A TIF1γ recombinant antigen, its preparation method and application

Abstract

This invention discloses a recombinant TIF1γ antigen, its preparation method, and its applications. Compared to existing antigens, the antigenic sites of this recombinant TIF1γ antigen are more fully exposed, thereby improving the sensitivity and specificity of anti-TIF1γ antibody detection. Applying this antigen to the diagnosis of cancer-associated myositis can significantly improve sensitivity and accuracy. Furthermore, this recombinant TIF1γ antigen also addresses the issue of easy precipitation of the full-length TIF1γ antigen, which is significant for increasing antigen expression levels, expanding antigen production and applications, and reducing production costs.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically, to a TIF1γ recombinant antigen, its preparation method, and its application. Background Technology

[0002] TIF1γ, also known as TRIM33 protein, belongs to the TRIM protein family and is one of the E3 ubiquitin ligase subfamily. It participates in various biological processes such as intracellular signal transduction, apoptosis, autophagy, and immunity by regulating the ubiquitination of target proteins. Studies have found that serum anti-TIF1γ antibody is a sensitive and specific serological marker for dermatomyositis complicated with tumors. It provides an effective means of screening for dermatomyositis with tumors. For patients with dermatomyositis, anti-TIF1γ antibody can be detected at the same time as diagnosis, which can be used for early tumor diagnosis and is of great significance for improving prognosis.

[0003] Currently, the detection of TIF1γ autoantibodies suffers from low signal values ​​and low sensitivity, and recombinant full-length TIF1γ antibodies are prone to precipitation, making them unsuitable for detection.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a TIF1γ recombinant antigen, its preparation method, and its application. The TIF1γ recombinant antigen provided by this invention greatly improves the sensitivity and accuracy of anti-TIF1γ antibody detection.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a TIF1γ recombinant antigen, the amino acids of which are shown in SEQ ID NO.1-2 or SEQ ID NO.5.

[0008] Because existing TIF1γ antigens have problems with low signal values ​​and low sensitivity when detecting TIF1γ autoantibodies, and recombinant full-length TIF1γ antibodies are prone to precipitation and cannot be well applied to detection, this invention provides a new recombinant IF1γ antigen whose amino acid sequence includes at least one of SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.5.

[0009] The amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.5 were obtained by optimizing the full-length amino acid sequence of the TIF1γ protein. The recombinant antigens shown in SEQ ID NO.1 and SEQ ID NO.2 have had non-epitope regions of the original TIF1γ protein removed, which sterically hinder antigen recognition, thus improving antibody recognition. The recombinant antigen shown in SEQ ID NO.5 is obtained by tandemly combining the recombinant antigens shown in SEQ ID NO.1 and SEQ ID NO.2, and is more easily recognized by antibodies compared to the truncated versions. These recombinant TIF1γ antigens can improve the steric hindrance problem in TIF1γ antibody detection, allowing for more complete exposure of antigen sites, thereby improving the sensitivity and specificity of TIF1γ antibody detection. Simultaneously, these recombinant TIF1γ antigens also improve the problem of easy precipitation of the full-length TIF1γ antigen, effectively increasing antigen expression levels.

[0010] Secondly, the present invention provides biological materials related to the above-mentioned TIF1γ recombinant antigen, which are any one of the following:

[0011] (1) Nucleic acid molecules encoding the above-mentioned TIF1γ recombinant antigen:

[0012] (2) An expression cassette containing the nucleic acid molecule described in (1);

[0013] (3) A recombinant vector containing the nucleic acid molecule described in (1) or the expression cassette described in (2);

[0014] (4) A cell containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant vector described in (3).

[0015] Based on the above-mentioned TIF1γ recombinant antigen, the present invention also provides a nucleic acid molecule that encodes the above-mentioned TIF1γ recombinant antigen.

[0016] Nucleic acid molecules can be DNA or RNA. In some cases, nucleic acid molecules can be modified for use in the vectors of this invention, such as for codon optimization. In some cases, for the purpose of cloning into the vector, the sequence can be designed to contain terminal restriction sites. Nucleic acid molecules can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of nucleic acids encoding nucleic acids from one or more given cells or isolated from said one or more given cells.

[0017] In specific implementation schemes, the aforementioned nucleic acid molecules can be optimized according to the codon preferences of the host cell before being used to artificially synthesize gene fragments. It should be understood that all nucleic acid molecules capable of being translated into the aforementioned amino acid sequences are within the scope of protection of this invention.

[0018] This invention provides an expression cassette comprising the aforementioned nucleic acid molecule. The expression cassette also contains regulatory sequences, such as promoters and terminators.

[0019] An expression cassette is a nucleic acid construct containing coding and regulatory sequences that are operable upon introduction into a host cell, leading to transcription and / or translation of RNA or polypeptide, respectively. An expression cassette should be understood to include a promoter that allows transcription to begin, an open reading frame of the target gene, and a transcription terminator. Typically, the promoter sequence is placed upstream of the target gene, at a distance compatible with expression control.

[0020] The present invention provides a recombinant vector comprising the above-mentioned nucleic acid molecules or expression cassettes.

[0021] Vectors include, but are not limited to: single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, or without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other polynucleotide types known in the art. The most commonly used vector type is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, through standard molecular cloning techniques. Recombinant expression vectors may contain a form suitable for expressing nucleic acids in host cells, meaning that recombinant expression vectors include one or more regulatory elements that can be selected based on the host cell used for expression and which can be operatively linked to the nucleic acid sequence to be expressed.

[0022] The present invention provides a cell comprising the above-mentioned nucleic acid molecule or the above-mentioned expression cassette or the above-mentioned expression vector, and the cell is capable of expressing the above-mentioned TIF1γ recombinant antigen.

[0023] The cells described above can be any cells useful in the production of the TIF1γ recombinant antigen of the present invention. To produce the TIF1γ recombinant antigen, the nucleic acid encoding the TIF1γ recombinant antigen can be isolated and inserted into one or more vectors for further cloning and / or expression in host cells. This nucleic acid can be readily isolated and sequenced using conventional techniques, e.g., by using oligonucleotide probes capable of specifically binding to the gene encoding the TIF1γ recombinant antigen. Methods for vector introduction into host cells are well known, and the present invention does not specifically limit them.

[0024] In the specific implementation plan, the host cells mentioned above can be prokaryotic cells or eukaryotic cells, specifically Chinese hamster ovary (CHO) cells, HeLa cells, human embryonic kidney cells, bacterial host cells such as Escherichia coli, Bacillus subtilis and Streptococcus cells, and yeast host cells such as Saccharomyces cerevisiae. The specific choice can be made according to the actual situation in the specific experiment.

[0025] Thirdly, the present invention provides a method for preparing the above-mentioned TIF1γ recombinant antigen, which includes culturing the above-mentioned cells.

[0026] Specifically, it may also include cloning a nucleic acid molecule encoding the TIF1γ recombinant antigen described in any one of SEQ ID NO. 1-3 into an expression vector to obtain a recombinant expression vector; transforming the recombinant expression vector into a host cell, inducing its expression, and then extracting and purifying it to obtain the TIF1γ recombinant antigen.

[0027] The above-mentioned TIF1γ recombinant antigen can be prepared using any appropriate method known in the art.

[0028] In some embodiments, nucleic acid molecules encoding the TIF1γ recombinant antigen described in any one of SEQ ID NO.1-2 and SEQ ID NO.5 can be prepared, then cloned into a suitable vector to obtain a recombinant vector; then the recombinant TIF1γ recombinant antigen can be expressed through a suitable host using a suitable expression system.

[0029] Fourthly, the present invention provides a complex comprising the above-described TIF1γ recombinant antigen.

[0030] The aforementioned complex also includes a solid support, a detectable marker, or a binding partner. The TIF1γ recombinant antigen in this complex can be obtained by directly or indirectly binding to the solid support, the detectable marker, or the binding partner.

[0031] In some embodiments, the solid support includes magnetic particles, microtiter plates, or cellulose membranes; the detectable label can be a metal particle, fluorescent label, chromophore label, electron-dense label, chemiluminescent label, radioactive label, or enzyme label, specifically colloidal gold, radioactive isotopes, fluorophores, spin labels, or phage labels, or it can be rhodamine, luciferin, acridinium ester, luciferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, sugar oxidase, glucose oxidase, galactose oxidase, or glucose-6-phosphate dehydrogenase label; the binding partner can be biotin, streptavidin, or avidin.

[0032] Fifthly, the present invention provides a composition comprising the above-described TIF1γ recombinant antigen, biological material, or complex. The composition may be a pharmaceutical composition or an immunomodulatory composition.

[0033] In some embodiments, the composition further includes one or more components for preserving the TIF1γ recombinant antigen, the biomaterial, or the complex, such as buffer, surfactant, protein stabilizer, preservative, etc. Those skilled in the art can obtain liquid or lyophilized formulations suitable for practical applications based on existing technology.

[0034] Sixthly, the present invention provides the use of the above-mentioned TIF1γ recombinant antigen, biomaterial, complex or composition in any one of the following (I) to (IV):

[0035] (I) Application in the preparation of products for detecting TIF1γ antibodies;

[0036] (II) Application in the preparation of products for the detection of dermatomyositis-related diseases;

[0037] (III) Used to prepare TIF1γ antibody;

[0038] (IV) is used for TIF1γ antibody purification.

[0039] Using the above-mentioned TIF1γ recombinant antigen and related biological materials, the application of the above-mentioned TIF1γ recombinant antigen, nucleic acid molecule, expression cassette, expression vector, host cell, complex or composition in the preparation of products for detecting TIF1γ antibodies can be further provided.

[0040] In some embodiments, the TIF1γ antibody in the test sample recognizes the epitope in the TIF1γ recombinant antigen of the present invention, and therefore the recombinant antigen of the present invention can be used in immunoassays to detect the TIF1γ antibody in the test sample.

[0041] In some embodiments, the TIF1γ recombinant antigen of the present invention can be used for immunoassays, such as immunoblotting, enzyme-linked immunosorbent assay (ELISA), fluorescence immunochromatography, colloidal gold immunochromatography, and chemiluminescence assay. The TIF1γ recombinant antigen of the present invention can be used as a capture antigen, a detection antigen, or both. The other antigen paired with the TIF1γ recombinant antigen of the present invention can be the same as or different from the TIF1γ recombinant antigen of the present invention, as long as it includes the TIF1γ recombinant antigen of the present invention.

[0042] Using the aforementioned TIF1γ recombinant antigen and related biological materials, the applications of the aforementioned TIF1γ recombinant antigen, nucleic acid molecules, expression cassettes, expression vectors, host cells, or complexes in the preparation of TIF1γ antibody products can be further provided. Through the TIF1γ recombinant antigen and related biological materials or complexes included in the above products, TIF1γ antibodies capable of specifically binding to the TIF1γ recombinant antigen can be prepared. For the preparation method of TIF1γ antibodies, any suitable method known in the art can be used, for example, immunizing animals with the TIF1γ recombinant antigen to induce the production of TIF1γ antibodies.

[0043] In some embodiments, the above-described products include kits.

[0044] In a seventh aspect, the present invention provides a kit comprising the above-mentioned TIF1γ recombinant antigen, biomaterial, complex or composition.

[0045] In some embodiments, the TIF1γ recombinant antigen or complex of the present invention is coated on a solid-phase support, which is a liquid-phase chip having superparamagnetic properties and composed of biocompatible polymers (Barcoded Magnetic Beads, BMB). Here, BMB refers to barcoded magnetic beads, which are made by incorporating paramagnetic materials into biocompatible polymers and etching 12-bit binary digital barcodes onto the magnetic beads using photolithography. Barcoded magnetic beads are also known as barcoded magnetic beads or digital magnetic beads. In this document, "K" is used as a unit to indicate a quantity of "thousands," so 50K BMB represents 50,000 barcoded magnetic beads.

[0046] In some embodiments, the kit of the present invention is used for the adjunctive diagnosis of myositis.

[0047] By using the TIF1γ recombinant antigen of the present invention as the detection antigen, the sensitivity and accuracy of anti-TIF1γ antibody detection can be greatly improved.

[0048] Using the above-described kit, the present invention can also provide a method for detecting TIF1γ antibodies. This method involves contacting the TIF1γ recombinant antigen of the present invention with the sample to be tested. If TIF1γ antibodies are present in the sample, a complex of TIF1γ antibody and TIF1γ recombinant antigen is formed. The presence of this complex is then detected, indicating the presence of TIF1γ antibodies in the sample. This method can accurately detect TIF1γ antibodies in the target sample, thus providing a novel approach for TIF1γ antibody detection.

[0049] In some embodiments, the myositis auxiliary diagnostic kit further includes an autoantibody detection reagent 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, SRP54, SSA / Ro52, PM-scl, PM-scl-100, SAE-2, and CN-1A.

[0050] The present invention has the following beneficial effects:

[0051] This invention provides a recombinant TIF1γ antigen and its preparation method. The antigenic sites of this recombinant TIF1γ antigen are more fully exposed, thereby improving the sensitivity and specificity of anti-TIF1γ antibody detection. Applying this antigen to the diagnosis of cancer-associated myositis can significantly improve sensitivity and accuracy. Simultaneously, the recombinant TIF1γ antigen of this invention also improves the problem of easy precipitation of the full-length TIF1γ antigen, which is of great significance for increasing antigen expression levels, expanding antigen production and application, and reducing production costs. Detailed Implementation

[0052] 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.

[0053] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0054] Example 1

[0055] TIF1γ nucleic acid fragments containing four truncated segments and one full-length segment were obtained through gene synthesis (the amino acid and nucleotide sequences of each antigen are shown in Table 1), and inserted into the pcDNA3.1+ vector. The fragments were then transformed, screened, and cultured to obtain expression plasmids.

[0056] Expression plasmids for four truncated and full-length proteins were transfected into 293F cells and cultured in suspension at 37°C and 5% CO2 for 6 days. Cells were collected by centrifugation, resuspended in lysis buffer, and sonicated until a watery consistency was achieved. The supernatant was collected by centrifugation at 12000 rpm for 15 min and purified using affinity chromatography. The eluted protein solution was concentrated by ultrafiltration, and the protein concentration was determined by UV spectrophotometry. The yield was calculated, and the results are shown below:

[0057] Table 1. Purification yield and protein precipitation of full-length TIF1γ and its truncated variants 1-4

[0058]

[0059]

[0060] As shown in the table above, the expression levels of truncated variants 1, 2, and 3 are increased compared to the full-length variant, while truncated variant 2 shows no precipitation.

[0061] Example 2

[0062] Furthermore, the truncated antigen and the full-length antigen from Example 1 were coated onto BMB using the same method, with a coating ratio of 25 μg / 50K. Using a phycoerythrin-labeled mouse anti-human IgG secondary antibody working solution, the detection efficacy of each antigen against TIF1γ-positive dermatomyositis was tested using a Livzon-developed fully automated multiplex immunoassay analyzer. The samples included 40 positive serum samples and 20 negative serum samples. The results are shown in Table 1 below.

[0063] Table 2. Detection results of full-length TIF1γ and its truncated forms 1-4 against TIF1γ-positive dermatomyositis.

[0064] Coating antigen Signal-to-noise ratio Sensitivity Specificity Full length (SEQ ID NO.11) 3.1 55％ 95％ Truncated body 1 (SEQ ID NO.1) 5.0 70％ 95％ Truncated body 2 (SEQ ID NO.2) 4.5 60％ 95％ Truncated body 3 (SEQ ID NO.3) 1.6 15％ 100％ Truncated body 4 (SEQ ID NO.4) 1.9 20％ 100％

[0065] Sensitivity is calculated as the number of positive samples detected divided by the number of positive samples; specificity is calculated as the number of negative samples detected divided by the number of negative samples. Signal-to-noise ratio is calculated as the mean signal value of positive samples divided by the mean signal value of negative samples.

[0066] As shown in the table above, truncated forms 3 and 4 exhibit better specificity than the full-length form, but their sensitivity is significantly reduced; while truncated forms 1 and 2 show improved sensitivity compared to the full-length form, without changing their specificity. Considering the overall protein precipitation, truncated form 2 shows the best results.

[0067] Example 3

[0068] As shown in Example 2, truncated versions 1 and 2 can improve the signal-to-noise ratio and sensitivity, while truncated versions 3 and 4 significantly reduce the signal-to-noise ratio and sensitivity. This indicates that the truncated sequences of truncated versions 1 and 2 do not participate in the formation of the TIF1γ antigenic epitope, but instead have a certain steric hindrance effect, while the truncated sequences of truncated versions 3 and 4 are the necessary sequences for the formation of the antigenic epitope.

[0069] Based on the above speculation, in this embodiment, truncated body 1 and truncated body 2 are combined and truncated to obtain truncated body 5. Using the same preparation and detection methods, the yield of truncated body 5 is shown below:

[0070] Table 3. Purification yield and protein precipitation of truncated form 5

[0071] Purified antigen amino acid sequence nucleotide sequence Yield Protein precipitation 5 truncated body SEQ ID NO.5 SEQ ID NO.10 4.5 mg / L No sediment

[0072] The antigen was coated onto the BMB as in Example 2 and tested. The results are shown in the table below:

[0073] Table 4. Results of truncation 5 test for anti-TIF1γ positive dermatomyositis

[0074] Coating antigen Signal-to-noise ratio Sensitivity Specificity Truncated body 5 (SEQ ID NO.5) 5.3 75％ 95％

[0075] As shown in Table 4, the optimized truncated protein 5 improved the signal-to-noise ratio by 71% and the sensitivity by 36% compared to the full-length protein, also outperforming truncated proteins 1 and 2. Furthermore, truncated protein 5 not only solved the precipitation problem during concentration of the full-length protein but also increased the yield by 36%, significantly improving the application of TIF1γ antigen in detection.

[0076] 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 TIF1γ recombinant antigen, characterized in that, The amino acid sequence of the TIF1γ recombinant antigen is selected from any one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.

5.

2. A biomaterial, characterized in that, It can be any of the following: (1) A nucleic acid molecule encoding the TIF1γ 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. The method for preparing the TIF1γ recombinant antigen as described in claim 1, characterized in that, This includes culturing the cells described in claim 2.

4. A complex, characterized in that, Includes the TIF1γ recombinant antigen as described in claim 1.

5. The complex according to claim 4, characterized in that, The complex also includes a solid support, a detectable marker, or a binding coupler that binds to the TIF1γ recombinant antigen.

6. A composition, characterized in that, It includes at least one of the TIF1γ recombinant antigen of claim 1, the biomaterial of claim 2, or the complex of claim 4 or 5.

7. The composition according to claim 6, characterized in that, The composition further includes one or more of the following: buffer solution, surfactant, protein stabilizer, and preservative.

8. The use of the TIF1γ recombinant antigen of claim 1, the biomaterial of claim 2, the complex of claim 4 or 5, or the composition of claim 6 or 7 in any one of the following (I) to (III): (I) Preparation of products for detecting TIF1γ antibodies; (II) To prepare products for the detection of dermatomyositis-related diseases; (III) Used to prepare TIF1γ antibody; (IV) Used for TIF1γ antibody purification.

9. A reagent kit, characterized in that, It comprises the TIF1γ recombinant antigen of claim 1, the biomaterial of claim 2, the complex of claim 4 or 5, or the composition of claim 6 or 7.

10. The reagent kit according to claim 9, 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, SRP54, SSA / Ro52, PM-scl, PM-scl-100, SAE-2, and CN-1A.

11. The use of the TIF1γ recombinant antigen of claim 1, the biomaterial of claim 2, the complex of claim 4 or 5, the composition of claim 6 or 7, or the kit of claim 9 or 10 in the detection of TIF1γ antibodies for non-diagnostic purposes.

Images