TIF1 gamma recombinant antigen and preparation method and application thereof

Abstract

The invention discloses a TIF1 gamma recombinant antigen and a preparation method and application thereof, compared with an existing antigen, an antigen site of the TIF1 gamma recombinant antigen is more fully exposed, then the sensitivity and specificity of TIF1 gamma antibody detection can be improved, the antigen is applied to diagnosis of cancer-related myositis, and the sensitivity and specificity of cancer-related myositis are improved. And the sensitivity and the accuracy can be greatly improved. Meanwhile, the TIF1gamma recombinant antigen also improves the problem that the full-length TIF1gamma antigen is easy to precipitate, and has important significance for improving the antigen expression quantity, expanding the production application of the antigen and reducing the production cost.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a TIF1γ recombinant antigen and a preparation method and application thereof. Background Art

[0002] TIF1γ, also known as TRIM33 protein, belongs to the three-domain (TRIM) protein family and is one of the E3 ubiquitin ligase subfamily. It participates in multiple 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 antibodies are sensitive and specific serological indicators for dermatomyositis combined with tumors. It provides an effective means for screening dermatomyositis combined with tumors. For patients with dermatomyositis, anti-TIF1γ antibodies can be detected at the same time as the diagnosis, which can be used for early diagnosis of tumors and is of great significance for improving prognosis.

[0003] Currently, the detection of TIF1γ autoantibodies has problems with low signal value and sensitivity, and recombinant full-length TIF1γ antibodies are prone to precipitation and cannot be well applied to detection.

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

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

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[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] Since the existing TIF1γ antigen has problems of low signal value and sensitivity when detecting TIF1γ autoantibodies, and the recombinant full-length TIF1γ antibody is easy to precipitate and cannot be well applied to detection, the present invention provides a new IF1γ recombinant antigen, whose amino acid sequence includes at least one of SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.5.

[0009] Among them, the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.5 are obtained after optimizing the full-length amino acids of TIF1γ protein. The amino acid sequences of the recombinant antigens shown in SEQ ID NO.1 and SEQ ID NO.2 have cut off the non-epitope region fragments of TIF1γ protein in the original protein that form steric hindrance to antigen recognition, thereby improving the recognition effect of the antibody; the recombinant antigen shown in SEQ ID NO.5 is obtained by connecting the recombinant antigens shown in SEQ ID NO.1 and SEQ ID NO.2 in series, and is easier to be recognized by the antibody than the truncated form. The above-mentioned TIF1γ recombinant antigen can improve the steric hindrance problem of TIF1γ antibody detection, so that the antigen site is more fully exposed, thereby improving the sensitivity and specificity of TIF1γ antibody detection. At the same time, the above-mentioned TIF1γ recombinant antigen also improves the problem of easy precipitation of the full-length TIF1γ antigen, effectively increasing the antigen expression amount.

[0010] In a second aspect, the present invention provides a biological material related to the above-mentioned TIF1γ recombinant antigen, which is any one of the following:

[0011] (1) Nucleic acid molecule 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] According to the above TIF1γ recombinant antigen, the present invention also provides a nucleic acid molecule encoding the above TIF1γ recombinant antigen.

[0016] Nucleic acid molecules can be DNA or RNA, and in some cases, can modify nucleic acid molecules for use in vectors of the present invention, such as for codon optimization. In some cases, for the purpose of being cloned into a vector, the sequence can be designed to contain a terminal restriction site sequence. Nucleic acid molecules can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of the encoding nucleic acid separated in one or more given cells or from the one or more given cells.

[0017] In a specific embodiment, the above nucleic acid molecules can be optimized according to the codon preference of the host cell and then artificially synthesized into gene fragments. It should be understood that nucleic acid molecules that can be translated into the above amino acid sequences are within the scope of protection of the present invention.

[0018] The present invention provides an expression cassette, which comprises the above nucleic acid molecule and also contains regulatory sequences, such as a promoter and a terminator.

[0019] An expression cassette refers to a nucleic acid construct comprising a coding sequence and a regulatory sequence that are operably connected when introduced into a host cell, resulting in transcription and / or translation of an RNA or polypeptide, respectively. An expression cassette is understood to include a promoter, a target gene open reading frame, and a transcription terminator that allow transcription to begin. Typically, the promoter sequence is placed upstream of the target gene, and the distance from the target gene is compatible with expression control.

[0020] The present invention provides a recombinant vector, which comprises the above nucleic acid molecule or expression box.

[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, no free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other polynucleotide species known in the art. Among the most commonly used vector types are "plasmids", which refer to circular double-stranded DNA loops into which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques. Recombinant expression vectors can contain a form suitable for expressing nucleic acids in host cells, which means that the recombinant expression vector includes one or more regulatory elements, which can be selected based on the host cell used for expression and can be operably linked to the nucleic acid sequence to be expressed.

[0022] The present invention provides a cell, which comprises the above nucleic acid molecule or the above expression box or the above expression vector, and the cell can express the above TIF1γ recombinant antigen.

[0023] The above-mentioned cells can be any cells useful in the production of the TIF1γ recombinant antigen of the present invention. In order 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 the host cell. Such nucleic acids can be easily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes that can specifically bind to the gene encoding the TIF1γ recombinant antigen). The method for introducing a vector into a host cell is well known, and the present invention does not specifically limit this.

[0024] In a specific embodiment, the above-mentioned host cells 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, yeast host cells such as Saccharomyces cerevisiae, which can be selected according to actual conditions in specific experiments.

[0025] In a third aspect, the present invention provides a method for preparing the above-mentioned TIF1γ recombinant antigen, which comprises culturing the above-mentioned cells.

[0026] Specifically, the method 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 the TIF1γ recombinant antigen.

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

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

[0029] In a fourth aspect, the present invention provides a complex comprising the above-mentioned TIF1γ recombinant antigen.

[0030] The above-mentioned complex also includes a solid support, a detectable marker or a binding partner. In the complex, the TIF1γ recombinant antigen can be directly or indirectly combined with the solid support, the detectable marker or the binding partner.

[0031] In some embodiments, the solid support comprises magnetic particles, a microtiter plate or a cellulose membrane; the detectable marker can be a metal particle, a fluorescent marker, a chromophore marker, an electron-dense marker, a chemiluminescent marker, a radioactive marker, or an enzyme marker, specifically colloidal gold, a radioisotope, a fluorophore, a spin marker, or a phage marker, or a rhodamine, fluorescein, an acridinium ester, luciferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidase, glucose oxidase, galactose oxidase or glucose-6-phosphate dehydrogenase marker; the binding partner can be biotin, streptavidin or avidin.

[0032] In a fifth aspect, the present invention provides a composition comprising the above-mentioned TIF1γ recombinant antigen, biological material or complex. The above-mentioned composition can be a pharmaceutical composition or an immune composition.

[0033] In some embodiments, the above-mentioned composition also includes components for preserving the above-mentioned TIF1γ recombinant antigen, the above-mentioned biological material or the above-mentioned complex, such as one or more of a buffer, a surfactant, a protein stabilizer, a preservative, etc.; those skilled in the art can obtain a liquid or lyophilized formula suitable for practical applications based on the prior art.

[0034] In a sixth aspect, 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 detecting dermatomyositis-related diseases;

[0037] (III) for preparing TIF1γ antibody;

[0038] (IV) Used for TIF1γ antibody purification.

[0039] By using the above-mentioned TIF1γ recombinant antigen and its related biological materials, the use 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 thus the recombinant antigen of the present invention can be used to detect the TIF1γ antibody in the test sample in an immunoassay.

[0041] In some embodiments, the TIF1γ recombinant antigen of the present invention can be used for immunoassays, such as immunoblotting, enzyme-linked immunosorbent assay, fluorescent immunochromatography, colloidal gold immunochromatography, and chemiluminescence assays. Among them, the TIF1γ recombinant antigen of the present invention can be used as a capture antigen or a detection antigen, or as a detection antigen and a capture antigen at the same time. The other antigen paired with the TIF1γ recombinant antigen of the present invention can be the same or different, as long as it includes the TIF1γ recombinant antigen of the present invention.

[0042] By using the above-mentioned TIF1γ recombinant antigen and its related biological materials, the use of the above-mentioned TIF1γ recombinant antigen, nucleic acid molecule, expression cassette, expression vector, host cell or complex in the preparation of products for preparing TIF1γ antibodies can be further provided. Through the TIF1γ recombinant antigen and its related biological materials or complexes included in the above-mentioned products, TIF1γ antibodies that can specifically bind to TIF1γ recombinant antigens can be prepared. For the preparation method of TIF1γ antibodies, any appropriate method known in the art can be used for preparation, for example, immunizing animals with TIF1γ recombinant antigens to produce TIF1γ antibodies.

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

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

[0045] In some embodiments, the TIF1γ recombinant antigen or complex of the present invention is coated on a solid phase carrier, which is a liquid phase chip, which has superparamagnetism and is composed of a biocompatible polymer (Barcoded Magnetic Beads, BMB). The BMB herein, i.e., barcoded magnetic particles, is a paramagnetic material incorporated into a biocompatible polymer, and a 12-bit binary digital barcode is engraved on the magnetic particles by photolithography. Barcoded magnetic particles are also called barcoded magnetic beads or digital magnetic beads. When "K" is used as a unit herein, it means the number is "thousands", and 50K BMB means 50,000 barcoded magnetic particles.

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

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

[0048] By using the above kit, the present invention can also provide a method for detecting TIF1γ antibodies, which can be: contacting the TIF1γ recombinant antigen of the present invention with a sample to be tested, forming a complex of TIF1γ antibodies and TIF1γ recombinant antigen when TIF1γ antibodies are present in the sample, and then detecting the presence of the complex, and indicating the presence of TIF1γ antibodies in the sample by the presence of the complex. This method can accurately detect TIF1γ antibodies in the target sample, so this method can provide a new way for TIF1γ antibody detection.

[0049] In some embodiments, the auxiliary diagnostic kit for myositis also includes an autoantibody detection reagent for at least one of the following markers: Jo-1, MDA-5, SAE-1, NXP-2, PL7, PL12, EJ, HMGCR, Ku, OJ, Mi-2α, Mi-2β, SRP54, SSA / Ro52, PM-scl, PM-scl-100, SAE-2 and CN-1A.

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

[0051] The present invention provides a TIF1γ recombinant antigen and a preparation method thereof, wherein the antigenic site of the TIF1γ recombinant antigen is more fully exposed, thereby being able to improve the sensitivity and specificity of anti-TIF1γ antibody detection, and applying the antigen of the present invention to the diagnosis of cancer-related myositis can greatly improve the sensitivity and accuracy. At the same time, the TIF1γ recombinant antigen of the present invention also improves the problem of easy precipitation of the full-length TIF1γ antigen, which is of great significance for increasing the expression amount of the antigen, expanding the production and application of the antigen, and reducing the production cost. DETAILED DESCRIPTION

[0052] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0053] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0054] Example 1

[0055] Four truncated and one full-length TIF1γ nucleic acid fragments were obtained by gene synthesis (amino acid and nucleotide sequences of each antigen are shown in Table 1), and inserted into the pcDNA3.1+ vector, transformed, screened, expanded and cultured, and the expression plasmid was extracted.

[0056] The expression plasmids of the four truncated proteins and the full-length protein were transfected into 293F cells respectively and cultured in suspension at 37°C and 5% CO2 for 6 days. The cells were collected by centrifugation, and the lysate was added to resuspend the lysed cells and treated by ultrasound until they became water-like; the supernatant was collected by centrifugation at 12000rpm for 15min and purified by affinity chromatography column. The eluted target protein solution was concentrated by ultrafiltration and the protein concentration was determined by UV spectrophotometry, and the yield was calculated. The results are shown below:

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

[0058]

[0059]

[0060] As can be seen from the above table, the expression levels of truncated form 1, truncated form 2, and truncated form 3 are increased relative to the full-length form, and truncated form 2 has no precipitation.

[0061] Example 2

[0062] Furthermore, the truncated antigen and the full-length antigen of Example 1 were coated on BMB in the same manner, with a coating ratio of 25 μg / 50K. The detection effect of each antigen on anti-TIF1γ positive dermatomyositis was tested using Livzon's self-developed fully automatic multiple immunoassay analyzer in combination with phycoerythrin-labeled mouse anti-human IgG secondary antibody working solution. 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 truncations 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 1 (SEQ ID NO.1) 5.0 70％ 95％ Truncated 2 (SEQ ID NO.2) 4.5 60％ 95％ Truncated 3 (SEQ ID NO.3) 1.6 15％ 100％ Truncated 4 (SEQ ID NO.4) 1.9 20％ 100％

[0065] Sensitivity is the number of positive samples detected / the number of positive samples; specificity is the number of negative samples detected / the number of negative samples. The signal-to-noise ratio is the mean signal value of positive samples / the mean signal value of negative samples.

[0066] From the table above, we can see that truncated form 3 and truncated form 4 have better specificity than the full length, but their sensitivity is significantly reduced; while truncated form 1 and truncated form 2 have improved sensitivity compared with the full length without changing their specificity. Considering the protein precipitation, truncated form 2 has the best effect.

[0067] Example 3

[0068] According to Example 2, both truncations 1 and 2 can improve the signal-to-noise ratio and sensitivity, while the signal-to-noise ratio and sensitivity of truncations 3 and 4 are greatly reduced; this indicates that the truncated sequences of truncations 1 and 2 do not participate in the formation of the TIF1γ antigenic epitope, but have a certain steric hindrance effect, while the truncated sequences of truncations 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. The same preparation method and detection method are used. The yield of truncated body 5 finally obtained is as follows:

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

[0071] Purification of antigens Amino acid sequence Nucleotide sequence Yield Protein precipitation Truncated body 5 SEQ ID NO.5 SEQ ID NO.10 4.5mg / L No precipitation

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

[0073] Table 4 Detection results of truncated form 5 against TIF1γ-positive dermatomyositis

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

[0075] As shown in the results in Table 4, the optimized truncated protein 5 has a 71% higher signal-to-noise ratio and a 36% higher sensitivity than the full-length protein, which is also better than truncated proteins 1 and 2. Moreover, the truncated protein 5 not only solves the problem of precipitation of the full-length protein during concentration, but also increases the yield by 36%, which greatly improves the problem of TIF1γ antigen application in detection.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A TIF1γ recombinant antigen, characterized in that: The amino acids of the TIF1γ recombinant antigen are shown in SEQ ID NO.1-2 or SEQ ID NO.

5.

2. Biomaterial, characterized in that Any of the following: (1) A nucleic acid molecule encoding the TIF1γ recombinant antigen according to claim 1: (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant vector containing the nucleic acid molecule described in (1) or the expression cassette described in (2); (4) A cell containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant vector described in (3).

3. The method for preparing the TIF1γ recombinant antigen according to claim 1, characterized in that: Comprising culturing the cell of claim 2.

4. A composite, characterized in that Comprising the TIF1γ recombinant antigen according to claim 1.

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

6. A composition, characterized in that Comprising at least one of the TIF1γ recombinant antigen according to claim 1, the biological material according to claim 2, or the complex according to claim 4 or 5; Optionally, the composition further comprises one or more of a buffer, a surfactant, a protein stabilizer, and a preservative.

7. Use of the TIF1γ recombinant antigen according to claim 1, the biomaterial according to claim 2, the complex according to claim 4 or 5, or the composition according to claim 6 in any one of the following (I) to (III): (I) preparing a product for detecting TIF1γ antibodies; (II) preparing products for detecting dermatomyositis-related diseases; (III) for preparing TIF1γ antibody; (IV) Used for TIF1γ antibody purification.

8. A kit, characterized in that: It comprises the TIF1γ recombinant antigen according to claim 1, the biomaterial according to claim 2, the complex according to claim 4 or 5, or the composition according to claim 6.

9. The kit according to claim 8, characterized in that The kit is used for auxiliary diagnosis of myositis and also includes an autoantibody detection reagent for at least one of the following markers: Jo-1, MDA-5, SAE-1, NXP-2, PL7, PL12, EJ, HMGCR, Ku, OJ, Mi-2α, Mi-2β, SRP54, SSA / Ro52, PM-scl, PM-scl-100, SAE-2 and CN-1A.

10. Use of the TIF1γ recombinant antigen according to claim 1, the biomaterial according to claim 2, the complex according to claim 4 or 5, the composition according to claim 6 or the kit according to claim 8 or 9 in detecting TIF1γ antibodies for non-diagnostic purposes.

Images