Cenpa fusion antigens, methods of making and uses thereof

Abstract

The application discloses a CENPA fusion antigen and a preparation method and application thereof, and belongs to the technical field of biotechnology.The CENPA fusion antigen contains a CENPA protein fragment and a histone fragment, the histone includes any one of a histone H3 and a histone H4, the CENPA fusion antigen is formed by connecting the CENPA protein fragment and the histone fragment, compared with single CENPA, the antigenicity of the CENPA fusion antigen is better, and the effect of detecting scleroderma is better; meanwhile, the fusion protein can be expressed in the form of secretion in an insect cell, the hydrophilicity of the fusion protein is good, and the fusion protein can be effectively applied to CENPA antibody detection, so that the CENPA fusion antigen has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a CENPA fusion antigen, its preparation method, and its application. Background Technology

[0002] Centromere proteins are a class of proteins that form or regulate centromere function. The central structure of chromosomes is attached with spindle fibers and tubulin, enabling chromosomes to separate during cell division. Currently, nine centromere proteins are known, and most are targets for autoantibodies and anti-centromere antibodies. CENPA (Centromere protein A, CENPA) is another important centromere autoantigen besides CENPB. Its molecular weight is close to 22 kDa, and it exhibits histone-like properties, forming aggregates with histones H3 and H4 to participate in the formation of centromere chromatin.

[0003] Systemic sclerosis (SSc) is a systemic connective tissue disorder characterized by peripheral arteriosclerosis and fibrosis of visceral structures, leading to fibrosis and functional decline in the heart, lungs, kidneys, and gastrointestinal tract. SSC is often associated with specific autoantibodies, with centromere antibodies being an important type. Therefore, detecting CENPA antibodies plays a crucial role in the auxiliary diagnosis of SSC.

[0004] Currently, the detection of CENPA antibodies is mainly performed through immunodiagnostics: CENPA protein immobilized on a vector can specifically bind to CENPA antibodies, and the immobilized CENPA antibodies can be further identified by labeled antibodies, thus achieving the detection of CENPA antibodies. However, recombinant CENPA protein has a series of problems, including low expression levels, high hydrophobicity, and low detection sensitivity.

[0005] Therefore, there is an urgent need to discover antigens that have good CENPA antibody recognition function and can be effectively applied to CENPA antibody detection.

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

[0007] The purpose of this invention is to provide a CENPA fusion antigen, its preparation method, and its application. This CENPA fusion antigen has higher detection specificity and sensitivity compared to the CENPA protein.

[0008] This invention is implemented as follows:

[0009] According to one aspect of the present invention, a CENPA fusion antigen is provided, the CENPA fusion antigen containing a CENPA protein fragment and a histone fragment.

[0010] The preferred amino acid sequence of the CENPA protein fragment is shown in SEQ ID NO.3.

[0011] The histone fragments in the CENPA fusion antigen provided by this invention are derived from histone H3 or histone H4. The amino acid sequence of histone H3 is shown in SEQ ID NO.4, and the amino acid sequence of histone H4 is shown in SEQ ID NO.5.

[0012] Histones can be attached to the C-terminus or N-terminus of CENPA. In some preferred embodiments, the CENPA protein fragment is fused to the N-terminus of the histone fragment. More preferably, the CENPA protein fragment and the histone fragment are linked by a first linker peptide. The linker peptide can improve the stability, expression level, and biological activity of the fusion protein. The amino acid sequence of the first linker peptide of this invention is shown in SEQ ID NO. 6.

[0013] In some preferred embodiments, the CENPA fusion protein further contains a signal peptide that enables the recombinant antigen to be efficiently secreted and expressed in insect cells. The signal peptide is preferably the GP67 signal peptide, whose amino acid sequence is shown in SEQ ID NO. 8, and is attached to the N-terminus of the fusion protein fragment.

[0014] In some preferred embodiments, the CENPA fusion protein also contains a tag for the isolation and purification of the CENPA fusion protein. The tag can be a conventional protein tag in the art, and can be added to the amino terminus and / or carboxyl terminus of the CENPA fusion protein. This invention selects a His tag (HHHHHH, SEQ ID NO. 7), which can be associated with Ni... + The His tag is short and has minimal impact on the structure of the CENPA fusion protein, thus resulting in good purification performance. Preferably, the His tag is attached to the C-terminus of the CENPA fusion protein.

[0015] In some preferred embodiments, the histone fragment and the His tag are linked by a second linker peptide, which facilitates tag display. From the N-terminus to the C-terminus, the sequence is: signal peptide, CENPA protein fragment, first linker peptide, histone fragment, second linker peptide, and His tag. The sequences of the first linker peptide and the second linker peptide can be the same or two different sequences. In some preferred embodiments, the sequence of the second linker peptide is SGG.

[0016] The preferred amino acid sequence of the CENPA fusion protein after expression with the above structure is the sequence shown in SEQ ID NO.1 or SEQ ID NO.2, which has better antigenicity and can be expressed solublely in insect cells.

[0017] According to another aspect of the invention, the invention also provides a gene encoding the aforementioned CENPA fusion protein. The gene is preferably a sequence as shown in SEQ ID NO. 9 or SEQ ID NO. 10, or a sequence that is more than 90% homologous to the sequence shown in SEQ ID NO. 9 or SEQ ID NO. 10. A sequence that is more than 90% homologous to the sequence shown in SEQ ID NO. 9 or SEQ ID NO. 10 refers to a nucleic acid sequence that differs from the sequence shown in SEQ ID NO. 9 or SEQ ID NO. 10 due to nucleotide deletion, mutation, reduction, or addition, but is at least 90% similar and has the same function. The homology can be, for example, but not limited to, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0018] According to another aspect of the present invention, a biomaterial is also provided, comprising at least one of a polynucleotide, an expression cassette, a vector, a recombinant microorganism, or a cell line. The biomaterial is capable of expressing the CENPA fusion protein and / or contains a gene encoding the CENPA fusion protein. The biomaterial may be, for example, a gene encoding the CENPA fusion protein; an expression cassette containing the aforementioned gene encoding the CENPA fusion protein; or, for example, a vector containing the gene encoding the CENPA fusion protein, enabling the gene encoding the CENPA fusion protein to replicate or be expressed in a host, or a medium capable of integrating the gene into a host cell. Examples of vectors include, but are not limited to, plasmids, phage particles, bacteriophages, and viral genomes. The biomaterial may also be a recombinant microorganism containing a vector encoding the CENPA fusion protein, the gene being cloned in the microorganism to increase the copy number of the gene. The biomaterial may also be a cell line containing an expression vector encoding the CENPA fusion protein, the cell line being capable of expressing the CENPA fusion protein.

[0019] According to another aspect of the present invention, a method for preparing the CENPA fusion protein is also provided, the method comprising expressing a gene encoding the CENPA fusion protein in a host. This method is simple to operate and suitable for large-scale production of the CENPA fusion protein.

[0020] According to another aspect of the invention, the invention also provides a polypeptide conjugate comprising the aforementioned CENPA fusion antigen and a conjugate linked thereto. The conjugate comprises a solid support. Preferably, the solid support is selected from magnetic microparticles, plates, and membranes. More preferably, the solid support is magnetic microparticles.

[0021] According to another aspect of the invention, the invention also provides the use of CENPA fusion antigens, biomaterials or peptide conjugates in the preparation of products for detecting CENPA antibodies.

[0022] According to another aspect of the invention, the invention also provides a kit comprising at least one of CENPA fusion antigen, biomaterial, and polypeptide conjugate.

[0023] In some embodiments, the kit contains CENPA fusion antigen for detecting CENPA antibodies. The kit may be an ELISA kit, and therefore the kit may also contain reagents and consumables for performing ELISA experiments.

[0024] According to another aspect of the present invention, the present invention also provides a method for detecting CENPA antibodies for non-diagnostic purposes, comprising: contacting a sample to be tested with a CENPA fusion antigen coated on a solid-phase support, and identifying CENPA antibodies captured by the CENPA fusion antigen by labeling an antibody.

[0025] The present invention has the following beneficial effects:

[0026] The CENPA fusion antigen of the present invention is composed of CENPA protein fragments and histone fragments linked together. Compared with single CENPA, the CENPA fusion antigen has better antigenicity and better detection effect on scleroderma. At the same time, the fusion protein of the present invention can be expressed in insect cells in a secretory form. The fusion protein has good hydrophilicity and can be effectively applied to CENPA antibody detection. Therefore, the CENPA fusion antigen of the present invention has good application prospects. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is an SDS-PAGE image of the CENPA_H3 protein in Example 1 of the present invention;

[0029] Figure 2This is an SDS-PAGE image of the CENPA_H4 protein in Example 1 of the present invention. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0031] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0032] Example 1

[0033] This invention relates to a method for preparing fusion proteins, comprising the following steps:

[0034] (1) Construction of fusion protein expression vector:

[0035] A gene fragment comprising the fusion protein as described in SEQ ID NO. 1 or 2 (with a signal peptide GP67 as shown in SEQ ID NO. 8 at the N-terminus) was obtained by gene synthesis. The nucleotide sequence of the gene fragment is shown in SEQ ID NO. 9 or 10. The gene fragment was inserted between the BamHⅠ and HindⅢ sites of the vector pFastBac1 using common molecular biology methods (including PCR, enzyme ligation, etc.) to construct the expression vectors GP67-CENPA_H3-6histag and GP67-CENPA_H4-6histag.

[0036] The above expression vector was transformed into E. coli DH10Bac competent cells and cultured on LB agar plates. Positive colonies were picked for colony PCR verification. After verification, the cells were expanded and the corresponding Bacmid DNA was extracted.

[0037] (2) Expression and purification of the fusion protein:

[0038] sf9 cells were transfected with the above Bacmid DNA, cultured at 27°C for 4 hours, the culture medium was changed, and the cells were cultured for another 5 days. The supernatant was then collected by centrifugation, which is the P1 generation virus.

[0039] P1 virus was re-infected into Sf9 cells and cultured at 27°C and 100 rpm in the dark. After 4 days of culture post-infection, the supernatant was collected by centrifugation, which is the P2 generation virus.

[0040] High Five cells were infected with P2 virus solution and cultured at 27°C in the dark at a rotation speed of 100 rpm / min. After 48 h of culture, the supernatant was collected by centrifugation and the target protein was purified using a Ni column. The supernatant was added to a Ni Resin affinity chromatography column (NiHiTrap column) and eluted with imidazole solutions at concentrations of 50 mM, 100 mM, 200 mM and 500 mM, respectively, and the elution peaks were collected.

[0041] (3) Ion exchange chromatography:

[0042] The elution peak was added to a Q HiTrap column, washed with low-salt buffer for 10 cycles, and then eluted with high salt to collect the protein. The obtained protein was verified by SDS-PAGE.

[0043] In this embodiment, two high-purity target fusion proteins were prepared: CENPA_H3 and CENPA_H4. Figure 1 This is an SDS-PAGE image of the CENPA_H3 protein. Figure 2 This is an SDS-PAGE image of the CENPA_H4 protein.

[0044] Experimental Example 1

[0045] This experiment evaluates and compares the fusion proteins CENPA_H3 and CENPA_H4.

[0046] The fusion proteins CENPA_H3 and CENPA_H4 prepared in Example 1 were coated onto ELISA plates, and then 10 SSC-positive serum samples were detected using IgG labeled with fluorescein phycoerythrin (PE) as the secondary antibody.

[0047] The corresponding fluorescence values ​​were detected under a fluorescence interpreter. The antigenicity of the two fusion proteins was determined based on the fluorescence cutoff value (a cutoff value of 1000 indicates a positive result, while a reaction value <1000 indicates a missed case of the antigen). The evaluation results are shown in Table 1 below.

[0048] Table 1 Evaluation results of CENPA_H3 and CENPA_H4

[0049]

[0050]

[0051] As shown in Table 1, for 10 SSC-positive serum samples, the detection sensitivity of CENPA_H3 was 100% (10 / 10), while that of CENPA_H4 was 80% (8 / 10). Furthermore, the reaction value of CENPA_H3 was on average 28.1% higher than that of CENPA_H4. Therefore, the antigenicity of recombinant protein CENPA_H3 is significantly superior to that of CENPA_H4.

[0052] Experiment Example 2

[0053] This experiment is for verifying the performance of the fusion antigen.

[0054] Using CENPA (amino acid sequence as shown in SEQ ID NO.11) as a control, the detection results of the fusion proteins CENPA_H4 (prepared in Example 1) and CENPA_H3 (prepared in Example 1) were verified.

[0055] The evaluation method is as follows: CENPA, fusion protein CENPA_H4, and CENPA_H3 were coated onto ELISA plates, and then IgG labeled with fluorescein phycoerythrin (PE) was used as the secondary antibody to detect the condition of 47 serum samples (including 25 SSC positive samples and 22 SSC negative samples).

[0056] The corresponding fluorescence values ​​were detected using a fluorescence interpreter, and the positive or negative result was determined based on the fluorescence cutoff value (a cutoff value of 1000 indicates a positive result, and a reaction value <1000 indicates a negative result). The verification results are shown in Tables 2 and 3 below.

[0057] Table 2. Validation results of positive samples

[0058] 1 14847 19247 24570 2 20036 17468 18475 3 22350 28463 32584 4 12375 14863 28438 5 812 895 956 6 21068 31473 38573 7 9842 97134 12583 8 1420 1256 3841 9 2873 3586 4532 10 12052 16453 21490 11 703 946 1190 12 10830 16210 24162 13 8455 8627 9832 14 460 753 856 15 12823 19432 24831 16 16217 25438 30599 17 916 1248 1521 18 1230 1127 1638 19 11916 20736 28754 20 20762 39867 44133 21 15838 34153 35382 22 821 968 1687 23 2185 3654 4419 24 1939 1843 4083 25 12470 17543 18354

[0059] Table 3. Validation results of negative samples

[0060] 1 711 743 785 2 265 565 664 3 823 834 778 4 718 810 521 5 815 756 724 6 498 583 644 7 720 712 815 8 1120 1038 958 9 523 653 716 10 463 653 724 11 396 381 528 12 453 589 621 13 1356 1510 1619 14 820 931 923 15 412 598 694 16 721 754 672 17 532 643 685 18 550 673 645 19 264 368 469 20 222 297 394 21 632 674 589 22 512 641 680

[0061] The results of the verification of positive and negative samples were statistically analyzed, and the results are shown in the table below:

[0062] Table 4 Statistical analysis of verification results

[0063]

[0064]

[0065] As shown in Table 4, both CENPA_H3 and CENPA_H4 exhibit better sensitivity than CENPA. Specifically, the detection sensitivity of CENPA_H3 is 92.0%, significantly higher than the 80.0% of CENPA alone; the detection specificity of CENPA_H3 also increases from 90.9% to 95.5%. In summary, both the fusion antigens CENPA_H3 and CENPA_H4 can replace CENPA for the diagnosis of systemic sclerosis, with CENPA_H3 being the optimal choice.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A CENPA fusion antigen, characterized in that, It consists of a CENPA protein fragment and a histone fragment linked to the C-terminus of the CENPA protein fragment via a first linker peptide; the histone is histone H3 or histone H4. The amino acid sequence of the CENPA protein fragment is shown in SEQ ID NO.3; The amino acid sequence of histone H3 is shown in SEQ ID NO.4; The amino acid sequence of histone H4 is shown in SEQ ID NO.

5.

2. The CENPA fusion antigen according to claim 1, characterized in that, The amino acid composition of the first linker peptide is shown in SEQ ID NO.

6.

3. A CENPA fusion antigen, characterized in that, The CENPA fusion antigen comprises the following: the CENPA fusion antigen as described in claim 1 or 2, and a tag attached to the N-terminus or C-terminus of the CENPA fusion antigen as described in claim 1 or 2.

4. The CENPA fusion antigen according to claim 3, characterized in that, The label includes the His label.

5. The CENPA fusion antigen according to claim 4, characterized in that, The tag is attached to the C-terminus of the CENPA fusion antigen.

6. The CENPA fusion antigen according to claim 5, characterized in that, The tag is attached to the C-terminus of the CENPA fusion antigen via a second linker peptide, the amino acid sequence of which is SGG.

7. The CENPA fusion antigen according to claim 6, characterized in that, The amino acid sequence of the CENPA fusion antigen is shown in SEQ ID NO.

1.

8. The CENPA fusion antigen according to claim 6, characterized in that, The amino acid sequence of the CENPA fusion antigen is shown in SEQ ID NO.

2.

9. A CENPA fusion antigen, characterized in that, The CENPA fusion antigen consists of the following components: The CENPA fusion antigen according to any one of claims 1 to 8, and the signal peptide attached to the N-terminus of the CENPA fusion antigen according to any one of claims 1 to 8.

10. The CENPA fusion antigen according to claim 9, characterized in that, The signal peptide includes the GP67 signal peptide, which is located at the N-terminus of the fusion antigen.

11. The CENPA fusion antigen according to claim 10, characterized in that, The amino acid sequence of the signal peptide is shown in SEQ ID NO.

8.

12. Biological material, wherein the biological material is any one of polynucleotides, expression cassettes, vectors, recombinant microorganisms, or cell lines; The polynucleotide encodes the CENPA fusion antigen according to any one of claims 1-11; The expression cassette, vector, recombinant microorganism, or cell line carries the polynucleotide.

13. The method for preparing the CENPA fusion antigen according to any one of claims 1-11, characterized in that, include: The CENPA fusion antigen is expressed in the host cells.

14. The preparation method according to claim 13, characterized in that, The host cell is an insect cell.

15. A polypeptide conjugate, characterized in that, It comprises the CENPA fusion antigen as described in any one of claims 1-11, and a conjugate thereto; The coupling material is a solid-phase carrier; the solid-phase carrier is magnetic microparticles.

16. The use of the CENPA fusion antigen of any one of claims 1-11, the biomaterial of claim 12, or the polypeptide conjugate of claim 15 in the preparation of a product for detecting CENPA antibodies.

17. A kit for detecting CENPA antibodies, characterized in that, It comprises at least one of the CENPA fusion antigen according to any one of claims 1-11, the biomaterial according to claim 12, and the polypeptide conjugate according to claim 15.

18. The kit according to claim 17, characterized in that, The amino acid sequence of the CENPA fusion antigen is shown in SEQ ID NO.1 or 2.

19. A method for detecting CENPA antibodies for non-diagnostic purposes, characterized in that, include: The sample to be tested is contacted with the CENPA fusion antigen as described in any one of claims 1 to 11 coated on a solid-phase support, and the CENPA antibody captured by the CENPA fusion antigen is identified by a labeled antibody.

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