A recombinant dust mite antigen and its application in allergen detection test strips

Abstract

The present invention provides a recombinant dust mite antigen and an application thereof in an allergen detection test strip. By constructing Derf1, Derf2 and Derf3 recombinant antigens and optimizing their amino acid sequences, especially in Derf3, the D58P, S106D and G231C sites thereof are mutated, thereby preparing a test strip capable of simultaneously detecting the three recombinant antigens, effectively solving the problems of erroneous detection results and false positives of Derf3 during simultaneous detection, realizing simultaneous detection of specific IgE antibodies of the three dust mite allergens in the test strip, avoiding the problem of cross-influence, providing direct allergen type judgment for patients allergic to dust mites before desensitization treatment, and facilitating more effective desensitization treatment for the symptoms.

Description

[0001] This application claims priority to the prior Chinese application, application number: 2024119735215, filing date December 30, 2024; all contents thereof are made part of the present invention. Technical Field

[0002] The invention relates to the technical field of antigen preparation, and in particular to a recombinant dust mite antigen and application thereof in an allergen detection test strip. Background Art

[0003] The condition of allergic diseases is becoming more complicated. Studies have shown that more than 50% of children with single allergies have the possibility of developing multiple allergies. Children who develop food allergies or atopic dermatitis at an early stage may develop other more difficult allergic diseases in the future. For allergic diseases, WHO proposes a "four-in-one" therapy: avoid contact with allergens, symptomatic drug treatment, specific immunotherapy, and doctor and patient education. Among them, avoiding contact with allergens means to do a good job of environmental prevention and control on the basis of identifying allergens and avoid contact with allergens as much as possible. In the treatment of allergic diseases, avoiding contact with allergens can not only reduce the probability of allergies in patients, but also improve the efficacy of drugs and help patients desensitize as soon as possible. Specific immunotherapy, that is, desensitization therapy, is the only "cause-targeted" therapy that can affect the natural course of allergic diseases and change the immune response mechanism. It uses gradually increasing doses of allergens to improve patients' tolerance to the allergens, reduce symptoms caused by exposure to the allergens, and ultimately achieve tolerance and even immune tolerance. It can be seen that identifying allergens is the key to preventing and treating allergic diseases. Avoiding contact with allergens and carrying out specific immunotherapy are inseparable from allergen diagnosis.

[0004] There are many types of allergens, among which dust mites are inhaled allergens. They are widely present in human living and working environments. Their excrement, metabolites and mites are highly allergenic. Dust mites have become an important allergen that induces allergic diseases. Dust mite allergens Der f 1 (Derf1 for short) and Der f 2 (Derf2 for short) are the main allergenic components, but dust mite extracts lacking Derf1 and Derf2 components can still induce asthma, indicating that other components also play an important role in the sensitization process. For example, dust mite allergen Der f 3 (Derf3 for short) can also mediate allergic reactions in the body, but relatively little research has been done on it.

[0005] Different allergy patients may be allergic to different dust mite allergens (such as Derf1, Derf2 or Derf3). Therefore, it is necessary to determine which dust mite allergen the patient is allergic to so that targeted treatment can be taken, such as using different drugs for desensitization treatment, to truly achieve the patient's tolerance to specific dust mite allergens. However, existing allergen detection test strips usually put all dust mite allergens on one test line for simultaneous testing, and cannot distinguish which dust mite allergen caused the patient's allergic reaction, which is very unfavorable for later treatment.

[0006] Therefore, there is an urgent need to find a detection method for IgE antibodies that can simultaneously distinguish the three allergens Derf1, Derf2 and Derf3, so as to help patients who need desensitization treatment to receive effective symptomatic treatment. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention provides a recombinant dust mite antigen and its application in an allergen detection test strip. By constructing Derf1, Der f2 and Der f3 recombinant antigens and optimizing their amino acid sequences, especially Derf3, the D58P, S106D and G231C sites therein are mutated, so as to prepare a test strip that can simultaneously detect and distinguish the three recombinant antigens, effectively solving the problems of erroneous detection results and false positives of Derf3 during simultaneous detection, realizing the simultaneous detection of IgE antibodies of three dust allergens in the test strip, avoiding the problem of cross-influence, and providing direct allergen type judgment for patients with dust mite allergy before desensitization treatment, so as to facilitate more effective desensitization treatment for the symptoms.

[0008] In one aspect, the present invention provides a Derf3 recombinant antigen, which is based on a wild-type Derf3 protein sequence and has a mutation site, wherein the mutation site comprises any one or more of D58P, S106D, and G231C.

[0009] The present invention targets the wild-type Derf3 protein (SEQ ID NO.8), takes the amino acid sequence of its mature protein, and mutates the amino acids at positions 28 to 259 of the wild-type Derf3 protein, hoping to form salt bridges, hydrophobic bonds or disulfide bonds in the Derf3 protein through the mutation sites, thereby increasing the stability of the Derf3 protein, thereby making it have higher stability, enhancing the ability to recognize antibodies, and improving the detection sensitivity and specificity when used to detect IgE antibodies of Derf3 allergens. It is understandable that any one or more mutations in the three mutation sites of D58P, S106D, and G231C in the Derf3 protein have the effect of improving the conformational stability of the Derf3 protein.

[0010] Among them, D58P can obtain a better signal peptide sequence and form new electrostatic interactions, leading to the formation of new salt bridge pairs. S106D increases the hydrophobicity of the space, and G231C increases the stability of the Derf3 protein antigen epitope.

[0011] Furthermore, the Derf3 recombinant antigen includes a combination of D58P, S106D, and G231C mutation sites.

[0012] At the same time, combined mutations of the three mutation sites D58P, S106D, and G231C have the effect of synergistically improving the conformational stability of the Derf3 protein, changing the spatial conformation, exposing more antigenic epitopes, and optimizing the ability of the Derf3 recombinant antigen to specifically recognize antibodies.

[0013] Furthermore, the amino acid sequence of the Derf3 recombinant antigen is shown in SEQ ID NO.3.

[0014] In the present invention, the Derf3 mutant with combined mutations of D58P, S106D and G231C is named Derf3 02. The amino acid sequence of Derf3 02 is shown in SEQ ID NO.3, and the encoding nucleotide sequence corresponding to the amino acid sequence is shown in SEQ ID NO.6.

[0015] Studies have shown that Derf3 02, as a recombinant antigen, can significantly improve the capture ability of IgE antibodies against Derf3 allergens.

[0016] In another aspect, the present invention provides a test strip for detecting dust mite antibodies, the test strip being provided with a detection line and a quality control line, the detection line comprising a Derf3 detection line, the Derf3 detection line being coated with a Derf3 recombinant antigen; the amino acid sequence of the Derf3 recombinant antigen being as shown in SEQ ID NO.3.

[0017] Furthermore, the detection line also includes a Derf1 detection line and / or a Derf2 detection line, and the Derf1 detection line and the Derf2 detection line are coated with Derf1 recombinant antigen and Derf2 recombinant antigen respectively; the amino acid sequences of the Derf1 and Derf2 recombinant antigens are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

[0018] The Derf1, Derf2 and Derf3 recombinant antigens purchased on the market are not only expensive, but when used to prepare test strips for IgE antibody testing, it is often found that not only are the test accuracy and sensitivity low, but also cross-influence is prone to occur when used to prepare recombinant antigens for simultaneous detection of Derf1, Derf2 and Derf3, and the test results are unstable.

[0019] In order to solve this problem, the present invention adopts self-made Derf1, Derf2 and Derf3 recombinant antigens, and codons are optimized for the encoding nucleotide sequences. The prepared recombinant antigens are used to prepare test strips, which can realize the simultaneous and accurate detection of IgE antibodies of the three recombinant antigens Derf1, Derf2 and Derf3, thereby improving the accuracy and sensitivity of detection.

[0020] The present invention targets a known Derf1 protein sequence (SEQ ID NO.7), takes the amino acid sequence of its mature protein, and constructs a Derf1 recombinant antigen by selecting the codons with the highest frequency of use according to the codon preference of the eukaryotic expression system, redesigns the coding sequence, obtains the coding gene of the Derf1 recombinant antigen as shown in SEQ ID NO.4, and artificially synthesizes the Derf1 recombinant antigen (SEQ ID NO.1).

[0021] The present invention targets the known Derf2 protein sequence (SEQ ID NO.2), takes the full-length amino acid sequence of the MD-2 related lipid recognition (ML) domain protein, constructs a Derf2 recombinant antigen, selects the codons with the highest usage frequency according to the codon preference of the eukaryotic expression system, redesigns the coding sequence, obtains the coding gene of the Derf2 recombinant antigen as shown in SEQ ID NO.5, and artificially synthesizes the Derf2 recombinant antigen (SEQ ID NO.2).

[0022] For the known Derf3 protein sequence (SEQ ID NO.8), the amino acid sequence of its mature protein was also selected at the beginning, located at the 28th to 259th amino acids of the wild-type Derf3 protein, and the Derf3 recombinant antigen was artificially synthesized. However, it was found that the IgE antibody of the Derf3 allergen often failed to be detected when the Derf3 recombinant antigen was prepared together with the Derf1 and Derf2 recombinant antigens to prepare the test strip. It was easily affected by the cross-influence of the Derf1 and Derf2 recombinant antigens and could not be detected smoothly. The reason may be that the Derf3 recombinant antigen formed a polymer, which changed the spatial conformation and made some antigen epitopes unable to be exposed. Therefore, the amino acid sequence was mutated to improve the conformational stability of the Derf3 protein, change the spatial conformation, expose more antigen epitopes, and optimize the ability of the Derf3 recombinant antigen to specifically recognize antibodies. The encoding nucleotide sequence was repeatedly adjusted many times to obtain the most preferred encoding nucleotide sequence of the Derf3 recombinant antigen (SEQ ID NO.6), when the prepared Derf3 recombinant antigen is used to prepare test strips for detection, it will not be cross-influenced by the Derf1 and Derf2 recombinant antigens, thus ensuring the accuracy and specificity of the test results.

[0023] Furthermore, the test strip contains three detection lines at the same time, including a Derf1 detection line, a Derf2 detection line and a Derf3 detection line.

[0024] By coating the Derf1, Derf2 and Derf3 recombinant antigens in different detection lines respectively, a test strip containing three detection lines is prepared, which can realize the simultaneous detection of IgE antibodies to the three allergens Derf1, Derf2 and Derf3.

[0025] Furthermore, it also includes a marking pad, on which mouse anti-human IgE antibodies are coated; and the quality control line is coated with DNP-BSA antigen.

[0026] Furthermore, the three detection lines are arranged in the order of Derf3, Derf1, and Derf2 detection lines, wherein the Derf3 detection line is closest to the sample addition pad.

[0027] Studies have shown that different arrangement orders of the three test lines will result in different detection sensitivities for IgE antibodies to the three allergens Derf1, Derf2 and Derf3. The most preferred arrangement order is Derf3, Derf1, and Derf2 test lines, among which the Derf3 test line is closest to the sample pad, and the sample added to the sample pad reaches the Derf3 test line first.

[0028] In yet another aspect, the present invention provides a method for detecting dust mite antibodies, wherein the method uses the test strip as described above for detection.

[0029] In another aspect, the present invention provides a method for preparing a recombinant dust mite antigen, the method comprising the following steps:

[0030] (1) inserting a nucleotide encoding a recombinant antigen into an empty vector to obtain a recombinant vector; the recombinant antigen comprises any one or more of a Derf1 recombinant antigen, a Derf2 recombinant antigen, and a Derf3 recombinant antigen, wherein the encoding nucleotide sequence of the Derf1 recombinant antigen is shown in SEQ ID NO.4, the encoding nucleotide sequence of the Derf2 recombinant antigen is shown in SEQ ID NO.5, and the encoding nucleotide sequence of the Derf3 recombinant antigen is shown in SEQ ID NO.6;

[0031] (2) Transfection of the recombinant vector into the eukaryotic cell expression system;

[0032] (3) Collecting the culture supernatant of the eukaryotic cell expression system;

[0033] (4) Purify the recombinant antigen using a chromatography column.

[0034] In the present invention, the three recombinant proteins Derf1, Derf2 and Derf3 all use the 293T cell eukaryotic expression system, the plasmid vector is the pcDNA3.4 vector provided by GenScript, and the transfection technology based on the cationic liposome transfection reagent is used. The 293T cell eukaryotic expression system can produce post-translational modifications and glycosylation consistent with natural cell membrane proteins, and can be well used as an antigen calibrator to meet the needs of IgE antibody detection reagents for Derf1, Derf2 and Derf3 allergens.

[0035] In some embodiments, step (1) selects the coding nucleotide sequences of Derf1, Derf2 and Derf3 respectively, and adds a deoxyribonucleic acid sequence that can be expressed as a His-Tag purification tag. The constructed recombinant protein deoxyribonucleic acid sequence is codon-optimized to make it more suitable for expression in human cells. Based on the pcDNA3.4 empty vector, which is a molecular biology vector that can regulate the expression of exogenous genes, the coding nucleotide sequences of Derf1, Derf2 and Derf3 are inserted into the AflII and XbaI restriction endonuclease sites of the expression vector respectively.

[0036] In some embodiments, the transfection method described in step (2) is a cationic liposome transfection reagent-mediated transfection technique.

[0037] In some embodiments, the culture medium in step (3) is 10% (v / v) premium fetal bovine serum and 90% (v / v) DMEM high glucose medium.

[0038] In some embodiments, the Ni-NTA agarose column purification described in step (4) is a commonly used His-tag recombinant protein purification method. The His-tag tag, i.e., the 6-10 histidine residue side chains, have a strong binding force with nickel ions and can be used for immobilized metal chelate affinity chromatography (IMAC) to separate and purify the recombinant protein.

[0039] On the other hand, the present invention provides a use of a recombinant antigen for preparing a reagent for improving the accuracy of detecting dust mite allergy IgE antibodies in serum samples, wherein the recombinant antigen comprises any one or more of a Derf1 recombinant antigen, a Derf2 recombinant antigen, and a Derf3 recombinant antigen, wherein the amino acid sequence of the Derf1 recombinant antigen is shown in SEQ ID NO.1, the amino acid sequence of the Derf2 recombinant antigen is shown in SEQ ID NO.2, and the amino acid sequence of the Derf3 recombinant antigen is shown in SEQ ID NO.3.

[0040] Beneficial effects achieved by the present invention:

[0041] 1. Prepare test strips with self-made Derf1, Derf2 and Derf3 recombinant antigens, which can realize the simultaneous detection of IgE antibodies of three allergens Derf1, Derf2 and Derf3;

[0042] 2. The known Derf3 protein sequence is mutated to improve the stability of the Derf3 protein conformation, change the spatial conformation, expose more antigenic epitopes, and optimize the ability of the Derf3 recombinant antigen to specifically recognize antibodies. When the prepared Derf3 recombinant antigen is used to prepare test strips for detection, it will not be cross-influenced by the Derf1 and Derf2 recombinant antigens, thus ensuring the accuracy and specificity of the test results;

[0043] 3. The arrangement order of the three test lines was optimized, and the most preferred arrangement order was adopted: Derf3 test line, Derf1 test line, and Derf2 test line, among which Derf3 was the front, which improved the detection accuracy of IgE antibodies of the three allergens Derf1, Derf2 and Derf3;

[0044] 4. The detection limit of specific IgE antibodies to Derf1 allergens reached 0.35 IU / mL, the detection limit of specific IgE antibodies to Derf2 allergens reached 0.35 IU / mL, and the detection limit of specific IgE antibodies to Derf3 allergens reached 0.35 IU / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 A schematic diagram of the test strip structure provided in Example 1;

[0046] Figure 2 A schematic diagram of the structure of the detection card provided in Example 1;

[0047] Figure 3 This is the compliance analysis diagram of Derf1-IgE clinical samples;

[0048] Figure 4 This is the compliance analysis diagram of Derf2-IgE clinical samples;

[0049] Figure 5 This is the compliance analysis diagram of Derf3-IgE clinical samples. DETAILED DESCRIPTION

[0050] To make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the art without making creative work are within the scope of protection of the present invention. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased through regular channels.

[0051] Example 1: Preparation and use of test strips

[0052] 1. Preparation of recombinant protein

[0053] 1.1 Design and synthesis of recombinant protein encoding genes

[0054] 1.1.1 Design and synthesis of Derf1 recombinant protein encoding gene

[0055] The amino acid sequence of the Derf1 gene published by NCBI (NCBI Reference Sequence accession number: BAC53948) was used as the optimization object, and the amino acid sequence of its mature protein sequence was selected as shown in Seq ID NO: 1. According to the codon preference of the eukaryotic expression system of human cells, the codons with the highest frequency of use were selected, and its nucleotide coding sequence was redesigned to obtain the coding gene of the Derf1 recombinant protein as shown in Seq ID NO: 4. A His-Tag sequence was added to the carboxyl terminus of its coding nucleotide sequence for artificial synthesis.

[0056] 1.1.2 Design and synthesis of Derf2 recombinant protein encoding gene

[0057] The amino acid sequence of the Derf2 gene published by NCBI (NCBI Reference Sequence accession number: BAA01239) was used as the optimization object, and the full-length amino acid sequence of its MD-2 related lipid recognition (ML) domain protein was selected (SEQ ID NO.2) to construct the Derf2 recombinant antigen. According to the codon preference of the eukaryotic expression system, the most frequently used codons were selected and the coding sequence was redesigned to obtain the coding gene of the Derf2 recombinant antigen as shown in SEQ ID NO.5. A His-Tag sequence was added to the carboxyl terminus and synthesized artificially.

[0058] 1.1.3 Design and synthesis of Derf3 recombinant protein encoding gene

[0059] Based on the amino acid sequence of Derf3, the experience of amino acid modification reported in the literature (Science, 2020, PMID:32703906; Trends Microbiol., 2018, PMID: 29681430) and the classic design principles of structural biology (including salt bridges, proline mutations, additional disulfide bonds, hydrogen bonds and hydrophobic pockets, etc.), this example obtains a more stable pre-fusion conformation by changing amino acids. In this embodiment, the structure of Derf3 protein is first predicted by Alfafold3, and it is found that there are 3 pairs of disulfide bonds in it. According to the spatial position of amino acids, new disulfide bond pairs are redesigned, and amino acids that may be paired to form disulfide bonds in spatial positions are found. The D at position 58 is mutated to P to form a new electrostatic interaction, resulting in the formation of a new salt bridge pair; at the same time, in order to maintain the pre-fusion conformation of Derf3 and avoid changes to the post-fusion conformation, the site G231 that can affect the conformational change in the loop region with variable conformation is transformed into G231C in this embodiment, which enhances the stability of the pre-fusion conformation and is more conducive to maintaining the antigen epitope; in addition, the site S106 is transformed into S106D, thereby enhancing the hydrophobic effect, increasing the hydrophobicity of the space, forming a salt bridge, and then stabilizing the pre-fusion conformation. Finally, this embodiment designs three mutation sites, D58P, S106D, and G231C. Any one or more mutations in the Derf3 recombinant protein at these three mutation sites can effectively improve the conformational stability of Derf3 and enhance the ability to recognize antibodies.

[0060] The amino acid sequence of the Derf3 gene published by NCBI (NCBI Reference Sequence Accession No.: BAA09920) was used as the optimization object, and the mature protein sequence was selected as shown in Seq ID NO: 8, and the corresponding mutated amino acid sequence (Seq ID NO: 3) was optimized to add a His-Tag sequence at the carboxyl end. According to the codon preference of the human cell eukaryotic expression system, the codon with the highest frequency of use was selected, and the coding sequence was redesigned to obtain the coding gene of the Derf3 recombinant protein shown in Seq ID NO: 6, and then artificially synthesized.

[0061] 1.2 Construction of vector using AflII and XbaI restriction endonuclease sites

[0062] (1) Use FastDigest AflII and FastDigest XbaI restriction endonucleases to treat the gene fragment of the recombinant protein and the pcDNA3.4 vector;

[0063] (2) 1% agarose gel electrophoresis was used to separate the gene fragments of the pcDNA3.4 vector and the recombinant protein treated with restriction endonucleases;

[0064] (3) Cut out the DNA fragment of the correct molecular weight from the gel and purify it;

[0065] (4) Use T4 DNA ligase to construct the recombinant vector;

[0066] (5) Transform Escherichia coli with the recombinant vector, extract the plasmid and verify it by sequencing.

[0067] 1.3 Transient transfection of 293T cells using the above three recombinant vectors

[0068] (1) One day before the experiment, 1.5 million cells were seeded into a cell culture flask. One day later, the cells grew to a confluence of more than 80%. (2) 6 μg of the recombinant plasmid and 12 μL of Lipofectamine™ were diluted in two 0.5 mL portions of Opti-MEM™ I Reduced Serum Medium. 2000 transfection reagent, mix the above two culture media, and incubate at room temperature for 30 minutes; (3) replace the original cell culture medium with 4mL DMEM high glucose medium, and then add 1mL of the above mixed medium; (4) culture at 37℃, 5% carbon dioxide for 4 hours, and replace the culture medium with 8mL complete culture medium containing 10% (v / v) premium fetal bovine serum and 90% (v / v) DMEM high glucose medium; (5) culture at 37℃, 5% carbon dioxide for 48-72 hours; (6) collect the culture medium by centrifugation for 5 minutes, the centrifugation conditions are 4℃, 2000G; (7) filter the culture medium with a 0.45μM filter membrane, and then purify the recombinant protein with a Ni-NTA agarose column. The amino acid sequence was verified by mass spectrometry, and three recombinant antigens, Derf1, Derf2, and Derf3, were successfully prepared.

[0069] 2. Preparation of test strips

[0070] like Figure 1 As shown, the test strip 10 comprises a sample adding pad 1, a marking pad 2, a detection pad 3 and a water absorbing pad 4. The sample adding pad, the marking pad, the detection pad and the water absorbing pad are overlapped with each other in sequence and adhered to a bottom plate 5.

[0071] Preparation method of absorbent pad 4: Place the filter paper in a 50°C oven to dry for later use.

[0072] Preparation method of label pad 2: Mouse anti-human IgE antibody and mouse anti-DNP antibody labeled with fluorescent microspheres were diluted with coating diluent and sprayed on glass fiber, and dried in a drying oven at 37° C. overnight.

[0073] The detection pad 3 is provided with a detection line and a quality control line. There are three detection lines in total. Starting from the sample pad 1, they are the Derf3 detection line 31 (T1 line), the Derf1 detection line 32 (T2 line), and the Derf2 detection line 33 (T3 line), which are coated with Derf3, Derf1, and Derf2 recombinant antigens respectively. The quality control line 34 (C line) is coated with DNP (1,3-dinitrobenzene)-BSA antigen. The material of the detection pad is selected from nitrocellulose membrane.

[0074] The test strip 10 is loaded into the test card 100. The structural diagram of the test card is shown in FIG. Figure 2 As shown, it comprises an upper cover 21 and a bottom plate 22, and the test strip 10 is placed between the upper cover and the lower cover. A sample addition hole 23 and an observation window 24 are provided on the upper cover.

[0075] During the test, the allergen-specific IgE antibody in the sample first binds to the mouse anti-human IgE antibody labeled with fluorescent microspheres, flows along the nitrocellulose membrane (detection pad) toward the absorbent pad under the action of chromatography, and binds to the coated allergen antigen at the detection line to form a complex of "allergen antigen-allergen-specific IgE antibody-mouse anti-human IgE antibody labeled with fluorescent microspheres"; when passing through the quality control line (line C), the mouse anti-DNP antibody labeled with fluorescent microspheres binds to the coated DNP-BSA antigen to form a quality control final reaction complex (DNP-BSA antigen-mouse anti-DNP antibody labeled with fluorescent microspheres). The fluorescent signal is read by a dry fluorescent immunoassay analyzer (iFIA-100 dry fluorescent immunoassay analyzer produced by Aikang Biotechnology (Hangzhou) Co., Ltd.), and the results are analyzed and displayed.

[0076] 3. Analysis of test results

[0077] The process of using the test card provided in this embodiment to perform the test is as follows:

[0078] 1) Turn on the dry fluorescence analyzer, and enter the test interface after the instrument self-checks. The specific operation of the instrument is carried out according to the instruction manual of the corresponding instrument model; 2) Accurately draw 40 μL of serum / plasma sample and add it to the sample well of the test card, and immediately vertically add 2 drops of sample diluent (about 70 μL), and start the timer; 3) After 15 minutes, read the test results. The diluent includes 20mMPBS, 0.02% Proclin-300 and 0.1% Tween-20.

[0079] 1. Clinical sample compliance analysis

[0080] The test card provided by the present invention is used to establish a standard curve of specific IgE antibodies (represented by Derf3-IgE, Derf1-IgE, and Derf2-IgE, respectively) of three allergens Derf3, Derf1, and Derf2. High-concentration Derf3-IgE positive serum, Derf2-IgE positive serum, and Derf1-IgE positive serum in clinical use are selected, and after gradient dilution, Derf3-IgE, Derf1-IgE, and Derf2-IgE standard products with concentrations of 0.5, 1, 5, 25, 50, and 100 IU / mL are prepared, respectively. After testing, the fluorescence value (T) of the corresponding test line and the fluorescence value (C) of the quality control line after adding different concentrations of standard products are read on a dry fluorescent immunoassay, and the ratio thereof is calculated, recorded as T / C. Each concentration of the standard product is tested 3 times, and the mean value (y) of the T / C ratio is calculated. The standard curve was prepared with the concentration of each standard and the mean T / C ratio (y). Based on the standard curve, the detection limits of Derf1-IgE, Derf2-IgE and Derf3-IgE were 0.35 IU / mL, 0.35 IU / mL and 0.35 IU / mL, respectively.

[0081] After adding Derf1-IgE, Derf2-IgE, and Derf3-IgE clinical samples with concentrations of 0, 0.3, 1, 3, 10, 30, and 100 IU / mL to the test card provided by the present invention, the detection concentration of each clinical sample is calculated according to the respective standard curves, so that the degree of conformity between the concentration value detected by the present invention and the actual concentration can be determined. The experimental results are shown in Tables 1, 2, and 3. At the same time, the actual concentrations of the clinical samples of Derf3-IgE, Derf1-IgE, and Derf2-IgE are taken as the X-axis, and the detected concentration values ​​are taken as the Y-axis, so as to draw a clinical conformity analysis diagram, as shown in FIG. Figure 3 , 4 and 5.

[0082] Table 1. Clinical sample test results with different Derf1-IgE concentrations

[0083]

[0084] Table 2. Clinical sample test results with different Derf2-IgE concentrations

[0085]

[0086] Table 3. Clinical sample test results with different Derf3-IgE concentrations

[0087]

[0088] Depend on Figure 3 , 4 As shown in Figures 5 and 5, no matter it is clinical samples with different Derf1-IgE concentrations, clinical samples with different Derf2-IgE concentrations, or clinical samples with different Derf3-IgE concentrations, the R value is greater than 0.99, which indicates that the detection concentration value of the present invention is well consistent with the actual concentration of the clinical sample, and the measurement result is relatively accurate.

[0089] 2. Analysis of sample test results

[0090] The T / C ratio obtained by the test was substituted into the corresponding standard curve to calculate the contents of Derf3-IgE, Derf1-IgE and Derf2-IgE in the serum samples.

[0091] 3. Accuracy & Precision

[0092] The fluorescence intensity of the analytes and their detection in low (L), medium (M), and high (H) concentration quality control samples (prepared using human positive serum, the low, medium, and high concentrations of Derf3-IgE, Derf1-IgE, and Derf2-IgE quality control products were 1 IU / mL, 10 IU / mL, and 50 IU / mL, respectively) were substituted into the established standard curve to calculate the concentrations of Derf3-IgE, Derf1-IgE, and Derf2-IgE in the quality control samples. The accuracy and precision results of each quality control sample of at least three analytical batches were then calculated. The acceptance criteria were that the clinical agreement rate between the mean of the measured value and the theoretical value was between 85.0% and 115.0%, and the precision (CV) of the three concentration levels (L, M, and H) was ≤15%. In addition, the specificity was also investigated, using about 20 endogenous and exogenous cross-interference substances (triglyceride 7 mg / mL, bilirubin 0.2 mg / mL, hemoglobin 2 mg / mL, rheumatoid factor 400 IU / m, antinuclear antibody 200 U / mL, antimitochondrial antibody 400 RU / mL, total immunoglobulin A 700 μg / mL, total immunoglobulin M 500 μg / mL, total immunoglobulin G 7000 μg / mL, total immunoglobulin D 60 μg / mL, total immunoglobulin E 60 IU / mL, yeast 10.2 μg / mL, Staphylococcus aureus 9.6 μg / mL, microhorn keratin 11.2 μg / mL, tyrophagoides putrescentiae 9.2 μg / mL, American cockroach 10.6 μg / mL, mouse hair dander 10.6μg / mL), no cross reaction was found, and the detection specificity was 100%, which met the clinical detection requirements.

[0093] Example 2: Screening of Derf3 recombinant protein coding sequence

[0094] At the beginning of this example, three allergen proteins Derf3 (Seq ID NO: 8), Derf1 (Seq ID NO: 7), and Derf2 (Seq ID NO: 2) prepared by original sequences were used to prepare test strips according to the method provided in Example 1. The detection line arrangement was as shown in Example 1. A low concentration quality control product (1 IU / mL) was used for detection. Each sample was repeated three times and the average value was taken. The detection method was as shown in Example 1. The detection results are shown in Table 4.

[0095] Table 4. Test results of existing recombinant antigen preparation test strips

[0096]

[0097] According to Table 4, it can be seen that when the test strips are directly prepared using the three recombinant proteins purchased, Derf3-IgE cannot be detected, and the accuracy of the detection values ​​of Derf1-IgE and Derf2-IgE is not high. The reason may be that the Derf3 recombinant antigen forms a polymer, which changes the spatial orientation and makes some antigen epitopes unable to be exposed. In addition, when the IgE of the three recombinant proteins are detected simultaneously, there is an obvious cross-influence.

[0098] In this example, homemade recombinant antigen protein was used, wherein the encoding nucleotide sequence of the Derf1 recombinant antigen was shown in Seq ID NO: 4, the encoding nucleotide sequence of the Derf2 recombinant antigen was shown in Seq ID NO: 5, and the encoding nucleotide sequence of the Derf3 recombinant antigen was shown in Seq ID NO: 6. The recombinant antigen was prepared according to the method provided in Example 1, the detection line sorting method was as shown in Example 1, and a low-concentration quality control product (1 IU / mL) was used for detection. Each sample was repeated three times and the average value was taken. The detection method was as shown in Example 1, and the detection results were shown in Table 5.

[0099] Table 5. Test results of existing recombinant antigen preparation test strips

[0100]

[0101] It can be seen from Table 5 that the three self-made recombinant antigens of the present invention can be used to prepare the test strips to achieve accurate simultaneous detection of Derf3-IgE, Derf1-IgE, and Derf2-IgE in serum samples. The reason may be that the self-made recombinant antigens of the present invention optimize the nucleotide sequence, and the prepared recombinant antigens can fully expose the antigen epitopes, improve the capture efficiency of specific IgE, and effectively solve the problem caused by cross-influence.

[0102] Example 3: Effects of different mutation sites on Derf3 detection results

[0103] In this example, the following seven mutants were constructed for the Derf3 recombinant antigen protein, mutant 1: mutation D58P, whose amino acid sequence is shown in Seq ID NO: 9, and whose encoding nucleotide sequence is shown in Seq ID NO: 10; mutant 2: mutation S106D, whose amino acid sequence is shown in Seq ID NO: 11, and whose encoding nucleotide sequence is shown in Seq ID NO: 12; mutant 3: mutation G231C, whose amino acid sequence is shown in Seq ID NO: 13, and whose encoding nucleotide sequence is shown in Seq ID NO: 14; mutant 4: mutations D58P and S106D, whose amino acid sequence is shown in Seq ID NO: 15, and whose encoding nucleotide sequence is shown in Seq ID NO: 16; mutant 5: mutations D58P and G231C, whose amino acid sequence is shown in Seq ID NO: 17, and whose encoding nucleotide sequence is shown in Seq ID NO: 18; mutant 6: mutations S106D and G231C, whose amino acid sequence is shown in Seq ID NO: 19, and the encoding nucleotide sequence is shown in Seq ID NO: 20; Mutant 7 (Example 1): mutations D58P, S106D and G231C, its amino acid sequence is shown in Seq ID NO: 3, and the encoding nucleotide sequence is shown in Seq ID NO: 6. The preparation methods of 7 kinds of Derf3 recombinant antigen proteins are as shown in Example 1.

[0104] The stability of the mutants was evaluated by measuring the thermal stability Tm value of the mutants using circular dichroism (CD) spectroscopy, and compared with the original Derf3 recombinant antigen protein. The test results are shown in Table 6. Tm represents the midpoint temperature of thermal denaturation of the protein, that is, the temperature at which the protein is 50% unfolded, reflecting the trend of protein conformation change during the temperature change process. The higher the Tm, the better the stability.

[0105] Table 6. Stability of Derf3 recombinant antigen protein

[0106]

[0107] According to Table 6, compared with the original Derf3 recombinant antigen, the thermal stability of the mutants has been significantly improved. Among the mutants with a single mutation site, the stability of mutant 3 has been improved most significantly, with a Tm of 54.3°C. The thermal stability of the mutants after combined mutations is higher than that of the single mutation site, and the highest thermal stability is mutant 7 (Seq IDNO: 3). The higher the thermal stability, the more stable the spatial conformation of the protein, and the more stable the prepared HA protein mutant.

[0108] At the same time, this example also found that when the mutant with higher thermal stability is used to detect antibodies, more antigen epitopes can be exposed, which significantly improves the ability of Derf3 recombinant antigen to specifically recognize antibodies. The above mutants were used as Derf3 recombinant antigens, and according to the method provided in Example 1, test strips for simultaneously detecting IgE of three allergens were prepared. The detection line arrangement method was as shown in Example 1. Low-concentration quality control products (1 IU / mL) were used for detection. Each sample was repeated three times and the average value was taken. The detection method was as shown in Example 1. The detection results are shown in Table 7.

[0109] Table 7. Test results of existing recombinant antigen preparation test strips

[0110]

[0111] It can be seen from Table 7 that the mutants obtained by mutating different sites are helpful to improve the detection accuracy of Derf3-IgE, and can also avoid the cross-influence between Derf1-IgE, Derf2-IgE, and Derf3-IgE. Among them, the most preferred mutation method is mutant 7. The Derf3 recombinant antigen prepared by mutating three sites at the same time can be used to prepare test strips. It can truly realize the simultaneous detection of Derf1-IgE, Derf2-IgE, and Derf3-IgE, and significantly improve the detection accuracy and sensitivity.

[0112] Example 4: Effects of different test line sorting methods on test results

[0113] In this example, the test strips were prepared by the method provided in Example 1, wherein the front and back order of the three test lines were respectively arranged in the four ways shown in Table 5, and a low concentration quality control product (1 IU / mL) was used for detection. Each sample was repeated three times and the average value was taken. The detection method was as shown in Example 1, and the detection results are shown in Table 8.

[0114] Table 8. Impact of different test line sorting methods

[0115]

[0116] It can be seen from Table 8 that different sorting methods for the three test lines will directly affect the accuracy of the test results, especially the position of the Derf3 test line, which has the most obvious impact. The most preferred sorting method is: Derf3- Derf1- Derf2, which can minimize the mutual influence among the three.

[0117] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. A Derf3 recombinant antigen, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

3.

2. A test strip for detecting dust mite antibodies, characterized in that: The test strip is provided with a detection line and a quality control line, wherein the detection line comprises a Derf3 detection line, and the Derf3 detection line is coated with a Derf3 recombinant antigen; the amino acid sequence of the Derf3 recombinant antigen is shown in SEQ ID NO.

3.

3. The test strip according to claim 2, characterized in that: The detection line also includes a Derf1 detection line and / or a Derf2 detection line, wherein the Derf1 detection line and the Derf2 detection line are coated with a Derf1 recombinant antigen and a Derf2 recombinant antigen respectively; the amino acid sequences of the Derf1 and Derf2 recombinant antigens are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

4. The test strip according to claim 3, characterized in that: The test strip also includes a marking pad, which contains mouse anti-human IgE antibodies.

5. The test strip according to claim 4, characterized in that: The detection lines are arranged in the order of Derf3, Derf1, and Derf2 detection lines, wherein the Derf3 detection line is closest to the sample addition pad of the test strip.

6. A method for detecting dust mite antibody Derf3, characterized in that: The method is used for non-disease diagnosis purposes and uses the test strip as claimed in claim 2 for detection.

7. A method for detecting dust mite antibodies Derf3, Derf1 and Derf2, characterized in that: The method is used for non-disease diagnosis purposes, and detection is performed using the test strip as described in any one of claims 3 to 5.

8. A method for preparing a recombinant dust mite antigen, characterized in that: The following steps are involved: (1) inserting a nucleotide encoding a recombinant antigen into an empty vector to obtain a recombinant vector; the recombinant antigens include Derf1 recombinant antigen, Derf2 recombinant antigen and Derf3 recombinant antigen, the encoding nucleotide sequence of the Derf1 recombinant antigen is shown in SEQ ID NO.4, the encoding nucleotide sequence of the Derf2 recombinant antigen is shown in SEQ ID NO.5, and the encoding nucleotide sequence of the Derf3 recombinant antigen is shown in SEQ ID NO.6; (2) Transfection of the recombinant vector into the eukaryotic cell expression system; (3) Collecting the culture supernatant of the eukaryotic cell expression system; (4) Purify the recombinant antigen using a chromatography column.

9. Use of a recombinant antigen for preparing a reagent for improving the accuracy of detecting dust mite allergy IgE antibodies in serum samples, characterized in that: The recombinant antigens include Derf1 recombinant antigen, Derf2 recombinant antigen and Derf3 recombinant antigen. The amino acid sequence of the Derf1 recombinant antigen is shown in SEQ ID NO.1, the amino acid sequence of the Derf2 recombinant antigen is shown in SEQ ID NO.2, and the amino acid sequence of the Derf3 recombinant antigen is shown in SEQ ID NO.3.

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