MAGE A1 recombinant antigen as well as preparation method and application thereof

Abstract

The invention discloses a MAGE A1 recombinant antigen as well as a preparation method and application thereof, and belongs to the technical field of biological detection. The amino acid sequence of the MAGE A1 recombinant antigen disclosed by the invention is as shown in SEQ ID NO. 1. Through verification, compared with a full-length MAGE A1 antigen, the MAGE A1 recombinant antigen disclosed by the invention has the advantage that the sensitivity of the MAGE A1 recombinant antigen in early lung cancer detection is improved. Therefore, the MAGE A1 recombinant antigen provides a new way for early auxiliary diagnosis of lung cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, in particular to a MAGE A1 recombinant antigen and a preparation method and application thereof. BACKGROUND

[0002] Lung cancer is the disease with the highest mortality rate among global cancer patients, especially because most patients are in the advanced stage and cannot be cured when diagnosed. Computed tomography (CT) screening of high-risk populations shows that early detection can reduce mortality. However, CT screening still has a high false positive rate, which may affect healthy populations due to unnecessary follow-up scans and invasive follow-up procedures. As a supplement to CT screening, non-invasive biomarkers can reduce the baseline false positive rate and false negative rate of CT screening, thereby reducing the number of patients who need to be followed up and making a diagnosis at an early stage of lung cancer. Lung cancer tissues produce lung cancer-related proteins, and the immune system may produce high-affinity autoantibodies against these proteins. This autoantibody response against tumor-associated antigens begins in the early stages of lung cancer and can last for several years. Therefore, identifying tumor-associated antigens or corresponding autoantibodies in body fluids as potential non-invasive biomarkers can be an effective method for early detection and monitoring of lung cancer.

[0003] MAGE A1 is usually detected in combination with six other antibodies (P53, PGP9.5, SOX2, GAGE7, GBU4-5, and CAGE) in clinical practice. The overall detection rate (i.e., sensitivity) of the combined detection for lung cancer early screening is not high, and the independent early screening for large-scale, asymptomatic populations is far from enough, still having a large room for improvement. The fundamental reason lies in the low diagnostic sensitivity of each individual autoantibody itself.

[0004] MAGE-A1 is a typical "cancer-testis antigen" (CTA), which is almost not expressed in normal adult tissues (except for testicular germ cells) but is highly ectopically activated in various solid tumors, especially lung cancer. This biological distribution determines that it has high tumor specificity. The expression of MAGE A1 is related to the invasiveness of non-small cell lung cancer (NSCLC), and improving its individual detection rate not only means more effectively identifying non-small cell lung cancer patients with stronger invasive biological behavior, but also is beneficial to making the combined detection have stronger identification ability for these patients, which has important significance for evaluating the malignancy of tumors and prognosis.

[0005] Currently, the detection method for anti-MAGE A1 antibody in serum of lung cancer patients on the market is ELISA, and the traditional ELISA relies on colorimetric method for signal output, and the detection limit (LoD) is relatively high, which is difficult to capture the extremely low concentration of biomarkers in the early stage of cancer. Therefore, there is a need to provide a method for improving the detection sensitivity of anti-MAGE A1 antibody in early lung cancer patients.

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

[0007] The purpose of the present application is to provide a MAGE A1 recombinant antigen and a preparation method and application thereof, and the detection of early lung cancer by using the MAGE A1 recombinant antigen of the present application can improve the detection sensitivity.

[0008] According to one aspect of the present application, the present application provides a MAGE A1 recombinant antigen, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0009] The research and analysis of the inventors on MAGE A1 antigen found that the reason why the detection sensitivity of full-length MAGE A1 antigen for early lung cancer is not high may be related to the poor exposure of the C-terminal antigen epitope. The inventors designed and screened to prepare a sequence in which part of the N-terminal sequence of the MAGE A1 antigen sequence was cut off, and the C-terminal sequence with the antigen epitope was retained. Tests show that compared with the full-length MAGE A1 antigen, the MAGE A1 recombinant antigen has a significant improvement in sensitivity for early lung cancer screening.

[0010] In some embodiments, the present application further modifies the MAGE A1 recombinant antigen shown in SEQ ID NO. 1, such as tag modification.

[0011] The optional tag includes but is not limited to His tag, Flag tag, HA tag, Myc tag, GST tag, MBP tag, SUMO tag, Avi Tag.

[0012] In some embodiments, the MAGE A1 recombinant antigen of the present application has an optimized His tag, and the corresponding amino acid sequence is shown in SEQ ID NO. 2.

[0013] According to another aspect of the present application, the present application provides a biological material related to the above-mentioned MAGE A1 recombinant antigen, which is any one of the following: (A1) a nucleic acid molecule encoding the above-mentioned MAGE A1 recombinant antigen; (A2) an expression cassette containing the nucleic acid molecule of (A1); (A3) A recombinant vector containing the nucleic acid molecule described in (A1) or the expression cassette described in (A2); (A4) A cell containing the nucleic acid molecule described in (A1), the expression cassette described in (A2), or the recombinant vector described in (A3).

[0014] Nucleic acid molecules 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, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases.

[0015] 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.

[0016] In some embodiments, the MAGE A1 recombinant antigen shown in SEQ ID NO.2 is codon-optimized for an expression system, and its nucleotide sequence is shown in SEQ ID NO.3.

[0017] Expression cassettes are DNA functional modules commonly used in the art to drive efficient and controlled transcription and translation of exogenous genes in host cells. They contain core elements such as promoters, target gene coding regions, and terminators, and are widely used in plasmid vectors, viral vectors, and gene editing systems to achieve gene expression regulation. These expression cassettes are preferably contained within a vector, which is preferably contained within a host cell.

[0018] Vectors are commonly used tools in this field for carrying nucleic acids or genetic material, and generally have the function of delivering nucleic acids or genetic material to cells. They typically include, but are not limited to, plasmids, viruses, and artificial chromosomes. The vector itself is usually a nucleotide sequence, often a DNA sequence. A recombinant vector is a nucleotide sequence containing a target gene, said sequence including an insert (target nucleic acid) and a larger sequence serving as the vector's "backbone." These recombinant vectors are preferably contained within host cells, and to adapt to the host cell, they may include one or more regulatory elements, the specific regulatory elements of which can be selected according to the host cell.

[0019] The host cell is the cell into which the target nucleic acid is introduced; it can be any cell useful in the production of the MAGE A1 recombinant antigen of this invention. To produce the MAGE A1 recombinant antigen, the nucleic acid encoding the MAGE A1 recombinant antigen can be isolated and inserted into a suitable vector for further cloning and / or expression in the host cell. This nucleic acid can be readily isolated and sequenced using conventional techniques. Methods for introducing the vector into the host cell are well known, and this invention does not specifically limit these methods.

[0020] In some implementations, the host cell can be Escherichia coli, yeast, insect cells, or mammalian cells, with Escherichia coli being preferred, but the specific cell can be selected according to the actual situation in the experiment.

[0021] The MAGE A1 recombinant antigen of the present invention can be prepared by any suitable method known in the art. For example, in some embodiments, a nucleic acid molecule encoding the above-described MAGE A1 recombinant antigen is introduced into an expression vector, and then the obtained recombinant plasmid is introduced into a host cell, followed by expression and purification to obtain the MAGE A1 recombinant antigen.

[0022] According to another aspect of the invention, the invention provides a complex comprising the MAGE A1 recombinant antigen.

[0023] In some embodiments, the complex further includes a solid support, a detectable marker, or a binding coupler conjugated to the MAGE A1 recombinant antigen.

[0024] Optionally, the solid support is selected from microspheres, plates, and membranes, such as magnetic microspheres, plastic microspheres, latex microparticles, microporous plates, glass, capillaries, nylon or nitrocellulose membranes, etc.

[0025] Optionally, the detectable marker is selected from fluorescent substances, quantum dots, digoxigenin-labeled probes, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescent markers, ultrasound contrast agents, photosensitizers, colloidal metals, or enzymes; alternatively, the detectable marker is selected from fluorescent microspheres, colored latex microspheres, acridinium esters, alkaline phosphatase, horseradish peroxidase, or colloidal gold.

[0026] Optionally, the conjugate body may be selected from biotin or avidin.

[0027] In some embodiments, the complex comprises the MAGE A1 recombinant antigen and magnetic microparticles coupled thereto.

[0028] In some embodiments, the magnetic microparticles are coated with tagged antibodies. Tagged antibodies are specific antibodies used to specifically recognize and bind to tags on recombinant proteins, including but not limited to histidine (His) tags, HA tags, Myc tags, FLAG tags, and V5 tags.

[0029] In some embodiments, the complex comprises a recombinant antigen with an amino acid sequence as shown in SEQ ID NO.2 and magnetic microparticles pre-coated with an anti-His-tagged antibody, wherein the recombinant antigen and the magnetic microparticles are coupled via a His tag and an anti-His-tagged antibody.

[0030] According to another aspect of the present invention, the present invention provides a method for preparing the above-mentioned complex, comprising mixing and incubating recombinant antigen and magnetic microparticles; the incubation temperature is generally carried out at 4°C to room temperature, and the incubation time is at least 30 min, which can be adjusted by those skilled in the art according to the protein characteristics, incubation temperature and abundance of target protein.

[0031] In some embodiments, a post-incubation sealing step is also included.

[0032] In some embodiments, the step of washing the magnetic particles before incubation is also included.

[0033] According to another aspect of the present invention, the present invention provides a composition comprising the above-described MAGE A1 recombinant antigen, biological material, or complex.

[0034] The above-described composition may be an expression product, a purified product, a pharmaceutical composition, or an immunoassay composition. In addition to containing the above-described MAGE A1 recombinant antigen or the above-described biological material or complex, the above-described composition may also include other components suitable for effectively preserving its structure and activity, such as buffer solutions, surfactants, protectants, metal salts, preservatives, etc. This invention does not limit the specific components; those skilled in the art can select and combine them according to actual needs.

[0035] The MAGE A1 recombinant antigen provided by this invention is a truncated version of the full-length MAGE A1 antigen. Compared to the full-length antigen, it more easily exposes the antigenic epitopes, so the conjugate obtained after coupling it with magnetic beads can more sensitively detect MAGE A1 antibodies. Therefore, based on the MAGE A1 recombinant antigen, biomaterials, or complexes provided by this invention, it has the following uses: preparing products for detecting MAGE A1 antibodies, preparing products for early auxiliary diagnosis of lung cancer, detecting MAGE A1 antibodies for non-diagnostic and therapeutic purposes, preparing MAGE A1 antibodies, and purifying MAGE A1 antibodies.

[0036] In some embodiments, the MAGE A1 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 MAGE A1 recombinant antigen of the present invention can be used as a capture antigen, a detection antigen, or both. The other antigen paired with the MAGE A1 recombinant antigen of the present invention can be the same as or different from the MAGE A1 recombinant antigen of the present invention, as long as it includes the MAGE A1 recombinant antigen of the present invention.

[0037] In some embodiments, the above composition may be a reagent or kit containing the above-described MAGE A1 recombinant antigen or complex. By using the MAGE A1 recombinant antigen of the present invention as the detection antigen, the sensitivity of anti-MAGE A1 antibody detection can be improved.

[0038] In some embodiments, the kit is used for the early auxiliary diagnosis of lung cancer and, in addition to the detection reagent containing the MAGE A1 recombinant antigen of the present invention or coated with the MAGE A1 recombinant antigen, also includes a detection reagent for detecting autoantibodies of at least one of the following indicators: p53, PGP9.5, SOX2, GAGE7, GBU4-5, and CAGE.

[0039] The above-mentioned MAGE A1 recombinant antigen and its related biological materials, complexes or compositions can be further applied to the purification of MAGE A1 antibodies. For example, the MAGE A1 recombinant antigen can be immobilized on a chromatography column, and the MAGE A1-containing antibody can be indirectly immobilized on the chromatography column after contact with it. Then, impurities can be removed and eluted with different elution solutions to obtain purified MAGE A1 antibody.

[0040] The above-mentioned MAGE A1 recombinant antigen and its related biological materials, complexes or compositions can be further applied to the preparation of MAGE A1 antibodies; for example, immunizing animals with an immune component containing the MAGE A1 recombinant antigen and obtaining the corresponding antibody through a known antibody preparation process.

[0041] Using the aforementioned MAGE A1 recombinant antigen and its related biological materials, complexes, or compositions, it can be further applied to the detection of MAGE A1 antibodies. The corresponding detection method can be as follows: The MAGE A1 recombinant antigen of this invention is contacted with the sample to be tested. If MAGE A1 antibodies are present in the sample, a complex of MAGE A1 antibody and MAGE A1 recombinant antigen is formed. The presence of this complex is then detected, indicating the presence of MAGE A1 antibodies in the sample. This method can accurately detect the presence of MAGE A1 antibodies in the target sample.

[0042] The present invention has the following beneficial effects: This invention extracts the sequence containing antigenic epitopes from the full-length MAGE A1 antigen, removes non-specific sequences, and obtains a truncated MAGE A1 antigen that more easily exposes the antigenic epitopes. This truncated antigen is then conjugated with magnetic microparticles coated with tagged antibodies, and the resulting conjugate can be used in MAGE A1 antibody detection. Verification has shown that, compared to the full-length MAGE A1 antigen, the recombinant MAGE A1 antigen of this invention improves the sensitivity in early lung cancer detection. Therefore, the recombinant MAGE A1 antigen of this invention provides a new approach for the early auxiliary diagnosis of lung cancer. Detailed Implementation

[0043] 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.

[0044] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0045] Example 1 This embodiment describes the expression and purification of the MAGE A1 truncated protein according to the present invention. S1: Synthesis of the target gene and construction of the recombinant vector Using a gene synthesis method, the gene containing the encoding MAGE A1 recombinant antigen (nucleotide sequence SEQ ID NO.3 of amino acid sequence SEQ ID NO.2) was ligated into the pET28a vector to construct a recombinant vector.

[0046] S2: Induced expression and purification of antigen 1) The recombinant vector in S1 was transformed into Escherichia coli BL21(DE3) competent cells and cultured on LB agar plates containing 50 μg / ml kanamycin at 37°C for 14-16 h. Positive recombinant bacteria were screened. Single colonies were picked and inoculated into LB liquid medium containing 50 μg / ml kanamycin and cultured at 37°C until OD600nm = 0.6-0.8. IPTG was added to a final concentration of 0.3 mM, and the cells were induced at 20°C for 14-16 h before collection.

[0047] 2) Add lysis buffer (lysis buffer formula: 20 mM Hepes, 1 M NaCl, 5% Glycerol, pH 8.0) to the bacterial cells and resuspend the cells on ice. Sonicate the cells: Sonicate at 400W for 4 seconds, pause for 3 seconds, until the bacterial solution is clear and transparent.

[0048] 3) After sonication, centrifuge at 12,000 rpm for 30 min at 4℃ (pre-cool the centrifuge beforehand) to separate the supernatant and precipitate. Filter the supernatant through a 0.22 μm membrane.

[0049] 4) Add 2 ml of equilibrated Ni resin to the supernatant separated in 3) and incubate on a shaker for 1 h.

[0050] 5) Pour the incubated supernatant into a gravity column, collect the flow-through liquid, and after all the supernatant has flowed out, add lysis buffer to clean the remaining supernatant. 6) Elution: The Ni column was eluted with 40 mM, 80 mM, 160 mM and 320 mM imidazole prepared by lysis buffer, respectively. The eluent was collected in separate tubes according to the peak, and 20 μl of the eluent was retained. The samples retained in the above steps were subjected to SDS-PAGE analysis to obtain high-purity target protein samples.

[0051] Example 2 In this embodiment, the antigen prepared in Example 1 is coated onto magnetic microparticles pre-coated with anti-His-tagged antibodies to prepare the corresponding antigen-capturing coating reagent.

[0052] The wrapping process is as follows: 1) Take the required volume of magnetic particle suspension; 2) Wash several times with coating solution; 3) Add the prepared antigen from Example 1 and incubate with shaking at room temperature for 60 min; 4) Blocking solution (coating solution containing 1% BSA bovine serum albumin and 0.1% Tween 20) for 60 min; 5) Resuspend the protein in a coating solution containing 1% BSA bovine serum albumin to obtain a capture antigen coating reagent for site-directed coupling of truncated MAGE A1 protein or full-length MAGE A1 protein.

[0053] The coating solution formulation is shown in Table 1: Table 1 Coating solution formulation

[0054] Comparative Example 1 This comparative example describes the expression and purification of the full-length MAGE A1 protein (amino acid and nucleotide sequences are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively). The steps are basically the same as in Example 1, except that the truncated MAGE A1 protein is replaced with the full-length MAGE A1 protein.

[0055] Comparative Example 2 This comparative example uses the antigen prepared in Comparative Example 1, coated onto magnetic microparticles pre-coated with anti-His-tagged antibodies. Except for the antigen composition, it is identical to Example 1.

[0056] Comparative Example 3 This comparative example uses the antigen prepared in Comparative Example 1, which is coated onto magnetic microparticles with abundant carboxyl groups on their surface.

[0057] The wrapping process is as follows: 1) Take the required volume of magnetic particle suspension; 2) Wash several times with an activation buffer containing sodium 2-(N-morpholino)ethanesulfonate; 3) Add a certain amount of EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) / NHS (N-hydroxysuccinimide), and shake at room temperature for 50 min to activate the carboxyl group; 4) Wash several times with coating solution; 5) Add the prepared antigen and incubate at room temperature with shaking for 60 min; 6) Block with blocking solution (coating solution containing 1% BSA bovine serum albumin and 0.1% Tween 20) for 60 min; 7) Resuspend the antigen in a coating solution containing 1% BSA bovine serum albumin to obtain a capture antigen coating reagent for random conjugation of the full-length MAGE A1 antigen.

[0058] The formulation of the activation buffer is shown in Table 2: Table 2 Activation Buffer Formulation

[0059] Test case This experiment is a reactivity test of the antigen-capturing coating reagents prepared in Examples 2 and 2 and 3.

[0060] The samples to be tested were mixed with the capture antigen coating reagents prepared in Examples 2, 2, and 3, respectively, and then mixed with phycoerythrin-labeled goat anti-human IgG-PE (in-house prepared by our company, working concentration of 0.5 μg / mL) and tested using a fully automated multiplex immunoassay analyzer.

[0061] This study used 115 early-stage lung cancer samples and 63 samples from individuals undergoing physical examinations as the test samples. The positive detection rate is the sensitivity, and the negative detection rate is the specificity. The test results are shown in Tables 3-5.

[0062] Table 3. Detection performance of the site-specific conjugation MAGE A1 truncated antigen-coating reagent in Example 2.

[0063] Table 4. Detection performance of the site-directed conjugation antigen-coating reagent for full-length MAGE A1 protein in Comparative Example 2.

[0064] Table 5. Detection performance of the capture antigen coating reagent for randomized conjugation of full-length MAGE A1 protein in Comparative Example 3.

[0065] As can be seen from the results in Tables 3-5, the specificity of each group for the detection of early lung cancer samples was basically the same, all greater than 95%. In terms of detection rate, the sensitivity of the capture antigen coating reagent of random conjugation with full-length MAGE A1 protein was the worst, at 10.4%. After site-specific conjugation, the sensitivity was improved to 13.9%. Further use of truncated MAGE A1 protein could increase the sensitivity to 19.1%, which is about 1.84 times the original value.

[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 MAGE A1 recombinant antigen, characterized in that, The amino acid sequence of the recombinant antigen is shown as SEQ ID NO.

1.

2. The recombinant antigen of claim 1, wherein, The recombinant antigen further comprises a tag. Preferably, the amino acid sequence of the recombinant antigen is shown as SEQ ID NO.

2.

3. Biomaterials related to the MAGE Al recombinant antigen of claim 1 or 2, characterized by, Any one of the following (A1)~(A4): (A1) a nucleic acid molecule encoding the MAGE A1 recombinant antigen of claim 1; (A2) an expression cassette comprising the nucleic acid molecule of (A1); (A3) a recombinant vector comprising the nucleic acid molecule of (A1), or the expression cassette of (A2); (A4) a host cell comprising the nucleic acid molecule of (A1), the expression cassette of (A2), or the recombinant vector of (A3); Preferably, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.

2.

4. The method of producing a MAGE Al recombinant antigen according to claim 1 or 2, wherein, Comprising: culturing a cell comprising the nucleic acid molecule of claim 3.

5. A composite, characterized by, The MAGE A1 recombinant antigen of claim 1 or 2.

6. The composite of claim 5, wherein, The complex further comprises a solid support, a detectable label, or a binding partner conjugated to the MAGE A1 recombinant antigen; Optionally, the complex comprises the MAGE A1 recombinant antigen of claim 1, and magnetic microparticles coupled thereto; Optionally, the complex comprises a recombinant antigen with an amino acid sequence shown as SEQ ID NO. 2 and magnetic microparticles pre-coated with an anti-His tag antibody on the surface, the recombinant antigen and the magnetic microparticles being coupled via the His tag and the anti-His tag antibody.

7. The method of claim 5 or 6, wherein the complex is prepared by the method comprising the steps of: Comprising mixing and incubating the recombinant antigen and the magnetic microparticles; Optionally, the preparation method further comprises blocking treatment after incubation; Optionally, the preparation method further comprises washing the magnetic microparticles before incubation.

8. A composition characterized in that, The MAGE A1 recombinant antigen of claim 1 or 2, the biomaterial of claim 3, or the complex of any one of claims 5~6.

9. The composition of claim 8, wherein, The composition is a kit for early auxiliary diagnosis of lung cancer, further comprising detection reagents for detecting at least one of the following index autoantibodies: p53, PGP9.5, SOX2, GAGE7, GBU4-5, and CAGE.

10. The MAGE A1 recombinant antigen of claim 1 or 2, the biomaterial of claim 3, the complex of any one of claims 5~6, or the composition of claim 8 or 9 for any one of the following (B1)~(B5): (B1) application in preparing a product for detecting MAGE A1 antibodies; (B2) application in preparing a product for early auxiliary diagnosis of lung cancer; (B3) detecting MAGE A1 antibodies for non-diagnostic and therapeutic purposes; (B4) for preparing MAGE A1 antibodies; (B5) for isolating or purifying MAGE A1 antibodies.

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