SRP54 recombinant antigen as well as preparation method and application thereof

Abstract

The invention discloses an SRP54 recombinant antigen as well as a preparation method and application thereof, compared with an existing antigen, the SRP54 recombinant antigen disclosed by the invention has the advantages that the protein expression quantity of the SRP54 is remarkably improved, and more required target proteins can be obtained; and the thermal stability of the protein is also improved, so that subsequent application and development are facilitated. Meanwhile, the optimization of the antigen also improves the steric hindrance problem of SRP54 antibody detection, and antigen sites are more fully exposed, so that the sensitivity of SRP54 antibody detection is improved. When the antigen is used for SRP54 antibody detection or polymyositis diagnosis, the accuracy can be greatly improved, and a new technical support is provided for production and application. Therefore, the recombinant antigen disclosed by the invention has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a SRP54 recombinant antigen, and a preparation method and application thereof. Background Art

[0002] Signal recognition particle (SRP) is a ribonucleoprotein complex that co-translationally targets proteins with signal sequences to the endoplasmic reticulum (ER) and protects their mRNA from degradation, playing a role in the proliferation and differentiation of granulocytes, the migration ability of neutrophils, and the development of the exocrine pancreas. The mammalian SRP consists of two parts, namely, an RNA of more than 300 bases (7SLRNA or SRP RNA) and six proteins of 9 kDa, 14 kDa, 19 kDa, 54 kDa, 68 kDa, and 72 kDa, respectively. SRP54 is a cytoplasmic ribonucleoprotein complex that directs the transport of newly synthesized secretory proteins from polysomes to the endoplasmic reticulum.

[0003] In 1986, Reeves et al. first discovered anti-SRP autoantibodies against the SRP54 subunit. In 2002, Miller et al. found that the muscle pathology of anti-SRP antibody-positive patients was severe necrosis and regeneration of muscle fibers, lack of inflammatory cell infiltration, and clinically characterized by relatively acute onset of severe myositis and poor response to glucocorticoid treatment. The concept of anti-SRP antibody myopathy (anti-SRP antibody syndrome) was first proposed.

[0004] In anti-signal recognition particle (SRP) myositis, a special subtype of idiopathic inflammatory myopathy (IIM), the detected anti-SRP54 antibodies are highly specific for the diagnosis of this disease. Patients with positive anti-SRP54 antibodies usually show severe myositis symptoms with acute or subacute onset, such as muscle weakness and pain, and often respond poorly to conventional immunotherapy. In some rare neuromuscular diseases or patients with unexplained muscle lesions, the detection of SRP54 antibodies can also help to rule out abnormal immune responses related to SRP54. At the same time, in the study of certain liver diseases or systemic autoimmune diseases, it was also found that SRP54 antibodies may be related to specific clinical manifestations or pathological processes of the disease.

[0005] The existing detection of SRP54 antibodies is usually carried out by ELISA and other methods, in which the antigen is coated on a carrier to capture the SRP54 antibodies in the sample to be tested. However, the existing coated antigens are still not sensitive enough for myositis detection.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a SRP54 recombinant antigen, and a preparation method and application thereof. The SRP54 recombinant antigen has a high detection sensitivity in detecting polymyositis, and its expression level is also significantly improved.

[0008] In a first aspect, the present invention provides a SRP54 recombinant antigen, the amino acids of which are shown in SEQ ID NO.1.

[0009] The present invention optimizes the existing SRP54 antigen protein (UniProtKB-P61011) to obtain a new SRP54 recombinant antigen, whose amino acid sequence is shown in SEQ ID NO.1.

[0010] The above-mentioned SRP54 recombinant antigen can improve the steric hindrance problem of SRP54 antibody detection, so that the antigen site is more fully exposed, thereby improving the sensitivity of SRP54 antibody detection. At the same time, through the optimization of the present invention, the obtained SRP54 recombinant antigen also improves the expression amount and thermal stability.

[0011] In some embodiments, the recombinant SRP54 antigen of the present invention may include further modifications, such as tag modifications. The tag modification of the recombinant SRP54 antigen of the present invention is not particularly limited, for example, it may be a protein purification tag, such as an affinity tag, such as a biotin tag.

[0012] In a second aspect, the present invention provides a biological material related to the above-mentioned SRP54 recombinant antigen, which is any one of the following:

[0013] (1) Nucleic acid molecules encoding the above-mentioned SRP54 recombinant antigen:

[0014] (2) an expression cassette containing the nucleic acid molecule described in (1);

[0015] (3) a recombinant vector containing the nucleic acid molecule described in (1) or the expression cassette described in (2);

[0016] (4) A cell containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant vector described in (3).

[0017] According to the above-mentioned SRP54 recombinant antigen, the present invention also provides a nucleic acid molecule encoding the above-mentioned SRP54 recombinant antigen.

[0018] Nucleic acid molecules can be DNA or RNA, and in some cases, can modify nucleic acid molecules for use in vectors of the present invention, such as for codon optimization. In some cases, for the purpose of being cloned into a vector, the sequence can be designed to contain a terminal restriction site sequence. Nucleic acid molecules can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of the encoding nucleic acid separated in one or more given cells or from the one or more given cells.

[0019] In a specific embodiment, the above nucleic acid molecules can be optimized according to the codon preference of the host cell and then artificially synthesized into gene fragments. It should be understood that nucleic acid molecules that can be translated into the above amino acid sequences are within the scope of protection of the present invention.

[0020] The present invention provides an expression cassette, which comprises the above nucleic acid molecule and also contains regulatory sequences, such as a promoter and a terminator.

[0021] An expression cassette refers to a nucleic acid construct comprising a coding sequence and a regulatory sequence that are operably connected when introduced into a host cell, resulting in transcription and / or translation of an RNA or polypeptide, respectively. An expression cassette is understood to include a promoter, a target gene open reading frame, and a transcription terminator that allow transcription to begin. Typically, the promoter sequence is placed upstream of the target gene, and the distance from the target gene is compatible with expression control.

[0022] The present invention provides a recombinant vector comprising the above nucleic acid molecule or expression box.

[0023] Vectors include, but are not limited to: single-stranded, double-stranded or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, no free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other polynucleotide species known in the art. Among the most commonly used vector types are "plasmids", which refer to circular double-stranded DNA loops into which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques. Recombinant expression vectors can contain a form suitable for expressing nucleic acids in host cells, which means that the recombinant expression vector includes one or more regulatory elements, which can be selected based on the host cell used for expression and can be operably linked to the nucleic acid sequence to be expressed.

[0024] The present invention provides a cell, which comprises the above nucleic acid molecule or the above expression box or the above expression vector, and the cell is capable of expressing the above SRP54 recombinant antigen.

[0025] The cell can be any cell useful in the production of the SRP54 recombinant antigen of the present invention. In order to produce the SRP54 recombinant antigen, the nucleic acid encoding the SRP54 recombinant antigen can be isolated and inserted into one or more vectors for further cloning and / or expression in the host cell. Such nucleic acids can be easily isolated and sequenced using conventional techniques. The method for introducing the vector into the host cell is well known and is not specifically limited in the present invention.

[0026] In a specific embodiment, the above-mentioned host cells can be prokaryotic cells or eukaryotic cells, including: bacterial hosts such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis; eukaryotic hosts such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, plant cells, etc., which can be selected according to actual conditions in specific experiments.

[0027] In a third aspect, the present invention provides a complex comprising the above-mentioned SRP54 recombinant antigen.

[0028] The present invention also provides a complex, which further comprises a solid support, a detectable marker or a binding partner conjugated to the SRP54 recombinant antigen. The SRP54 recombinant antigen in the complex can be conjugated or coupled directly or indirectly.

[0029] In some embodiments, the solid support comprises magnetic particles, a microtiter plate or a cellulose membrane; the detectable marker can be a metal particle, a fluorescent marker, a chromophore marker, an electron-dense marker, a chemiluminescent marker, a radioactive marker, or an enzyme marker, specifically colloidal gold, a radioisotope, a fluorophore, a spin marker, or a phage marker, or a rhodamine, fluorescein, an acridinium ester, luciferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidase, glucose oxidase, galactose oxidase or glucose-6-phosphate dehydrogenase marker; the binding partner can be biotin, streptavidin or avidin.

[0030] In a fourth aspect, the present invention provides a composition comprising the above-mentioned SRP54 recombinant antigen or the above-mentioned biological material or the above-mentioned complex.

[0031] The above composition may be a pharmaceutical composition or an immune composition. In addition to the above SRP54 recombinant antigen or the above biological material or the above complex, the above composition may also include other components suitable for effectively preserving its structure and activity, such as buffer, surfactant, protective agent, metal salt, preservative, etc., which can be selected and matched by those skilled in the art according to actual needs.

[0032] In some embodiments, the composition is in liquid form. In other embodiments, the composition can also be in a freeze-dried form.

[0033] The preparation method of SRP54 recombinant antigen can be prepared by any appropriate method known in the art, such as direct synthesis, such as transformation expression; one of which is listed below.

[0034] In a fifth aspect, the present invention provides a method for preparing the above-mentioned SRP54 recombinant antigen, which comprises culturing the above-mentioned cells under suitable conditions, and then isolating and obtaining the SRP54 recombinant antigen from the culture medium and / or cells.

[0035] In some embodiments, the above cells can be cultured under appropriate conditions, and then the SRP54 recombinant antigen can be isolated from the culture medium and / or cells.

[0036] In some embodiments, a baculovirus / insect cell expression system is used to produce SRP54 recombinant antigen, specifically comprising: connecting a nucleic acid molecule encoding the SRP54 recombinant antigen to a vector to obtain a recombinant vector; then transforming the recombinant vector into competent cells to obtain a recombinant baculovirus plasmid; then transfecting the recombinant baculovirus plasmid into insect cells, and culturing and purifying to obtain the SRP54 recombinant antigen.

[0037] In some embodiments, insect cells include: Sf9 cells, Hi5 cells, and Sf21 cells.

[0038] In a sixth aspect, the present invention provides the use of the above-mentioned SRP54 recombinant antigen, biological material or complex in the preparation of a product for detecting SRP54 antibodies.

[0039] In a seventh aspect, the present invention provides the use of the above-mentioned SRP54 recombinant antigen, biomaterial or complex in the preparation of a product for auxiliary diagnosis of myositis.

[0040] In an eighth aspect, the present invention provides the use of the above-mentioned SRP54 recombinant antigen, biomaterial or complex in the preparation of a product for the preparation of SRP54 antibodies.

[0041] By using the above-mentioned SRP54 recombinant antigen and its related biological materials and complexes, we can further provide the use of the above-mentioned SRP54 recombinant antigen and its related biological materials or complexes in the preparation of products for detecting SRP54 antibodies.

[0042] In some embodiments, the SRP54 antibody in the test sample recognizes an epitope in the SRP54 recombinant antigen of the present invention, and thus the recombinant antigen of the present invention can be used to detect the SRP54 antibody in the test sample in an immunoassay.

[0043] In some embodiments, the SRP54 recombinant antigen of the present invention can be used for immunoassays, such as immunoblotting, enzyme-linked immunosorbent assay, fluorescent immunochromatography, colloidal gold immunochromatography, and chemiluminescence assay, etc. Among them, the SRP54 recombinant antigen of the present invention can be used as a capture antigen or a detection antigen, or as a detection antigen and a capture antigen at the same time. The other antigen paired with the SRP54 recombinant antigen of the present invention can be the same or different, as long as it includes the SRP54 recombinant antigen of the present invention.

[0044] By using the above-mentioned SRP54 recombinant antigen and its related biological materials and complexes, the use of the above-mentioned SRP54 recombinant antigen and its related biological materials or complexes in the preparation of products for auxiliary diagnosis of SRP54 antibody-positive diseases, especially myositis, can be further provided. The SRP54 recombinant antigen and its related biological materials or complexes in the above-mentioned products can be used to assist in the identification of whether a subject is a myositis patient.

[0045] By using the above-mentioned SRP54 recombinant antigen and its related biological materials and complexes, the use of the above-mentioned SRP54 recombinant antigen and its related biological materials or complexes in preparing products for preparing SRP54 antibodies can be further provided. The SRP54 recombinant antigen and its related biological materials or complexes included in the above-mentioned products can be used to prepare SRP54 antibodies that can specifically bind to the SRP54 recombinant antigen. For the preparation method of the SRP54 antibody, any appropriate method known in the art can be used for preparation, for example, immunizing an animal with the SRP54 recombinant antigen to produce the SRP54 antibody.

[0046] In another embodiment, recombinant SRP54 antigen can be used to purify SRP54 antibody; for example, recombinant SRP54 antigen is fixed on a chromatography column, and the SRP54 antibody is contacted with the recombinant SRP54 antigen and then indirectly fixed on the chromatography column, and then further purified and eluted with different eluents to obtain purified SRP54 antibody.

[0047] In a seventh aspect, the present invention provides a kit comprising the above-mentioned SRP54 recombinant antigen or complex.

[0048] Based on the above scheme, the present invention can also provide a kit, which comprises the above SRP54 recombinant antigen or complex. By using the SRP54 recombinant antigen of the present invention as a detection antigen, the sensitivity and accuracy of anti-SRP54 antibody detection can be greatly improved.

[0049] In some embodiments, the kit may further include a calibrator or control, such as a calibrator or control SRP54 antibody.

[0050] In some embodiments, the SRP54 recombinant antigen or complex of the present invention is contained in a container such as a test tube, a microplate or a test strip in a kit. The kit may also include a solid support such as magnetic beads, a test tube, a microplate, a cuvette, a film, a filter paper, a syringe, a pipette, a buffer such as an assay buffer, a wash buffer, a pretreatment reagent, a detectable label such as an enzyme-labeled substrate solution, etc.

[0051] In some embodiments, the SRP54 recombinant antigen or complex of the present invention is coated on a solid phase carrier, which is a liquid phase chip, which has superparamagnetism and is composed of a biocompatible polymer (Barcoded Magnetic Beads, BMB). The BMB herein, i.e., barcoded magnetic particles, is a paramagnetic material incorporated into a biocompatible polymer, and a 12-bit binary digital barcode is engraved on the magnetic particles by photolithography. Barcoded magnetic particles are also called barcoded magnetic beads or digital magnetic beads. When "K" is used as a unit herein, it means the number is "thousands", and 50K BMB means 50,000 barcoded magnetic particles.

[0052] By using the above kit, the present invention can also provide a method for detecting SRP54 antibodies, which can be: contacting the SRP54 recombinant antigen of the present invention with a sample to be tested, forming a complex of the SRP54 antibody and the SRP54 recombinant antigen when the SRP54 antibody is present in the sample, and then detecting the presence of the complex, wherein the presence of the complex indicates the presence of the SRP54 antibody in the sample. This method can accurately detect whether the SRP54 antibody is present in the target sample.

[0053] The present invention has the following beneficial effects:

[0054] The present invention provides a SRP54 recombinant antigen and a preparation method thereof. The SRP54 recombinant antigen has a significantly improved expression efficiency compared to the original protein (2.46 times the original). At the same time, the recombinant antigen site is more fully exposed, solving the steric hindrance problem of the original antigen during SRP54 antibody detection. The recombinant antigen of the present invention has a significant improvement in signal-to-noise ratio, sensitivity, specificity, reagent stability, etc. for the recognition of SRP54 antibodies compared to the original antigen. It can be used for SRP54 antibody detection to greatly improve the accuracy, provide new technical support for the production and application of SRP54 antibody detection reagents, and have good application prospects for the auxiliary diagnosis of polymyositis. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0056] Figure 1 The SDS-PAGE detection results of the recombinant protein SRP54 in Example 1. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0058] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0059] Example 1

[0060] This example is the expression and purification of SRP54 recombinant protein

[0061] S1. Insert the nucleic acid fragment (nucleotide sequence shown in SEQ ID NO.2) encoding the SRP54 recombinant protein (amino acid sequence shown in SEQ ID NO.1) into the pFastBac1 vector, with the restriction enzyme sites of BamHI and HindⅢ inserted.

[0062] S2. The recombinant plasmid was transformed into E. coli DH5α competent cells by heat shock method, and then spread on LB solid agar medium and cultured inverted at 37℃ overnight. Positive clones were picked and expanded after identification and plasmid extraction.

[0063] S3. Plasmid transfection and packaging of P1 virus:

[0064] S31. Sf9 cells (in the logarithmic growth phase and with a viability greater than 95%) were seeded into a six-well plate and cultured at 28° C. for 1 h to allow the cells to adhere (SIM SF, serum-free, 3 mL / well).

[0065] S32. Discard the culture medium, add new culture medium (SIM SF, without serotonin) and continue culturing to prepare for transfection.

[0066] S33.Bacmid DNA and transfection reagent mixture preparation:

[0067] (1) Take a 1.5 mL sterile centrifuge tube, add 20 μg of Bacmid DNA (the expression plasmid prepared by S1 and S2 above) into 100 μL of SIM SF medium (serum-free), gently blow with a gun to mix, and let stand at room temperature for about 2-5 minutes.

[0068] (2) Take another 1.5 mL sterile centrifuge tube, pipette 10 μL of transfection reagent into 100 μL of SIM SF medium (serum-free), gently blow evenly with a gun, and let stand at room temperature for about 2-5 minutes.

[0069] (3) Use a pipette to gently add the bacmid diluted in step (1) to the transfection reagent diluted in step (2), gently blow with a gun to mix, and let stand at room temperature for 20 minutes.

[0070] (4) Add the mixture prepared in step (3) evenly dropwise to the cells in step S32 and gently shake to mix.

[0071] (5) After culturing at 28°C for 4 h, replace with fresh culture medium (SIM SF, 10% FBS), continue culturing for 6 days, and then collect the supernatant by centrifugation. This is the P1 virus and store it in a cryopreservation tube away from light.

[0072] S4. Virus Amplification:

[0073] S41.P2 virus preparation: Inoculate Sf9 cells (in logarithmic growth phase and viability greater than 95%) in a T75 culture flask, with a culture medium of SIM + 5% FBS, with a total volume of 15 mL. Add 1.5 mL of P1 virus and continue to culture at 28°C in the dark. After 4 days of culture, centrifuge and collect the supernatant, which is the P2 virus.

[0074] S42.P3 virus preparation: Sf9 cells (in logarithmic growth phase and viability greater than 95%) were inoculated in a culture flask, and the culture medium was SIM+double antibody+2% FBS (50 mL). 3 mL of P2 virus was added and cultured at 28°C in the dark. After the cell viability reached 70%-80%, the supernatant was collected by centrifugation, i.e., P3 virus.

[0075] S5. Determine the P3 virus titer.

[0076] S6. Protein expression:

[0077] In a shake flask, the inoculation density of 1 L is 4.0 × 10 6 To SF9 cells with a viability of more than 95% cells / mL, add an appropriate amount of P3 virus (MOI=2-5), culture at 28°C in the dark, and rotate at 100 rpm.

[0078] S7. Protein purification:

[0079] (1) Take out the collected cells, add lysis buffer (lysis buffer formula: 1× PBS, 1 M NaCl, 10 mM imidazole, 5% glycerol, pH 7.4), and resuspend the cells on ice.

[0080] (2) Ultrasonic disruption: The bacteria were disrupted at an ultrasonic power of 520 W, with a cycle of 4 s on, 3 s off, and 200 cycles until the bacterial solution became clear and transparent.

[0081] (3) After the ultrasound, centrifuge at 4°C, 12,000 rpm for 30 min (the centrifuge is pre-cooled in advance) to separate the supernatant and the precipitate, and filter the supernatant using a 0.45 μM membrane.

[0082] (4) Take 2 mL of Ni-TED resin and equilibrate the column with lysis buffer for 5 CV, then add it to the filtered supernatant, mix well, and place it in an ice box on a shaker for incubation for 1-2 hours.

[0083] (5) Pour the incubated mixture into the gravity column and collect the flow-through in a clean centrifuge tube. After the flow-through has completely flowed out, add 10 CV of lysis buffer to clean the residue in the tube.

[0084] (6) Elution: Use 20 mL of elution buffer 1 (elution buffer 1 formula: 1×PBS, 20 mM imidazole, 5% glycerol, pH 8.0), 20 mL of elution buffer 2 (elution buffer 2 formula: 1×PBS, 50 mM imidazole, 5% glycerol, pH 8.0), 20 mL of elution buffer 3 (elution buffer 3 formula: 1×PBS, 150 mM imidazole, 5% glycerol, pH 8.0), and 20 mL of elution buffer 4 (elution buffer 4 formula: 1×PBS, 250 mM imidazole, 5% glycerol, pH 8.0) for elution and collect the eluate.

[0085] (7) Protein electrophoresis was used to verify the purity of the target protein in the eluate. The eluate with a purity ≥ 90% was dialyzed into a protein preservation solution (the formula of the protein preservation solution was: 1× PBS, 5% glycerol, pH 8.0).

[0086] (8) The protein concentration is concentrated to 1-2 mg / mL and can be used for antigen verification.

[0087] Figure 1 The figure is the SDS-PAGE detection result of the SRP54 recombinant protein obtained by the above preparation method.

[0088] Comparative Example 1

[0089] This example is the expression and purification of SRP54 proprotein, and its steps are basically the same as those in Example 1, except that in this experiment, the protein sequence inserted in S1 is replaced with SRP54 proprotein (amino acid sequence as shown in SEQ ID NO.3, nucleic acid sequence as shown in SEQ ID NO.4), to obtain purified SRP54 proprotein.

[0090] Example 2

[0091] Comparison of the expression levels of the original SRP54 protein in Comparative Example 1 and the recombinant SRP54 protein in Example 1

[0092] The protein concentration obtained after purification was detected using the Biyuntian BCA protein concentration assay kit, and the total protein expression was calculated, and then the protein yield was estimated based on the amount of cells expressing the protein. Two parallel experiments were performed for each antigen, and the results are shown in Table 1:

[0093] Table 1. Protein expression results of wild type and optimized

[0094]

[0095]

[0096] *WT refers to the SRP54 original protein of Comparative Example 1; DE refers to the SRP54 recombinant protein of Example 1

[0097] It can be seen from the results in Table 1 that the protein expression level and protein yield of the SRP54 recombinant protein (DE) of Example 1 are significantly improved compared with the original protein, and the average yield is about 2.44 times that of the original.

[0098] Example 2

[0099] This example is a test of the effect of using SRP54 original protein and recombinant SRP54 protein for SRP54 antibody detection

[0100] 1. Preparation of antigen-liquid phase chip BMB complex

[0101] The solid phase carrier is a digital liquid chip (Digital Liquid Chip) from Applied BioCode, which has superparamagnetism and is composed of biocompatible polymers (Barcoded Magnetic Beads, BMB). Each chip has a unique 12-bit binary digital code. The BMB surface is covalently cross-linked with abundant carboxyl groups.

[0102] The recombinant SRP54 protein of Example 1 and the original SRP54 protein of Comparative Example 1 were coated with BMB magnetic beads in the same manner. The coating process was as follows:

[0103] 1) Pipette the required volume of BMB suspension;

[0104] 2) Wash several times with activation buffer;

[0105] 3) Add a certain amount of EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) / NHS (Chinese name for N-hydroxysuccinimide) and react at room temperature with shaking for 50 minutes to activate the carboxyl group;

[0106] 4) Wash with coating solution several times;

[0107] 5) adding the prepared antigen (the recombinant SRP54 protein of Example 1 or the original SRP54 protein of Comparative Example 1) and incubating at room temperature with shaking for 60 minutes;

[0108] 6) Block BMB with blocking solution (containing 1% BSA bovine serum albumin and 0.1% Tween 20) for 60 min;

[0109] 7) Remove the blocking solution and resuspend with coating solution containing 1% BSA bovine serum albumin.

[0110] 2. Testing process

[0111] 1) Take the diluted sample to be tested and add it to the prepared antigen-liquid phase chip BMB complex, and incubate at 37°C for 15 minutes (to form a magnetic barcode-antigen-antibody complex);

[0112] 2) Wash three times in a magnetic plate washer to remove unbound substances and remove residual liquid;

[0113] 3) Add 50 μL of fluorescent labeled mouse anti-human IgG antibody IgG-PE (made by our company) to each well and incubate at 37°C for 15 min to form a magnetic barcode-antigen-antibody-secondary antibody complex;

[0114] 4) Wash the plate three times using a magnetic plate washer to remove unbound substances and remove residual liquid; detect the corresponding fluorescence value under a fluorescence reader and determine the positive or negative value based on the fluorescence value.

[0115] This implementation includes 75 SRP54 antibody positive samples and 25 SRP54 antibody negative serum samples to be tested, and the positive detection rate of each protein is verified as sensitivity, and the negative detection rate is verified as specificity. The test results of each antigen protein are shown in Table 2.

[0116] Table 2 Comparison of detection results of wild-type and optimized SRP54 antigen

[0117]

[0118] *WT refers to the SRP54 original protein of Comparative Example 1; DE refers to the SRP54 recombinant protein of Example 1

[0119] According to the data in Table 2 , among the 75 positive samples, DE detected 14 more cases than WT, the sensitivity could be increased to 96%, the specificity to 100%, and no false positives occurred.

[0120] Six SRP54 antibody-positive samples (PC) and two SRP54 antibody-negative samples (NC) were further randomly selected to compare the signal-to-noise ratios of different antigen detections. The results are shown in Table 3.

[0121] Table 3 Signal-to-noise ratio of wild-type and optimized SRP54 antigen detection

[0122]

[0123]

[0124] *Signal-to-noise ratio = positive sample signal value / negative sample average signal value.

[0125] According to the results in Table 3, it can be seen that the optimized SRP54 antigen has a better signal-to-noise ratio, can better resist noise interference, and ensure signal clarity and stability.

[0126] Example 3

[0127] Stability test

[0128] The test method is as follows:

[0129] 1) Prepare antigen-liquid chip BMB complexes coated with different antigens according to the method of Example 2. Each complex is divided into two parts, one is stored at 4°C for 18 hours, and the other is stored at 37°C for 18 hours. After treatment, the diluted samples to be tested are added to each of them and incubated at 37°C for 15 minutes (to form a magnetic barcode-antigen-antibody complex).

[0130] 2) Wash three times in a magnetic plate washer to remove unbound substances and remove residual liquid;

[0131] 3) Add 50 μL of fluorescently labeled mouse anti-human IgG antibody IgG-PE to each well and incubate at 37°C for 15 min to form a magnetic barcode-antigen-antibody-secondary antibody complex;

[0132] 4) Wash the plate three times with a magnetic plate washer to remove unbound substances and remove residual liquid; detect the corresponding fluorescence under a fluorescence reader.

[0133] The samples to be tested in this example include 14 SRP54 antibody positive samples (PC) and 6 SRP54 antibody negative samples (NC). The test results of each protein after heat treatment are compared, and the results are shown in Table 4.

[0134] Table 4 Thermal accelerated stability results of wild-type and optimized SRP54 recombinant antigens

[0135]

[0136] *Signal retention rate = signal value of sample treated at 37°C / signal value of sample treated at 4°C.

[0137] It can be concluded from Table 4 that, by comparing the reactivity of each protein treated at 37°C and 4°C, the average DE signal retention rate was 89% in 14 positive samples, which was 39% higher than WT; the average DE signal retention rate was 105% in 6 negative samples, which was 30% higher than WT. This shows that the SRP54 recombinant antigen is more stable than the original protein.

[0138] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A SRP54 recombinant antigen, characterized in that: The amino acid sequence of the SRP54 recombinant antigen is shown in SEQ ID NO.

1.

2. The biological material related to the SRP54 recombinant antigen according to claim 1, characterized in that: Any of the following: (1) A nucleic acid molecule encoding the SRP54 recombinant antigen according to claim 1; (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant vector containing the nucleic acid molecule described in (1) or the expression cassette described in (2); (4) A cell containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant vector described in (3).

3. A composite, characterized in that Comprising the SRP54 recombinant antigen according to claim 1.

4. The composite according to claim 3, characterized in that The complex also includes a solid support, a detectable label or a binding partner conjugated to the SRP54 recombinant antigen.

5. A composition, characterized in that It comprises the recombinant antigen according to claim 1 or the biological material according to claim 2 or the complex according to claim 3 or 4.

6. The method for preparing the SRP54 recombinant antigen according to claim 1, characterized in that: The cells of claim 2 are cultured under appropriate conditions, and then the SRP54 recombinant antigen is isolated from the culture medium and / or the cells.

7. Use of the SRP54 recombinant antigen according to claim 1, the biomaterial according to claim 2, or the complex according to claim 3 or 4 in any one of the following (I) to (III): (I) Application in the preparation of products for detecting SRP54 antibodies; (II) Application in the preparation of products for auxiliary diagnosis of myositis; (III) Use in the preparation of products for the preparation of SRP54 antibodies.

8. A kit, characterized in that: It comprises the SRP54 recombinant antigen according to claim 1 or the complex according to claim 3 or 4.

9. The kit according to claim 8, characterized in that The kit is used for auxiliary diagnosis of myositis and also includes an autoantibody detection reagent for at least one of the following markers: Jo-1, MDA-5, SAE-1, NXP-2, PL7, PL12, EJ, HMGCR, Ku, OJ, Mi-2α, Mi-2β, TIF1-γ, SSA / Ro52, PM-scl, PM-scl-100, SAE-2 and CN-1A.

10. Use of the SRP54 recombinant antigen according to claim 1, the biomaterial according to claim 2, the complex according to claim 3 or 4, or the kit according to claim 9 in any one of the following (a) or (b): (a) Detection of SRP54 antibodies for non-diagnostic and therapeutic purposes; (b) Used to purify SRP54 antibodies.

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