Anti-double-stranded DNA antibody, nucleotide fragment, magnetic beads and method for extracting free DNA from peripheral blood

Abstract

The present application relates to the field of biotechnology, and discloses an anti-double-stranded DNA antibody, a nucleotide fragment, a magnetic bead, and a method for extracting free DNA from peripheral blood. The anti-double-stranded DNA antibody comprises a heavy chain variable region and a light chain variable region, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2. In the present application, the specificity and specificity of monoclonal antibodies are utilized to extract free DNA from peripheral blood, which is not limited by the size of DNA fragments and is particularly suitable for the purification of small fragments of DNA. The recovery rate of free DNA from peripheral blood can be significantly improved during extraction, especially for samples containing small DNA fragments or with low DNA content.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and mainly to an anti-double-stranded DNA antibody, a nucleotide fragment, a magnetic bead, and a method for extracting free DNA from peripheral blood. Background Art

[0002] Anti-DNA antibodies are antibodies that are produced in large quantities in animals with autoimmune diseases. These antibodies bind to nucleic acid molecules in the body, including double-stranded DNA, single-stranded DNA, and some non-specific self-antigens, thereby inducing cell apoptosis, inflammatory responses, and tissue fibrosis. Anti-double-stranded DNA antibodies are antibodies that are present in large quantities in the serum of patients with systemic lupus erythematosus (SLE). These self-produced antibodies can bind to nucleic acids in the body to form immune complexes, which can cause damage to multiple organs in SLE patients. The most common cause is lupus nephritis caused by the accumulation of immune complexes in the basement membranes of the glomeruli and tubules.

[0003] Cell-free DNA (cfDNA) is a kind of free and fragmented DNA existing in human blood, cerebrospinal fluid and periosteal fluid. It was first reported by Mandel et al. in 1948, but it did not receive much attention at that time. It was not until the 1980s that Leon et al. reported the existence of high concentrations of free DNA in tumor patients. Subsequently, many scholars studied the free DNA in these tumor patients and found that these free DNAs carried tumor-specific mutations or epigenetic changes. They were tumor DNA fragments released into the peripheral blood by necrotic or apoptotic tumor cells. Later, they were named circulating tumor DNA (ctDNA). In 1997, Lo et al. reported the presence of Y chromosomes in the peripheral blood of pregnant women with male babies, thus proving the presence of fetal free DNA in the peripheral blood of pregnant women. Subsequent studies have shown that the content of fetal free DNA in the peripheral blood of pregnant women increases with the increase of gestational age. This finding is of great significance for clinical non-invasive prenatal testing of pregnant women. cfDNA has two obvious characteristics. First, its concentration in peripheral blood is extremely low. Usually, only about 50 ng of cfDNA can be extracted from 1 mL of plasma. Second, the length of cfDNA is very short. Most cfDNA is about 180 bp in length, while ctDNA in tumor patients is even shorter, with most of the length being <150 bp.

[0004] Genetic typing of blood type compatibility abroad requires the extraction of nucleic acid from blood for PCR amplification. The quality of the extracted nucleic acid is a key factor affecting the success or failure of the experiment, especially the quality requirements of nucleic acid extraction reagents for fetal blood type identification after extracting free nucleic acid from maternal peripheral blood. In principle, the carriers used for nucleic acid extraction at home and abroad are silicon-based materials, which utilize the phenomenon that the silicon hydroxyl groups on the surface of the material can bind to free DNA non-specifically through intermolecular forces such as ionic bonds in a high-salt, low-pH environment; and can dissociate under low-salt, high-pH conditions, thereby achieving nucleic acid purification and extraction, and has been widely used. However, this DNA extraction method has a very low efficiency for the extraction of small DNA fragments. The recovery rate of DNA fragments less than 200 bp in peripheral blood free DNA is usually less than 30%. Even if some kits are assisted by carrier RNA (ribonucleic acid carrier), polymers and other substances to co-precipitate with small fragments of free DNA to achieve the purpose of improving the recovery efficiency, the total recovery rate can only reach about 60%.

[0005] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide an anti-double-stranded DNA antibody, nucleotide fragments, magnetic beads and a method for extracting free DNA from peripheral blood, aiming to solve the problem of low recovery efficiency of existing free DNA from peripheral blood.

[0007] The technical solution of this application is as follows:

[0008] An anti-double-stranded DNA antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1;

[0009] The amino acid sequence of the light chain variable region is shown in SEQ ID NO:2.

[0010] The present application provides an anti-double-stranded DNA antibody that can non-specifically bind to all double-stranded DNAs and can be used to extract free DNA from peripheral blood.

[0011] A nucleotide fragment, wherein it is used to encode the production of the anti-double-stranded DNA antibody as described above.

[0012] The present application also provides a nucleotide sequence encoding the above-mentioned anti-double-stranded DNA antibody, which can be used to encode and generate a recombinant anti-double-stranded DNA antibody.

[0013] The nucleotide fragment, wherein the nucleotide sequence encoding the amino acids in the heavy chain variable region is shown in SEQ ID NO: 3.

[0014] The nucleotide fragment, wherein the nucleotide sequence encoding the light chain variable region amino acids is shown in SEQ ID NO:4.

[0015] A magnetic bead, wherein the anti-double-stranded DNA antibody as described above is connected to the magnetic bead.

[0016] A method for extracting free DNA from peripheral blood, comprising the following steps:

[0017] Adding the magnetic beads as described above to peripheral blood serum, the free DNA in the peripheral blood serum specifically binds to the anti-double-stranded DNA antibody to form a peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex;

[0018] The peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex is washed with a washing solution to remove the peripheral blood serum; the washed peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex is eluted to separate the peripheral blood free DNA and the magnetic beads to obtain the enriched peripheral blood free DNA.

[0019] The method for extracting free DNA from peripheral blood comprises adding the magnetic beads described above to peripheral blood serum, and the final concentration of the magnetic beads is 2 mg / ml.

[0020] The method for extracting free DNA from peripheral blood, wherein the washing solution is PBST buffer.

[0021] The method for extracting free DNA from peripheral blood, wherein the process of washing the free DNA from peripheral blood-anti-double-stranded DNA antibody magnetic bead complex with a washing solution comprises the following steps:

[0022] The PBST buffer is added to disperse and wash the peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex, and the complex is placed in a magnetic rack until the solution is clarified, and the supernatant is discarded.

[0023] The method for extracting free DNA from peripheral blood, wherein the process of eluting the washed free DNA from peripheral blood-anti-double-stranded DNA antibody magnetic bead complex comprises the following steps:

[0024] Add Gly-HCl at pH 3.0 for elution, stir to disperse, place in a magnetic rack until the solution is clear, and retain the supernatant.

[0025] Beneficial effects: This application uses the property of anti-double-stranded DNA antibodies that can bind to double-stranded DNA to establish a nucleic acid extraction method based on anti-double-stranded DNA antibodies. By using the specificity and specificity of anti-double-stranded DNA antibodies to double-stranded DNA, the recovery rate of small fragments of DNA in complex body fluid environments can be improved, and cfDNA in peripheral blood can be efficiently captured and enriched to achieve extraction and purification of cfDNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a diagram showing the gel electrophoresis results of the anti-double-stranded DNA antibody before purification, after crude purification, and after fine purification in Example 3 of the present application.

[0027] Figure 2 This is a graph showing the gel electrophoresis results of the DNA extracted from the example and the comparative example in Example 5 of the present application. DETAILED DESCRIPTION

[0028] The present application provides an anti-double-stranded DNA antibody, a nucleotide fragment, a magnetic bead, and a method for extracting free DNA from peripheral blood. In order to make the purpose, technical solution, and effect of the present application clearer and more specific, the present application is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0029] In this application, the specificity and specificity of monoclonal antibodies are used to extract free DNA from peripheral blood, which is not limited by the size of DNA fragments and is particularly suitable for the purification of small fragments of DNA. The recovery rate of free DNA from peripheral blood can be significantly improved, especially for samples containing small fragments of DNA or with low DNA content. In addition, due to the change in the principle of DNA extraction, the entire extraction and purification process does not use dangerous chemicals such as guanidine salts or isopropanol in the conventional DNA extraction process.

[0030] Specifically, the present application provides an anti-double-stranded DNA antibody that can non-specifically bind to all double-stranded DNA and can be used to extract free DNA from peripheral blood. The anti-double-stranded DNA antibody of the present application comprises a heavy chain variable region and a light chain variable region;

[0031] Among them, the amino acid sequence of the heavy chain variable region (SEQ ID NO: 1) is Signal peptide -FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (140 aa), and the specific amino acid sequence is as follows:

[0032] MERHWIFLFLLSVTGGVHSQVQLQQSEAELARPGASVKMSCKASGYTFTRYWMHWVKQRPGQALEWIGAIYPGNSDTSYNQKFKGKAKLTAVTSASTAYMELSSLTSEDSAVYYCARGEEIGVRRWFAYWGQGTLVTVSA;

[0033] Among them, the amino acid sequence corresponding to the signal peptide is MERHWIFLFLLSVTGGVHS; the amino acid sequence corresponding to FR1 is QVQLQQSEAELARPGASVKMSCKASGYTFT; the amino acid sequence of CDR1 is RYWMH; the amino acid sequence corresponding to FR2 is WVKQRPGQALEWIG; the amino acid sequence corresponding to CDR2 is AIYPGNSDTSYNQKFKG; the amino acid sequence corresponding to FR3 is KAKLTAVTSASTAYMELSSLTSEDSAVYYCAR; the amino acid sequence corresponding to CDR3 is GEEIGVRRWFAY; the amino acid sequence corresponding to FR4 is WGQGTLVTVSA.

[0034] The amino acid sequence of the light chain variable region (SEQ ID NO: 2) is Signal peptide -FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (127 aa). The specific amino acid sequence is as follows

[0035] MVFTPQILGLMLFWISASRGDIVLTQSPATLSVTPGDRVSLSCRASQSISNYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPLTFGAGTKLELK.

[0036] Among them, the amino acid sequence corresponding to the signal peptide is MVFTPQILGLMLFWISASRG; the amino acid sequence corresponding to FR1 is DIVLTQSPATLSVTPGDRVSLSC; the amino acid sequence corresponding to CDR1 is RASQSISNYLH; the amino acid sequence corresponding to FR2 is WYQQKSHESPRLLIK; the amino acid sequence corresponding to CDR2 is YASQSIS; the amino acid sequence corresponding to FR3 is GIPSRFSGSGSGTDFTLSINSVETEDFGMYFC; the amino acid sequence corresponding to CDR3 is QQSNSWPLT; the amino acid sequence corresponding to FR4 is FGAGTKLELK.

[0037] The present application also provides a nucleotide fragment encoding the anti-double-stranded DNA antibody, which can be used to encode the heavy chain variable region of the recombinant anti-double-stranded DNA antibody. The nucleotide sequence (SEQ ID NO: 3) encoding the amino acids of the heavy chain variable region is Signal sequence -FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (420 bp). The specific nucleotide sequence is as follows:

[0038] ATGGAAAGGCACTGGATCTTTCTCTTCCTGTTATCAGTAACTGGAGGTGTCCACTCCCAGGTCCAGCTGCAGCAGTCAGAGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGATACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGCTCTGGAATGGATTGGCGCTATT TATCCTGGAAATAGTGATACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCAAACTGACTGCAGTCACATCTGCCAGCACTGCCTACATGGAGCTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGAGGGGAGGAAATAGGGGTACGACGCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA;

[0039] Among them, the nucleotide sequence corresponding to the signal sequence is ATGGAAAGGCACTGGATCTTTCTCTTCCTGTTATCAGTAACTGGAGGTGTCCACTCC; the nucleotide sequence corresponding to FR1 is CAGGTCCAGCTGCAGCAGTCAGAGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACC; the nucleotide sequence corresponding to CDR1 is AGATACTGGATGCAC; the nucleotide sequence corresponding to FR2 is TGGGTAAAACAGAGGCCTGGACAGGCTCTGGAATGGATTGGC; CDR2 The corresponding nucleotide sequence is GCTATTTATCCTGGAAATAGTGATACTAGCTACAACCAGAAGTTCAAGGGC; the corresponding nucleotide sequence of FR3 is AAGGCCAAACTGACTGCAGTCACATCTGCCAGCACTGCCTACATGGAGCTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGA; the corresponding nucleotide sequence of CDR3 is GGGGAGGAAATAGGGGTACGACGCTGGTTTGCTTAC; the corresponding nucleotide sequence of FR4 is TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA.

[0040] The present application also provides a nucleotide sequence encoding the above-mentioned anti-double-stranded DNA antibody, which can be used to encode the light chain variable region of the recombinant anti-double-stranded DNA antibody. The nucleotide sequence (SEQ ID NO: 4) encoding the amino acids of the light chain variable region is Signal sequence -FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (381 bp). The specific nucleotide sequence is as follows:

[0041] ATGGTTTTTCACACCTCAGATTCTTGGACTTATGCTTTTCTGGATTTCAGCCTCCAGAGGTGATATTGTGCTAACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGAGTCAGTCTTTCCTGCAGGGCCAGCCAAAGTATTAGCAACTACCTACACTGGTATCAACAAAAATCACATGAGTCTCC AAGGCTTCTCATCAAGTATGCTTCCCAGTCCATCTCTGGGATCCCCTCCAGGTTTCAGTGGCAGTGGATCAGGGACAGATTTCACTCTCAGTATCAACAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGTCAACAGAGTAACAGCTGGCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA.

[0042] The nucleotide sequence corresponding to the signal sequence is ATGGTTTTCACACCTCAGATTCTTGGACTTATGCTTTTCTGGATTTCAGCCTCCAGAGGT; the nucleotide sequence corresponding to FR1 is GATATTGTGCTAACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGAGTCAGTCTTTCCTGC; the nucleotide sequence corresponding to CDR1 is AGGGCCAGCCAAAGTATTAGCAACTACCTACAC; the nucleotide sequence corresponding to FR2 is TGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCAT CAAG; the nucleotide sequence corresponding to CDR2 is TATGCTTCCCAGTCCATCTCT; the nucleotide sequence corresponding to FR3 is GGGATCCCCTCCAGGTTCAGTGGCAGTGGATCA GGGACAGATTTCACTCTCAGTATCAACAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGT; the nucleotide sequence corresponding to CDR3 is CAACAGAGTAACAGCTGGCCGCTCACG; the nucleotide sequence corresponding to FR4 is TTCGGTGCTGGGACCAAGCTGGAGCTGAAA.

[0043] In the present application, the amino acid sequences and corresponding nucleotide sequences of the heavy chain variable region and the light chain variable region of the anti-double-stranded DNA antibody are provided. The anti-double-stranded DNA antibody also includes a constant region, and the constant region is the constant region sequence of the mouse IgG antibody. Basically, the constant region antibodies of all mouse IgG are consistent and will not be repeated here.

[0044] The present application also provides a magnetic bead, to which the anti-double-stranded DNA antibody of the present application is connected.

[0045] In the present application, magnetic beads and anti-double-stranded DNA antibodies are directly combined to obtain an antibody-carrier complex that can specifically bind to nucleic acids, and then the antibody-carrier complex is used to extract nucleic acids from peripheral blood. The high specificity and high affinity of anti-double-stranded DNA antibodies for DNA enable the antibody-carrier complex to efficiently capture and enrich DNA in peripheral blood, achieving the purpose of extracting DNA in a complex peripheral blood environment. For small fragments of cfDNA that are difficult to recover, the extraction method and extraction medium of the present application still have a fairly high recovery efficiency, which can meet the subsequent experimental requirements in clinical samples.

[0046] Furthermore, the present application also provides a method for extracting free DNA from peripheral blood, comprising the following steps:

[0047] Preparation of antibodies: Select mice that can produce anti-double-stranded DNA antibodies for cell fusion, screen to obtain positive cell lines, and purify them with purification columns to obtain anti-double-stranded DNA antibodies;

[0048] Coupling of magnetic beads with anti-double-stranded DNA antibodies: coupling the purified anti-double-stranded DNA antibodies with magnetic beads to obtain magnetic beads connected with the anti-double-stranded DNA antibodies;

[0049] Enrichment of peripheral blood free DNA: Add magnetic beads connected with anti-double-stranded DNA antibodies to peripheral blood serum. The free DNA in the peripheral blood serum specifically binds to the anti-double-stranded DNA antibodies to form a peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex;

[0050] Elution of peripheral blood free DNA: washing the peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex with a washing solution to obtain a peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex from which peripheral blood serum has been removed; eluting the washed peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex to separate the peripheral blood free DNA and the magnetic beads connected with the anti-double-stranded DNA antibody to obtain the enriched peripheral blood free DNA.

[0051] In the process of enriching free DNA in peripheral blood, the final concentration of the magnetic beads connected with the anti-double-stranded DNA antibody can be 2 mg / ml.

[0052] In the elution process of peripheral blood free DNA, the washing solution can be PBST buffer. The washing process can be adding PBST buffer, dispersing and washing the peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex, placing it in a magnetic rack until the solution is clarified, and discarding the supernatant. After washing, the elution process can be adding Gly-HCl with pH 3.0 for elution, stirring and dispersing, placing it in a magnetic rack until the solution is clarified, and retaining the supernatant.

[0053] The method for extracting free DNA from peripheral blood disclosed in the present application utilizes the property of anti-double-stranded DNA antibodies that can bind to double-stranded DNA to establish a nucleic acid extraction method based on anti-double-stranded DNA antibodies. By utilizing the specificity and specificity of anti-double-stranded DNA antibodies to double-stranded DNA, the recovery rate of small fragments of DNA in complex body fluid environments can be improved, cfDNA in peripheral blood can be efficiently captured and enriched, and cfDNA can be extracted and purified.

[0054] The present application is further described below through specific embodiments.

[0055] Embodiment 1:

[0056] Preparation of mouse anti-double-stranded DNA antibody:

[0057] Select 8-12 weeks old NZW×NZB F1 mice with SLE symptoms, take spleen cells under sterile conditions, put spleen cells in a culture dish, add 10mL complete DMEM culture medium (dulbecco's modified eagle medium) and gently squeeze to obtain spleen cell suspension, centrifuge at 1000rpm for 5min, and discard the supernatant. Resuspend spleen cells in DMEM, mix spleen cells and myeloma cells at a cell number ratio of 2:1, place in a 50mL centrifuge tube, centrifuge at 1000rpm for 10min, and discard the supernatant. Then put the centrifuge tube in a 37℃ water bath, add 1mL of PEG (polyethylene glycol) with a mass volume ratio of 50% to promote fusion. Then add 25mL of DMEM complete culture medium to terminate fusion, incubate at 1000rpm at room temperature for 10min, and discard the supernatant. Then add 20mL of complete DMEM culture medium containing HAT (H-Hypoxanthine, A-Aminopterin, T-Thymidine), and dispense 100ul per well into a 96-well culture plate with feeder cells, and place in a carbon dioxide incubator for culture. After 3 days, half of the medium was replaced with HAT selective culture medium (a mixture of xanthine, aminopterin, and thymidine) and cultured for 2 weeks; HT adaptive culture medium (a mixture of hypoxanthine and thymidine) was used instead, and when the hybridoma clones grew to 1 / 4 of the field of view (i.e., to 1 / 4 of the bottom area of ​​the well), positive clones were screened. The hybridoma colonies were diluted to 0.5 cells per well by limiting gradient dilution. Continue to culture with HAT selection medium for 5-7 days. When it grows into a cluster (about 20-30 cells), the cell supernatant was taken to screen positive hybridoma cell lines. After confirming the positive cell line, expand the culture and collect the positive cell supernatant.

[0058] Embodiment 2:

[0059] Screening of positive cell supernatant:

[0060] Coat 96-well ELISA plates with 0.5 mg / mL poly-L-lysine (molecular weight 70000-150000), 200ul per well, stand at 37℃ for 2h, wash 3 times with PBST buffer, then add 50ul calf thymus DNA with a concentration of 20ng / ul to each well of the plate, and bake overnight at 37℃. The next day, add 5% skim milk to each well of the ELISA plate for blocking for 2h, wash 3 times with PBST, and the ELISA plate is ready. Then add the supernatant of positive cells to the wells, react at 37℃ for 1h, and wash 3 times with PBST buffer. Then add HRP-goat anti-mouse IgG at a ratio of 1:10000 (volume ratio of HRP-goat anti-mouse IgG to PBS buffer), react for 1h and wash 5 times, then add TMB colorimetric solution to the wells for color development, and read after termination.

[0061] Embodiment 3:

[0062] Antibody purification:

[0063] Crude purification: Take 1L of positive cell supernatant and place it in a beaker. Place the beaker on a magnetic stirrer and put a magnetic rotor in it. Slowly drip an equal volume of saturated ammonium sulfate solution into the beaker, and then place it at 4°C for precipitation overnight. The next day, discard part of the supernatant, centrifuge at 6000rpm for 30min, discard all the supernatant, and then dissolve the precipitate with PBS buffer. Then put the dissolved liquid into a dialysis bag and seal it tightly, and put the dialysis bag into PBS buffer for dialysis. Change the PBS buffer every 8h, and change it 3 times.

[0064] Purification: Use 5mL Protein A 4FF pre-loaded gravity column purchased from BBI for purification. Fix the gravity column, add 20mL of PBS buffer to the column for balance, slowly add 10mL of the dialyzed solution to the column after balance, then elute the non-specifically adsorbed impurities with 20mL of PBS buffer, collect the effluent, and finally elute with 10mL of 0.1M pH 2.5 citric acid, collect the eluate and immediately neutralize with 1 / 20 volume of 1M pH 9.0 Tris-HCl. Finally, the purified anti-double-stranded DNA antibody is obtained.

[0065] The prepared anti-double-stranded DNA antibody was sent to a third-party sequencing company for sequencing to obtain the amino acid sequence (SEQ ID NO: 1-2) and nucleotide sequence (SEQ ID NO: 3-4) of the anti-double-stranded DNA antibody.

[0066] Gel electrophoresis was performed on the anti-double-stranded DNA antibody before purification, after crude purification, and after fine purification. The sample loading amount was 2 μg, and the gel was a 4-12% gradient non-reducing polyacrylamide gel. The results are as follows: Figure 1As shown, the marker is Thermolot #26619, the original is the anti-double-stranded DNA antibody before purification, the crude is the anti-double-stranded DNA antibody after crude purification, and the refined is the anti-double-stranded DNA antibody after purification. Figure 1 It can be concluded that the anti-double-stranded DNA antibody was successfully purified from the cell supernatant.

[0067] Embodiment 4:

[0068] Coupling of anti-double-stranded DNA antibody to magnetic beads: The magnetic beads used were Dynabeads®MyOne™ Carboxylic Acid purchased from thermofisher.

[0069] Roll the vial for 30 minutes to resuspend the Dynabeads® beads, then transfer 1 mL to a new tube. Place the tube on the magnet for 2 minutes and remove the supernatant. Remove the tube from the magnet, add 1 mL of 15 mM MES buffer (pH 6.0), vortex for 5-10 seconds, place the tube on the magnet for 2 minutes, and remove the supernatant. Repeat this step once. Resuspend the Dynabeads® beads in 100 μL of 15 mM MES buffer (pH 6.0), add 100 μL of EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) and incubate on a roller at room temperature for 30 minutes, place the tube on the magnet for 2 minutes, and remove the supernatant. Add up to 400 μg of anti-dsDNA antibody diluted to a total volume of 200-500 μL in 15 mM MES buffer (pH 6.0) and incubate on a roller at room temperature overnight. The next day, place the test tube in the magnet for 2 minutes, remove the supernatant, remove the test tube from the magnet, add 1mL of PBS buffer containing 0.1% Tween®-20; place the test tube on a roller mixer for 10 minutes. Place the test tube on the magnet for 2 minutes, then remove the supernatant to obtain magnetic beads bound to the anti-double-stranded DNA antibody. Resuspend the magnetic beads bound to the antibody in 200-500μL PBS buffer containing 0.1% Tween®-20 and 0.1% BSA (bovine serum albumin) to achieve the desired concentration of magnetic beads bound to the anti-double-stranded DNA antibody, which can generally be 1-2mg / ml.

[0070] Example 5: Binding efficiency of anti-double-stranded DNA antibody magnetic beads to DNA of different lengths

[0071] 100 bp double-stranded DNA, 200 bp double-stranded DNA and calf thymus DNA were taken as three DNA test samples, and one experimental example was set for each DNA test sample. Three 1 μL DNA test samples were added into three different 200 μL EP tubes at a concentration of 500 ng / μL, and 3 μL of the anti-double-stranded DNA antibody magnetic beads with a concentration of 2 mg / ml prepared in Example 4 were added to each EP tube. Finally, PBS was added to each EP tube to a total volume of 10 μL.

[0072] One control sample was set for each DNA test sample. Three 1 μL DNA test samples were added into three different 200 μL EP tubes at a concentration of 500 ng / μL. 3 μL magnetic beads coupled with mouse anti-CD36 monoclonal antibody were added into the EP tube of each control sample.

[0073] Among them, calf thymus DNA was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd. (Cat. No. D85790). The nucleotide sequences of 100 bp and 200 bp calf thymus DNA are as follows:

[0074] 5'- CCAGAAGAGGAAACCGG TATGTTCTTAGTTTTAAATAGTTGCTCTGGAGTCATATGCCTCCTTTCTTCTCATTATTTTTTA CATGTTTTAAAAATGCATT -3' (SEQ ID NO:5);

[0075] 5'- CCAGAAGAGGAAACCG GTATGTTCTTAGTTTTAAATAGTTGCTCTGGAGTCATTGTTGTGATTGAACTCTATTTACACGAGCTGTAACTCATGACAGTTCCGCATGACCCTTCTTTGCATGGGACTGGCATCTCTGTGGAGTAATGGCTCCATATGCCTCCTTTCTTCTCATTATTTTTTA CATGTTTTAAAAATGCATT -3' (SEQ ID NO:6);

[0076] The underlined part is the primer sequence.

[0077] Place the EP tube in a vortex oscillator, use the vortex oscillator to disperse the magnetic beads in the EP tube into the liquid, and then mix it under a roller stirrer at room temperature for 30 minutes. Then place the centrifuge tube in a magnetic stand for 2 minutes until the solution is clear, aspirate the supernatant, and use the purchased Helixyte Green™ double-stranded DNA fluorescent quantitative kit (Cat. No. 17650) according to the method in its instructions to dilute the liquid in the supernatant to 100μL with the buffer in the kit, and then dilute the fluorescent substrate in the kit 200 times with the buffer, mix it with the diluted supernatant in an equal volume in a black ELISA plate, and after 5-10 minutes at room temperature away from light, use a fluorescent ELISA reader to read the fluorescence signal with an excitation wavelength of 490nm and an emission wavelength of 525nm. The results are shown in Table 1, where "before reaction" refers to the fluorescence signal of each experimental example before adding anti-double-stranded DNA antibody magnetic beads, the experimental group refers to the fluorescence signal of each experimental example after adding anti-double-stranded DNA antibody magnetic beads for reaction, and the control group refers to the fluorescence signal of each control example after adding mouse anti-CD36 monoclonal antibody magnetic beads for reaction. It can be seen from Table 1 that the fluorescence intensity in the supernatant after adsorption of anti-double-stranded DNA antibody magnetic beads is very low, proving that the anti-double-stranded DNA antibody magnetic beads can effectively adsorb double-stranded DNA of different lengths in the solution.

[0078] Table 1

[0079] 100bp 200bp Calf thymus Before reaction 194 433 1116 Experimental Group 9 9 20 Control group 149 302 1181

[0080] Example 6: Purification of free DNA from peripheral blood using anti-double-stranded DNA antibody magnetic beads

[0081] Take 2 mL of plasma purchased from the market that has been free of endogenous DNA, and add 1 μL of 100 bp double-stranded DNA with a concentration of 500 ng / μL. The nucleotide sequence of this double-stranded DNA is as follows:

[0082] 5'- CCAGAAGAGGAAACCGG TATGTTCTTAGTTTTAAATAGTTGCTCTGGAGTCATATGCCTCCTTTCTTCTCATTATTTTTTA CATGTTTTAAAAATGCATT -3'; (SEQ ID NO:5)

[0083] Among them, the underlined part is the primer.

[0084] This double-stranded DNA is used as a substitute for cfDNA in plasma. After mixing evenly, centrifuge at 4°C and 1600g for 10 minutes using a refrigerated centrifuge, and take the supernatant and put it into a new centrifuge tube. Dilute the supernatant with an equal volume of PBST buffer, and then add 10 μL of the anti-double-stranded DNA antibody magnetic beads prepared in Example 4 to a final concentration of 2 mg / ml. Disperse the anti-double-stranded DNA antibody magnetic beads into the liquid using a vortex oscillator, and then mix under a roller agitator at room temperature for 30 minutes. Then place the centrifuge tube in a magnetic stand for 2 minutes until the solution is clear, and discard the supernatant. Add 1 mL of PBST buffer, cover the lid, and use a vortex oscillator to disperse and wash the anti-double-stranded DNA antibody magnetic beads for 15 seconds, place the centrifuge tube in a magnetic stand for 2 minutes until the solution is clear, discard the supernatant, and repeat this step 3 times. Finally, 100 μL of 0.1 M pH 3.0 Gly-HCl was added for elution, and the anti-double-stranded DNA antibody magnetic beads were dispersed with a vortex oscillator, mixed and stirred for 5 minutes on a roller stirrer, and then the centrifuge tube was placed in a magnetic rack for 2 minutes until the solution was clarified, and the centrifuge liquid was taken out to a new tube and immediately neutralized with 1 / 15 volume of 1 M pH 9.0 Tris-HCl.

[0085] Two control cases were set up, and the same plasma containing double-stranded DNA was tested using the Thermo kit and the Qiagen kit respectively. The Thermo kit was Thermofisher MagMAX TM Cell-Free DNA Isolation Kit. Qiagen kit is Qiagen QIAamp® Circulating Nucleic Acid Kit.

[0086] The DNA extracted in the examples and the comparative examples was subjected to agarose gel electrophoresis, the sample loading amount was 5ul, and the gel was 1% agarose gel. The results are shown in FIG. Figure 2 As shown. Among them, the marker is dl2000 DNA marker, 1 is the DNA result extracted by the embodiment of the present application, 2 is the DNA result extracted by the Thermo kit, and 3 is the DNA result extracted by the Qiagen kit. Figure 2 It can be concluded that the recovery efficiency of this method for small molecule fragments of 100 bp is better than that of Thermo and Qiagen kits, and is suitable for the purification of small molecule free DNA.

[0087] It should be understood that the application of the present application is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made according to the above description, and all these improvements and changes should fall within the scope of protection of the present application.

Claims

1. An anti-double-stranded DNA antibody comprising a heavy chain variable region and a light chain variable region, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1; The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

2. A nucleotide fragment, characterized in that: Used to encode and produce the anti-double-stranded DNA antibody as claimed in claim 1.

3. The nucleotide fragment according to claim 2, characterized in that The nucleotide sequence encoding the amino acids in the heavy chain variable region is shown in SEQ ID NO:

3.

4. The nucleotide fragment according to claim 2, characterized in that The nucleotide sequence encoding the amino acids in the light chain variable region is shown in SEQ ID NO:

4.

5. A magnetic bead, characterized in that: The anti-double-stranded DNA antibody according to claim 1 is connected to the magnetic beads.

6. A method for extracting free DNA from peripheral blood, characterized in that: The following steps are involved: Adding the magnetic beads as claimed in claim 5 to peripheral blood serum, wherein the free DNA in the peripheral blood serum specifically binds to the anti-double-stranded DNA antibody to form a peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex; The peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex is washed with a washing solution to remove the peripheral blood serum; the washed peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex is eluted to separate the peripheral blood free DNA and the magnetic beads to obtain the enriched peripheral blood free DNA.

7. The method for extracting free DNA from peripheral blood according to claim 6, characterized in that: The magnetic beads as claimed in claim 5 are added to peripheral blood serum, and the final concentration of the magnetic beads is 2 mg / ml.

8. The method for extracting free DNA from peripheral blood according to claim 6, characterized in that: The washing solution is PBST buffer.

9. The method for extracting free DNA from peripheral blood according to claim 8, characterized in that: The process of washing the peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex with a washing solution comprises the following steps: The PBST buffer is added to disperse and wash the peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex, and the complex is placed in a magnetic rack until the solution is clarified, and the supernatant is discarded.

10. The method for extracting free DNA from peripheral blood according to claim 6, characterized in that: The process of eluting the washed peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex comprises the following steps: Add Gly-HCl at pH 3.0 for elution, stir to disperse, place in a magnetic rack until the solution is clear, and retain the supernatant.

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