CN115819604A - Anti-double-stranded DNA antibody, nucleotide fragment, magnetic beads and method for extracting free DNA from peripheral blood - Google Patents

Abstract

The application relates to the field of biotechnology, and discloses an anti-double-stranded DNA antibody, nucleotide fragments, magnetic beads 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, 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 As shown in SEQ ID NO:2. 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 especially suitable for the purification of small fragments of DNA, which can significantly improve the extraction time of free peripheral blood DNA. The recovery rate is high, especially for samples containing small fragments of DNA or low DNA content.

Description

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

technical field

The application relates to the field of biotechnology, and mainly relates to an anti-double-stranded DNA antibody, a nucleotide fragment, magnetic beads and a method for extracting free DNA from peripheral blood.

Background technique

Anti-DNA antibodies (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 triggering apoptosis, inflammation and tissue fibrosis. Anti-double Stranded DNA Antibodies (Anti-double Stranded DNA Antibodies) are antibodies that exist in large amounts 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. It can cause damage to multiple organs in SLE patients, the most common being lupus nephritis due to the accumulation of immune complexes in the glomerulus and tubular basement membrane.

Cell-free DNA (cfDNA) is a kind of free fragmented DNA that exists 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 that there was a high concentration of free DNA in tumor patients. Subsequently, many scholars have studied the free DNA in these tumor patients and found that these free DNA carry tumor-specific mutations or epigenetic changes, which are tumor DNA fragments released into the peripheral blood by necrotic or apoptotic tumor cells. It was later named circulating tumor DNA (circulating tumor DNA, ctDNA). In 1997, Lo et al. reported that the presence of Y chromosome was detected in the peripheral blood of pregnant women who were pregnant with male babies, thus proving that there were fetal cell-free DNA in the peripheral blood of pregnant women. Later studies showed that fetal cell-free DNA in the peripheral blood of pregnant women The content of the protein increases with the increase of gestational age. This finding has important significance for the non-invasive prenatal detection of pregnant women in clinical practice. cfDNA has two distinct features. One is that its concentration in peripheral blood is extremely low. Usually, only about 50 ng of cfDNA can be extracted from 1 mL of plasma. The other is that the length of cfDNA is very short, most of which are around 180 bp. The ctDNA in tumor patients is shorter, most of which are <150bp in length.

The genotyping of blood type compatibility in foreign countries needs to extract nucleic acid from blood for PCR amplification. The quality of the extracted nucleic acid is the key factor affecting the success of the experiment, especially the nucleic acid for fetal blood type identification after extracting free nucleic acid from maternal peripheral blood. The quality requirements of the extraction reagents are extremely high. In principle, at present, the carrier used for nucleic acid extraction at home and abroad is a silicon-based material, which utilizes the silanol on the surface of the material and free DNA to perform non-specific interactions through intermolecular forces such as ionic bonds in a high-salt, low-pH environment. Combination; It can dissociate under the conditions of low salt and high pH, so as to realize the purification and extraction of nucleic acid, and has been widely used. However, this DNA extraction method is very inefficient for extracting small fragments of DNA, and the recovery rate of DNA fragments less than 200 bp in peripheral blood free DNA is usually less than 30%. Even if some kits assist the co-precipitation of carrier RNA (ribonucleic acid carrier), polymers and other substances with small fragments of free DNA to improve the recovery efficiency, the total recovery rate can only reach about 60%.

Therefore, the prior art still needs to be improved and developed.

Contents of the invention

In view of the above-mentioned deficiencies in the prior art, the purpose of this 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 at solving the recovery efficiency of free DNA from peripheral blood. lower question.

The technical scheme of the application is as follows:

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 shown in SEQ ID NO:1;

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

The application provides an anti-double-stranded DNA antibody, which can non-specifically bind to all double-stranded DNAs, and can be used to extract free DNA from peripheral blood.

A nucleotide fragment, wherein, it is used for coding to generate the above-mentioned anti-double-stranded DNA antibody.

This application also provides the nucleotide sequence encoding the above anti-double-stranded DNA antibody, which can be used to encode and generate recombinant anti-double-stranded DNA antibody.

The nucleotide fragment, wherein the nucleotide sequence encoding the amino acid of the heavy chain variable region is shown in SEQ ID NO:3.

The nucleotide fragment, wherein the nucleotide sequence encoding the amino acid of the light chain variable region is shown in SEQ ID NO:4.

A magnetic bead, wherein the above-mentioned anti-double-stranded DNA antibody is linked to the magnetic bead.

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

Add the above-mentioned magnetic beads to peripheral blood serum, and 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 ;

Washing the peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex with a washing solution to remove the peripheral blood serum; the washed peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex Eluting and separating the free peripheral blood DNA and the magnetic beads to obtain the enriched free peripheral blood DNA.

The method for extracting free DNA from peripheral blood, wherein, the above-mentioned magnetic beads are added to peripheral blood serum, and the final concentration of the magnetic beads is 2 mg/ml.

The method for extracting free DNA from peripheral blood, wherein the washing liquid is PBST buffer.

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 washing solution comprises the following steps:

Add the PBST buffer, disperse and wash the peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex, put it into a magnetic stand until the solution is clear, and discard the supernatant.

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

Add Gly-HCl with pH 3.0 for elution, stir to disperse, put in a magnetic stand until the solution is clear, and keep the supernatant.

Beneficial effect: the present application utilizes the property that the anti-double-stranded DNA antibody can bind to double-stranded DNA, and establishes a nucleic acid extraction method based on the anti-double-stranded DNA antibody. Using the specificity and specificity of anti-double-stranded DNA antibodies to double-stranded DNA, it can improve the recovery rate of small fragments of DNA in complex body fluid environments, efficiently capture and enrich cfDNA in peripheral blood, and realize the extraction and purification of cfDNA.

Description of drawings

Fig. 1 is a graph of gel electrophoresis results of anti-double-stranded DNA antibodies before purification, after crude purification and after purification in Example 3 of the present application.

Fig. 2 is a graph of gel electrophoresis results of DNA extracted from the example and the comparative example in Example 5 of the present application.

Detailed ways

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

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 especially suitable for the purification of small fragments of DNA, which can significantly improve the time of extraction of free DNA from peripheral blood. The recovery rate is high, especially for samples containing small fragments of DNA or low DNA content. In addition, due to the change of the DNA extraction principle, the entire extraction and purification process does not use hazardous chemicals such as guanidinium salt or isopropanol in the conventional DNA extraction process.

Specifically, the present application provides an anti-double-stranded DNA antibody, which 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 includes a heavy chain variable region and a light chain variable region;

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:

MERHWIFLFLLSVTGGVHSQVQLQQSEAELARPGASVKMSCKASGYTFTRYWMHWVKQRPGQALEWIGAIYPGNSDTSYNQKFKGKAKLTAVTSASTAYMELSSLTSEDSAVYYCARGEEIGVRRWFAYWGQGTLVTVSA;

Among them, the amino acid sequence corresponding to Signal peptide is MERHWIFLFLLSVTGGVHS; the amino acid sequence corresponding to FR1 is QVQLQQSEAELARPGASVKMSCKASGYTFT; the amino acid sequence corresponding to 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 CDR3 is GEEIGVRRWFAY; the amino acid sequence corresponding to FR4 is WGQGTLVTVSA.

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

MVFTPQILGLMLFWISASRGDIVLTQSPATLSVTPGDRVSLSCRASQSISNYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPLTFGAGTKLELK.

Among them, the amino acid sequence corresponding to 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 CDR3 is QQSNSWPLT; the amino acid sequence corresponding to FR4 is FGAGTKLELK.

This 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, and encodes the nucleotide sequence corresponding to the amino acid of the heavy chain variable region ( SEQ ID NO:3) is Signal sequence -FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (420 bp), the specific nucleotide sequence is as follows:

ATGGAAAGGCACTGGATCTTTCTCTTCCTGTTATCAGTAACTGGAGGTGTCCACTCCCAGGTCCAGCTGCAGCAGTCAGAGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGATACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGCTCTGGAATGGATTGGCGCTATTTATCCTGGAAATAGTGATACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCAAACTGACTGCAGTCACATCTGCCAGCACTGCCTACATGGAGCTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGAGGGGAGGAAATAGGGGTACGACGCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA；

其中，Signal sequence对应的核苷酸序列为ATGGAAAGGCACTGGATCTTTCTCTTCCTGTTATCAGTAACTGGAGGTGTCCACTCC；FR1对应的核苷酸序列为CAGGTCCAGCTGCAGCAGTCAGAGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACC；CDR1对应的核苷酸序列为AGATACTGGATGCAC；FR2对应的核苷酸序列为TGGGTAAAACAGAGGCCTGGACAGGCTCTGGAATGGATTGGC；CDR2对应的核苷酸序列为GCTATTTATCCTGGAAATAGTGATACTAGCTACAACCAGAAGTTCAAGGGC；FR3对应的核苷酸序列为AAGGCCAAACTGACTGCAGTCACATCTGCCAGCACTGCCTACATGGAGCTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGA；CDR3对应的核苷酸序列为GGGGAGGAAATAGGGGTACGACGCTGGTTTGCTTAC；FR4对应的核苷酸序列为TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA。

The application also provides the 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, and encodes the nucleotide sequence corresponding to the amino acid of the light chain variable region ( SEQ ID NO:4) is Signal sequence -FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (381 bp), the specific nucleotide sequence is as follows:

ATGGTTTTCACACCTCAGATTCTTGGACTTATGCTTTTCTGGATTTCAGCCTCCAGAGGTGATATTGTGCTAACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGAGTCAGTCTTTCCTGCAGGGCCAGCCAAAGTATTAGCAACTACCTACACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAGTATGCTTCCCAGTCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACTCTCAGTATCAACAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGTCAACAGAGTAACAGCTGGCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA。

Signal sequence对应的核苷酸序列为ATGGTTTTCACACCTCAGATTCTTGGACTTATGCTTTTCTGGATTTCAGCCTCCAGAGGT；FR1对应的核苷酸序列为GATATTGTGCTAACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGAGTCAGTCTTTCCTGC；CDR1对应的核苷酸序列为AGGGCCAGCCAAAGTATTAGCAACTACCTACAC；FR2对应的核苷酸序列为TGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCAT CAAG；CDR2对应的核苷酸序列为TATGCTTCCCCAGTCCATCTCT; the nucleotide sequence corresponding to FR3 is GGGATCCCCTCCAGGTTCAGTGGCAGTGGATCA GGGACAGATTTCACTTCAGTATCAACAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGT; the nucleotide sequence corresponding to CDR3 is CAACAGAGTAACAGCTGGCCGCTCACG; the nucleotide sequence corresponding to FR4 is TTCGGTGCTGGGACCAAGCTGGAGCTGAAA.

In this 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, which is a mouse IgG The antibody constant region sequence is basically the same for all murine IgG constant region antibodies, and will not be described here.

The present application also provides a magnetic bead, on which the anti-double-stranded DNA antibody of the present application is linked.

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

Further, the application also provides a method for extracting free DNA from peripheral blood, comprising the steps of:

Antibody preparation: Select mice capable of producing 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;

Coupling of magnetic beads and anti-double-stranded DNA antibodies: Coupling the purified anti-double-stranded DNA antibodies with magnetic beads to obtain magnetic beads linked with anti-double-stranded DNA antibodies;

Enrichment of free DNA in peripheral blood: the magnetic beads linked with anti-double-stranded DNA antibody are added to peripheral blood serum, and the free DNA in peripheral blood serum binds specifically to anti-double-stranded DNA antibody to form peripheral blood free DNA-antibody Double-stranded DNA antibody magnetic bead complex;

The elution of peripheral blood free DNA: wash the peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex with washing solution to obtain the peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex from which the peripheral blood serum is removed; After washing, the peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex is eluted, and the peripheral blood free DNA and the magnetic beads connected with the anti-double-stranded DNA antibody are separated to obtain the enriched peripheral blood free DNA .

During the enrichment process of peripheral blood free DNA, the final concentration of the magnetic beads linked with the anti-double-stranded DNA antibody can be 2 mg/ml.

During the elution of peripheral blood free DNA, the washing solution can be PBST buffer. The washing process can be adding PBST buffer solution to disperse and wash the peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex, put it in a magnetic stand until the solution is clear, and discard the supernatant. After washing, the elution process can be elution by adding Gly-HCl with pH 3.0, stirring to disperse, putting into a magnetic stand until the solution is clear, and retaining the supernatant.

The method for extracting free DNA from peripheral blood disclosed in this application utilizes the property that anti-double-stranded DNA antibodies can bind to double-stranded DNA, and establishes a nucleic acid extraction method based on anti-double-stranded DNA antibodies. Using the specificity and specificity of anti-double-stranded DNA antibodies to double-stranded DNA, it can improve the recovery rate of small fragments of DNA in complex body fluid environments, efficiently capture and enrich cfDNA in peripheral blood, and realize the extraction and purification of cfDNA.

The present application will be further described below through specific examples.

Example 1:

Preparation of mouse anti-double-stranded DNA antibody:

Select 8-12 weeks of NZW×NZB F1 mice with SLE symptoms, take spleen cells under sterile conditions, put the spleen cells into a petri dish, add 10mL of complete DMEM culture medium (dulbecco's modified eagle medium) Squeeze gently to obtain the spleen cell suspension, centrifuge at 1000rpm for 5min, and discard the supernatant. The spleen cells were then suspended in DMEM, and the spleen cells and myeloma cells were mixed according to the cell number ratio of 2:1, placed in a 50 mL centrifuge tube, centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. Then put the centrifuge tube into a 37°C water bath, and add 1 mL of PEG (polyethylene glycol) with a mass-volume ratio of 50% to promote fusion. Then 25 mL of DMEM complete culture solution was added to terminate the fusion, room temperature was 1000 rpm for 10 min, and the supernatant was discarded. Then add 20mL of complete DMEM medium containing HAT (H-Hypoxanthine, A-Aminopterin, T-Thymidine thymidine), and distribute 100ul per well into a 96-well culture plate with feeder cells cultured in a carbon dioxide incubator. After 3 days, use the HAT selective medium (a mixture of xanthine, aminopterin, and thymidine) to change the medium once in half, and cultivate continuously for 2 weeks; use HT adaptive medium (hypoxanthine and thymidine) instead. Glycoside mixture), when the hybridoma clone grows to 1/4 of the field of view (that is, grows to 1/4 of the bottom area of the well), the screening of positive clones is carried out. Small hybridoma colonies were diluted to 0.5 cells per well by limiting serial dilution. Continue to culture with HAT selection medium for 5-7 days. After it grows into a cluster (about 20-30 cells), the cell supernatant is taken to screen positive hybridoma cell lines. After confirming the positive cell line, expand the culture and collect the positive cell supernatant.

Example 2:

Screening of positive cell supernatants:

Coat 96-well ELISA plate with 0.5mg/mL poly-L-lysine (molecular weight 70000-150000), 200ul per well, let stand at 37°C for 2h, wash 3 times with PBST buffer, and then apply to the plate Add 50ul of calf thymus DNA at a concentration of 20ng/ul to each well, and oven overnight at 37°C. The next day, 5% skim milk was added to each well of the microplate for blocking for 2 hours, and after washing with PBST three times, the microplate was completed. Then the positive cell supernatant was added to the wells, reacted at 37°C for 1 h, and washed 3 times with PBST buffer. Then add HRP-goat anti-mouse IgG at a ratio of 1:10000 (the volume ratio of HRP-goat anti-mouse IgG to PBS buffer), react for 1 hour and wash 5 times, then add TMB chromogenic solution to the well for color development, and stop reading.

Example 3:

Antibody purification:

Crude and pure: Take 1L of positive cell supernatant in a beaker, place the beaker on a magnetic stirrer and put a magnetic rotor into it, slowly drop an equal volume of saturated ammonium sulfate solution into the beaker, and then place it at 4°C Precipitate overnight. Discard part of the supernatant the next day, centrifuge at 6000 rpm for 30 min, discard all the supernatant, and then dissolve the precipitate with PBS buffer. Afterwards, put the dissolved liquid into a dialysis bag and seal it tightly, and put the dialysis bag into PBS buffer solution for dialysis, and change the PBS buffer solution every 8 hours for 3 times.

Purification: 5mL Protein A 4FF prepacked gravity column purchased from BBI was used for purification. Fix the gravity column, add 20mL of PBS buffer to the column for equilibrium, slowly add 10mL of the above-mentioned dialyzed solution to the column after equilibrium, then use 20mL of PBS buffer to elute non-specifically adsorbed foreign proteins, and collect The liquid was flowed out and finally eluted with 10 mL of 0.1M pH 2.5 citric acid, and the eluate was collected and immediately neutralized with 1/20 volume of 1M pH 9.0 Tris-HCl. Finally, the purified anti-double-stranded DNA antibody was obtained.

Submit the prepared anti-double-stranded DNA antibody 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 ).

Gel electrophoresis was performed on anti-double-stranded DNA antibodies before purification, after crude purification, and after purification. The sample load was 2 μg, and the gel was a 4-12% gradient non-reducing polyacrylamide gel. The results are shown in the figure As shown in 1, 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 purified is the purified anti-double-stranded DNA antibody. It can be concluded from Figure 1 that the anti-double-stranded DNA antibody was successfully purified from the cell supernatant.

Example 4:

Coupling of anti-double-stranded DNA antibody to magnetic beads: Dynabeads® MyOne™ Carboxylic Acid purchased from thermofisher was used as magnetic beads.

Resuspend the Dynabeads® Magnetic Beads by rolling the vial for 30 minutes, then transfer 1 mL to a new tube. Place the tube in the magnet for 2 min, then remove the supernatant. Remove the tube from the magnet, add 1mL of 15mM MES buffer (pH6.0), vortex for 5-10 seconds, put the tube on the magnet for 2 minutes, then remove the supernatant, repeat this step 1 time. Then resuspend Dynabeads® magnetic beads in 100 μL 15 mM MES buffer (pH6.0), add 100 μL EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide ) and incubate for 30 min at room temperature on a roller, place the tube in the magnet for 2 min, then 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 overnight on a roller at room temperature. The next day, remove the supernatant after placing the tube in the magnet for 2 minutes, remove the tube from the magnet, and add 1 mL of PBS buffer containing 0.1% Tween®-20; place the tube on a roller mixer for 10 minutes. Place the tube on the magnet for 2 minutes, then remove the supernatant to obtain magnetic beads bound to the anti-dsDNA antibody. Resuspend the magnetic beads bound to the antibody in 200-500 µL of PBS buffer containing 0.1% Tween®-20 and 0.1% BSA (bovine serum albumin) such that the concentration of the magnetic beads bound to the anti-double-stranded DNA antibody To achieve the required concentration, it can generally be 1-2mg/ml.

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

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

One control example was set up for each DNA test sample, and three 1 μL DNA test samples were added to three different 200 μL EP tubes at a concentration of 500 ng/μL, and 3 μL of even Magnetic beads linked to mouse anti-CD36 monoclonal antibody.

Among them, calf thymus DNA was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd. (product number #D85790). The nucleotide sequences of 100bp and 200bp calf thymus DNA are as follows:

5'- CCAGAAGAGGAAACCGG TATGTTTCTTAGTTTTAAATAGTTGCTCTGGAGTCATATGCCTCCTTTCTTCTCATTATTTTTTA CATGTTTTAAAAATGCATT -3' (SEQ ID NO: 5);

5'- CCAGAAGAGGAAACCG GTATGTTCTTAGTTTTAAATAGTTGCTCTGGAGTCATTGTTGTGATTGAACTCTATTTACACGAGCTGTAACTCATGACAGTTCCGCATGACCCTTCTTTGCATGGGACTGGCATCTCTGTGGAGTAATGGCTCCATATGCCTCCTTTCTTCTCATTATTTTTTACATGTTTTAAAAATGCATT -3' (SEQ ID NO: 6)

Wherein, the underlined part is the primer sequence.

Put the EP tube into the vortex shaker, use the vortex shaker to disperse the magnetic beads in the EP tube into the liquid, and then mix under the roller stirrer at room temperature for 30min. Then put the centrifuge tube in the magnetic stand for 2 minutes until the solution is clear, draw the supernatant, and use the purchased Helixyte Green™ Double-Stranded DNA Fluorescence Quantification Kit (Catalog No. Dilute to 100 μL with the buffer in the kit, then dilute the fluorescent substrate in the kit 200 times with the buffer, mix it with the diluted supernatant in equal volume on a black microtiter plate, keep away from light for 5-10 minutes at room temperature, Fluorescent signals with an excitation wavelength of 490 nm and an emission wavelength of 525 nm were read with a fluorescent microplate reader. The results are shown in Table 1, wherein, before the reaction refers to the fluorescent signal of each experimental example before adding anti-double-stranded DNA antibody magnetic beads, and the experimental group refers to each experimental example after adding anti-double-stranded DNA antibody magnetic beads. The fluorescent signal after reaction, the control group refers to the fluorescent signal of each control example after adding the magnetic beads of mouse anti-CD36 monoclonal antibody for reaction. It can be seen from Table 1 that the fluorescence intensity in the supernatant after the adsorption of the anti-double-stranded DNA antibody magnetic beads is very low, which proves that the anti-double-stranded DNA antibody magnetic beads can effectively adsorb double-stranded DNA of different lengths in the solution.

Table 1

100bp 200bp calf thymus before reaction 194 433 1116 test group 9 9 20 control group 149 302 1181

Example 6: Purification of peripheral blood cell-free DNA by anti-double-stranded DNA antibody magnetic beads

Take 2 mL of plasma purchased from the market from which endogenous DNA has been removed, and add 1 μL of 100 bp double-stranded DNA with a concentration of 500 ng/μL to it. Wherein, the nucleotide sequence of this double-stranded DNA is as follows:

5' - CCAGAAGAGGAAACCGG TATGTTTCTTAGTTTTAAATAGTTGCTCTGGAGTCATATGCCTCCTTTCTTCTCATTATTTTTTACATGTTTTAAAAATGCATT-3'; (SEQ ID NO: 5)

Among them, the underlined part is the primer.

This double-stranded DNA was used as a substitute for cfDNA in plasma. After mixing evenly, centrifuge at 4°C and 1600g for 10min with a refrigerated centrifuge, and put the supernatant 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 the final concentration of 2 mg/ml. Use a vortex shaker to disperse the anti-double-stranded DNA antibody magnetic beads into the liquid, and then mix for 30 minutes at room temperature under a roller agitator. Then put the centrifuge tube into the magnetic stand for 2 min until the solution is clear, and discard the supernatant. Add 1mL of PBST buffer to it, cover it with a vortex shaker to disperse and wash the anti-double-stranded DNA antibody magnetic beads for 15 seconds, put the centrifuge tube in the magnetic stand for 2 minutes until the solution is clear, discard the supernatant, and repeat this step 3 Second-rate. Finally, add 100 μL of 0.1M Gly-HCl at pH 3.0 for elution, disperse the anti-double-stranded DNA antibody magnetic beads with a vortex shaker, mix and stir for 5 minutes under a roller stirrer, and then put the centrifuge tube into Put it on the magnetic stand for 2 minutes until the solution is clear, take out the centrifugate into a new tube, and immediately neutralize it with 1/15 volume of 1M pH9.0 Tris-HCl.

Two control examples were set up, and the same double-stranded DNA-containing plasma as above was detected with a Thermo kit and a Qiagen kit, respectively. The Thermo kit is Thermofisher MagMAX ^™ Cell-Free DNA Isolation Kit. The Qiagen kit is Qiagen QIAamp® Circulating Nucleic Acid Kit.

Agarose gel electrophoresis was performed on the DNA extracted in Examples and Comparative Examples. The sample loading volume was 5 ul, and the gel was 1% agarose gel. The results are shown in FIG. 2 . Wherein, Marker is the 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. It can be concluded from Figure 2 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.

It should be understood that the application of the present application is not limited to the above examples, and those skilled in the art may make improvements or changes based on the above descriptions, and all these improvements and changes shall belong to the protection scope of the present application.

Claims (10)

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 as 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, it is used to encode and produce the anti-double-stranded DNA antibody as claimed in claim 1. 3. The nucleotide fragment according to claim 2, wherein the nucleotide sequence encoding the amino acid of the heavy chain variable region is as shown in SEQ ID NO:3. 4. The nucleotide fragment according to claim 2, wherein the nucleotide sequence encoding the amino acid of the light chain variable region is as 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 bead. 6. A method for extracting free DNA from peripheral blood, comprising the following steps: The magnetic beads as claimed in claim 5 are added to peripheral blood serum, and the free DNA in the peripheral blood serum is specifically combined with the anti-double-stranded DNA antibody to form a peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complexes; Washing the peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex with a washing solution to remove the peripheral blood serum; the washed peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex Eluting and separating the free peripheral blood DNA and the magnetic beads to obtain the enriched free peripheral blood DNA. 7. the method for extracting peripheral blood free DNA according to claim 6, is characterized in that, described magnetic bead as claimed in claim 5 is added in the peripheral blood serum, and the final concentration of described magnetic bead is 2mg/ ml. 8. The method for extracting free DNA from peripheral blood according to claim 6, wherein the washing solution is PBST buffer. 9. the method for extracting peripheral blood free DNA according to claim 8 is characterized in that, the process of washing described peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex with washing solution comprises the following steps: Add the PBST buffer, disperse and wash the peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex, put it into a magnetic stand until the solution is clear, and discard the supernatant. 10. the method for extracting peripheral blood free DNA according to claim 6, is characterized in that, the described process of eluting described peripheral blood free DNA-anti-double-stranded DNA antibody magnetic bead complex after washing comprises The following steps: Add Gly-HCl with pH 3.0 for elution, stir to disperse, put in a magnetic stand until the solution is clear, and keep the supernatant.

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