A recombinant srp54 antigen, and preparation method and application thereof

Abstract

The application discloses an SRP54 recombinant antigen as well as a preparation method and application thereof. Compared with existing antigens, the SRP54 recombinant antigen of the application not only significantly improves the protein expression amount of SRP54, but also can obtain more required target proteins; and the thermal stability of the protein is improved, thereby facilitating subsequent application and development. Meanwhile, optimization of the antigen also improves the steric hindrance problem of SRP54 antibody detection, and antigen sites are more fully exposed, thereby improving the sensitivity of SRP54 antibody detection. The antigen of the application can be used for SRP54 antibody detection or diagnosis of multiple myeloma, and the accuracy can be greatly improved, thereby providing new technical support for production and application. Therefore, the recombinant antigen of the application has a good application prospect.

Description

TECHNICAL FIELD

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

[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, and plays a role in granulocyte proliferation and differentiation, neutrophil migration ability and exocrine pancreas development. The SRP of mammals is composed of two parts, i.e. a 300-base RNA (7SL RNA 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 guides newly synthesized secretory proteins from the polysome to the endoplasmic reticulum.

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

[0004] In a special subtype of idiopathic inflammatory myopathy (IIM), anti-signal recognition particle (SRP) myositis, the detected anti-SRP54 antibody has high specificity for the diagnosis of the disease. Anti-SRP54 antibody-positive patients usually exhibit symptoms of severe myositis with acute or subacute onset, such as muscle weakness, pain, etc., and often have poor response to conventional immunotherapy. In some rare neuromuscular diseases or patients with unexplained muscle lesions, detecting SRP54 antibodies can also help to rule out the presence of abnormal immune responses related to SRP54. Meanwhile, in the study of some liver diseases or systemic autoimmune diseases, it has also been 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 the like, and the SRP54 antibodies in the sample to be detected are captured by coating the antigen on a carrier. However, the existing coated antigen for myositis detection still has the problem of low sensitivity.

[0006] In view of this, the present application is proposed. SUMMARY

[0007] The present application aims to provide a SRP54 recombinant antigen, a preparation method and application thereof, which has higher detection sensitivity in detecting polymyositis and its expression amount is also significantly improved.

[0008] In a first aspect, the present application provides a SRP54 recombinant antigen, whose amino acid sequence is shown as SEQ ID NO. 1.

[0009] The present application obtains a new SRP54 recombinant antigen based on the optimization of the existing SRP54 antigen protein (UniProtKB-P61011), whose amino acid sequence is shown as 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 application, the expression amount and thermal stability of the obtained SRP54 recombinant antigen are also improved.

[0011] In some embodiments, the recombinant SRP54 antigen of the present application can comprise further modifications, such as tag modifications. The tag modification of the recombinant SRP54 antigen of the present application is not particularly limited, which can be a protein purification tag, such as an affinity tag, such as a biotin tag.

[0012] In a second aspect, the present application provides biological materials related to the above-mentioned SRP54 recombinant antigen, which are any of the following:

[0013] (1) a nucleic acid molecule encoding the above-mentioned SRP54 recombinant antigen:

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

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

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

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

[0018] The nucleic acid molecule can be DNA or RNA, and in some cases, the nucleic acid molecule can be modified for use in the vectors of the present application, such as for codon optimization. In some cases, the sequence can be designed to contain terminal restriction site sequences for the purpose of cloning into a vector. The nucleic acid molecule can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of the encoding nucleic acid within or isolated from the given cell or cells.

[0019] In specific embodiments, the nucleic acid molecule described above can be synthesized artificially after optimization of the relevant gene sequence according to the codon preference of the host cell. It should be understood that the nucleic acid molecule capable of being translated into the amino acid sequence described above is within the scope of the present application.

[0020] The present application provides an expression cassette comprising the nucleic acid molecule described above. Meanwhile, the expression cassette also contains regulatory sequences, such as promoters and terminators.

[0021] The expression cassette refers to a nucleic acid construct comprising a coding sequence and regulatory sequences operably linked when introduced into a host cell, resulting in transcription and / or translation of RNA or polypeptide, respectively. The expression cassette should be understood to include a promoter that allows the initiation of transcription, an open reading frame of the gene of interest, and a transcription terminator. Typically, the promoter sequence is placed upstream of the gene of interest, compatible with expression control.

[0022] The present application provides a recombinant vector comprising the nucleic acid molecule or the expression cassette described above.

[0023] The vector includes, but is not limited to, a single-stranded, double-stranded, or partially double-stranded nucleic acid molecule; a nucleic acid molecule comprising one or more free ends, without free ends (e.g., circular); a nucleic acid molecule comprising DNA, RNA, or both; and other polynucleotide species known in the art. The most commonly used vector type is "plasmid", which refers to a circular double-stranded DNA ring that can insert additional DNA fragments, such as by standard molecular cloning techniques. The recombinant expression vector can comprise a form suitable for expression of the nucleic acid in a host cell, 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, which can be operably linked to the nucleic acid sequence to be expressed.

[0024] The present application provides a cell comprising the nucleic acid molecule described above or the expression cassette described above or the expression vector described above, and the cell is capable of expressing the SRP54 recombinant antigen described above.

[0025] The cell can be any cell useful in the production of the SRP54 recombinant antigen of the present application. To produce the SRP54 recombinant antigen, a nucleic acid encoding the SRP54 recombinant antigen can be isolated and inserted into one or more vectors for further cloning / expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional techniques. Methods for the introduction of vectors into host cells are well known and the present application is not limited in this respect.

[0026] In specific embodiments, the above-mentioned host cell can be a prokaryotic cell or a eukaryotic cell, including: bacterial hosts such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis, etc.; 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 application provides a complex comprising the above-mentioned SRP54 recombinant antigen.

[0028] The present application also provides a complex further comprising a solid support, a detectable label or a binding partner conjugated with 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 a magnetic particle, a microtiter plate or a cellulose membrane; the detectable label can be a metal particle, a fluorescent label, a chromophore label, an electron-dense label, a chemiluminescent label, a radioactive label, or an enzyme label, specifically a colloidal gold, a radioisotope, a fluorophore, a spin label, or a bacteriophage label, and can also be a rhodamine, a fluorescein, an acridinium ester, a luciferase, a horseradish peroxidase, an alkaline phosphatase, a beta-galactosidase, a glucoamylase, a lysozyme, a sugar oxidase, a glucose oxidase, a galactose oxidase or a glucose-6-phosphate dehydrogenase label; the binding partner can be biotin, streptavidin or avidin.

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

[0031] The above-mentioned composition can be a pharmaceutical composition or an immunological composition. In the above-mentioned composition, in addition to containing the above-mentioned SRP54 recombinant antigen or the above-mentioned biological material or the above-mentioned complex, one or more of other components suitable for effectively preserving the structure and activity thereof, such as a buffer, a surfactant, a protective agent, a metal salt, a preservative, etc., can be included, which can be selected and combined according to actual needs by those skilled in the art.

[0032] In some embodiments, the above-mentioned composition is in a liquid state, and in other embodiments, the above-mentioned composition can also be in a lyophilized state. In some embodiments, the above-mentioned composition is in a liquid state, and in other embodiments, the above-mentioned composition can also be in a lyophilized state.

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

[0034] In some embodiments, the SRP54 recombinant antigen can be prepared by culturing the above-mentioned cell under suitable conditions, and then isolating the SRP54 recombinant antigen from the culture solution and / or the cell.

[0035] In some embodiments, the SRP54 recombinant antigen can be prepared by culturing the above-mentioned cell under suitable conditions, and then isolating the SRP54 recombinant antigen from the culture solution and / or the cell.

[0036] In some embodiments, the SRP54 recombinant antigen is produced by using a baculovirus / insect cell expression system, which specifically includes: connecting the nucleic acid molecule encoding the SRP54 recombinant antigen to a vector to obtain a recombinant vector; then transforming the recombinant vector into a competent cell to obtain a recombinant baculovirus plasmid; and then transfecting the recombinant baculovirus plasmid into an insect cell, and after culturing, isolating and purifying to obtain the SRP54 recombinant antigen.

[0037] In some embodiments, the insect cell includes: Sf9 cell, Hi5 cell and Sf21 cell.

[0038] In some embodiments, the SRP54 recombinant antigen, biological material or complex can be used for preparing a product for detecting SRP54 antibody.

[0039] In some embodiments, the SRP54 recombinant antigen, biological material or complex can be used for preparing a product for assisting in the diagnosis of myositis.

[0040] In some embodiments, the SRP54 recombinant antigen, biological material or complex can be used for preparing a product for preparing SRP54 antibody.

[0041] In some embodiments, the SRP54 recombinant antigen, biological material or complex can be used for preparing a product for detecting SRP54 antibody.

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

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

[0044] The SRP54 recombinant antigen and related biological materials and complexes thereof can be further used for preparing products for the auxiliary diagnosis of SRP54 antibody positive diseases, in particular myositis. Through the SRP54 recombinant antigen and related biological materials or complexes thereof in the above products, it can be used to assist in identifying whether a subject is a myositis patient.

[0045] The SRP54 recombinant antigen and related biological materials and complexes thereof can be further used for preparing products for the preparation of SRP54 antibodies. Through the SRP54 recombinant antigen and related biological materials or complexes thereof included in the above products, SRP54 antibodies capable of specifically binding to the SRP54 recombinant antigen can be prepared. For the preparation method of SRP54 antibodies, any suitable method known in the art can be used for preparation, for example, immunizing animals with SRP54 recombinant antigen to generate SRP54 antibodies.

[0046] In another embodiment, the SRP54 recombinant antigen can be used for the purification of SRP54 antibodies; for example, the SRP54 recombinant antigen is immobilized on a chromatography column, and the SRP54 antibodies are indirectly immobilized on the chromatography column after being contacted with the SRP54 recombinant antigen, and then the purified SRP54 antibodies are obtained by further removing impurities and eluting with different eluents.

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

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

[0049] In some embodiments, the kit can also include a calibrator or a control, such as a calibration or control SRP54 antibody.

[0050] In some embodiments, the SRP54 recombinant antigen or complex of the present application is contained in a container such as a test tube, microplate or test strip in a kit. The kit can also contain a solid support such as magnetic beads, test tubes, microplates, cuvettes, membranes, filter paper, syringes, pipettes, buffers such as assay buffers, washing buffers, pretreatment reagents, detectable labels such as enzyme-labeled substrate solutions, etc.

[0051] In some embodiments, the SRP54 recombinant antigen or complex of the present application is coated on a solid support, which is a liquid chip, has superparamagnetic properties and is composed of a biocompatible high polymer (Barcoded Magnetic Beads, BMB). The BMB herein, i.e., barcoded magnetic microspheres, are obtained by incorporating paramagnetic materials into a biocompatible high polymer and etching a 12-bit binary digital barcode onto the magnetic microspheres by photolithography. The barcoded magnetic microspheres are also referred to as barcoded magnetic beads or digital magnetic beads. In the present application, "K" as a unit means "thousands", and 50K BMB means 50,000 barcoded magnetic microspheres.

[0052] Using the above kit, the present application also provides a method for detecting SRP54 antibodies, which can be as follows: contacting the SRP54 recombinant antigen of the present application with a sample to be tested, forming a complex of SRP54 antibodies and SRP54 recombinant antigen in the presence of SRP54 antibodies in the sample, and then detecting the presence of the complex, which indicates the presence of SRP54 antibodies in the sample. This method can accurately detect whether SRP54 antibodies are present in the target sample.

[0053] The present application has the following advantages:

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

[0055] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be considered as limiting the scope. For those skilled in the art, other related drawings can also be obtained without creative labor.

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

[0057] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the following will clearly and completely describe the technical solutions in the embodiments of the present application. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not mentioned by the manufacturers are all conventional products which can be obtained by market purchase.

[0058] The following will further illustrate the present application through specific embodiments. It should be understood that these embodiments are only used for more detailed illustration, and should not be understood as limiting the present application in any form.

[0059] Example 1

[0060] Expression and purification of SRP54 recombinant protein in this embodiment

[0061] S1. Insert the nucleic acid fragment (nucleotide sequence as shown in SEQ ID NO. 2) containing the coding sequence of SRP54 recombinant protein (amino acid sequence as shown in SEQ ID NO. 1) into pFastBac1 vector, and the inserted enzyme cutting sites are BamH I and Hind III.

[0062] S2. Transform the recombinant plasmid into E. coli DH5α competent cells by heat shock method, then coat on LB solid agar medium, cultivate at 37℃ overnight, pick positive clone colonies, and identify and extract plasmid after expansion culture.

[0063] S3. Transfect the plasmid to package P1 generation virus:

[0064] S31. Inoculate Sf9 cells (in logarithmic growth phase and with a viability of more than 95%) in a six-well plate, and cultivate at 28℃ for 1h to make the cells adhere (SIM SF, without serum, 3mL / well).

[0065] S32. Discard the culture medium, add new culture medium (SIM SF, without serum) to continue cultivation, and 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 Bacmid DNA (expression plasmid prepared by S1, S2 above) into 100 μL SIM SF medium (serum-free), mix gently with a gun, and stand at room temperature for about 2-5 min.

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

[0069] (3) Dilute the bacmid of step (1) with a pipette and add it to the transfection reagent of step (2), mix gently with a gun, and stand at room temperature for 20 min.

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

[0071] (5) After 4 h of culture at 28°C, replace the fresh medium (SIM SF, 10% FBS), continue to culture for 6 d, and then centrifuge to collect the supernatant, which is the P1 generation virus, and store it in a light-proof cryogenic tube.

[0072] S4. Virus amplification:

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

[0074] S42. P3 virus preparation: inoculate Sf9 cells (in the logarithmic growth phase and with a viability of greater than 95%) in a culture flask with 50 mL of SIM + double antibody + 2% FBS medium. Add 3 mL of P2 virus, and continue to culture at 28°C in the dark. After the cell viability is 70%-80%, centrifuge to collect the supernatant, which is the P3 virus.

[0075] S5. Determination of P3 virus titer.

[0076] S6. Protein expression:

[0077] Inoculate 1 L of SF9 cells with a density of 4.0 x 10 6 cells / mL and a viability of greater than 95% in a flask, add an appropriate amount of P3 virus (MOI = 2-5), and culture at 28°C in the dark at a rotation speed of 100 rpm.

[0078] S7. Protein purification:

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

[0080] (2) Ultrasonic disruption: disrupt the bacteria under the conditions of ultrasonic power 520 W, ultrasonic 4 s, stop 3 s, cycle 200 times, until the bacterial solution is clear and transparent.

[0081] (3) After ultrasonic, centrifuge at 4°C, 12000 rpm for 30 min (the centrifuge is pre-cooled) to separate the supernatant and the precipitate, and the supernatant is filtered using a 0.45 μM membrane.

[0082] (4) Take 2 mL Ni-TED resin and equilibrate the column with 5 CV of lysis Buffer, then add the filtered supernatant, mix well, and incubate in an ice box on a shaking bed for 1-2 h.

[0083] (5) Pour the incubated mixture into a gravity column, collect the flow-through liquid with a clean centrifuge tube, and after the flow-through liquid is completely drained, add 10 CV of lysis Buffer to wash the residual in the pipeline.

[0084] (6) Elution: use 20 mL of elution Buffer 1 (elution Buffer 1 formula: 1 x PBS, 20 mM imidazole, 5% glycerol, pH 8.0), 20 mL of elution Buffer 2 (elution Buffer 2 formula: 1 x PBS, 50 mM imidazole, 5% glycerol, pH 8.0), 20 mL of elution Buffer 3 (elution Buffer 3 formula: 1 x PBS, 150 mM imidazole, 5% glycerol, pH 8.0), and 20 mL of elution Buffer 4 (elution Buffer 4 formula: 1 x PBS, 250 mM imidazole, 5% glycerol, pH 8.0) to elute and collect the eluate.

[0085] (7) Protein electrophoresis verifies the purity of the target protein in the eluate, and the eluate with a purity of ≥90% is dialyzed into protein storage solution (protein storage solution formula: 1 x PBS, 5% glycerol, pH 8.0).

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

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

[0088] Comparative Example 1

[0089] The expression and purification of the SRP54 proprotein in this example were basically the same as in Example 1, except that the protein sequence inserted in S1 was replaced by the SRP54 proprotein (the amino acid sequence is shown as SEQ ID NO. 3, and the nucleic acid sequence is shown as SEQ ID NO. 4). The purified SRP54 proprotein was obtained.

[0090] Example 2

[0091] Comparison of the expression amount of the SRP54 proprotein in Comparative Example 1 and the recombinant SRP54 protein in Example 1

[0092] The protein concentration obtained after purification was detected using the Biyun Tian BCA protein concentration determination kit, and the total protein expression amount was calculated, and then the protein yield was calculated according to the cell amount of the expressed protein. Each antigen was subjected to two parallel experiments, and the results are shown in Table 1:

[0093] Table 1: Detection results of the expression amount of wild-type and optimized proteins

[0094]

[0095]

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

[0097] As can be seen from the results in Table 1, the expression amount and protein yield of the SRP54 recombinant protein (DE) in Example 1 are both significantly improved compared to the proprotein, with an average yield of about 2.44 times that of the proprotein.

[0098] Example 2

[0099] This example tests the effect of using the SRP54 proprotein and the recombinant SRP54 protein for SRP54 antibody detection, respectively

[0100] 1. Antigen-BMB complex preparation

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

[0102] The recombinant SRP54 protein of Example 1 and the SRP54 proprotein of Comparative Example 1 were coated on the BMB magnetic beads in the same way, and the coating process was as follows:

[0103] 1) Take 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 (N-hydroxysuccinimide), shake at room temperature for 50 min to activate the carboxyl group;

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

[0107] 5) Add the prepared antigen (recombinant SRP54 protein of Example 1 or SRP54 proprotein of Comparative Example 1), shake at room temperature for 60 min for incubation;

[0108] 6) Block the BMB with blocking solution (coating solution containing 1% BSA bovine serum albumin, 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. Test process

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

[0112] 2) Wash 3 times with a magnetic plate washer to remove unbound substances, and aspirate the residual liquid;

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

[0114] 4) Wash 3 times with a magnetic plate washer to remove unbound substances, and aspirate the residual liquid; detect the corresponding fluorescence value under a fluorescence reader, and determine the positive and negative according to the size of the fluorescence value.

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

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

[0117]

[0118] * WT refers to the SRP54 proprotein 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, 14 more samples were detected by DE than by WT, the sensitivity was increased to 96%, the specificity was increased to 100%, and no false positives occurred.

[0120] Further, 6 SRP54 antibody positive samples (PC) and 2 SRP54 antibody negative samples (NC) were randomly selected, and the signal-to-noise ratio of different antigens was compared, and 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, the signal-to-noise ratio of the optimized SRP54 antigen is better, which can better resist noise interference and ensure the clarity and stability of the signal.

[0126] Example 3

[0127] Stability test

[0128] The test method is as follows:

[0129] 1) Prepare the antigen-liquid phase chip BMB complex coated with different antigens according to the method of Example 2, and divide each complex into two parts, one part is stored at 4°C for 18h, and the other part is placed at 37°C for 18h; After treatment, add the diluted sample to be tested to each part, and incubate at 37°C for 15min (to form a magnetic barcode-antigen-antibody complex),

[0130] 2) Wash 3 times with a magnetic plate washer to remove unbound substances, and aspirate the residual liquid;

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

[0132] 4) Wash 3 times with a magnetic plate washer to remove unbound substances, and aspirate the residual liquid; detect the corresponding fluorescence under a fluorescence reader,

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

[0134] Table 4 Heat accelerated stability results of wild type and optimized SRP54 recombinant antigen

[0135]

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

[0137] From Table 4, by comparing the reactivity of each protein treated at 37℃ and 4℃, in the 14 positive samples, the average signal retention rate of DE is 89%, which is increased by 39% compared with WT; in the 6 negative samples, the average signal retention rate of DE is 105%, which is increased by 30% compared with WT. Therefore, it is proved that the stability of SRP54 recombinant antigen is better than that of the original protein.

[0138] The preferred embodiments of the present application have been described above by way of example only, not for limiting the present application, and various changes and modifications can be made by those skilled in the art based on the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

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

1.

2. The biomaterial associated with the SRP54 recombinant antigen of claim 1, characterized in that, It can be any of the following: (1) A nucleic acid molecule encoding the SRP54 recombinant antigen of 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 complex, characterized in that, Includes the SRP54 recombinant antigen as described in claim 1.

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

5. A composition, characterized in that, This includes the recombinant antigen of claim 1, the biomaterial of claim 2, or the complex of claim 3 or 4.

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

7. The use of the SRP54 recombinant antigen of claim 1, the biomaterial of claim 2, or the complex of 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 the auxiliary diagnosis of myositis; (III) Application in the preparation of products for the preparation of SRP54 antibody.

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

9. The reagent kit according to claim 8, characterized in that, The kit is used for the auxiliary diagnosis of myositis and also includes autoantibody detection reagents for at least one of the following biomarkers: 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. The use of the SRP54 recombinant antigen of claim 1, the biomaterial of claim 2, the complex of claim 3 or 4, or the kit of claim 9 in any one of the following (a) or (b): (a) Detection of SRP54 antibodies for non-diagnostic and non-therapeutic purposes; (b) Used for purifying SRP54 antibody.

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