A method for biotin labeling antigen protein

Abstract

The application relates to a method for labeling an antigen protein with biotin, which comprises the following steps: (1) providing a ligase complex formed by a polypeptide chelated with Ni 2+ and a recombinant TurboID protein; (2) providing a recombinant antigen protein with a HisTag; and (3) reacting the recombinant antigen protein with the HisTag with biotin under the condition that the ligase complex, ATP and Mg 2+ exist to obtain the antigen protein labeled with biotin, wherein the amino acid sequence of the polypeptide in the step (1) is shown in SEQ ID NO: 1, and the amino acid sequence of the recombinant TurboID protein is shown in SEQ ID NO: 2. The method improves the labeling efficiency of the antigen protein labeled with biotin, improves the biotin utilization rate, and improves the positive detection rate of specific antibody detection in a clinical sample.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagents, and specifically relates to a method for biotin-labeled antigen proteins. Background Technology

[0002] The binding between biotin and streptavidin is one of the most well-known non-covalent interactions in biochemistry. Its extremely high affinity makes it one of the strongest known biomolecular interactions, a property that makes it the cornerstone of protein-directed immobilization, high-sensitivity detection, and targeted delivery systems.

[0003] The efficiency of the biotin-streptavidin signal amplification system is entirely limited by the efficiency and specificity of the biotin-labeled protein step. Existing biotin-labeled protein methods mainly include enzymatic biotin ligase systems and chemical coupling methods.

[0004] Enzymatic methods, exemplified by E. coli-derived BirA enzymes, require modification of the target protein gene and fusion with a 15-aaAviTag peptide (such as GLNDIFEAQKIEWHE). Then, under ATP-driven conditions, biotin is covalently linked to a specific lysine residue in AviTag. This method requires a long biotin labeling reaction time (at least 4 hours even under optimal reaction conditions) and has low labeling efficiency. Furthermore, AviTag insertion may disrupt the native conformation of the target protein, leading to insertional inactivation.

[0005] Chemical coupling methods, exemplified by the use of NHS ester-based biotin reagents (such as EZ-Link NHS-Biotin), involve the covalent binding of NHS ester groups to the ε-amino group of lysine residues on the protein surface. This reaction is non-specific, meaning biotin can bind to any exposed lysine residue on the protein surface. Due to its random modification characteristics, this can indeed lead to uncontrollable protein function and even protein inactivation. Furthermore, due to the competitive hydrolysis reaction, the coupling efficiency is low, requiring an excess of 5-10 times the reagent for compensation, significantly increasing costs.

[0006] Therefore, there is an urgent need to develop more efficient methods for biotin-labeled antigen proteins. Summary of the Invention

[0007] The purpose of this invention is to provide a more efficient method for biotin-labeling antigen proteins.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] The first aspect of this invention provides a method for biotin-labeling antigen proteins, the method being as follows:

[0010] (1) Provided by chelated Ni2+ The ligase complex formed by the polypeptide and recombinant TurboID protein;

[0011] (2) Provide a recombinant antigen protein with HisTag;

[0012] (3) The recombinant antigen protein with HisTag is placed in the ligase complex and ATP and Mg 2+ Under the presence of these conditions, the antigen reacts with biotin to obtain the biotin-labeled antigen protein.

[0013] In step (1), the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1, and the amino acid sequence of the recombinant TurboID protein is shown in SEQ ID NO: 2.

[0014] In some embodiments, in step (1), the cysteine ​​residues of the polypeptide have a chelating effect on Ni. 2+ Modification with maleimide groups.

[0015] In some embodiments, the chelated Ni 2+ The polypeptide and recombinant TurboID protein form the ligase complex under the action of Sortase A enzyme.

[0016] According to some specific embodiments, step (1) specifically involves: reducing the polypeptide to disulfide to thiol group under the action of a reducing agent, then reacting it with excess maleimide, purifying it to remove unreacted maleimide to obtain maleimide-modified polypeptide, reacting the maleimide-modified polypeptide with excess NiCl2, and removing free Ni by desalting. 2+ The chelated Ni was obtained 2+ The polypeptide, chelating Ni 2+ The polypeptide was reacted with recombinant TurboID protein and Sortase A enzyme (SrtA) in a reaction buffer, and the reaction product was purified to obtain the ligase complex.

[0017] Furthermore, the reducing agent is a reducing agent capable of reducing disulfide bonds (-SS-) to thiol groups (-SH), such as tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol, DTT, etc., with TCEP, which has better stability, being preferred.

[0018] Furthermore, all reactions and purifications in step (1) were carried out at a pH of 7.0 to 8.5 and a temperature of 20 to 30°C.

[0019] Furthermore, the reaction between the maleimide-modified polypeptide and NiCl2 is carried out under light-protected conditions.

[0020] Furthermore, the molar ratio of maleimide to peptide is (1.5~3):1, for example 1.5:1, 2:1, 2.5:1, 3:1 or any molar ratio between any two of the above molar ratios.

[0021] Furthermore, maleimide is fed in solution form, with dimethyl sulfoxide (anhydrous DMSO) as the solvent.

[0022] Furthermore, anti-DYKDDDDK affinity beads were used to purify and remove unreacted maleimide. Anti-DYKDDDDK affinity beads are affinity chromatography media capable of purifying and immunoprecipitating DYKDDDDK peptides, thereby separating them from unreacted maleimide.

[0023] Furthermore, the molar ratio of NiCl2 to the maleimide-modified polypeptide is (4.5~9):1, for example, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1 or any molar ratio between any two of the above molar ratios.

[0024] Furthermore, a PD MidiTrap G-10 desalting column was used to desalt the light-protected reaction products to remove free Ni. 2+ .

[0025] Furthermore, the chelated Ni 2+ The molar ratio of the polypeptide to the recombinant TurboID protein is (5~10):1, for example, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1 or any molar ratio between any two of the above.

[0026] Furthermore, the recombinant TurboID protein is produced by an Escherichia coli expression system, obtained by lysing recombinant Escherichia coli cells with bacterial lysis buffer, and purified by metal ion affinity chromatography and Superdex 75 Increase 10 / 300GL size exclusion chromatography.

[0027] Sortase A (SrtA) is a transpeptidase derived from Gram-positive bacteria (such as Staphylococcus aureus) and belongs to the C60 peptidase family. The molar ratio of the recombinant TurboID protein to the Sortase A enzyme is (10~20):1, for example, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, or any molar ratio between any two of the above.

[0028] Furthermore, the reaction buffer is 40-80 mM Tris-HCl containing 100-200 mM NaCl and 5-15 mM CaCl2.

[0029] More preferably, the reaction buffer is 40-60mM Tris-HCl containing 140-160mM NaCl and 8-12mM CaCl2.

[0030] In some embodiments, in step (3), the Mg 2+ Feed in the form of MgOAc.

[0031] In some embodiments, the HisTag-containing Bet v1 protein and the ligase complex are mixed at a molar ratio of (80-120):1, and 80-120 mM ATP, 80-120 mM MgOAc, and 400-600 μM biotin are added to the mixture, and the reaction is carried out at 20-30°C.

[0032] More preferably, the Bet v1 protein with HisTag and the ligase complex are mixed in a molar ratio of (90~110):1, and 90~110mM ATP, 90~110mM MgOAc, and 450~550μM biotin are added to the mixture. The mixture is then reacted at 20~30°C for 20~60 min.

[0033] Furthermore, imidazole at a final concentration of 200-300 mM was added to the reaction system, and the biotin-labeled antigen protein was obtained by purification using an SEC column.

[0034] In some implementations, steps (1) and (2) are not in any particular order.

[0035] In some embodiments, the recombinant antigen protein is a recombinant protein of an allergen protein. For example, Bet v1 protein, an allergen protein primarily found in birch pollen, is one of the main components causing respiratory allergic reactions. It belongs to the PR-10 protein family, whose members are widely distributed in plants and have homologous allergens in various plant pollens and foods.

[0036] A second aspect of the present invention provides the use of the biotin-labeled antigen protein prepared by the above method in the preparation of allergen-specific antibody detection reagents or kits.

[0037] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0038] This invention utilizes mutated biotin ligase G-TurboID and Ni 2+ The ion-chelated modified polypeptide complex forms a biotin ligase complex, which facilitates the biotin labeling of recombinant antigen proteins containing HisTag in vitro. Existing experiments have verified that the biotinylated recombinant antigen proteins prepared by the biotinylation method of this invention exhibit good biological activity and can improve the positive detection rate of antibody-specific detection in clinical samples. The biotinylation method of this invention demonstrates high labeling efficiency for target proteins in vitro, requiring only the addition of HisTag without the need for additional genetic modification of the target protein. The overall process is simple, convenient, efficient, and has high biotin utilization, providing more and better options for the preparation of antigen reagents in high-throughput labeling and specific antibody detection kits. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the biotin labeling method used in Example 1;

[0040] Figure 2 Image showing the purification results of G-TurboID;

[0041] Figure 3 This is a graph showing the results of streptavidin gel translocation assay. Detailed Implementation

[0042] In order to improve the labeling efficiency of biotin-labeled antigen proteins, increase biotin utilization, and improve the positive detection rate of antibody-specific detection in clinical samples, the inventors of this application have conducted extensive research and experimental verification, and designed an in vitro biotin-labeled antigen protein method using TurboID.

[0043] TurboID is a modified biotin ligase primarily used for proximity labeling in living cells. By fusing TurboID with the target protein, biotin labeling can be rapidly performed intracellularly in just 10 minutes. The inventors of this application utilize a mutated biotin ligase G-TurboID and Ni... 2+ The modified polypeptide complex chelated by ions forms a biotin ligase complex. This complex facilitates the biotin labeling of recombinant antigen proteins containing HisTag in vitro under the action of ATP. Existing experiments have verified that this labeling method is highly efficient and has high biotin utilization. The resulting biotinylated antigen protein can improve the positive detection rate of antibody-specific detection in clinical samples. Specifically, the biotinylated antigen protein labeling method of the present invention is as follows:

[0044] (1) Provided by chelated Ni 2+ The ligase complex formed by the polypeptide and recombinant TurboID protein;

[0045] (2) Provide a recombinant antigen protein with HisTag;

[0046] (3) The recombinant antigen protein with HisTag is placed in the ligase complex and ATP and Mg 2+ Under the presence of these conditions, the antigen reacts with biotin to obtain the biotin-labeled antigen protein.

[0047] In step (1), the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1, and the amino acid sequence of the recombinant TurboID protein is shown in SEQ ID NO: 2.

[0048] The present invention will be further described below with reference to embodiments and comparative examples. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0049] In the following examples and comparative examples, the implementation conditions not specified are standard conditions in the industry, and the reagents not specified are all commercially available products. The detection instrument was a SMART6500 fully automated chemiluminescence analyzer from Jiangsu Haooubo Biomedical Co., Ltd. (HOB).

[0050] TCEP was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0051] NTA-Maleimide was purchased from AAT Bioquest.

[0052] The bacterial lysis buffer was prepared in-house with the following formula: 20 mM Tris, 500 mM NaCl, 1% NP40, 5 mM imidazole, pH 7.9.

[0053] Anti-DYKDDDDK Affinity Beads are from Changzhou Tiandi Renhe Biotechnology Co., Ltd.

[0054] The PD MidiTrap G-10 desalination column is a pre-loaded, disposable gravity column from Cytiva.

[0055] The Superdex 75 Increase 10 / 300 GL is a pre-packed size exclusion chromatography (SEC) column from Cytiva.

[0056] The HisTrap FF metal ion affinity chromatography column is from Cytiva.

[0057] HiTrap desalination columns are from Cytiva.

[0058] Sortase A (SrtA) was purchased from Aikon Biotechnology (Suzhou) Co., Ltd.

[0059] sulfo-NHS-PEG12-Biotin was purchased from Thermo Fisher Scientific.

[0060] The Bet v1 protein solution with HisTag (histidine tag) was purchased from OriGene China (AoriGene (Beijing) Technology Co., Ltd.), catalog number TP750226. The HisTag is located at the N-terminus of the Bet v1 protein.

[0061] Example 1

[0062] This embodiment provides a method for biotinylated Bet v1 protein. Details are as follows:

[0063] 1. Preparation of biotin ligase complex

[0064] The peptide was designed and synthesized by Nanjing Genscript Biotech Co., Ltd., with the amino acid sequence DYKDDDDKCCCLEPTG (SEQ ID NO: 1).

[0065] The specific labeling of cysteine ​​residues in the peptide using NTA-maleimide is as follows:

[0066] The peptide was dissolved in degassed PBS (pH 7.0) (peptide concentration 5 mM), TCEP (final concentration 5 mM) was added, and the mixture was incubated at room temperature for 30 minutes to reduce disulfide bonds and expose free -SH, thus obtaining the peptide solution.

[0067] Dissolve NTA-maleimide in anhydrous DMSO (NTA-Maleimide concentration is 50 mM) to obtain an NTA-maleimide solution, which should be prepared and used immediately.

[0068] To ensure over-modification, the NTA-maleimide:peptide ratio was 2:1 (molar ratio). The NTA-maleimide solution was added dropwise to the peptide solution, and the mixture was stirred at room temperature in the dark for 3 hours in an ice bath. L-cysteine ​​(final concentration 5 mM) was added, and the mixture was incubated at room temperature for 15 min to quench unreacted maleimide groups. The peptide was then purified using Anti-DYKDDDDK affinity beads according to the manufacturer's instructions to remove free NTA-maleimide, yielding the modified peptide.

[0069] 1.3 Chelating Ni with modified peptides 2+ Following a NiCl2:peptide molar ratio of 6:1, NiCl2 solution was added dropwise to the modified peptide solution while magnetically stirring. The pH was maintained at 7.5-8.5 using 1M NaOH solution, and the reaction was carried out at room temperature in the dark for 60 minutes. Following the manufacturer's instructions, the product was desalted using PD MidiTrap G-10 to remove free Ni. 2+ Chelated Ni after desalting 2+ The modified peptides can be stored at -80°C for later use.

[0070] 1.4 Preparation of optimized TurboID enzyme, the specific steps are as follows:

[0071] The sequence of the TurboID enzyme was obtained from the UniProt database. After analysis, an optimized TurboID enzyme (abbreviated as G-TurboID) was designed, and its amino acid sequence is as follows:

[0072] GGGKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADK VRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEKENLYFQGHHHHHHH (SEQ ID NO: 2).

[0073] The gene was submitted to Nanjing GenScript Biotech Co., Ltd. for codon optimization and gene synthesis. The synthesized gene was inserted into the pET-24a(+) vector to form a plasmid containing the G-TurboID target gene.

[0074] The plasmid containing the G-TurboID target gene was transformed into the expression strain BL21(DE3). The transformation steps were as follows: Take 50 μL of competent Escherichia coli BL21(DE3) cells, add an appropriate amount of plasmid, incubate on ice for 30 min, heat shock at 42℃ for 90 s, immediately return to ice, and incubate on ice for 2 min; add 400 μL of LB medium, and culture on a shaker at 37℃ for 60 min; take 100 μL and spread it on LB solid medium containing kanamycin (50 μg / mL), and incubate upside down at 37℃ overnight.

[0075] Single clones of the bacterial strain were picked and added to 100 mL of LB medium containing kanamycin, and cultured overnight at 37°C and 220 rpm. 10 mL of *E. coli* BL21(DE3) containing the recombinant plasmid was added to 1000 mL of LB medium and cultured. The appropriate antibiotic was added at a 1:1000 ratio, and the culture was shaken at 37°C and 220 rpm until the OD600 reached 0.6. IPTG was then added to a final concentration of 0.5 mmol / L, and the culture was shaken at 37°C and 220 rpm for 4 h. The bacterial pellet was collected by centrifugation at 4°C and 8000 rpm for 10 min.

[0076] After resuspending the bacterial cells in bacterial lysis buffer, the cells were lysed using an ultrasonic cell disruptor. The lysed cells were centrifuged for 30 min, and the supernatant was collected. Following the manufacturer's instructions, the collected supernatant was subjected to HisTrap FF metal ion affinity chromatography and Superdex 75 Increase 10 / 300GL size exclusion chromatography to obtain high-purity ( ) Figure 2 The concentration of G-TurboID enzyme solution was 1 mg / ml.

[0077] 1.5 Chelated Ni 2+ The modified polypeptide is coupled with G-TurboID enzyme to synthesize a ligase complex. The specific operation is as follows:

[0078] Prepare Sortase A reaction buffer: 50 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl and 10 mM CaCl2.

[0079] Chelating Ni according to the manufacturer's instructions 2+The modified peptide and G-TurboID enzyme were desalted using a HiTrap column and the buffer was changed to Sortase A reaction buffer. The final peptide concentration was ≥1mM and the G-TurboID enzyme concentration was ≥0.1mg / mL. The peptide:G-TurboID enzyme (molar ratio) was controlled at 10:1 to ensure over-modification of the peptide. The G-TurboID enzyme:Sortase A (molar ratio) was controlled at 20:1. Sortase A reaction buffer was added to make the final G-TurboID enzyme concentration 20μM. The reaction was carried out at room temperature for 60min. The high-purity ligase complex was obtained by using Superdex 75 Increase 10 / 300GL.

[0080] 2. Biotin-labeled Bet v1 protein

[0081] A Bet v1 protein solution (80 μM) containing HisTag was mixed with the ligase complex at a molar ratio of 100:1 (Bet v1 protein containing HisTag: ligase complex). 100 mM ATP, 100 mM MgOAc, and 500 μM D-biotin were added to the mixture, and the mixture was reacted at room temperature for 30 min to complete biotinylation. Imidazole buffer was added to bring the final imidazole concentration to 250 mM. The biotinylated Bet v1 protein reagent was purified using Superdex 75 Increase 10 / 300GL.

[0082] 3. Streptavidin gel transfer assay

[0083] Prepare the following samples and incubate at room temperature for 5-15 min: Betv1 protein containing HisTag; biotinylated Betv1 protein; a mixture of biotinylated protein and streptavidin in a 1:1 molar ratio; and a mixture of biotinylated protein and streptavidin in a 1:2 molar ratio. Take an appropriate amount of 4×SDS-PAGE buffer (without reducing agent), add the sample, and perform SDS-PAGE analysis without boiling. See [image attached]. Figure 3 The presence of bands corresponding to free streptavidin in lanes containing biotinylated proteins and streptavidin verified the oversupply of streptavidin, indicating that all biotinylated Bet v1 proteins were bound and moved on the gel.

[0084] Comparative Example 1

[0085] This comparative example provides another method for biotinylated Bet v1 protein, as follows:

[0086] Sulfo-NHS-PEG12-Biotin was dissolved in DMSO to obtain a 10 mg / mL sulfo-NHS-PEG12-Biotin reagent. Bet v1 protein was dissolved in 20 mM PB buffer (pH 7.4) containing 150 mM NaCl to obtain a 25 μM Bet v1 protein solution. The sulfo-NHS-PEG12-Biotin reagent was continuously added dropwise to the Bet v1 protein solution, and the reaction was carried out at room temperature for 1 h. After the reaction, the free sulfo-NHS-PEG12-Biotin was removed by dialyzing with 0.1 M PBS buffer (pH 7.4) to obtain the biotinylated Bet v1 protein reagent.

[0087] Example 2

[0088] This embodiment provides a Bet v1 protein allergen-specific IgE quantitative detection kit, which includes reagent 1, reagent 2, reagent 3, reagent 4, calibrator, quality control, and luminescent substrate solution.

[0089] Reagent 1 is a 0.6 mg / mL streptavidin-coated magnetic microparticle suspension, which is prepared as follows:

[0090] After mixing 10 mg / mL streptavidin magnetic beads, magnetic separation was performed. The mixture was then resuspended in phosphate buffer (pH 7.4) containing 2 wt% BSA, 3 wt% glycerol, and 5 wt% mannitol. After washing three times, magnetic separation was performed again. Finally, the mixture was resuspended in phosphate buffer (pH 7.4) containing 2 wt% BSA, 3 wt% glycerol, and 5 wt% mannitol to obtain a 0.6 mg / mL streptavidin-coated magnetic microparticle suspension.

[0091] Reagent 2 is a biotin-labeled Bet v1 protein allergen solution, which is prepared as follows:

[0092] The biotin-labeled Bet v1 protein reagent prepared in Example 1 was diluted to a concentration of 5 μg / mL using 0.02 M phosphate buffer containing 1 wt% bovine serum albumin at pH 7.5 to obtain the biotin-labeled Bet v1 protein allergen solution.

[0093] Reagent 3 is an alkaline phosphatase-labeled anti-human IgE antibody, which is prepared using conventional methods in the art: the alkaline phosphatase to be activated is dialyzed, then a coupling agent is added to react, the free coupling agent is separated and removed, then the activated alkaline phosphatase is added to the activated antibody solution to perform a coupling reaction, and then the free alkaline phosphatase is separated and removed again.

[0094] Reagent 4 is a biotin-labeled anti-human IgE antibody, which is prepared by adding the anti-human IgE antibody to Sulfo-NHS-LC-biotin dissolved in dimethylformamide, and then dialyzing with phosphate buffer containing 2 wt% BSA at pH 7.4.

[0095] Preparation of calibrators: IgE protein derived from WHO standards was diluted with 0.1M Tris-HCl buffer at pH 7.4 to prepare calibrators of 0 IU / mL, 0.35 IU / mL, 0.7 IU / mL, 3.5 IU / mL, 17.5 IU / mL, and 100 IU / mL.

[0096] Preparation of quality control samples: IgE protein from WHO standards was used to prepare quality control samples of 0.7 IU / mL and 17.5 IU / mL using 0.1 M Tris-HCl buffer at pH 7.4.

[0097] The luminescent substrate solution is an alkaline phosphatase chemiluminescent substrate based on AMPPD luminescence, which is produced in-house. For details, please refer to the invention patent "An Enzymatic Chemiluminescent Substrate for Alkaline Phosphatase", authorized announcement number CN 104990912B.

[0098] The detection method of the Bet v1 protein allergen-specific IgE quantitative detection kit in this embodiment is as follows:

[0099] (1) The calibrator was mixed with streptavidin-coated magnetic microparticle suspension and biotin-labeled anti-human IgE antibody, respectively, and incubated at 37°C for 15 min. The supernatant was removed by magnetic separation and the mixture was washed three times.

[0100] (2) Add alkaline phosphatase-labeled anti-human IgE antibody, mix, incubate at 37°C for 15 min, remove supernatant by magnetic separation, and wash three times;

[0101] (3) Add the luminescent substrate solution, incubate at 37°C for 5 min, measure the chemiluminescence intensity, and plot a standard curve with the concentration of the calibrator as the x-axis and the chemiluminescence intensity as the y-axis.

[0102] (4) Mix the sample to be tested with streptavidin-coated magnetic microparticle suspension and biotin-labeled Bet v1 protein allergen solution, incubate at 37°C for 15 min, magnetically separate the supernatant, and wash three times.

[0103] (5) Add alkaline phosphatase-labeled anti-human IgE antibody, mix, incubate at 37°C for 15 min, remove supernatant by magnetic separation, and wash three times;

[0104] (6) Add the luminescent substrate solution, incubate at 37°C for 5 min, measure the chemiluminescence intensity, substitute it into the standard curve, and calculate the concentration of Bet v1 protein allergen-specific IgE in the sample to be tested.

[0105] Comparative Example 2

[0106] A Bet v1 protein allergen-specific IgE quantitative detection kit is provided, which includes reagent 1, reagent 2, reagent 3, reagent 4, calibrator, quality control and luminescent substrate solution, wherein reagent 1, reagent 3, reagent 4, calibrator, quality control and luminescent substrate solution are the same as in Example 2, and reagent 2 is the Bet v1 protein allergen solution labeled with chemibiotin reagent in Comparative Example 1.

[0107] The test kits of the above examples and comparative examples were configured to be compatible with the SMART 6500 fully automated chemiluminescence analyzer, and labels containing corresponding information were affixed to the test tubes of the kits and each reagent.

[0108] Performance comparison:

[0109] The Bet v1 allergen-specific IgE quantitative detection kits of Example 2 and Comparative Example 2 were used to quantitatively detect the Bet v1 allergen-specific IgE content in clinical serum samples (30 positive samples and 15 negative samples). The software automatically generated the corresponding luminescence value and calculated concentration value for each sample. The detection concentration value ≤0.35 IU / mL was interpreted as negative, and the detection concentration value >0.35 IU / mL was interpreted as positive. The results were compared with those of the Immuno CAP detection kit from PHADIA. The results are shown in Table 1, unit: IU / mL.

[0110] Table 1

[0111]

[0112] In Table 1, P1~P30 are positive samples, and N1~N15 are negative samples.

[0113] Table 1 shows that the quantitative detection results of the Bet v1 protein allergen-specific IgE quantitative detection kit in Example 2 are closer to the PHADIA detection data than those of the Bet v1 protein allergen in Comparative Example 2.

[0114] The positive and negative concordance rates of Example 2 and Comparative Example 1 with PHADIA are shown in Table 2.

[0115] Table 2

[0116]

[0117] Table 2 shows that the biotinylated Bet v1 protein reagents used in Example 2 and Comparative Example 2 both achieved positive concordance rates of over 83% and overall concordance efficiency of over 84%, demonstrating good correlation with the industry gold standard (PHADIA) reagent. The biotinylated Bet v1 protein allergen of Example 2 exhibited better allergen performance, showing better biological activity, a higher negative concordance rate, and higher positive detection sensitivity compared to the biotinylated allergen obtained by the chemical conjugation method in Comparative Example 1.

[0118] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for biotin-labeling antigen proteins, characterized in that, The steps of the method are as follows: (1) Provided by chelated Ni 2+ The ligase complex formed by the polypeptide and recombinant TurboID protein; (2) Provide recombinant Bet v1 protein with HisTag; (3) The recombinant Bet v1 protein with HisTag is placed in the ligase complex and ATP and Mg 2+ Under the presence of these conditions, the antigen reacts with biotin to obtain the biotin-labeled antigen protein. In step (1), the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1, and the amino acid sequence of the recombinant TurboID protein is shown in SEQ ID NO:

2. The cysteine ​​residues of the polypeptide carry chelation for Ni 2+ The maleimide group, the chelated Ni 2+ The polypeptide and recombinant TurboID protein form the ligase complex under the action of Sortase A enzyme.

2. The method for biotin-labeled antigen proteins according to claim 1, characterized in that, Step (1) specifically involves: reducing the disulfide bonds of the polypeptide to thiol groups under the action of a reducing agent, then reacting it with excess maleimide, purifying it to remove unreacted maleimide to obtain a maleimide-modified polypeptide, reacting the maleimide-modified polypeptide with excess NiCl2, and removing free Ni by desalting. 2+ The chelated Ni was obtained 2+ The polypeptide, chelating Ni 2+ The polypeptide was reacted with recombinant TurboID protein and Sortase A enzyme in a reaction buffer, and the reaction product was purified to obtain the ligase complex.

3. The method for biotin-labeled antigen proteins according to claim 2, characterized in that, All reactions and purifications in step (1) were carried out at a pH of 7.0 to 8.5 and a temperature of 20 to 30°C. And / or, the reaction of the maleimide-modified polypeptide with NiCl2 is carried out under light-protected conditions; And / or, the molar ratio of the maleimide to the polypeptide is (1.5~3):1; And / or, the molar ratio of NiCl2 to the maleimide-modified polypeptide is (4.5~9):

1.

4. The method for biotin-labeled antigen proteins according to claim 2, characterized in that, The chelated Ni 2+ The molar ratio of the polypeptide to the recombinant TurboID protein was (5~10):1; And / or, the molar ratio of the recombinant TurboID protein to the Sortase A enzyme is (10~20):1; And / or, the reaction buffer is 40-80mM Tris-HCl containing 100-200mM NaCl and 5-15mM CaCl2.

5. The method for biotin-labeled antigen proteins according to claim 1, characterized in that, In step (3), the Mg 2+ Feeding in the form of MgOAc; And / or, the recombinant Bet v1 protein with HisTag and the ligase complex are mixed at a molar ratio of (80~120):1, and 80~120mM ATP, 80~120mM MgOAc, and 400~600μM biotin are added to the mixture, and the reaction is carried out at 20~30°C.

6. The method for biotin-labeled antigen proteins according to claim 5, characterized in that, Imidazole at a final concentration of 200-300 mM was added to the reaction system, and the biotin-labeled antigen protein was obtained by purification using an SEC column.

7. The method for biotin-labeled antigen proteins according to claim 1, characterized in that, The steps (1) and (2) are not in any particular order.

8. The use of the biotin-labeled antigen protein prepared by any one of claims 1 to 7 in the preparation of allergen-specific antibody detection reagents or kits.

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