Centromere protein- modifying proteins, methods of making, biomaterials, and uses thereof

Abstract

The application provides a kinetochore protein engineered protein, a preparation method thereof, a biomaterial and application, and relates to the technical field of protein engineering. The kinetochore protein engineered protein sequentially comprises a fragment B5, a fragment B6, a fragment B2 and a fragment A1 from an N-terminal to a C-terminal, and the amino acid sequences of the fragment B5, the fragment B6, the fragment B2 and the fragment A1 are shown in Seq_1-4. The kinetochore protein engineered protein contains CENP-A antigen and CENP-B antigen at the same time, and the problem that the sensitivity and specificity of detecting kinetochore antibodies by using one kind of antigen alone in the prior art are poor are solved.

Description

Technical Field

[0001] This invention relates to the field of protein engineering technology, and in particular to a centromere protein remodeling protein, its preparation method, biomaterials, and applications. Background Technology

[0002] Systemic sclerosis (SSc) is a systemic disorder of connective tissue characterized by peripheral atherosclerosis and fibrosis of visceral structures. SSC affects the skin and internal organs and is often associated with specific autoantibodies, especially centromere antibodies.

[0003] Centromere proteins are a class of proteins that form or regulate centromere function, influencing chromosome stability and normal segregation. The central structure of chromosomes is attached with spindle fibers and tubulin, enabling chromosome separation during cell division. Centromere protein B (CENP-B) is a major target for autoantibodies and anti-centromere antibodies. CENP-B antibodies are detectable in the serum of 80% of patients with systemic sclerosis and are commonly used as a primary diagnostic antigen for the disease. CENP-A is another important centromere autoantigen besides CENP-B: its molecular weight is close to 22 kDa, and due to its histone-like properties, it participates in the formation of centromere chromatin.

[0004] Experiments have shown that using CENP-A antigen alone to detect systemic sclerosis (SCS)-negative serum produces fewer false positives than using CENP-B antigen alone, but produces more false negatives when detecting SCS-positive serum. Conversely, using CENP-B antigen alone to detect SCS-positive serum produces fewer false negatives than using CENP-A antigen alone, but produces more false positives when detecting SCS-negative serum. Therefore, improving the CENP antigen to enhance its sensitivity and specificity in detecting centromere antibodies is a pressing issue that needs to be addressed.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The primary objective of this invention is to provide a centromere protein modified to simultaneously contain antigens of both CENP-A and CENP-B, thereby improving the sensitivity and specificity of centromere antibody detection. Based on the modification of the centromere protein, biomaterials related to the modified protein and their applications are also provided.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: According to one aspect of the present invention, a centromere protein remodeling protein is provided, which comprises, from the N-terminus to the C-terminus, fragments B5, B6, B2 and A1 in sequence; The amino acid sequence of fragment B5 is shown in Seq_1; the amino acid sequence of fragment B6 is shown in Seq_2; the amino acid sequence of fragment B2 is shown in Seq_3; and the amino acid sequence of fragment A1 is shown in Seq_4.

[0008] Preferably, the peptide segments are linked together by linker peptides; Preferably, the amino acid sequence of the linker peptide is shown in Seq_5; Preferably, the amino acid sequence of the centromere protein remodeling protein is shown in Seq_6.

[0009] Preferably, the centromere protein remodeling protein carries a protein tag; Preferably, the protein tag is a 6×Histag; Preferably, the amino acid sequence of the centromere protein remodeling protein is shown in Seq_7.

[0010] Preferably, the centromere protein-modified protein is expressed by insect cells; Preferably, the centromere protein is a protein expressed by insect cells with an amino acid sequence as shown in Seq_7.

[0011] According to another aspect of the present invention, the present invention also provides a biomaterial selected from any one of (A) to (C) below; (A) A polynucleotide encoding a centromere protein modified protein according to any one of claims 1-4; (B) A vector containing the polynucleotide; (C) Cells, which are transformed by the vector; Preferably, the vector includes a viral vector, and more preferably a baculovirus vector; Preferably, the cells comprise insect cells, and more preferably insect cells sf9.

[0012] According to another aspect of the present invention, the present invention also provides a method for preparing the above-described centromere protein remodeling protein, the method comprising expressing a gene encoding the centromere protein remodeling protein in a host.

[0013] Preferably, the preparation method includes expressing the centromere protein modified protein using a baculovirus insect cell expression system.

[0014] According to another aspect of the present invention, the present invention also provides the application of the above-described centromere protein modified protein, the above-described biomaterial, or the above-described preparation method in any one of the following (a) to (e): (a) Detection of centromere antibodies for non-diagnostic and non-therapeutic purposes; (b) Preparation of a kit for centromere antibody detection; (c) Preparation of centromere antibodies; (d) Prepare products for the detection of systemic sclerosis; (e) Prepare medicines for the prevention and / or treatment of systemic sclerosis.

[0015] According to another aspect of the invention, the invention also provides a composition comprising the centromere protein remodeling protein and the marker described above.

[0016] According to another aspect of the present invention, the present invention also provides a kit for centromere antibody detection, the kit comprising the above-described centromere protein modified protein, or the above-described composition.

[0017] Compared with the prior art, the present invention has the following beneficial effects: By performing bioinformatics analysis on the antigenic epitopes of centromere proteins CENP-A and CENP-B, respectively, the antigenic domains of the centromere proteins of this invention are extracted. The extracted antigenic domains form the centromere protein modified protein of this invention, which contains both CENP-A and CENP-B antigenic epitopes. The centromere protein modified protein provided by this invention comprises fragments B5, B6, B2, and A1 sequentially from the N-terminus to the C-terminus. Fragments B5, B6, and B2 contain the CENP-B antigen, while fragment A1 contains the CENP-A antigen. This invention screens fragments A1, B5, B6, and B2 from multiple antigenic domains of CENP-A and CENP-B, respectively, and optimizes their connection sequence. The resulting centromere protein modified protein is significantly more effective in detecting centromere antibodies than using CENP-A or CENP-B alone. When detecting positive and negative sera for systemic sclerosis, it can simultaneously reduce false positive and false negative results, exhibiting high sensitivity (93.5%) and specificity (92.6%). Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 An SDS-PAGE electrophoresis image of CENP-M, a centromere protein remodeling protein provided in an embodiment of the present invention. Detailed Implementation

[0020] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] According to one aspect of the present invention, a modified centromere protein (CENP) is provided. Named CENP-M, CENP-M contains antigenic epitopes of both CENP-A and CENP-B.

[0022] The antigenic epitope of CENP-B is provided by fragments B5, B6, and B2, with amino acid sequences shown in Seq_1~3, respectively; the antigenic epitope of CENP-A is provided by fragment A1, with an amino acid sequence shown in Seq_4. The connection order of the above four fragments from the N-terminus to the C-terminus is fragment B5, fragment B6, fragment B2, and fragment A1, respectively.

[0023] This invention screened fragments from multiple antigenic domains of CENP-A and CENP-B, and optimized the position of each fragment in the modified protein to obtain the centromere protein modified protein CENP-M. Experimental verification showed that peptides with sequences such as fragments B5, B6, B2, and A1 reacted with systemic sclerosis positive serum with better sensitivity than peptides from other antigenic domains in centromere proteins. Due to steric hindrance, the sensitivity of peptides connected in different sequences varied, with the modified protein constructed in the order of fragments B5, B6, B2, and A1 from the N-terminus to the C-terminus exhibiting the best sensitivity. Furthermore, the CENP-M provided by this invention can simultaneously reduce false positives and false negatives when detecting systemic sclerosis positive and negative sera, exhibiting high sensitivity (93.5%) and specificity (92.6%).

[0024] The technical effect of the centromere protein remodeling protein provided by this invention is based on the improved sensitivity of the antigenic epitopes in fragments B5, B6, B2, and A1 to antibodies, as well as their specific arrangement order. Those skilled in the art can connect the above fragments in the order of fragments B5, B6, B2, and A1, and further modify the centromere protein remodeling protein using mature and well-known existing techniques; this invention does not limit such modifications.

[0025] In some optional embodiments, the above-mentioned linkage can be direct linkage or spaced linkage. Direct linkage means that there are no amino acid residues between adjacent segments; spaced linkage means that there are several amino acid residues between adjacent segments, such as, but not limited to, linking peptides between adjacent segments. Optionally, the above-mentioned centromere protein remodeling protein may contain both directly linked adjacent segments and spaced adjacent segments; optionally, all adjacent proteins in the above-mentioned centromere protein remodeling protein are linked by linking peptides, and the linking peptides can be the same or different. In some preferred embodiments, all adjacent proteins in the above-mentioned centromere protein remodeling protein are linked by the same type of linking peptide, preferably by the flexible peptide GGGGSSSS (Seq_5), and the amino acid sequence of the centromere protein remodeling protein linked by the flexible peptide GGGGSSSS is shown in Seq_6.

[0026] In some optional embodiments, the centromere protein remodeling protein described above also carries a protein tag acceptable in the art. Examples of protein tags include, but are not limited to, one or more of the following: Flag tag, 3×Flag tag, His tag, GST tag, HA tag, Sumo tag, Avi tag, MBP tag, NusA tag, SNAP tag, and C-Myc tag. The protein tag may optionally be located at the N-terminus and / or C-terminus. In an optional embodiment, the protein tag is selected from the 6×His tag, which is a flexible peptide GGGGSSSS linked to the C-terminus of the remodeling protein. The amino acid sequence of the centromere protein remodeling protein with the 6×His tag is shown in Seq_7.

[0027] The aforementioned centromere protein modified proteins can be prepared by methods well known in the art and described in various general and more specific textbooks, references, process manuals, product instructions, and standard documents, and the present invention does not limit this. When expressing the centromere protein modified proteins using a host, the host includes, but is not limited to, *Escherichia coli*, yeast, insect cells, or mammalian cells. In a preferred embodiment, the aforementioned centromere protein modified proteins are expressed using a baculovirus insect cell expression system. In a more preferred embodiment, the centromere protein modified protein is a modified protein with the amino acid sequence shown in Seq_7 expressed by the baculovirus insect cell expression system.

[0028] Based on the above-mentioned inventive concept of centromere protein remodeling protein, the present invention also provides related biological materials, wherein the biological materials are selected from any one of (A) to (C) below.

[0029] (A) Polynucleotides: Polynucleotides refer to polymeric forms of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acids include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. Polynucleotides encode the aforementioned centromere protein-modified proteins, optionally encoding the sense or antisense strand.

[0030] (B) Vector: The vector is a medium that enables the above-mentioned polynucleotides to replicate or be expressed in the host, or that enables the above-mentioned genes to be integrated into the host cell, including but not limited to plasmids, phage particles, bacteriophages, artificial chromosomes, or viral vectors. Viruses derived from viral vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, baculoviruses, herpesviruses, poxviruses, or papillomaviruses. The vector also includes optional regulatory elements acceptable in the art, including but not limited to non-coding sequences, promoters, terminators, enhancers, ribosome binding sites, polyA, and other regulatory elements.

[0031] (C) Cell: The cell is transformed by the above-described vector to enable the cloning or expression of the vector; or, the host cell's genome integrates the nucleic acid encoding the centromere protein remodeling protein. The cell may be, for example, but not limited to, Escherichia coli, yeast, insect cells, or mammalian cells.

[0032] According to another aspect of the present invention, the present invention also provides a method for preparing the above-mentioned centromere protein remodeling protein, comprising expressing a gene encoding the centromere protein remodeling protein in a host, the host including but not limited to Escherichia coli, yeast, insect cells or mammalian cells, preferably using a baculovirus insect cell expression system to express the centromere protein remodeling protein, wherein the insect cell is preferably insect cell sf9 (Spodoptera frugiperda).

[0033] According to another aspect of the present invention, the present invention also provides the application of the above-described centromere protein modified protein, the above-described biomaterial, or the method for preparing the above-described centromere protein modified protein in any one of the following (a) to (e): (a) Detection of centromere antibodies for non-diagnostic and non-therapeutic purposes; (b) Preparation of kits for the detection of centromere antibodies; (c) Preparation of centromere antibodies; (d) Preparation of products for the detection of systemic sclerosis; (e) Preparation of medicines for the prevention and / or treatment of systemic sclerosis.

[0034] Examples of applications include, but are not limited to: the above-mentioned centromere protein modification protein being used to prepare centromere antibodies for non-diagnostic and therapeutic purposes, which can be used as a capture antigen, for example, by coating it onto an ELISA plate as a capture antigen in an ELISA detection method; or using a composition of centromere protein modification protein labeled with colloidal gold, fluorescent microspheres, or colored microspheres as a capture antigen in an immunochromatographic detection method to detect centromere antibodies; or immunizing animals with the above-mentioned centromere protein modification protein to produce antibodies, which are then further used in detection methods or to prepare detection products; the above-mentioned biological materials can be used as raw materials for producing centromere protein modification protein or its antibodies, and the prepared proteins and antibodies can be applied to the above five aspects (a) to (e); examples of kits for centromere antibody detection, or products for detecting or assisting in the detection of systemic sclerosis, include, but are not limited to, ELISA detection kits or immunochromatographic detection cards, or packaging the above-mentioned centromere protein modification protein separately as a reagent to be used in conjunction with other kits based on the detection principle of antigen-antibody binding reaction. In the preparation of drugs for the prevention and / or treatment of systemic sclerosis, the aforementioned centromere protein modified proteins or related biological materials can be used directly as active ingredients of the drugs, or optionally as raw materials for the production of the drugs.

[0035] According to another aspect of the invention, a composition is also provided comprising the centromere protein and a label, wherein the centromere protein and the label are optionally linked or not linked. The label includes, but is not limited to, one or more of enzymes, latex particles, fluorescent molecules, quantum dots, fluorescent microspheres, colored microspheres, colloidal gold, colloidal silver, and colloidal carbon; the label may also be selected as a solid-phase carrier, such as magnetic microparticles. The label is used to provide a detectable signal to the centromere protein, or an immune complex formed with the centromere protein, such as an antigen-antibody or an antigen-antibody-antibody complex, enabling the centromere protein to be used in detection.

[0036] According to another aspect of the present invention, a kit for detecting centromere antibodies is also provided, comprising the aforementioned centromere protein modification protein or the aforementioned composition. This kit utilizes the reaction of the aforementioned centromere protein modification protein with antibodies in the test sample to detect centromere antibodies in the test sample, exhibiting good sensitivity and specificity. When the kit contains the aforementioned composition, the marker in the composition provides an observable detection signal, facilitating the acquisition, statistical analysis, and interpretation of the detection results. Examples of the kit include, but are not limited to, ELISA kits; immunochromatographic assay cards, such as colloidal gold assay cards; or immunomagnetic bead assay kits, etc. It is understood that the kit may optionally also include other reagents and consumables for detection, including but not limited to buffer reagents, diluents, solvents, detergents, chromogenic agents, antibodies, blocking reagents, pH adjusters, markers, enzymes, magnetic microparticles, ELISA plates, or nitrocellulose membranes, etc. Those skilled in the art can adjust the specific composition of the kit according to the specific detection method of the kit, based on methods well known in the art and described in various general and more specific textbooks, references, process manuals, product instructions, and standard documents.

[0037] The technical solution and beneficial effects of the present invention will be further described below with reference to preferred embodiments.

[0038] Example This embodiment provides a centromere protein remodeling protein, named CENP-M, which includes fragments B5, B6, B2 and A1 sequentially from the nitrogen terminus to the carbon terminus. The amino acid sequence of fragment B5 is shown in Seq_1: GFGGGPNATITTSLKSEGEEEEEEEEEEEEGEGEEEEEEGEEEEGGEGEELGEEEEVEEEGDVDSDEEEEEDE; The amino acid sequence of fragment B6 is shown in Seq_2: EGLEAEDWAQGVVEAGGSFGAYGAQEEAQCPTLHFLEGGEDSDSDSEEEDDEEEDDEDEDDDDDEEDGDEVPVPSF; The amino acid sequence of fragment B2 is shown in Seq_3: RRRHGVVSCSGVARARARNAAPRTPAAPASPAAVPSEGSGGSTTGWRAREEQPPSVAEGYASQ; The amino acid sequence of fragment A1 is shown in Seq_4: RRRSRKPEAPRRRSPSPTPTPGPSRRGPSLGASSHQHSRRRQGWLKEIRKLQKSTHLLI.

[0039] Fragments B5, B6, B2, and A1 are linked by a flexible peptide linker (GGGGSSSS, Seq_5), and the modified protein with an amino acid sequence as shown in Seq_7 is obtained by attaching the aforementioned flexible peptide linker protein tag 6×histag at the end. It contains 314 amino acids and has the structure: B5-linker peptide-B6-linker peptide-B2-linker peptide-A1-linker peptide-6×Histag, named CENP-M.

[0040] The CENP-B gene was amplified by PCR to obtain the gene sequences of fragments B2, B5, and B6, respectively. The human CENP-A gene was amplified by PCR to obtain the gene sequence of fragment A1. The corresponding amplification primers are shown in Table 1. Table 1 Primer sequences for antigen fragments

[0041] Fragments B2, B5, and B6 are linked together to obtain fragment B5-linker peptide-B6-linker peptide-B2, which is then linked to fragment A1. Finally, a 6×Histag is added to the end to obtain B5-linker peptide-B6-linker peptide-B2-linker peptide-A1-linker peptide-6×Histag.

[0042] The preparation method is as follows: Construction of recombinant plasmids containing antigenic domains and preparation of Bacmid DNA: The gene sequence, which was ligated by PCR, was inserted between the BamHⅠ and HindⅢ sites of the vector pFastBac1 to construct an expression plasmid.

[0043] The constructed expression plasmid was transformed into E. coli DH10Bac competent cells. The transformed competent cells were plated on LB solid agar medium containing the corresponding antibiotics. The solid agar plates contained the following ingredients: Kana 50 μg / mL, Gentamicin 7 μg / mL, Tetracycline 10 μg / mL, X-Gal 40 μg / mL, and IPTG 40 μg / mL.

[0044] After spreading the plating evenly, incubate at 37℃ for 2 days. Once colonies have grown, select a few white single colonies and perform colony PCR verification using M13 primers. M13 primers are shown in Table 2. After positive PCR verification, extract Bacmid DNA.

[0045] Table 2 M13 Primer Sequences

[0046] Virus preparation: P1 virus preparation (1) Inoculation of approximately 0.8 × 10 6 Sf9 cells with a viability greater than 95% in the logarithmic growth phase were placed in a six-well plate and incubated at 28°C for 1 hour in a constant temperature incubator.

[0047] (2) Aspirate the culture medium from the 6-well plate, and add 1.5 ml of culture medium to wash the cells to remove residual antibiotics. Then add 800 μL of antibiotic-free and serum-free culture medium and continue to incubate at 28°C until transfection.

[0048] (3) Preparation of Bacmid DNA and transfection reagent mixture (3.1) In a sterile laminar flow hood, take a 1.5ml sterile EP tube, add 20μg Bacmid DNA, then add 100μl culture medium, gently pipette to mix evenly, and then let stand at room temperature for about 2~5min.

[0049] (3.2) Take another 1.5ml sterile EP tube, add 10μl of transfection reagent, then add 100μl of culture medium, gently mix with pipette tip, and then let stand at room temperature for about 2~5min.

[0050] (3.3) Gently transfer the diluted Bacmid DNA to the diluted transfection reagent using a pipette, gently mix with a pipette tip, and let stand at room temperature for about 20 minutes.

[0051] (4) Slowly add the above-mentioned well-mixed 200 μl Bacmid DNA and transfection reagent mixture into the cell wells and gently shake the 6-well plate to mix.

[0052] (5) Place in a constant temperature incubator and incubate at 28℃ for 4 hours. After the incubation is completed, replace with fresh complete culture medium (2% FBS, 1% penicillin and streptomycin), and continue to incubate for 5 days. After the incubation is completed, centrifuge and collect the supernatant, which is the P1 generation virus.

[0053] P2 virus preparation and protein expression (1) Take a sterile cell culture shake flask and add 30 ml of a solution with a density of 2×10⁻⁶ cells / ml. 6 / ml Sf9 cells with a viability greater than 95%. Add 500μl of the above-mentioned P1 virus. Place in a cell culture shaker and culture at 28℃ and 100rpm. Monitor cell density and diameter changes daily. After 4 days of culture, centrifuge and collect the supernatant, which is the P2 generation virus.

[0054] (2) Take a 1000ml sterile cell culture shake flask and add 400ml of water with a density of approximately 2×10⁻⁶. 6 Add 5 ml of the above-mentioned P2 virus to Sf9 cells with a viability greater than 95%. Incubate in a cell culture shaker at 28°C and 100 rpm. Count cells at 24 h, 48 h, 72 h, and 96 h after virus addition, observing changes in cell number, viability, and diameter. Stop culture when cell viability decreases and diameter no longer changes, and collect cells by centrifugation.

[0055] Protein purification: (1) Pretreatment: Take out the cells collected in the previous step and resuspend the cells in lysis buffer.

[0056] (2) Ultrasonic disruption: Ultrasonic power 300W, ultrasonic for 4 seconds, stop for 4 seconds, disrupt cells for 20 minutes.

[0057] (3) After the ultrasound, the centrifuge was centrifuged at 4℃ and 12000rpm for 15min. After centrifugation, the supernatant was collected and filtered through a 0.45uM membrane.

[0058] (4) Equilibrate the Ni HiTrap purification column with Lysis Buffer for 5 CV.

[0059] (5) Sample loading: Load the sample at a flow rate of 1.5 mL / min. After loading, reequilibrate with Lysis Buffer until the UV280 baseline is stable.

[0060] (6) Wash with 50 mM imidazole solution, then elute with 500 mM imidazole solution and collect the target protein.

[0061] (7) Ion exchange chromatography was performed using a Q HiTrap column. The column was washed with a low ionic strength buffer containing 50 mM NaCl for 10 cycles, and then eluted with a high ionic strength buffer containing 500 mM NaCl and the target protein was collected.

[0062] (8) SDS-PAGE gel was run to verify protein purity and size. The results are as follows: Figure 1 As shown, electrophoretic analysis indicated that its molecular weight was approximately 35 kDa and its purity was greater than 90%. After the protein passed the purity test, it was aliquoted and frozen at -80°C for antigenicity assessment.

[0063] The antigenic epitopes in the centromere protein remodeling protein provided in this embodiment were determined in the following manner: (a) Screening for antigenic epitopes: 1. CENP-A antigenic epitope screening: Bioinformatics analysis was performed on the antigenic epitopes of CENP-A (UniProtKB: P49450-1), and two antigenic domains were extracted. The amino acid sequences of the extracted fragments are as follows: A1(Seq_4) RRRSRKPEAPRRRSPSPTPTPGPSRRGPSLGASSHQHSRRRQGWLKEIRKLQKSTHLLI A2 (Seq_12) SRLAREICVKFTRGVDFNWQAQALLALQEAAEAFLVHLFEDAYLLTLHAGRVTLFPKDVQLARRIRGLEEG 2. CENP-B antigenic epitope screening: Bioinformatics analysis was performed on the antigenic epitopes of CENP-B (UniProtKB:P07199), and seven antigenic domains were extracted sequentially. The amino acid sequences of the seven extracted fragments are as follows: B1 (Seq_8): EVEENPDLRKGEIARRFNIPPSTLSTILKNKRAILASERKYGVASTCRKTNKLSPYDKLEGLLIAWFQQIRAAGLPVKGIILKEKALRIAEE; B2 (Seq_3): RRRHGVVSCSGVARARARNAAPRTPAAPASPAAVPSEGSGGSTTGWRAREEQPPSVAEGYASQ; B3 (Seq_9): GDGRPRQATQRLSVLLCANADGSEKLPPLVAGKSAKPRAGQAGLPCDYTANSKGGVTTQALAKYLKALDTRMAAESRRVLLLAG; B4 (Seq_10): DTSGLRHVQLAFFPPGTVHPLERGVVQQVKGHYRQAMLLKAMAALEGQDPSGLQLGLTEALHFV; B5 (Seq_1): GFGGGPNATITTSLKSEGEEEEEEEEEEEEGEGEEEEEEGEEEEGGEGEELGEEEEVEEEGDVDSDEEEEEDE; B6 (Seq_2): EGLEAEDWAQGVVEAGGSFGAYGAQEEAQCPTLHFLEGGEDSDSDSEEEDDEEEDDEDEDDDDDEEDGDEVPVPSF; B7 (Seq_11): MAYFAMVKRYLTSFPIDDRVQSHILHLEHDLVHVTRKNHARQAGVRGLGHQS.

[0064] Nine peptides were synthesized by a third-party peptide synthesis company. The antigen sensitivity of each peptide was evaluated using the following method: The peptides to be tested were coated onto ELISA plates. Serum samples from 31 patients with high levels of centromere antibodies (positive for systemic sclerosis) were added and reacted. After blocking and washing, the plates were incubated with a secondary antibody containing fluorescein jujube (PE), washed again, and finally, the corresponding fluorescence values ​​were detected using a fluorescence analyzer. The positive or negative results were determined based on the fluorescence values. The evaluation results are shown in Table 3 below. Table 3 Evaluation results of positive samples from which antigen domain peptides were extracted.

[0065] The evaluation results show that the four peptides A1, B2, B5 and B6 have good sensitivity.

[0066] (II) PCR amplification of the CENP-B gene yielded the gene sequences of B2, B5, and B6, respectively. The corresponding amplification primers are shown in Table 1. The three CENP-B gene fragments were linked in different sequences using a flexible peptide linker (GGGGSSSS, Seq_5), and a 6×histag was added to the end to form the following six proteins: CENP-Ba:B2-linker peptide-B5-linker peptide-B6-linker peptide-6×histag.

[0067] CENP-Bb: B2-linker peptide-B6-linker peptide-B5-linker peptide-6×histag.

[0068] CENP-Bc: B5-linker peptide-B2-linker peptide-B6-linker peptide-6×histag.

[0069] CENP-Bd:B5-linker peptide-B6-linker peptide-B2-linker peptide-6×histag.

[0070] CENP-Be:B6-linker peptide-B2-linker peptide-B5-linker peptide-6×histag.

[0071] CENP-Bf: B6-linker peptide-B5-linker peptide-B2-linker peptide-6×histag.

[0072] Expression plasmids for the above six proteins were constructed and expressed in insect cell expression systems, followed by protein purification, using the same steps as the preparation method for CENP-M described above. Due to steric hindrance, the sensitivity of peptides linked in different sequences will vary.

[0073] The sensitivity of these six antigens was evaluated as follows: The peptides to be validated were coated onto ELISA plates, and then serum samples from 31 patients with high levels of centromere antibodies were added to react with them. After blocking and washing, the plates were incubated with secondary antibody containing fluorescein jujube red (PE), washed again, and finally the corresponding fluorescence values ​​were detected under a fluorescence interpreter. The positive or negative results were determined based on the fluorescence values. The evaluation results are shown in Table 4. From the evaluation results, CENP-Bd showed the best sensitivity.

[0074] Table 4. Evaluation results of positive samples of CENP-B truncated protein.

[0075] (III) The human CENP-A gene was amplified by PCR to obtain the gene sequence of A1 mentioned above. The amplification primers are shown in Table 1. The CENP-Bd gene and the above gene fragment were linked in different sequences using a flexible peptide linker (GGGGSSSS), and a 6×Histag was added to the end to form the following two proteins: CENP-A1-CENP-Bd: A1-linker peptide-B5-linker peptide-B6-linker peptide-B2-linker peptide-6×Histag.

[0076] CENP-Bd-CENP-A1: B5-linker peptide-B6-linker peptide-B2-linker peptide-A1-linker peptide-6×Histag.

[0077] Expression plasmids for the two proteins were constructed, and the proteins were expressed using insect cell expression systems. The proteins were then purified, following the same steps as the preparation method for CENP-M described above.

[0078] The antigen sensitivity of these two antigens was evaluated separately. The evaluation method is as follows: The peptides to be validated were coated onto ELISA plates, and then serum samples from 31 cases of systemic sclerosis positive (with high centromere antibody) were added to react with them. After blocking and washing, the plates were incubated with secondary antibody containing fluorescein jujube red (PE), washed again, and finally the corresponding fluorescence values ​​were detected under a fluorescence interpreter. The positive and negative results were determined based on the fluorescence values. The evaluation results are shown in Table 5. From the evaluation results, CENP-Bd-CENP-A1 showed better sensitivity. The new modified protein CENP-Bd-CENP-A1 was named CENP-M, which is the modified protein provided in this embodiment.

[0079] Table 5. Evaluation results of positive samples of truncated protein.

[0080] Example of effect The CENP-M antigen and the CENP-A and CENP-B antigens were evaluated separately.

[0081] The evaluation method was as follows: The antigen to be validated was coated onto an ELISA plate. Serum samples from patients with high levels of centromere antibodies (31 positive cases) and those with low levels of centromere antibodies (27 negative cases) were then added and reacted. After blocking and washing, the plate was incubated with a secondary antibody containing fluorescein jujube (PE), washed again, and finally the corresponding fluorescence values ​​were detected using a fluorescence interpreter. The positivity or negativity was determined based on the fluorescence cutoff value (a cutoff value of 1100 indicates a positive result, and a reaction value <1100 indicates a negative result). The validation results are shown in Tables 6 and 7 below, and the statistical results are shown in Table 8.

[0082] Table 6 Validation Results of Positive Samples

[0083] Table 7. Validation results of negative samples

[0084] The results of the positive and negative sample verifications above were statistically analyzed, and the results are shown in Table 8: Table 8 Statistical analysis of verification results

[0085] Statistical analysis of the test results shows that the detection sensitivity of CENP-M is significantly higher than that of CENP-A / CENP-B, reaching 93.5%, and the detection specificity of CENP-M is also improved to 92.6%. Overall, the fusion antigen CENP-M is significantly more effective than CENP-A or CENP-B alone in detecting centromere antibodies.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A centromere protein remodeling protein, characterized in that, From the nitrogen end to the carbon end, the fragments are B5, B6, B2, and A1, respectively. The amino acid sequence of fragment B5 is shown in Seq_1; The amino acid sequence of fragment B6 is shown in Seq_2; The amino acid sequence of fragment B2 is shown in Seq_3; The amino acid sequence of fragment A1 is shown in Seq_4; The peptide segments are linked together by linker peptides; The amino acid sequence of the linker peptide is shown in Seq_5; The amino acid sequence of the centromere protein remodeling protein is shown in Seq_6.

2. A centromere protein remodeling protein, characterized in that, It consists of the centromere protein remodeling protein and protein tag as described in claim 1.

3. The centromere protein remodeling protein according to claim 2, characterized in that, The protein tag is a 6×Histag.

4. The centromere protein remodeling protein according to claim 2, characterized in that, The amino acid sequence of the centromere protein remodeling protein is shown in Seq_7.

5. The centromere protein remodeling protein according to any one of claims 1-4, characterized in that, The centromere protein is modified and expressed by insect cells.

6. The centromere protein remodeling protein according to claim 5, characterized in that, The centromere protein is a protein expressed by insect cells with an amino acid sequence as shown in Seq_7.

7. A biomaterial, characterized in that, The biomaterial is selected from any one of (A) to (C) below; (A) A polynucleotide encoding a centromere protein modified protein according to any one of claims 1-6; (B) A vector containing the polynucleotide; (C) Cells, which are transformed by the vector.

8. The biomaterial according to claim 7, characterized in that, The vector includes a viral vector.

9. The biomaterial according to claim 8, characterized in that, The vector is a baculovirus vector.

10. The biomaterial according to claim 7, characterized in that, The cells include insect cells.

11. The biomaterial according to claim 10, characterized in that, The cells in question are insect cells, specifically sf9.

12. The method for preparing the centromere protein modified protein according to any one of claims 1-6, characterized in that, The preparation method includes expressing a gene encoding the centromere protein remodeling protein in a host.

13. The preparation method according to claim 12, characterized in that, The preparation method includes expressing the centromere protein modified protein using a baculovirus insect cell expression system.

14. The use of the centromere protein modified protein according to any one of claims 1-6 or the biomaterial according to any one of claims 7-11 in any one of the following (a) and (b): (a) Preparation of a kit for centromere antibody detection; (b) Prepare products for the detection of systemic sclerosis.

15. A composition, characterized in that, It includes the centromere protein remodeling protein and marker as described in any one of claims 1-6.

16. A kit for detecting centromere antibodies, characterized in that, It comprises the centromere protein modified protein according to any one of claims 1-6, or the composition according to claim 15.

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