CN109317083B - Application of nano-selenium in preparation of DNA immunoadsorbent - Google Patents
Application of nano-selenium in preparation of DNA immunoadsorbent Download PDFInfo
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Abstract
The invention discloses an application of nano-selenium in preparation of a DNA immunoadsorbent. The invention is based on the discovery that the DNA immunoadsorbent obtained by loading DNA with nano selenium has the advantages of good adsorption performance and better safety performance, and the invention is created. The DNA-loaded nano-selenium immunoadsorbent provided by the invention is obtained based on the application, and has the advantages of no need of pre-flushing, resource saving, high safety and the like; SeNPs are used as a carrier to modify DNA on the surface, and the method has the advantages of large DNA immobilization amount, good adsorption performance and the like. In the DNA immunoadsorbent provided by the invention, DNA molecules are fully contacted with anti-dsDNA antibodies, and the specific binding capacity of the DNA immunoadsorbent can be exerted to the maximum extent.
Description
Technical Field
The invention belongs to the field of blood purification, and particularly relates to application of nano-selenium in preparation of a DNA immunoadsorbent.
Background
Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease with multiple system lesions, the exact cause is unknown, irreversible lesions of multiple organ systems are often caused, skin, serosa, joints, kidneys and central nervous system can be affected, and the pathological manifestations are deposition of autoantibodies and immune complexes, which affect the life span and quality of life of patients. The prevalence rate of SLE varies with the population, and most women are ill in China, especially women of childbearing age. Patients plasma present with nuclear antibodies (ANA), anti double chain DNA antibodies (anti dsDNA antibody) as representative of a variety of antibodies, wherein the anti dsDNA antibodies are involved in SLE pathogenesis of the main antibody, its specificity can reach 90%, SLE specific antibody.
Early clearance of autoantibodies from the plasma of patients, control of epidemic disease activity, and protection of kidney function are key to treatment. The immunoadsorption therapy is a new blood purification technology developed in recent years, which uses an adsorbing material to remove pathogenic antibodies (such as anti-dsDNA antibodies) in plasma, thereby achieving the purpose of treating epidemic diseases. The immune adsorption removes pathogenic factors through antigen-antibody immune reaction or physical and chemical action, mainly adopts plasma adsorption, thereby avoiding the defect of massive plasma discarding in centrifugal type and one-time membrane plasma separation. In recent years, immunoadsorption has been increasingly applied to the treatment of immune diseases.
Among them, the application is more widely DNA immunoadsorption, and the DNA immunoadsorption therapy can specifically adsorb and remove anti-dsDNA antibodies, thereby effectively treating SLE patients. European patent EP0272792A1 discloses DNA immunoadsorbent based on activated carbon or carbonized resin, which uses activated carbon or carbonized resin as carrier, calf thymus DNA (hereinafter referred to as DNA) as ligand and collodion to embed and immobilize calf thymus DNA. However, both activated carbon and carbonized resin have the problem that carbon particles are easy to fall off, so that time is required to be added before use for fully pre-flushing until no particles exist, and serious adverse reactions such as thrombus, allergy, irritation and the like caused by the fact that the particles enter a human body along with blood circulation during treatment are avoided. In addition, there are two disadvantages to the immobilization of DNA by collodion embedding: firstly, partial DNA can fall off in the subsequent production process due to insufficient embedding, so that the DNA immobilization amount is reduced; secondly, the DNA partially embedded too deeply can not contact with the antibody. Therefore, it is a problem to be solved to provide a DNA immunoadsorbent having high adsorption performance and high safety.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the application of nano-selenium in preparing a DNA immunoadsorbent.
The invention also aims to provide the DNA-loaded nano-selenium immunoadsorbent.
The invention further aims to provide a preparation method and application of the DNA-loaded nano-selenium immunoadsorbent.
The purpose of the invention is realized by the following technical scheme: the application of nano-selenium in the preparation of DNA immunoadsorbent is based on the discovery of the inventor that the DNA immunoadsorbent obtained by loading DNA with nano-selenium has the advantages of good adsorption performance and better safety performance.
The application of the nano selenium in the preparation of the DNA immunoadsorbent comprises the following steps: mixing nano selenium and DNA, and reacting to obtain the DNA immunoadsorbent.
The nano selenium is prepared by a redox method, and is preferably prepared by the following steps: mixing inorganic selenium and a reducing agent, and reacting to obtain the nano-selenium.
The inorganic selenium is preferably one or at least two of selenious acid, selenite and selenium dioxide.
The selenite is sodium selenite (Na)2SeO3) Potassium selenite (K)2SeO3) And zinc selenite (ZnSeO)3) One or at least two of them.
The reducing agent is preferably one or at least two of vitamin C, sodium borohydride, mercaptoethanol, reduced glutathione, glucose and sodium thiosulfate pentahydrate.
The dosage of the inorganic selenium and the reducing agent is proper according to the excess of the reducing agent, so that the inorganic selenium can be fully reduced; more preferably, the molar ratio is 1: 3-10.
The immune adsorbent for loading DNA by nano selenium is obtained by realizing the application, and comprises nano selenium and DNA for binding with anti-dsDNA antibodies, wherein the DNA for binding with the anti-dsDNA antibodies is loaded on the nano selenium.
The DNA used for binding to the anti-dsDNA antibody is preferably double stranded DNA.
The nano selenium is prepared by a redox method, and is preferably prepared by the following steps: mixing inorganic selenium and a reducing agent, and reacting to obtain the nano-selenium.
The inorganic selenium is preferably one or at least two of selenious acid, selenite and selenium dioxide.
The selenite is sodium selenite (Na)2SeO3) Potassium selenite (K)2SeO3) And zinc selenite (ZnSeO)3) One or at least two of them.
The reducing agent is preferably one or at least two of vitamin C, sodium borohydride, mercaptoethanol, reduced glutathione, glucose and sodium thiosulfate pentahydrate.
The dosage of the inorganic selenium and the reducing agent is proper according to the excess of the reducing agent, so that the inorganic selenium can be fully reduced; more preferably, the molar ratio is 1: 3-10.
The reaction temperature is preferably 0-10 ℃; more preferably 3-5 ℃; most preferably 4 deg.c.
The mixing mode is preferably as follows: mixing inorganic selenium solution and reducing agent solution; more preferably by mixing as follows: and dropwise adding the inorganic selenium solution into the reducing agent solution, or dropwise adding the reducing agent solution into the inorganic selenium solution.
The concentration of the inorganic selenium solution is preferably 5-15 mM; more preferably 10 mM.
The concentration of the reducing agent solution is preferably 30-50 mM; more preferably 40 mM.
The preparation method of the DNA-loaded nano-selenium immunoadsorbent comprises the following steps: and mixing the DNA solution for combining with the anti-dsDNA antibody and the nano-selenium solution, reacting and purifying to obtain the nano-selenium DNA-loaded immunoadsorbent.
The concentration of the DNA solution for combining with the anti-dsDNA antibody is preferably 1-10 mg/mL; more preferably 1 to 5 mg/mL.
The solvent in the DNA solution for combining with the anti-dsDNA antibody is preferably a buffer solution with the pH value of 7-9; more preferably a buffer solution with a pH value of 7.2-8.2.
The buffer solution is preferably Tris-HCl buffer solution.
The concentration of the Tris-HCl buffer solution is preferably 0.01-0.1M; more preferably 0.01 to 0.05M.
The concentration of the nano selenium solution is preferably 1-10 mM; more preferably 2 to 5 mM.
The amount of the DNA for binding with the anti-dsDNA antibody and the nano-selenium is preferably 0.1-5mg: 1. mu. mol, more preferably 0.5-2mg: 1. mu. mol.
The reaction temperature is preferably 0-10 ℃; more preferably 3-5 ℃; most preferably 4 deg.c.
The reaction time is 0.5-24 h.
The purification mode is preferably dialysis by using a dialysis bag.
The specification of the dialysis bag is preferably 6000-8000 kDa.
The dialysis time is 12-48 h.
The application of the DNA-loaded nano-selenium immunoadsorbent in the preparation of blood purification preparations.
The application of the DNA-loaded nano-selenium immunoadsorbent in preparation of drugs for treating diseases caused by increase of anti-dsDNA antibodies.
Preferably, the disease caused by the increase of the anti-dsDNA antibody is systemic lupus erythematosus.
Compared with the prior art, the invention has the following advantages and effects:
1. the SeNPs carrier in the DNA immunoadsorbent provided by the invention is a beneficial substance which can supplement selenium element for a human body, remove excessive active oxygen free radicals in the body and has high safety. Therefore, the immunoadsorbent of the invention does not need to be flushed in advance, saves resources and improves the safety of the use of the immunoadsorbent.
2. According to the invention, SeNPs are used as carriers, DNA is modified on the surface of the SeNPs, the DNA immobilization amount of the prepared immune adsorbent of nano-selenium loaded DNA reaches 928 mu g/mL, and the immune adsorbent has good adsorption performance.
3. In the DNA immunoadsorbent provided by the invention, DNA molecules are fully contacted with anti-dsDNA antibodies, and the specific binding capacity of the DNA immunoadsorbent is exerted to the maximum extent.
4. The SeNPs of the carrier in the DNA immunoadsorbent provided by the invention also have a certain removing effect on dsDNA antibodies.
Drawings
FIG. 1 is a graph of the characterization of DNA-SeNPs before and after adsorption of anti-dsDNA antibodies in the plasma of a patient; wherein, the graph a is a distribution graph of the nanometer particle size before and after adsorption, the graph b is a potential graph before and after adsorption, and 1-4 in the graphs a and b are DNA-SeNPs after adsorbing No. 61, No. 28, No. 70 and No. 23 case plasma respectively; FIG. c is a graph of ultraviolet spectra before and after adsorption, in which curve 1 is DNA-SeNPs, and curves 2 to 4 are DNA-SeNPs after adsorbing plasma of cases 19, 69 and 109, respectively; FIG. d is an infrared spectrum before and after adsorption, in which curve 1 is DNA-SeNPs, and curves 2 to 4 are DNA-SeNPs after adsorption of plasma from cases 14, 30 and 34, respectively.
FIG. 2 is a transmission electron microscope photograph of DNA-SeNPs before and after adsorption of anti-dsDNA antibodies in the plasma of a patient; in this figure, a is a TEM image of DNA-SeNPs, b is a TEM image of DNA-SeNPs adsorbed to the plasma of case 17, c is a TEM image of DNA-SeNPs adsorbed to the plasma of case 31, and d is a TEM image of DNA-SeNPs adsorbed to the plasma of case 58.
FIG. 3 is an atomic force microscope and corresponding thickness analysis before and after adsorption of anti-dsDNA antibodies in the patient's plasma by DNA-SeNPs; in the figure, a shows an AFM (upper) and a thickness analysis chart (lower) for DNA-SeNPs, b shows an AFM (upper) and a thickness analysis chart (lower) for DNA-SeNPs after adsorbing plasma of 32 cases, and c shows an AFM (upper) and a thickness analysis chart (lower) for DNA-SeNPs after adsorbing plasma of 33 cases.
FIG. 4 is a graph showing the results of adsorption of anti-dsDNA antibodies by DNA-SeNPs, wherein, panel a is a statistical graph showing the clearance of anti-dsDNA antibodies in plasma of 128 cases by DNA-SeNPs; panel b is a statistical plot of anti-dsDNA antibody titers in plasma after clearance of anti-dsDNA antibodies in plasma from 128 cases of DNA-SeNPs; FIG. c is a graph showing the change in the clearance of dsDNA antibodies in the plasma of patients in the same case by DNA-SeNPs, commercial product 1 and commercial product 2; FIG. d is a graph showing the change in the anti-dsDNA antibody titer in plasma of patients in the same case in the course of clearance of anti-dsDNA antibodies in plasma of DNA-SeNPs, commercial product 1 and commercial product 2.
FIG. 5 is a statistical plot of the clearance of anti-dsDNA antibodies in plasma from SeNPs and DNA-SeNPs for 3 cases.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The present invention will be described in further detail below with reference to specific examples and drawings, but the embodiments of the present invention are not limited thereto.
EXAMPLE 1 preparation of DNA-SeNPs nanoparticles
1mL of Na having a concentration of 10mM was added at 4 deg.C2SeO3The solution was placed in a small beaker, 1mL of 40mM vitamin C (Vc, alatin) solution was slowly added, 1mL of 5mg/mL DNA (D4522, Sigma) Tris-HCl solution (0.05M, pH 7.2) was added after the dropwise addition, the volume was adjusted to 5mL with ultrapure water, the reaction was carried out at 4 ℃ for 12 hours, and the solution was dialyzed in ultrapure water for 24 hours with a dialysis bag (6000 to 8000kDa) to obtain DNA-SeNPs nanoparticles.
Preparation of SenPs: 1mL of Na having a concentration of 10mM was added at 4 deg.C2SeO3Putting the solution into a small beaker, slowly adding 1mL of vitamin C with the concentration of 40mM, then adding ultrapure water to a constant volume of 5mL, reacting for 12h at 4 ℃, and dialyzing for 24h in the ultrapure water by using a dialysis bag (6000-8000 kDa) to obtain the SeNPs nano particles.
Example 2 characterization of DNA-SeNPs before and after adsorption of anti-dsDNA antibodies
mu.L of each of the DNA immunoadsorbents prepared in example 1 was added to 300. mu.L of plasma (from 128 SLE cases, respectively) and adsorbed for 2h at room temperature (20rpm, tube spinner mixer, TZL-5010, Perceval laboratories, Inc., Suzhou). The DNA-SeNPs before and after the adsorption of the anti-dsDNA antibody were characterized by particle size and potential (Malvern particle sizer), ultraviolet spectrophotometer, infrared spectrophotometer, Transmission Electron Microscope (TEM) and Atomic Force Microscope (AFM), and the results are shown in FIGS. 1-3. The hydrated particle size of the DNA-SeNPs before adsorption was about 126nm, and after adsorbing 4 patients' plasma (supplied from three houses in Zhongshan), they were centrifuged (12000rpm, 10min), the supernatant was removed, resuspended, and the hydrated particle size of the DNA-SeNPs after adsorption was increased to 189nm (case 1: titer of anti-dsDNA antibody before adsorption 563), 254nm (case 2: titer of anti-dsDNA antibody before adsorption 353), 405nm (case 3: titer of anti-dsDNA antibody before adsorption 600) and 259nm (case 4: titer of anti-dsDNA antibody before adsorption 336), respectively, as measured (FIG. 1 a). However, since the surface potentials of the anti-dsDNA antibody and DNA-SeNPs are similar, there is no significant change in the surface potentials of the DNA-SeNPs before and after adsorption (FIG. 1 b). The UV-visible and IR spectra of the DNA-SeNPs before and after adsorption were identical (FIGS. 1c and d). TEM images of DNA-SeNPs before adsorption appeared as monodisperse spherical nanoparticles with a size of about 107nm, and a layer of modification of the DNA was clearly visible on the surface (FIG. 2 a). After adsorption of the patient's plasma, the DNA-SeNPs were essentially unchanged in size, but the DNA layer on the nanoparticle surface was thickened (FIGS. 2b-d, corresponding to cases 17, 31 and 58, respectively). The AFM results showed a significant increase in the height of the DNA-SeNPs after adsorption (FIG. 3). The above results all indicate that anti-dsDNA antibodies are adsorbed to the nanoparticle surface.
Example 3 evaluation of the ability of DNA-SeNPs to adsorb anti-dsDNA antibodies
The plasma after adsorption in example 2 was subjected to enzyme-linked immunosorbent assay kit (QUANTA)
dsDNA ELISA, 708510, Inova Diagnostics, a Werfen Company, detection procedures as per instructions) quantitative detection of anti-dsDNA antibodies was performed, and finally values were read at 450nm and 620nm with a microplate reader. Sample value ═ sample OD450nmSample OD620nm) /(dsDNA ELISA calibrator OD450nm-dsDNA ELISA calibrator OD620nm)*375. The clearance rate of anti-dsDNA antibodies in the sample was calculated as follows: anti-dsDNA antibody clearance ═ (pre-adsorption sample number-post-adsorption sample number)/pre-adsorption sample number 100%. As shown in FIGS. 4 a-4 b, the clearance of anti-dsDNA antibodies by DNA-SeNPs was greater than 50% in most of the 128 cases, and the titer of anti-dsDNA antibodies in plasma was significantly reduced after adsorption of DNA-SeNPs to plasma.
1mL of each of the DNA-SeNPs prepared in example 1 and 40mg of a commercially available DNA immunoadsorbent was added to 2mL of plasma from the same 1 SLE patient, the mixture was shaken at room temperature (20rpm, a tube rotation mixer, TZL-5010, Perceval laboratories, Inc., Suzhou) for 2 hours of adsorption, samples were taken every 15min, the plasma before and after adsorption was subjected to quantitative detection against dsDNA antibody using an enzyme linked immunosorbent assay kit, and the values were read at 450nm and 620nm using an microplate reader. As shown in FIGS. 4 c-4 d, it can be seen that when the immunoadsorbent of DNA loaded with nano-selenium provided by the present invention is compared with commercially available immunoadsorbent products (commercially available product 1: DNA immunoadsorbent column, carbonized resin immobilized calf thymus DNA; commercially available product 2: selective plasma component adsorber, phenylalanine immobilized polyvinyl alcohol gel), the clearance of DNA-SeNPs reaches 75% at 15min, whereas commercially available product 1 has no substantial clearance within two hours, and the clearance of commercially available product 2 reaches 35% at two hours. Changes in anti-dsDNA antibody titers in plasma were consistent with changes in clearance.
mu.L of SeNPs (prepared in example 1) and 150. mu.L of DNA-SeNPs were added to 300. mu.L of plasma (from 3 SLE cases, repeat number 3), mixed at room temperature at 20rpm, adsorbed for 2h, and the clearance of anti-dsDNA antibodies in the adsorbed plasma was measured as described above. The results are shown in FIG. 5, where the SeNPs alone also had some clearance effect against the dsDNA antibody.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (6)
1. The application of nano selenium in the preparation of DNA immunoadsorbent is characterized by comprising the following steps: mixing a DNA solution for combining with the anti-ds DNA antibody and a nano-selenium solution, reacting and purifying to obtain the DNA-loaded nano-selenium immunoadsorbent;
the concentration of the DNA solution for combining with the anti-dsDNA antibody is 1-10 mg/mL;
the concentration of the nano selenium solution is 1-10 mM;
the dosage of the DNA used for combining with the anti-dsDNA antibody and the nano selenium is 0.1-5mg:1 mu mol;
the reaction temperature is 0-10 ℃;
the reaction time is 0.5-24 h;
the nano selenium is prepared by the following steps: mixing inorganic selenium and a reducing agent, and reacting to obtain nano selenium;
the inorganic selenium is one or at least two of selenious acid, selenite and selenium dioxide;
the reducing agent is one or at least two of vitamin C, sodium borohydride, mercaptoethanol, reduced glutathione, glucose and sodium thiosulfate pentahydrate;
the dosage of the inorganic selenium and the reducing agent is calculated according to the molar ratio of 1: 3-10.
2. A DNA-loaded nano-selenium immunoadsorbent, which is characterized in that: the application of the nano-selenium in preparing a DNA immunoadsorbent of claim 1 is realized, the nano-selenium comprises nano-selenium and DNA for binding with anti-dsDNA antibodies, and the DNA for binding with the anti-dsDNA antibodies is loaded on the nano-selenium;
the preparation method comprises the following steps: mixing a DNA solution for combining with the anti-dsDNA antibody and a nano-selenium solution, reacting and purifying to obtain the DNA-loaded nano-selenium immunoadsorbent;
the concentration of the DNA solution for combining with the anti-dsDNA antibody is 1-10 mg/mL;
the concentration of the nano selenium solution is 1-10 mM;
the dosage of the DNA used for combining with the anti-dsDNA antibody and the nano selenium is 0.1-5mg:1 mu mol;
the reaction temperature is 0-10 ℃;
the reaction time is 0.5-24 h.
3. The DNA-loaded nanoselenium immunoadsorbent of claim 2, wherein:
the solvent in the DNA solution for combining with the anti-dsDNA antibody is a buffer solution with the pH value of 7-9;
the purification mode is dialysis by using a dialysis bag.
4. Use of the DNA-loaded nano-selenium immunoadsorbent of claim 2 in the preparation of a blood purification formulation.
5. Use of the DNA loaded nanoselenium immunoadsorbent of claim 2 in the manufacture of a medicament for the treatment of disease caused by elevated anti-dsDNA antibodies.
6. Use of the nanoselenium-loaded DNA immunoadsorbent of claim 5 in the manufacture of a medicament for the treatment of diseases caused by elevated anti-dsDNA antibodies, wherein: the disease caused by the increase of the anti-dsDNA antibody is systemic lupus erythematosus.
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| PCT/CN2019/115559 WO2020093989A1 (en) | 2018-11-05 | 2019-11-05 | Dna immunoadsorbent using nano-selenium as adsorbent carrier, preparation method therefor, and application thereof |
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| CN102183662A (en) * | 2011-03-22 | 2011-09-14 | 浙江大学 | Method for establishing colon cancer prognosis prediction model |
| CN103616503A (en) * | 2013-11-05 | 2014-03-05 | 辛竹 | Method for detecting anti-dsDNA antibody |
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| CN101165491A (en) * | 2006-10-19 | 2008-04-23 | 陕西西大北美基因股份有限公司 | Method for detecting double-chain DNA resistant antibody using gold magnetic particle as carrier |
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