of polypeptides with amino acid sequences shown as SEQ ID No.5-12, wherein Xaa represents norleucine and Xaa represents homoarginine in the sequence shown as SEQ ID No.6 and 9.
14. 14. Any one of the following (1) - (3) of the A, B, E and botulinum toxin type F specific substrate pepsets of claim 11, or the biological material of claim 12, or the detection A, B, E and botulinum toxin type F characteristic polypeptids of claim 13:

    (1) use in the manufacture of a product for detecting botulinum toxin types A, B, E and F;

    (2) use in the manufacture of a product for screening A, B, E for botulinum toxin type F;

    (3) use in the detection of botulinum toxin A, B, E and botulinum toxin type F for non-disease diagnostic purposes.
15. 15. A botulinum toxin detection kit, characterized in that the kit comprises the following (1) and (2):

    (1) the set of A, B, E botulinum toxin type F specific substrate peptides of claim 11;

    (2) the panel of polypeptides of claim 13 for detecting botulinum toxin A, B, E and type F.
16. 16. The botulinum toxin detection kit of claim 15, wherein the kit further comprises a sample enrichment reagent and/or a reaction buffer;

    the sample enrichment reagent comprises a streptavidin-biotin magnetic bead coupled antibody;

    the reaction buffer contains 0.05-0.1M Hepes, 20-30mM DTT and 15-25. mu.M ZnCl ₂ 。
17. 17. The botulinum toxin detection kit of claim 15 or 16, wherein the kit further comprises one or more selected from the group consisting of PBST, BSA, alpha-hydro-4-hydroxycinnamic acid, mass spectrometer standard calibration solutions.
18. 18. A method for detecting botulinum toxin types A, B, E and F for non-disease diagnostic purposes, the method comprising:

    performing enzyme digestion treatment on A, B, E and F type botulinum toxin specific substrate peptide group according to claim 11 by using a sample to be tested to obtain an enzyme digestion product;

    performing mass spectrometry on the enzyme digestion product, and obtaining at least one of the following results according to the amino acid sequence of the enzyme digestion product or the mass-to-charge ratio m/z of a mass spectrum peak:

    (1) whether the sample to be tested contains botulinum toxin;

    (2) the sample to be tested contains the serotype of botulinum toxin.
19. 19. The method for detecting A, B, E and F botulinum toxins according to claim 18, wherein if the enzyme-cleaved product contains a polypeptide with an amino acid sequence as shown in SEQ ID No.5-6, or the mass spectrum peak contains characteristic peaks with a mass/charge ratio m/z of 1427 ± 1 and 999 ± 1, the sample to be tested contains A botulinum toxin;

    if the enzyme digestion product contains polypeptide with an amino acid sequence shown as SEQ ID NO.7-8, or the mass spectrum peak contains characteristic peaks with mass-to-charge ratios m/z of 1760 +/-1 and 2283 +/-1, the sample to be detected contains botulinum toxin type B;

    if the enzyme digestion product contains a polypeptide with an amino acid sequence shown as SEQ ID NO.9-10, or the mass spectrum peak contains characteristic peaks with mass-to-charge ratios m/z of 2501 +/-1 and 1133 +/-1, the sample to be detected contains the botulinum toxin E;

    if the enzyme digestion product contains polypeptide with an amino acid sequence shown as SEQ ID NO.11-12, or the mass spectrum peak contains characteristic peaks with mass-to-charge ratios m/z of 3784 +/-1 and 1346 +/-1, the sample to be detected contains the botulinum toxin F;

    wherein Xaa represents norleucine in the sequence shown in SEQ ID NO.6, and Xaa represents homoarginine in the sequence shown in SEQ ID NO. 9.
20. 20. The method for detection of botulinum toxin A, B, E and F for non-disease diagnostic purposes according to claim 18 or 19, comprising:

    mixing a sample to be detected with a PBST solution to obtain a first mixed solution;

    mixing the first mixed solution with a sample enrichment reagent to obtain a second mixed solution;

    incubating the second mixed solution at 15-30 ℃ for 30min-2h, and collecting magnetic bead precipitate to obtain first magnetic bead precipitate;

    mixing the first magnetic bead precipitate with BSA (bovine serum albumin), incubating at 25-40 ℃, and collecting the magnetic bead precipitate to obtain a second magnetic bead precipitate;

    mixing the second magnetic bead precipitate with a reaction buffer solution and the A, B, E and F botulinum toxin specific substrate peptide group according to claim 11, and reacting at 30-40 ℃ for 30min-4 h;

    after the reaction is finished, collecting reaction supernatant for mass spectrometry;

    wherein the sample enrichment reagent comprises streptavidin-biotin magnetic bead coupled antibody;

    the reaction buffer contains 0.05-0.1M Hepes, 20-30mM DTT and 15-25. mu.M ZnCl ₂ 。

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