ATRX antigen epitope peptide and its application in detecting brain glioma

Abstract

The present invention discloses an ATRX antigen epitope peptide and its application in detecting brain glioma. The amino acid sequence of the ATRX antigen epitope peptide is shown in the sequence table SEQ ID NO: 1 or SEQ ID NO: 2. The ATRX antigen epitope peptide of the present invention has good antigenicity, and the antigen prepared by the peptide can produce highly specific monoclonal antibodies and polyclonal antibodies in animals immunized with the peptide. The human ATRX in vitro diagnostic kit of the present invention can effectively detect the expression level of ATRX in brain tissue samples, and can be used to assess the risk of brain glioma.

Description

Technical Field

[0001] The invention belongs to the technical field of immunology, and particularly relates to an ATRX antigen epitope peptide and an application thereof in detecting brain glioma. Background Art

[0002] Gliomas are relatively common primary tumors of the central nervous system, with an incidence of approximately 0.7 per 10,000 cases in my country. The different histological subtypes of gliomas are as follows: astrocytomas account for 70% of all cases, oligodendrogliomas account for 9%, including classic oligodendrogliomas and mixed oligodendrogliomas, and ependymomas account for 6%.

[0003] ATRX gene is a gene located at Xq21.1 that regulates chromosome remodeling, nucleosome assembly and maintenance of telomere length. It encodes a 280kDa nuclear protein. Its mutation was first found in X-linked a-thalassemia and intellectual development disorder. Recent studies have found ATRX mutations in at least 15 types of cancer, including neuroblastoma, osteosarcoma and pancreatic neuroendocrine tumors. ATRX protein plays an important epigenetic role. It can deposit histones on heterochromatin and telomeric DNA, and it is also closely related to the maintenance of telomere length.

[0004] ATRX protein strongly binds to the nucleus pulposus of promyelocytic leukemia. When ATRX interacts with DAXX, the complex deposits the histone variant H3.3 around the center of heterochromatic repeats, including telomeres and ribosomal DNA repeats, in the form of a histone chaperone complex. Functional mutations in this gene are associated with defects in its sister chromatid constitution, abnormal DNA methylation, and telomerase-independent maintenance of telomeres, leading to alternative elongation of telomeres. ATRX mutations and reduced ATRX nuclear expression have been observed in grade II and III astrocytic tumors, IDH-mutant gliomas, IDH-mutant GBMs, and pediatric high-grade gliomas. ATRX mutations are present in 31% of primary GBM patients, accompanied by TP53 mutations and point mutations in the gene encoding the histone H3.3 variant H3F3A.

[0005] The ATRX gene plays a key role in the development of the nervous system. Berube NG et al. found that ATRX gene mutations are closely related to the invasiveness of central nervous system tumors. As a tumor derived from the peripheral neuroectoderm, NB also needs to determine whether NB patients have ATRX mutations and whether NB with ATRX gene mutations have longer telomeres. Further research is needed on the mechanism of the loss of ATRX protein expression in the development and invasion of neuroblastoma. The case report of WadaT et al. pointed out that ATRX gene mutations affect two functionally important areas: the ADD domain and the helicase domain.

[0006] ATRX has been repeatedly proven to have potential value as a biomarker. The most ideal method for detecting ATRX levels in serum is immunoassay, which uses ATRX antibodies and advanced enzyme immunoassay and chemiluminescence technology to check ATRX levels through routine blood draws. However, due to the large molecular weight of ATRX protein, it is difficult to prepare antibodies using its whole antigen, and the prepared antibodies have poor specificity. Therefore, finding suitable immunogenic ATRX epitope peptides and preparing specific ATRX antibodies have become a focus of research. Summary of the invention

[0007] The purpose of the present invention is to provide an ATRX antigen epitope peptide and its application in detecting brain glioma.

[0008] An ATRX antigen epitope peptide, whose amino acid sequence is shown in SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing.

[0009] An antigen is formed by coupling the ATRX antigen epitope peptide with a carrier protein.

[0010] The present invention also provides ATRX monoclonal antibody or polyclonal antibody prepared from the antigen.

[0011] The ATRX antibody is used in preparing a reagent for detecting brain glioma.

[0012] The ATRX antibody is used to detect the expression level of ATRX protein in vivo; compared with normal people, the expression level of ATRX protein is reduced in patients with brain glioma.

[0013] A kit for detecting brain glioma, comprising the ATRX monoclonal antibody or polyclonal antibody.

[0014] Preferably, it is used for quantitative detection of human ATRX protein in serum.

[0015] Beneficial effects of the present invention: The ATRX antigen epitope peptide of the present invention has good antigenicity, and the antigen prepared by the peptide can produce highly specific monoclonal antibodies and polyclonal antibodies in animals. The human ATRX in vitro diagnostic kit of the present invention can effectively detect the ATRX expression level in brain tissue samples and can be used to assess the risk of glioma. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the western blot detection diagram of monoclonal antibody and polyclonal antibody;

[0017] In the figure, 1 is a monoclonal antibody and 2 is a polyclonal antibody.

[0018] Figure 2is the concentration of ATRX protein in glioma tissue samples and normal brain tissue samples. DETAILED DESCRIPTION

[0019] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0020] Specific techniques or conditions not specified in the examples are all performed according to the literature in the art or conventional techniques in the art.

[0021] Example 1 Antigenic epitope peptide screening

[0022] The antheprot software was used to perform sequence analysis based on the human ATRX protein sequence published by NCBI. The analysis content included transmembrane differentiation, signal peptide analysis, modification site analysis and full sequence analysis to find possible antigenic epitopes, and add cysteine ​​to the end of the sequence that does not contain cysteine ​​in the above antigenic epitope. The amino acid sequence of the ATRX antigenic epitope peptide finally screened is shown in the sequence table SEQ ID NO: 1 or SEQ ID NO: 2.

[0023] The antigen epitope peptide was synthesized by artificial synthesis method by GenScript Biotech Co., Ltd. through the amino acid direct synthesis method.

[0024] Conjugation of antigen epitope peptides to carrier proteins:

[0025] 1) Dissolve 20 mg SMCC in 2 ml DMF and add 0.8 ml KLH into a 25 ml round-bottom flask, and add 1× PBS to make the final protein concentration 15 mg / ml;

[0026] 2) Slowly add the dissolved SMCC solution to the 120 mg KLH protein system and stir at room temperature for 1 hour;

[0027] 3) Dialyze with 1 L of 1× PBS solution at 4°C for 6 h to remove free SMCC;

[0028] 4) Pour the dialyzed KLH protein into a 50 ml centrifuge tube, determine its volume by the scale of the centrifuge tube, calculate the concentration of the dialyzed protein based on the amount of KLH protein added before the reaction, and then transfer 2.5 mg of KLH-SMCC solution into a 5 ml centrifuge tube based on its concentration; the amount of KLH protein added is 120 mg, and the volume of the dialyzed KLH protein is 20 ml;

[0029] 5) Dissolve 3.0 mg of antigen epitope peptide in 0.6 ml of 1× PBS solution;

[0030] 6) Use Ellman's reagent to detect thiol groups in the peptide: add 100 μl of Ellman's reagent stock solution to a 96-well plate, then add 10 μl of antigen epitope peptide solution, and measure its ultraviolet absorption value at λ=412nm using a Nano spectrophotometer. If the OD value is >0.15, proceed to the next step; if the OD value is <0.05, add additional antigen epitope peptide until the requirement is met; if the OD value is <0.03, it means that the cross-linking rate between the antigen epitope peptide and the KLH protein has reached more than 80%; if the OD value is >0.03, add SMCC-activated KLH protein to continue cross-linking; if the Ellman's reagent turns yellow, it means that the antigen epitope peptide is not completely coupled to the KLH protein; if the Ellman's reagent does not turn yellow, it means that the antigen epitope peptide has been completely coupled to the KLH protein.

[0031] Example 2 Polyclonal Antibody Preparation

[0032] As the immunized animals, healthy male New Zealand white rabbits aged 3 months and weighing 2.5±0.1 kg were selected, and the antigen prepared in Example 1 was used for animal immunization.

[0033] Before the first immunization, blood was collected from the ear vein as a negative control. 2 HPO 4 , 2 mM KH 2 PO 4 , pH7.4) to 1mg / mL, and store at -20℃ after aliquoting. Take 500μL of 1mg / mL antigen (i.e. 0.5mg, add 300μL PBS to dilute again, and then add an equal volume of Freund's complete adjuvant (first immunization) or Freund's incomplete adjuvant (2nd-4th immunization). Multiple injections were made to the rabbit's limbs, armpits and back subcutaneously. The second immunization was carried out three weeks after the first immunization, and then boosted every two weeks (a total of 4 immunizations). 7d after the third immunization, blood was collected from the ear vein to determine the antibody titer. For those who met the requirements, blood was drawn from the carotid artery 11d after the fourth immunization, and blood samples were collected. The blood samples were placed at 4℃ overnight or incubated at 37℃ for 3h, centrifuged at 6000rpm for 10min at 4℃, serum was collected, and stored at -80℃ after aliquoting.

[0034] Ammonium sulfate precipitation method to concentrate protein samples: dilute 20 mL of serum to 100 mL with TBS buffer solution, then add saturated ammonium sulfate solution to the solution while stirring until the concentration of the ammonium sulfate solution reaches 55%. Stir overnight at 4°C using a magnetic stirrer. The next day, centrifuge the solution containing a large amount of precipitate, discard the supernatant, and retain the precipitate. Add 10 mL of PBS buffer solution containing sodium azide to dissolve the precipitate, and dialyze it with a dialysis bag to remove ammonium sulfate. After dialysis, centrifuge the solution in the dialysis bag and collect the supernatant.

[0035] Purification of anti-peptide antibodies by affinity chromatography: Add the above protein solution after dialysis and centrifugation to the chromatography column (protein A FF) at a rate of 0.5 mL / min. To ensure that the antibody protein is fully combined with the filler, it is necessary to load the column twice in succession. After washing the chromatography column with TBS until the A280nm of the eluent is less than 0.008, elute with elution buffer (50 mM glycine, pH 2.7) at the same rate, collect the eluent in a centrifuge tube pre-added with neutralization buffer (1 M Tris-HCl, pH 8.0, 1.5 M NaCl, 1 mM EDTA), mix well, and check the pH of the solution with pH test paper. If the pH is lower than 7.4, adjust it to about pH 7.4 with neutralization buffer to prevent antibody denaturation, and detect the concentration of the antibody at the same time. Finally, add an equal volume of glycerol and store at -20°C to obtain the purified polyclonal antibody.

[0036] Example 3 Monoclonal Antibody Preparation

[0037] Ten healthy 6-8 week old female BALB / c mice were selected for the first immunization. The antigen prepared in Example 1 was dissolved in physiological saline and mixed with an equal volume of Freund's complete adjuvant. After sufficient emulsification, the mixture was subcutaneously injected at four points on the back of the mice. Each mouse was injected with 40 ug of antigen. On the 14th day, boost immunization was performed by dissolving the antigen in physiological saline and mixing it with an equal volume of Freund's incomplete adjuvant. After sufficient emulsification, the mixture was subcutaneously injected at 4 points on the back of the mouse, and 40ug of antigen was injected into each mouse. On the 21st day, blood was collected from the medial canthus vein of the mouse, and the serum was separated. The antiserum titer was detected by indirect ELISA. On the 28th day, boost immunization was performed by dissolving the antigen in physiological saline and mixing it with an equal volume of Freund's incomplete adjuvant. After sufficient emulsification, the mixture was subcutaneously injected at 4 points on the back of the mouse, and 40ug of antigen was injected into each mouse. On the 35th day, blood was collected from the medial canthus vein of the mouse, and the serum was separated. The antiserum titer was detected by indirect ELISA. On the 42nd day, the mouse with the highest antiserum titer was selected and intraperitoneally injected with 100μg of antigen. On the 45th day, spleen cells were harvested on a clean bench for fusion.

[0038] Mice with high antiserum antibody titers were selected, and the mice were boosted with the last immunization. After 3 days, the spleen cells were taken and fused with SP2 / 0 myeloma cells to prepare hybridoma cell lines. Positive hybridoma cell lines were screened by indirect ELISA method, identified by western blot method, subcloned by limiting dilution method, and the antiserum titers were detected by indirect ELISA method. The positive cells were expanded and cultured, and finally a hybridoma cell line that stably secreted monoclonal antibodies was obtained.

[0039] Three healthy BALB / c mice were selected, and each mouse was intraperitoneally injected with 0.5 ml of pristane. After 14 days, hybridoma cells were intraperitoneally injected. One day before injection, the hybridoma cells in the logarithmic growth phase were replaced with fresh culture medium. On the day of injection, the hybridoma cells were mixed to make a cell suspension. After counting, the cell suspension was added to a 15 ml centrifuge tube, centrifuged at 200 × g for 5 minutes at room temperature, and the supernatant was discarded. The cells were suspended with PBS to a cell concentration of 5 × 106 cells / ml, and 1 ml was injected into each mouse; the health status of the mice and the production of ascites were observed, and the ascites was collected and the titer was determined after 12 days. The collected ascites was centrifuged at 1500 × g for 10 minutes, and the supernatant was purified using a protein A affinity column.

[0040] Example 4 Determination of polyclonal antibody and monoclonal antibody detection ability

[0041] In the three gases (CO 2 / N 2 / O 2 ) in a cell culture incubator. Human endothelial HUVEC cells growing in the logarithmic phase were plated in a 6-well plate and incubated at 37°C with 5% CO 2 After the cells adhered to the wall, they were transferred to a three-gas incubator at 37°C and 5°C CO 2 , 2% O 2 The cells were cultured for 24 h under saturated humidity and 37 °C, 5% CO 2 The cells were cultured for 24 h under saturated humidity conditions and the cells were lysed.

[0042] Cell lysis and protein electrophoresis: Wash the cultured cells with ice-cold PBS, collect them and lyse them in RIPA buffer containing a mixture of protease inhibitors. The lysis process is carried out on ice for 30 minutes. After centrifugation at 12,000r / min for 10 minutes at 4°C, collect the supernatant, and then use the conventional BCA method for protein quantification. The quantified samples are mixed with 5× loading buffer and denatured in a 100°C water bath for 5 minutes. Protein electrophoresis is performed in SDS-polyacrylamide gel and buffer system, and samples are loaded at 50μg / well and electrophoresed at 80 / 120V constant voltage.

[0043] 60 μg of total protein from human endothelial HUVEC cells was separated by 6% SDS-PAGE and transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked with a TBST solution containing 10% skimmed milk powder at room temperature for 2 h, and then immersed in a TBST solution containing 1 μg / ml polyclonal antibody and monoclonal antibody at 4°C overnight. The nitrocellulose membrane was taken out the next day, washed three times with TBST solution by shaking, and then immersed in a TBST solution containing HRP-labeled goat anti-mouse IgG (dilution: 1:5000). After being in a 27°C water bath for 1 h45 min, the membrane was washed three times with TBST solution by shaking (240 rpm, 15 min each time), and washed once with TBS solution by shaking (240 rpm, 5 min). Protein bands were observed in a chemiluminescence imaging system (Syngene, Cambridge, UK) using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific, Waltham, USA).

[0044] The results of the test are shown in Figure 1 As shown, a single band appeared around 280 kDa, which was consistent with the theoretical molecular weight of ATRX protein. Both monoclonal antibodies and polyclonal antibodies could recognize ATRX protein.

[0045] Example 5

[0046] 38 brain glioma tissue samples and 35 normal brain tissue samples were used, which were taken from pathological specimens of patients who were treated in Peking University People's Hospital from January 2015 to January 2022 and confirmed as brain glioma by surgical pathology. The World Health Organization divides central nervous system tumors into grades I to IV based on histological and molecular characteristics. Among the 38 brain glioma tissue samples selected in the present invention, there are 10 cases of grade I, 8 cases of grade II, 10 cases of grade III, and 10 cases of grade IV; the patients participating in the trial of the present invention signed informed consent forms and were approved by the hospital ethics committee.

[0047] The total protein of the samples was extracted by conventional protein extraction method, and the ATRX protein concentration was detected using self-prepared monoclonal antibody and polyclonal antibody detection kits.

[0048] The preparation and operation of the kit are as follows:

[0049] Coating buffer preparation: 0.050M, pH 9.6 CB (carbonate buffer), Na 2 CO 3 16.0 g, NaHCO 3 29.0g, dissolved in distilled water to 1000ml.

[0050] Sample / wash buffer preparation: 10× PBS-Tween 20, pH 7.2, Na 2HPO 4 12H 2 O 58g, KH 2 PO 4 4g, NaCl 100g, KCl 4g, Tween 20 20ml, dissolve in distilled water to 1000ml.

[0051] Preparation of enzyme marker diluent: 10 ml of 10×PBS-Tween 20, 20 ml of FCS (calf serum), 1 g of enzyme stabilizer, 1 ml of biological preservative, dissolved in distilled water to 1000 ml.

[0052] Color developer A preparation: 35.5 g citric acid, 10 g urea peroxide, 10 ml Tween 20, dissolved in distilled water to 1000 ml.

[0053] Color developer B preparation: 120 g citric acid, 1 g EDTA-2Na, 2 g TMB·2HCl, dissolved in distilled water to 1000 ml.

[0054] Stop solution preparation: 2M H 2 SO 4 22.2 ml of concentrated sulfuric acid and 177.3 ml of distilled water. When preparing, slowly drop the concentrated sulfuric acid into the distilled water and shake while adding.

[0055] Preparation of pre-coated plates: Dissolve ATRX monoclonal antibody in 0.05 M carbonate buffer at pH = 9.6 to prepare a pre-coating solution, add 100 μl to each well of the ELISA plate at 0.1 μg / well, place at 4°C for 24 hours, take out, discard the coating solution, wash with sample / washing buffer, block with 1 (w / v)% BSA-0.05 M ethanolamine for 16 hours, dry overnight, put into an aluminum platinum bag, vacuum seal, and store at 4°C.

[0056] The dilution ratios of the binding antibody (ATRX polyclonal antibody) and the enzyme conjugate (horseradish peroxidase-labeled goat anti-rabbit IgG antibody) were determined by square array titration experiments, and the horseradish peroxidase-labeled goat anti-rabbit IgG antibody was diluted using enzyme marker diluent.

[0057] Composition of the kit:

[0058] Pre-coated plates: 48 / 96 wells

[0059] ATRX calibrators: 6 pieces: 6×0.5ml (concentrations are 0.0ng / ml, 8.0ng / ml, 15.0ng / ml, 50.0ng / ml, 100.0ng / ml, 200.0ng / ml)

[0060] ATRX binding antibody: 1×2.5ml / 1×5ml (diluted 1:5000)

[0061] Enzyme conjugate: 2×5ml / 2×5ml (diluted 1:5000)

[0062] ATRX quality control: 1×0.5ml (serum with ATRX concentration of 60.0ng / mL)

[0063] Concentrated washing solution (25×PBS-Tween 20): 1×20ml

[0064] Color developer A: 1×5.0ml

[0065] Color developer B: 1×5.0ml

[0066] Stop solution: 1×5.0ml.

[0067] Kit operation steps:

[0068] Add 50 μl / well of the total protein sample to be tested, calibrator and quality control to each well of the pre-coated plate, all in double wells, incubate at 37°C for 30 min, wash each well with 300ul of 1× washing buffer 5 times, and pat dry. Add 100 μl / well of ATRX binding antibody to each well, incubate at 37°C for 30 min, wash each well with 300ul of 1× washing buffer 5 times, and pat dry. Then add 100 μl / well of enzyme conjugate to each well, incubate at 37°C for 30 min, wash each well with 200-300ul of 1× washing buffer 5 times, and pat dry. Add chromogen A and B solution, 50 μl per well, mix well, and incubate at 37°C for 15 minutes. Add 50 μl / well of stop solution to terminate the reaction, and use an enzyme-linked detector (model RT-6000, Raydu Company) to detect absorbance at dual wavelengths (450nm, 620nm).

[0069] A standard curve was drawn using the logarithmic values ​​of the calibration sample concentration and the corresponding absorbance. The ATRX concentration in the sample tested was calculated based on the standard curve. The measurement results were as follows: Figure 2 As shown, the ATRX concentration in glioma patients was significantly lower than that in normal human brain tissue.

[0070] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An ATRX antigen epitope peptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO: 1 in the sequence listing.

2. An antigen, characterized in that The antigen is the ATRX antigen epitope peptide according to claim 1 coupled with the carrier protein KLH.

3. An ATRX polyclonal antibody prepared from the antigen according to claim 2.

4. A kit for detecting brain glioma, characterized in that: Comprising the ATRX polyclonal antibody according to claim 3.

5. The kit for detecting brain glioma according to claim 4, characterized in that: It is used to quantitatively detect human ATRX protein in serum.

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