Artemisia argyi and ragweed pollen allergen detection kit and detection method thereof

Abstract

The invention discloses a mugwort and ragweed pollen allergen detection kit and a detection method thereof. The kit comprises an incubation tank, a sample diluent, a sample cleaning solution, an enzyme-labeled antibody binding solution, a substrate solution and a test strip, the test strip is provided with a positive control area, a negative control area and a plurality of test areas for pollen allergens of mugwort, ragweed and absinese wormwood; the detection method comprises the following steps: S1, balancing to room temperature; s2, performing incubation; s3, cleaning is conducted; s4, binding an antibody; s5, cleaning; s6, mixing the substrates; and S7, obtaining a color development result. The artemisia argyi and ragweed pollen allergen detection kit has the beneficial effects that the artemisia argyi and ragweed pollen allergen detection kit disclosed by the invention can be used for qualitatively detecting various allergen components of allergic patients, can meet the requirements of the patients and fully reduces the detection cost, and the kit is low in manufacturing cost, can be widely popularized and used and is simple and convenient to prepare; the detection method is simple, easy to operate, time-saving and labor-saving in detection and high in detection efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, and particularly relates to a detection kit for Artemisia argyi and ragweed pollen allergen and a detection method thereof. BACKGROUND

[0002] At present, commercially available allergen-specific IgE antibody detection kits mostly use allergen factor protein extracts, and cannot go deep into specific allergen monomers, that is, cannot perform more detailed allergen component analysis, which means that it is impossible to accurately assess which one or several allergen components in the allergen have an allergic reaction in patients, so as to accurately perform clinical diagnosis.

[0003] The patent No. 201810913872.5 discloses a combination of Artemisia pollen allergens, application and kit. Through research on 240 cases of Artemisia pollen allergy, it is found that when at least three of Art v 1, Art ar 2, Art v 3 and Art an 7 in the detection results of the Artemisia pollen allergy cases are positive, the incidence of allergic asthma is 57%, and when the number of positive allergens is less than 3, the incidence of allergic asthma is 24%, and the former is 2.4 times the latter. Through detection of allergic patients, patients with high risk of allergic asthma are detected and judged, and only through drug control and desensitization treatment can the allergic asthma be eradicated, so as to avoid the occurrence of allergic asthma. However, the above-mentioned patent has the following problems: 1. The above-mentioned content only detects the content of Art v 1, Art ar 2, Art v 3 and Art an 7 in the Artemisia pollen in the serum of patients, and the detection type cannot meet the needs of patients; 2. The above-mentioned content uses ImmunoCAP-100 instrument for detection, and the ImmunoCAP-100 instrument is expensive, and can only detect the content of one kind of allergen-specific IgE in the serum of patients at a time. When the detection type is too many, the detection cost is increased, the use of the instrument is increased, the service life of the instrument is reduced, and the detection process is complicated, time-consuming and laborious, and the detection efficiency is low. SUMMARY

[0004] Therefore, the present application aims to provide an Artemisia argyi and ragweed pollen allergen detection kit and a detection method thereof. The kit provided by the present application can qualitatively detect multiple allergen components of allergic patients, can meet the needs of patients, sufficiently reduces the detection cost, and has low manufacturing cost, can be widely used, is simple and convenient to prepare, the detection method is simple and easy to operate, saves time and labor, and has high detection efficiency.

[0005] The present invention discloses a reagent kit for detecting pollen allergens of Artemisia argyi and ragweed, comprising an incubation tank, a sample dilution solution, a sample washing solution, an enzyme-labeled antibody binding solution, a substrate solution, and a test strip. The test strip is provided with a positive control area, a negative control area, and several test areas for pollen allergens of Artemisia argyi, ragweed, and Artemisia scoparia. The pollen allergens of Artemisia argyi include Art an 1, Art an 2, Art an 3, Art an 4, and Art an 7; the pollen allergens of ragweed include Amb a 1, Amb a 3, Amb a 9, Amb a 11, and Amb a 12.

[0006] Among the pollen allergens of Artemisia argyi, Art an 1 (GenBank ID ANC85006.1), Art an 2 (GenBank ID AVD29822.1), Art an 3 (GenBank ID ANC85017.1), and Art an 4 (GenBank ID UYS93123.1) were all obtained by recombinant expression of SF9 in insect cells, while Art an 7 (from Chinese invention patent publication number CN117821408A) was also obtained. Among the pollen allergens of Ragweed, Amb a 1 (GenBank ID AAA32665.1), Amb a 3 (GenBank ID P00304), Amb a 9 (GenBank ID AAX77684.1), Amb a 11 (GenBank ID AHA56102.1), and Amb a... 12 (GenBank ID ANZ22900.1) were obtained by recombinant expression in Escherichia coli.

[0007] Furthermore, the sample diluent has a pH of 5.0-6.0, with a concentration of 0.1-0.5 mol / L for 2-(N-morpholino)ethanesulfonic acid (MES) and a concentration of 0.1-1.0 mol / L for NaCl; the sample washing solution has a pH of 7.4-8.0, with a concentration of 10-50 mmol / L for Tris-HCl and a concentration of 100-150 mmol / L for NaCl; the enzyme-labeled antibody conjugate is an anti-human IgE antibody solution labeled with alkaline phosphatase at a final concentration of 0.1-1 μg / mL; and the substrate solution contains 0.15-0.30 mg / mL for 5-bromo-4-chloro-3-indole phosphate and 0.3-0.5 mg / mL for nitroblue tetrazolium.

[0008] Furthermore, the preparation method of the test strip includes the following steps: (1) Allergen protein dissolution: The pollen allergens of Artemisia argyi, ragweed and wormwood are dissolved in MES buffer, wherein the concentration of the allergen is 0.1-1.0 mg / mL, to obtain an allergen solution.

[0009] (2) Activation of allergen protein: Add EDC zero-length crosslinking agent and N-hydroxysuccinimide (NHS) to the allergen solution in step (1) to make their final concentrations 1-10 mmol / L and 1-10 mmol / L, respectively; then vortex mix and incubate at room temperature in the dark for 15-30 min to obtain an allergen protein activation solution; wherein 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is a zero-length crosslinking agent and N-hydroxysuccinimide (NHS) in a synergistic reaction mechanism. Specifically, EDC reacts with the carboxyl group (-COOH) in proteins or other molecules to generate an O-acylisourea intermediate. This intermediate has high reactivity but poor stability and is prone to hydrolysis failure. NHS then reacts further with the O-acylisourea intermediate to generate an NHS ester intermediate. The stability of NHS ester is much higher than that of O-acylisourea, which can reduce hydrolysis side reactions and prolong the existence time of the active intermediate.

[0010] (3) Activation termination: Add 2-mercaptoethanol to the allergen protein activation solution in step (2) to a final concentration of 10-50 mmol / L, incubate for 12-17 min to quench unreacted EDC zero-length cross-linking agent; then use a desalting column to elute and purify, wherein the elution buffer is a coupling buffer to remove unreacted 2-mercaptoethanol and inactivated EDC zero-length cross-linking agent, to obtain an allergen activated protein solution.

[0011] (4) Coupling of allergen-activated proteins on amino nylon membrane: The amino nylon membrane is laid flat in the reaction vessel, and then the allergen-activated protein solution in step (3) is added dropwise until it completely covers the surface of the amino nylon membrane. The NHS ester in the allergen-activated protein solution undergoes a nucleophilic substitution reaction with the primary amino group of the adjacent protein to form a stable amide bond (peptide bond). This step achieves intermolecular covalent connection, promotes the covalent cross-linking of protein carboxyl and amino groups, and couples the allergen protein to the surface of the amino nylon membrane. Then, a shaker is used to promote uniform contact of the solution with the membrane surface. After incubation at room temperature for 1-3 h or at 3-5℃ for 7-9 h, the membrane is finally rinsed and washed with PBS buffer to obtain the allergen nylon membrane.

[0012] (5) Blocking non-specific sites: Immerse the allergen nylon membrane from step (4) in bovine serum albumin blocking solution and incubate at room temperature for 0.5-1.5 h or at 3-5 °C for 7-9 h; then rinse with washing solution 2-4 times, and finally dry to obtain the test strip.

[0013] Furthermore, the pH of the coupling buffer in step (3) is 7.0-8.0, wherein the concentration of sodium phosphate is 50-100 mmol / L and the concentration of NaCl is 100-200 mmol / L.

[0014] Furthermore, in step (4), rinse with PBS buffer 2-4 times, each time for 4-6 minutes; then rinse with PBS buffer 1-3 times.

[0015] Furthermore, the preparation method of the bovine serum albumin blocking solution in step (5) is as follows: Bovine serum albumin is added to PBS buffer to prepare a bovine serum albumin blocking solution with a mass concentration of 3-5%. The phosphate buffer maintains the pH value through acid-base balance to ensure the structural stability of biomolecules (such as antibodies and proteins) and prevent denaturation and inactivation.

[0016] Furthermore, in step (5), the pH of the washing solution is 7-8, the concentration of NaCl is 7-9 g / L, the concentration of KCl is 0.1-0.3 g / L, the concentration of Na2HPO4 is 1-2 g / L, the concentration of KH2PO4 is 0.1-0.3 g / L, and the concentration of surfactant Tween-20 is 0.05-0.15%. The Na⁺ and Cl⁻ in the washing solution shield the electrostatic charge on the sample surface, reducing the electrostatic adsorption of non-target molecules to the solid support (amino nylon membrane). The function of Tween-20 is to reduce the surface tension of the liquid, promote the peeling of residual reagents from the surface of the solid support, and reduce background noise. It works synergistically with bovine serum albumin blocking solution to cover unbound hydrophobic regions on the solid support, further suppressing false positive signals.

[0017] Another aspect of the technical solution of the present invention discloses a detection method for a reagent kit for detecting Artemisia argyi and ragweed pollen allergens, which includes the following steps: S1: Equilibrate the serum sample, sample diluent, sample washing solution, enzyme-labeled antibody conjugate solution, and substrate solution to room temperature; S2: Place the test strip into the incubation tank, then place the incubation tank on a shaking incubator. Add 0.5-2 ml of the sample diluent from step S1 to the incubation tank, followed by 100-200 μL of the serum sample from step S1. Then start the shaking incubator and incubate at room temperature for 60-80 minutes, after which stop the shaking incubator. The MES in the sample diluent is used to maintain an acidic environment, inhibit protease activity, and prevent protein denaturation or aggregation. NaCl enhances protein solubility and reduces non-specific binding by shielding charges and regulating the hydration layer, thus regulating ionic strength. Simultaneously, NaCl maintains an osmotic pressure close to that of serum, preventing cell rupture or dehydration and maintaining sample integrity. The acidic environment and ionic strength synergistically reduce matrix effects (such as lipid and enzyme interference), adapting to subsequent detection requirements.

[0018] S3: Drain the liquid from the incubation tank in step S2, add 2-5 ml of the sample washing solution from step S1, then start the shaking incubator and wash for 10-20 minutes. Repeat this process 2-4 times to complete the washing. The Tris-HCl in the sample washing solution has a pH range of 7.4-8.0, which is close to the physiological pH of human blood (7.4). This pH range can resist pH fluctuations caused by metabolic products, impurities, or chemical reactions in the serum sample during the washing process, preventing proteins (such as antibodies, enzymes, albumin, etc. in serum) from denaturing, agglomerating, or becoming inactive due to drastic pH changes, thus ensuring the structural stability of the target components. The NaCl concentration is close to that of human physiological saline (approximately 150 mmol / L), forming an isotonic solution. This prevents cells in the serum (such as residual red blood cells) from rupturing due to osmotic pressure changes, and prevents the release of cell contents (such as hemoglobin) from interfering with subsequent detection.

[0019] S4: Add 1-2 ml of the enzyme-labeled antibody conjugation solution from step S1 to the incubation tank in step S3, then start the shaking incubator and incubate at room temperature for 30-40 minutes before stopping the shaking incubator; the enzyme-labeled anti-human IgE antibody binds, specifically recognizes IgE, and forms a complex.

[0020] S5: Pour out the liquid in the incubation tank in step S4, add 2-5 ml of the sample cleaning solution in step S1, then start the shaking table and clean for 10-20 minutes. Stop the shaking table and repeat 2-4 times to complete the cleaning.

[0021] S6: Add 1-2 ml of the substrate solution from step S1 to the incubation tank in step S5, then start the shaking incubator and incubate at room temperature for 30-60 min before stopping the shaking incubator; 5-bromo-4-chloro-3-indole phosphate (BCIP) in the substrate solution is hydrolyzed by alkaline phosphatase to 5-bromo-4-chloro-3-indolephenol (reduced state). Since 5-bromo-4-chloro-3-indolephenol is unstable and easily oxidized by oxygen to dimer indole ether (colorless or light-colored), it cannot directly form a stable signal. Nitroblue tetrazolium (NBT) in the substrate solution, as a strong oxidant, can quickly capture the electrons released by 5-bromo-4-chloro-3-indolephenol and reduce it to insoluble purple formazan precipitate.

[0022] S7: Remove the test strip from the incubation tank in step S6, use clean absorbent paper to dry the remaining moisture in the incubation tank, and let the test strip dry. Visually compare the color development results of the test area of ​​the test strip through the positive control area.

[0023] Furthermore, the swing frequency of the swinging bed is 30-60 times / min.

[0024] Advantages of this invention: 1. This invention discloses a test kit for allergens in Artemisia argyi and ragweed pollen, which can qualitatively detect multiple allergen components such as Art an 1, Art an 2, Art an 3, Art an 4 and Art an 7 in Artemisia argyi pollen and Amba 1, Amba 3, Amba 9, Amba 11 and Amba 12 in ragweed pollen in allergy patients, thus meeting the needs of patients.

[0025] 2. This invention discloses a detection kit for Artemisia argyi and ragweed pollen allergens. One kit can be used to detect 10 allergen-specific IgE in the serum of patients, which significantly reduces the detection cost. Moreover, the kit has low manufacturing cost and can be widely used.

[0026] 3. This invention discloses a detection kit for Artemisia argyi and ragweed pollen allergens. In its preparation method, EDC zero-length crosslinking agent and N-hydroxysuccinimide are added to the allergen solution to activate the allergen protein. Then, the activation is stopped, and the activated allergen protein is coupled on the amino nylon membrane. The allergen protein is fully coupled to the surface of the amino nylon membrane with good binding. Moreover, the above operation is simple and convenient.

[0027] 4. This invention discloses a detection method for a test kit for Artemisia argyi and ragweed pollen allergens. The detection method is simple and easy to operate. By visually comparing the color development results of the test area of ​​the test strip with the positive control area, the allergen components of the allergic patient can be quickly identified. Therefore, the detection is time-saving, labor-saving, and highly efficient. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a schematic diagram of the test strip in Embodiment 1 of the present invention. Detailed Implementation

[0030] The present invention will be further described in detail below through embodiments.

[0031] Example 1: A kit for detecting Artemisia argyi and ragweed pollen allergens, comprising an incubator, sample dilution buffer, sample washing buffer, enzyme-labeled antibody binding buffer, substrate solution, and test strips, such as... Figure 1 As shown, the test strip has a positive control area, a negative control area, and several test areas for pollen allergens of Artemisia argyi, ragweed, and Artemisia argyi; the pollen allergens of Artemisia argyi include Artan 1, Artan 2, Artan 3, Artan 4, and Artan 7; the pollen allergens of ragweed include Amb a 1, Amb a 3, Amb a 9, Amb a 11, and Amb a 12.

[0032] The sample dilution buffer had a pH of 5.0-6.0, with a concentration of 0.1-0.5 mol / L for 2-(N-morpholino)ethanesulfonic acid (MES) and 0.1-1.0 mol / L for NaCl; the sample washing buffer had a pH of 7.4-8.0, with a concentration of 10-50 mmol / L for Tris-HCl and 100-150 mmol / L for NaCl; the enzyme-labeled antibody conjugate was an anti-human IgE antibody solution labeled with alkaline phosphatase at a final concentration of 0.1-1 μg / mL; the substrate solution contained 0.15-0.30 mg / mL of 5-bromo-4-chloro-3-indole phosphate and 0.3-0.5 mg / mL of nitroblue tetrazolium.

[0033] The preparation method of the test strip includes the following steps: (1) Dissolution of allergen proteins: The pollen allergens of Artemisia argyi, ragweed and wormwood were dissolved in MES buffer, with the concentration of allergens being 0.1-1.0 mg / mL, to obtain allergen solutions.

[0034] (2) Activation of allergen protein: Add EDC zero-length crosslinking agent and N-hydroxysuccinimide (NHS) to the allergen solution in step (1) to make their final concentrations 1-10 mmol / L and 1-10 mmol / L, respectively; then vortex mix and incubate at room temperature in the dark for 15-30 min to obtain the allergen protein activation solution.

[0035] (3) Activation termination: Add 2-mercaptoethanol to the allergen protein activation solution in step (2) to a final concentration of 10-50 mmol / L, incubate for 12-17 min to quench unreacted EDC zero-length cross-linking agent; then use a desalting column to elute and purify, wherein the eluent is a coupling buffer to remove unreacted 2-mercaptoethanol and inactivated EDC zero-length cross-linking agent, to obtain an allergen activated protein solution; wherein the pH of the coupling buffer is 7.0-8.0, the concentration of sodium phosphate is 50-100 mmol / L, and the concentration of NaCl is 100-200 mmol / L.

[0036] (4) Coupling of allergen-activated protein on amino nylon membrane: Spread the amino nylon membrane flat in the reaction vessel, and then add the allergen-activated protein solution from step (3) until it completely covers the surface of the amino nylon membrane; then use a shaker to promote uniform contact of the solution with the membrane surface, and then incubate at room temperature for 1-3 h or at 3-5℃ for 7-9 h, and finally rinse and wash with PBS buffer. Specifically, rinse with PBS buffer 2-4 times, each time for 4-6 min; then rinse with PBS buffer 1-3 times to obtain the allergen nylon membrane.

[0037] (5) Blocking non-specific sites: Immerse the allergen nylon membrane from step (4) in bovine serum albumin blocking solution and incubate at room temperature for 0.5-1.5 h or at 3-5 °C for 7-9 h; then rinse with washing solution 2-4 times, and finally dry to obtain the test strip.

[0038] The preparation method of bovine serum albumin blocking solution is as follows: Bovine serum albumin is added to PBS buffer to prepare a bovine serum albumin blocking solution with a mass concentration of 3-5%.

[0039] The washing solution has a pH of 7-8, with NaCl concentration of 7-9 g / L, KCl concentration of 0.1-0.3 g / L, Na2HPO4 concentration of 1-2 g / L, KH2PO4 concentration of 0.1-0.3 g / L, and surfactant Tween-20 concentration of 0.05-0.15%.

[0040] Example 2: The detection method using the Artemisia argyi and ragweed pollen allergen detection kit shown in Example 1 includes the following steps: S1: Equilibrate the serum sample, sample diluent, sample washing solution, enzyme-labeled antibody binding solution, and substrate solution to room temperature.

[0041] S2: Place the test strip into the incubation tank, then place the incubation tank on a shaking table. Add 0.5-2 ml of the sample diluent from step S1 to the incubation tank, and then add 100-200 μL of the serum sample from step S1. Then start the shaking table and incubate at room temperature for 60-80 minutes before stopping the shaking table.

[0042] S3: Pour out the liquid in the incubation tank in step S2, add 2-5 ml of the sample cleaning solution in step S1, then start the shaking incubator and clean for 10-20 minutes. Stop the shaking incubator and repeat 2-4 times to complete the cleaning.

[0043] S4: Add 1-2 ml of the enzyme-labeled antibody conjugate solution from step S1 to the incubation tank in step S3, then start the shaking incubator and incubate at room temperature for 30-40 minutes before stopping the shaking incubator.

[0044] S5: Pour out the liquid in the incubation tank in step S4, add 2-5 ml of the sample cleaning solution in step S1, then start the shaking incubator and clean for 10-20 minutes. Stop the shaking incubator and repeat 2-4 times to complete the cleaning.

[0045] S6: Add 1-2 ml of the substrate solution from step S1 to the incubation tank in step S5, then start the shaking incubator and incubate at room temperature for 30-60 minutes before stopping the shaking incubator.

[0046] S7: Remove the test strip from the incubation tank in step S6, use clean absorbent paper to dry the remaining moisture in the incubation tank, and let the test strip dry. Visually compare the color development results of the test area of ​​the test strip through the positive control area.

[0047] The oscillation frequency of the rocking table in steps S2-S6 above is 30-60 times / min.

[0048] Example 3: The preparation method of recombinant expressed allergen proteins Artan 1, Artan 2, Artan 3 and Artan 4 from the Artemisia pollen allergen in Example 1 is as follows: 1. Construction of recombinant expression plasmids The original nucleic acid sequences encoding the genes for Art an 1, Art an 2, Art an 3, and Art an 4 were obtained through WHO / IUIS Allergen Nomenclature Subcommittee data (www.allergn.org) and NCBI searches (https: / / www.ncbi.nlm.nih.gov / nuccore / 1190354091). Signal peptides were predicted using the SignalP-5.0 website (https: / / services.healthtech.dtu.dk / services / SignalP-5.0 / ), and nucleic acid sequences were optimized using the NetNGlyc-1.0 website (https: / / services.healthtech.dtu.dk / services / NetNGlyc-1.0 / ) and the UniProt website (https: / / www.uniprot.org / uniprotkb / A0A2H4HHY6 / entry#ptm_processing). The sequences were then fully synthesized by General Biotech (Anhui) Co., Ltd. His and Flag tags were introduced at the C-terminus and N-terminus of the sequences, respectively. The optimized gene sequences were then inserted into the Pfastbac1 expression vector with EcoRI(GAATTC)-XhoI(CTCGAG) restriction enzyme sites to construct recombinant expression plasmids Art an 1, Art an 2, Art an 3, and Art an 4, respectively.

[0049] 2. Recombinant protein expression 2.1 Plasmid transfection Approximately 10 µl of each of the constructed Art an 1, Art an 2, Art an 3, and Art an 4 recombinant expression plasmids were added to *E. coli* DH10Bac competent cells. After slight mixing, the cells were incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and then cultured in 500 µL of SOC medium for 4 h. The cells were then plated on triple-antibody LB agar plates (containing 7 µg / ml gentamicin, 100 µg / ml kanamycin, 10 µg / ml tetracycline, and 40 µg / ml IPTG, and pre-coated with 40 µl of 2% X-gal) and incubated upside down at 37°C for 48 h. White-white colonies were selected using blue-white screening and added to 3 mL of SOC medium. The cells were incubated overnight at 37°C, 200 rpm. 1.5 mL of the overnight bacteria was collected by centrifugation at 10000 × g for 1 min, and the supernatant was discarded. Add another 1.5 mL of overnight bacteria, centrifuge again to collect 3 mL of overnight bacteria per tube.

[0050] Recombinant expression plasmids were extracted using the M5 HiPer Multi-color Plasmid Miniprep Kit (with column) and stored at -20°C. PCR identification and verification were performed using universal primer sequences M13F (5'-CCCAGTCACGACGTTGTAAAACG-3') and M13R (5'-AGCGGATAACAATTTCACACAGG-3'), analyzed by agarose gel electrophoresis. The recombinant expression plasmids were then double-digested with EcoRI (GAATTC)-XhoI (CTCGAG), and the products were analyzed by agarose gel electrophoresis.

[0051] 2.2 Insect Cell Culture and Transfection SF9 insect cells stored in liquid nitrogen were revived in a 37 ℃ water bath and resuspended in 10 mL of insect cell culture medium (containing 10% serum) (SIM SF Expression Medium (For SF9, SF21) (Serum free, animal free), MSF-AF). After centrifugation at 1000 r / min for 5 min, the cells were transferred to a 250 mL flask, and 15 mL of insect cell culture medium (containing 10% serum) was added to prepare a 25 mL cell suspension. The cells were cultured at 27 ℃ and 125 r / min. When the cell density reached 3.0-3.5×106 cells / mL (usually within 24-48 h), the cell density was adjusted to 0.7-1.0×106 cells / mL, and the cells were passaged in suspension.

[0052] The double-digested recombinant plasmid was transfected into SF9 insect cells using a bacmid liposome packaging transfection method to prepare P1 and P2 generation baculoviruses. The recombinant expression plasmids pFastBac-Art an 1, pFastBac-Art an 2, pFastBac-Art an 3, and pFastBac-Art an 4 were diluted to 2 μg per 100 μL in serum-free insect cell culture medium, followed by incubation with 3 times the volume of transfection reagent for 15 min. 100 μL of SF9 cells were seeded into 96-well plates, and 10 μL of transfection reagent containing the recombinant expression plasmid was added to the cells to be transfected, followed by incubation for 24 h. The P1 virus strain was extracted and isolated, and the strain was amplified to produce a high-titer P2 strain, which was then collected. During the expression process, cell lysis was observed under a microscope, indicating successful baculovirus infection and a normal expression process. Finally, 400 mL of expression cells were collected for purification.

[0053] 3. Protein purification Centrifuge at 4000 r / min for 10 min at 4℃, collect the expressed insect SF9 cells, resuspend in lysis buffer, and sonicate. Sonication conditions: 300 W, 3 s sonication, 5 s interval, 30 min. Centrifuge the sonicated samples at 18000 r / min for 15 min at 4℃.

[0054] The supernatant was purified by Ni affinity chromatography. 1 mL of Ni IDA Beads was added and incubated at 4 °C for 30 h. After incubation, the sample was loaded onto the column and flowed through the nickel column at a rate of 0.5 mL / min. After flow-through, the column was washed with 60 mL of washing buffer. After washing away any contaminating proteins, the sample was eluted with 10 mL of elution buffer to obtain the target proteins, namely the recombinant Artemisia pollen allergens Art an1, Art an2, Art an3, and Art an4.

[0055] Example 4: The method for preparing recombinant expressed allergen proteins of Amb a 1, Amb a 3, Amb a 9, Amb a 11 and Amb a 12 from the ragweed pollen allergen in Example 1 is as follows: 1. Construction of recombinant expression plasmids The original nucleic acid sequences encoding the genes Amb a 1, Amb a 3, Amb a 9, Amb a 11, and Amb a 12 were obtained through WHO / IUIS Allergen Nomenclature Subcommittee data (www.allergn.org) and NCBI searches (https: / / www.ncbi.nlm.nih.gov / nuccore / MH220501.1). The nucleic acid sequences were optimized and synthesized by General Biotechnology (Anhui) Co., Ltd. His tags and Flag tags were introduced at the C-terminus and N-terminus of the sequences, respectively. The optimized gene sequences were inserted into the pcDNA3.1(+) expression vector, with BamHI-XhoI restriction enzyme sites, to construct recombinant expression plasmids for Amb a1, Amb a 3, Amb a 9, Amb a 11, and Amb a 12, respectively.

[0056] 2. Recombinant protein expression 2.1 Plasmid transfection Approximately 10 µl of each of the constructed recombinant expression plasmids Amb a 1, Amb a 3, Amb a 9, Amb a 11, and Amb a 12 were added to *E. coli* T1 competent cells. After slight mixing, the cells were incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and immediately placed on ice for 2 min. 400 µl of preheated LB medium (antibiotic-free) was added, and the cells were incubated at 37°C with shaking for 1 h. 100 µl of the bacterial culture was evenly spread onto LB agar plates containing Ampicillini antibiotic resistance and incubated overnight at 37°C with shaking. Half a single colony was added to LB liquid medium containing Ampicillini antibiotic resistance and incubated overnight at 37°C with shaking. 1.5 mL of the overnight bacteria was centrifuged at 10000 x g for 1 min, and the supernatant was discarded. Another 1.5 mL of the overnight bacteria was added, and the cells were centrifuged again to collect the bacteria. 3 mL of overnight bacteria were collected from each tube.

[0057] Recombinant expression plasmids were extracted using a plasmid miniprep kit (DP103), analyzed by agarose gel electrophoresis, and stored at -20°C. The recombinant expression plasmids were then digested with BamHI-XhoI, and the products were analyzed by agarose gel electrophoresis.

[0058] 2. Protein purification Bacterial culture was carried out in LB liquid medium containing 50 µg / ml of Ampicillini antibiotic resistance. When the OD value reached 0.6, IPTG inducer was added, and the culture was carried out overnight at 20°C for high-level expression. Cells were collected by centrifugation. Small-scale samples were collected, processed, and subjected to affinity purification. Crude protein was collected: cell cells were dissolved in buffer C, sonicated, and the supernatant was collected by centrifugation. 5 ml of Ni-NTA was used to wash and equilibrate the column with 5 column volumes of binding buffer. The crude protein was incubated with the equilibrated column packing for 1 h, and the eluent was collected. The column was washed and equilibrated with binding buffer. The column was washed with washing buffer, and the eluent was collected. Elution buffer was used to elute, and the eluent was collected. The crude protein and eluent fractions were processed separately, and samples were prepared for SDS-PAGE detection. The target proteins, namely recombinant ragweed pollen allergens Amb a1, Amb a3, Amb a9, Amb a11 and Amb a12, were obtained.

[0059] The above are preferred embodiments of the present invention. For those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A kit for detecting Artemisia argyi and ragweed pollen allergens, comprising an incubation tank, sample dilution buffer, sample washing buffer, enzyme-labeled antibody binding buffer, and substrate solution, characterized in that, It also includes test strips, which have a positive control area, a negative control area, and several test areas for pollen allergens of Artemisia, ragweed, and Artemisia argyi; the pollen allergens of Artemisia include Art an 1, Art an 2, Art an 3, Art an 4, and Art an 7; the pollen allergens of ragweed include Amba 1, Amba 3, Amba 9, Amba 11, and Amba 12.

2. The Artemisia argyi and ragweed pollen allergen detection kit according to claim 1, characterized in that, The sample diluent has a pH of 5.0-6.0, with 2-(N-morpholino)ethanesulfonic acid at a concentration of 0.1-0.5 M and NaCl at a concentration of 0.1-1.0 M; the sample washing solution has a pH of 7.4-8.0, with Tris-HCl at a concentration of 10-50 mmol / L and NaCl at a concentration of 100-150 mmol / L; the enzyme-labeled antibody conjugate is an anti-human IgE antibody solution labeled with alkaline phosphatase at a final concentration of 0.1-1 μg / mL; the substrate solution contains 5-bromo-4-chloro-3-indole phosphate at a concentration of 0.15-0.30 mg / mL and nitroblue tetrazolium at a concentration of 0.3-0.5 mg / mL.

3. The Artemisia argyi and ragweed pollen allergen detection kit according to claim 1, characterized in that, The preparation method of the test strip includes the following steps: (1) Allergen protein dissolution: The pollen allergens of Artemisia argyi, ragweed and Artemisia argyi were dissolved in MES buffer, wherein the concentration of the allergens was 0.1-1.0 mg / mL, to obtain allergen solutions; (2) Activation of allergen protein: Add EDC zero-length crosslinking agent and N-hydroxysuccinimide to the allergen solution in step (1) to make their final concentrations 1-10 mmol / L and 1-10 mmol / L, respectively; then vortex mix and incubate at room temperature in the dark for 15-30 min to obtain allergen protein activation solution; (3) Activation termination: Add 2-mercaptoethanol to the allergen protein activation solution in step (2) to a final concentration of 10-50 mmol / L, incubate for 12-17 min to quench unreacted EDC zero-length cross-linking agent; then use a desalting column to elute and purify, wherein the elution buffer is a coupling buffer to remove unreacted 2-mercaptoethanol and inactivated EDC zero-length cross-linking agent, to obtain an allergen activated protein solution; (4) Coupling of allergen-activated protein on amino nylon membrane: The amino nylon membrane is laid flat in the reaction vessel, and then the allergen-activated protein solution in step (3) is added dropwise until it completely covers the surface of the amino nylon membrane. Then, a shaker is used to promote uniform contact of the solution with the membrane surface. After incubation at room temperature for 1-3 h or at 3-5℃ for 7-9 h, the membrane is finally rinsed and washed with PBS buffer to obtain the allergen nylon membrane. (5) Blocking non-specific sites: Immerse the allergen nylon membrane from step (4) in bovine serum albumin blocking solution and incubate at room temperature for 0.5-1.5 h or at 3-5 °C for 7-9 h; then rinse with washing solution 2-4 times, and finally dry to obtain the test strip.

4. The Artemisia argyi and ragweed pollen allergen detection kit according to claim 3, characterized in that, The coupling buffer in step (3) has a pH of 7.0-8.0, a sodium phosphate concentration of 50-100 mmol / L, and a NaCl concentration of 100-200 mmol / L.

5. The Artemisia argyi and ragweed pollen allergen detection kit according to claim 3, characterized in that, In step (4), rinse with PBS buffer 2-4 times, each time for 4-6 minutes; then rinse with PBS buffer 1-3 times.

6. The Artemisia argyi and ragweed pollen allergen detection kit according to claim 3, characterized in that, The method for preparing the bovine serum albumin blocking solution in step (5) is as follows: Bovine serum albumin is added to PBS buffer to prepare a bovine serum albumin blocking solution with a mass concentration of 3-5%.

7. The Artemisia argyi and ragweed pollen allergen detection kit according to claim 3, characterized in that, In step (5), the pH of the washing solution is 7-8, the concentration of NaCl is 7-9 g / L, the concentration of KCl is 0.1-0.3 g / L, the concentration of Na2HPO4 is 1-2 g / L, the concentration of KH2PO4 is 0.1-0.3 g / L, and the concentration of surfactant Tween-20 is 0.05-0.15%.

8. A detection method for the Artemisia argyi and ragweed pollen allergen detection kit as described in any one of claims 1-7, characterized in that, It includes the following steps: S1: Equilibrate the serum sample, sample diluent, sample washing solution, enzyme-labeled antibody conjugate solution, and substrate solution to room temperature; S2: Place the test strip into the incubation tank, then place the incubation tank on a shaking table, add 0.5-2 ml of the sample diluent from step S1 into the incubation tank, then add 100-200 μL of the serum sample from step S1, then start the shaking table and incubate at room temperature for 60-80 min before stopping the shaking table. S3: Pour out the liquid in the incubation tank in step S2, add 2-5 ml of the sample cleaning solution in step S1, then start the shaking table and clean for 10-20 minutes. Stop the shaking table and repeat 2-4 times to complete the cleaning. S4: Add 1-2 ml of the enzyme-labeled antibody conjugation solution from step S1 to the incubation tank in step S3, then start the shaking incubator and incubate at room temperature for 30-40 min before stopping the shaking incubator. S5: Pour out the liquid in the incubation tank in step S4, add 2-5 ml of the sample cleaning solution in step S1, then start the shaking table and clean for 10-20 minutes. Stop the shaking table and repeat 2-4 times to complete the cleaning. S6: Add 1-2 ml of the substrate solution from step S1 to the incubation tank in step S5, then start the shaking incubator and incubate at room temperature for 30-60 minutes before stopping the shaking incubator. S7: Remove the test strip from the incubation tank in step S6, use clean absorbent paper to dry the remaining moisture in the incubation tank, and let the test strip dry. Visually compare the color development results of the test area of ​​the test strip through the positive control area.

9. The detection method of the Artemisia argyi and ragweed pollen allergen detection kit according to claim 8, characterized in that, The swing frequency of the swinging bed is 30-60 times / min.

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