A method for broad-spectrum separation of antibodies using magnetic nano-mycoplasma protein

Abstract

The present application discloses a method for broad-spectrum separation of antibodies using magnetic nano-mycoplasma proteins, which specifically relates to the field of antibody purification methods. The method comprises the following steps: S01, preparation of magnetic nano-materials Fe3O4@SiO2–PIDA–Ni 2+ S02, expression and purification of mycoplasma protein M; S03, immobilization of mycoplasma protein M on NiNP. S04, separation and purification of target antibody by MNiNP. This method can achieve the advantages of one material method for separating different types of antibody proteins.

Description

Technical Field

[0001] The present application relates to the field of antibody purification methods, and in particular to a method for broad-spectrum separation of antibodies using magnetic nano-mycoplasma proteins. Background Art

[0002] Antibodies are produced by B cells on the plasma membrane and have a similar structure, consisting of two heavy chains and two light chains. The production of recombinant antibodies faces many challenges. These modifications include: recombinant expression, purification, and possible post-translational modifications. The purification of recombinant antibodies is one of the most important production steps, and the purification effect directly affects the final application.

[0003] To date, many antibody purification systems based on antibody binding proteins have been constructed for antibody purification, such as protein A, protein G, protein L and protein M. Among them, protein M has an obvious advantage over other antibody binding proteins: it can bind to both κ and λ chains at the same time, and can be widely applied to immunoglobulin G (IgG), immunoglobulin (IgA), immunoglobulin M (IgM) and its derivatives such as antigen binding fragment (Fab), bivalent antibody fragment F(ab′)2 or single-chain variable fragment (scFv), but protein M is the least studied of all antibody binding proteins because when protein M was discovered, it was shown that its binding to antibodies was irreversible or required very harsh conditions.

[0004] In order to improve protein stability, activity and reusability, protein immobilization technology has been applied to different fields, such as biocatalysis, toxin adsorption, biosensing and other important industries. Traditional immobilization materials include nanoparticles (NPs), nanoflowers (NFs), porous carbohydrate-based materials, covalent organic frameworks (COFs) and metal organic frameworks (MOFs). However, the above-mentioned immobilization materials all have the following problems: low biocompatibility, poor stability and difficulty in separation from the system. At the same time, when applying the above-mentioned materials, it is difficult to control how to prevent the solid materials from blocking the active sites of the protein.

[0005] Regarding magnetic nanomaterials, China's invention patent application No. 2024105132405 discloses a method for preparing Fe3O4@SiO2@EDTA-PDA magnetic nanomaterials, which prepares Fe3O4 nanoparticles with a core-shell structure for extracting malachite green from samples. However, the above-mentioned magnetic nanomaterials, after chelating EDTA-PDA, are prone to ion exchange with antibodies and destroy the structure of the antibodies.

[0006] At the same time, China's invention patent application number 2021116455685 discloses a highly selective core-shell magnetic nanoparticle for antibody separation and a preparation method thereof. In this scheme, ferroferric oxide / silicon oxide nanoparticles are designed to be coupled with IgG2a and coated on their surface. Subsequently, by blocking nonspecific binding sites and removing IgG2a, the roughness of the surface of the magnetic nanoparticles is increased, and good binding ability with antibodies can be achieved. However, in the process of applying the above-mentioned magnetic particles to separate and purify antibodies, only IgG can be separated, and antibodies of different sizes such as immunoglobulin A (IgA), immunoglobulin M (IgM), antigen binding fragment (Fab), bivalent antibody fragment F (ab') 2 and single-chain variable fragment (scFv) cannot be separated and purified. This method has great limitations during use. Therefore, how to design a broad-spectrum antibody separation method is an urgent problem for technicians. Summary of the invention

[0007] The present invention provides a method for separating antibodies using magnetic nano-mycoplasma proteins in a broad spectrum, which can achieve the advantage of separating different types of antibody proteins using a single material method.

[0008] The objective of the present invention is achieved through the following technical solutions:

[0009] A method for separating antibodies using magnetic nano-mycoplasma protein in a broad spectrum, the method comprising the following steps:

[0010] S01, Magnetic Nanomaterials Fe3O4@SiO2–PIDA–Ni 2+ The synthesis is denoted as NiNP;

[0011] S02, expression and purification of mycoplasma protein M;

[0012] S03, Mycoplasma protein M in magnetic nanomaterials Fe3O4@SiO2–PIDA–Ni 2+ The immobilization on is denoted as MNiNP;

[0013] S04. Separating the MNiNP adsorbed with the target antibody by an external magnet, eluting, and obtaining the purified target antibody; the MNiNP can be reused after the above elution.

[0014] Preferably, the synthesis step of NiNP in step S01 comprises:

[0015] S01.1. Synthesis of Fe3O4 nanoparticles

[0016] Dissolve FeCl3·6H2O and FeCl2·4H2O in deionized water to obtain a mixed solution;

[0017] Under argon protection, ammonia water was added dropwise to the mixed solution, and the mixture was stirred at 250 rpm for 10-12 h at 60-65 °C to obtain crude Fe3O4 nanoparticles;

[0018] The crude Fe3O4 nanoparticles were collected by an external magnet, washed with deionized water until neutral, and then washed with a 50% ethanol solution for 10-15 times, and vacuum dried at 60°C for 12-24 h to obtain the Fe3O4 nanoparticles;

[0019] S01.2. Synthesis of Fe3O4@SiO2

[0020] The Fe3O4 nanoparticles were dispersed in ethanol, and deionized water and ammonia water were added. 1.5 mL of tetraethyl silicate was added dropwise under ultrasonic conditions at room temperature for 30 min, and then ultrasonicated for 15 min.

[0021] The mixture obtained above was stirred at 300 rpm at 25 °C for 12 h to obtain a crude Fe3O4@SiO2 product;

[0022] The crude Fe3O4@SiO2 was collected by an external magnet, washed with deionized water for 10-15 times, and vacuum dried at 60°C for 12-24 h to obtain Fe3O4@SiO2;

[0023] S01.3. Synthesis of Fe3O4@SiO2–APTMS

[0024] Fe3O4@SiO2 was dispersed in toluene, 3-aminopropyltrimethoxysilane was added dropwise, and the mixture was refluxed at 95 °C for 12 h under argon protection to obtain a crude Fe3O4@SiO2–APTMS product;

[0025] The crude Fe3O4@SiO2–APTMS was collected by an external magnet, washed with pure methanol for 3–5 times, and dried under vacuum at 60 °C for 12 h to obtain Fe3O4@SiO2–APTMS;

[0026] S01.4, Fe3O4@SiO2–PIDA–Ni 2+ Synthesis

[0027] The obtained Fe3O4@SiO2-APTMS was dispersed in a mixed solution of ethanol and triethylamine in which sodium chloroacetate was dissolved, and refluxed at 80°C for 12 h under argon protection to obtain a crude Fe3O4@SiO2-PIDA product;

[0028] The crude Fe3O4@SiO2-PIDA was washed with pure methanol and deionized water for 5-10 times to obtain pure Fe3O4@SiO2-PIDA;

[0029] Pure Fe3O4@SiO2-PIDA was dispersed in NiSO4·6H2O solution and shaken at 25℃ for 12 h to obtain Fe3O4@SiO2–PIDA–Ni 2+ crude product;

[0030] Fe3O4@SiO2–PIDA–Ni 2+ The crude product was washed with deionized water for 3-5 times to obtain pure Fe3O4@SiO2–PIDA–Ni 2+ .

[0031] Preferably, the above step S02 is specifically as follows:

[0032] S02.1. Transform the pET28a-M-8his plasmid into Escherichia coli cells, spread the Escherichia coli cells on an LB plate containing 50 μg / mL kanamycin, and incubate at 37°C overnight to obtain Escherichia coli colonies;

[0033] S02.2. Take a single E. coli colony and transfer it to 5 mL of culture medium containing 50 μg / mL kanamycin. Incubate overnight at 37°C and 200 rpm.

[0034] S02.3, transfer the culture obtained above into 2YT medium and incubate until OD 600 At 0.5-0.6, isopropyl-β-D-thiogalactoside was added to promote induction, and the mixture was incubated at 20°C and 200 rpm for 16-18 h to obtain Escherichia coli colonies that could efficiently express the target protein;

[0035] S02.4, collect the above-mentioned E. coli cells, centrifuge at 4°C and 8000 rpm for 10 min, resuspend with buffer solution, and lyse by high-pressure homogenization to obtain cell lysate;

[0036] S02.5. The cell lysate was centrifuged at 8000 rpm for 1 h at 4°C, and the supernatant was filtered with a 0.22 μm filter membrane to obtain a solution containing mycoplasma protein M.

[0037] Preferably, in step S02.1, the amino acid sequence of the pET28a-M-8his plasmid is SEQ ID NO: 1:

[0038] MGSSHHHHHHSSGLVPRGSHMSLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGHYDHVASDGSLLSYHQS TEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSFSGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLII RGLSGNGSQLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVIYDLHASKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDI YNYRRFERQFQGHHAGGHIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAHREGDNTYYRVNENYHPGASIYENERASRDSEFQNEILKRGGSGGSWSHPQFEKC.

[0039] Preferably, in step S02.3, the YT medium is prepared from the following components: 1000 mL of distilled water, 5.0 g of sodium chloride, 10 g of yeast extract, and 16 g of peptone.

[0040] Preferably, in step S02.4, the centrifugation conditions of the Escherichia coli colonies are: rotation speed 8000 rpm, temperature 4°C, centrifugation time 10 min; and the binding buffer is 5 mM imidazole buffer.

[0041] Preferably, in step S02.5, the centrifugation conditions of the cell lysate are a speed of 8000 rpm, a temperature of 4°C, and a centrifugation time of 1 h;

[0042] The elution conditions of the cell lysate were as follows: the Ni-NTA-agarose column was precalibrated with 2 column volumes of binding buffer, then washed with 5 column volumes of wash buffer, and finally the protein was eluted with elution buffer;

[0043] The washing buffer was 20 mM imidazole buffer, and the elution buffer was 250 mM imidazole buffer.

[0044] Preferably, the above step S03 is specifically as follows:

[0045] S03.1. Put Fe3O4@SiO2–PIDA–Ni 2+ Wash 1-2 times with deionized water and then with mycoplasma protein M solution;

[0046] S03.2, add 1% Fe3O4@SiO2–PIDA–Ni to the mycoplasma protein M solution 2+ The immobilized mycoplasma protein M was prepared by ultrasonication at 4°C for 5-40 min and external magnetic separation, which was recorded as MNiNP.

[0047] Preferably, step S04 is specifically as follows:

[0048] S04.1 Disperse the E. coli containing the target antibody to be purified into the binding buffer, lyse the cells and release the target antibody by high-pressure homogenization, add 1-2% MNiNP by mass ratio, shake at 4°C, and after 5-40 minutes, separate the MNiNP by an external magnet, and at the same time separate the target antibody from the cell lysate;

[0049] S04.2 Re-disperse the separated MNiNPs into the elution buffer, maintain the MNiNP concentration at 2-4%, shake at room temperature for 5 min, release the target antibody from the MNiNPs, separate the MNiNPs by an external magnet, and obtain the purified target antibody; the MNiNPs are reused after separation.

[0050] Preferably, in step S04.1, the antibodies available for separation and purification of magnetic nanomaterials include: antibody S2P6, antibody 76E1 and trastuzumab scFv. The binding buffer is a mixture of 300 mM NaCl, 50 mM NaH2PO4 and 5 mM imidazole.

[0051] Preferably, in step S04.2, the elution buffer is a citric acid buffer, wherein the concentration of the citric acid buffer is 0.1 M.

[0052] Compared with the prior art, the advantages or beneficial effects of the technical solution of the present application include:

[0053] 1. This method can replace the traditional affinity chromatography antibody purification technology and achieve antibody purification without using purification tag expression.

[0054] 2. This method can use mycoplasma protein M to replace traditional protein A, G, L and other selective antibody binding proteins to achieve the purpose of separating different types of antibody proteins with one material method.

[0055] 3. Antibody proteins of different molecular weights can be separated simultaneously.

[0056] 4. The extraction effect of different antibodies can be improved through parameter optimization, which is universal. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Flowchart for nanomaterial preparation;

[0058] Figure 2 TEM image of Fe3O4 superparamagnetic nanoparticles;

[0059] Figure 3 for Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2-APTMS, Fe3O4@SiO2-PIDA and Fe3O4@SiO2-PIDA-Ni 2+ XRD pattern;

[0060] Figure 4 for Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2-APTMS, Fe3O4@SiO2-PIDA and Fe3O4@SiO2-PIDA-Ni 2+ Decomposition rate TGA spectrum;

[0061] Figure 5 The data diagram of fixation and elution of mycoplasma protein M;

[0062] Figure 6 This is a diagram showing the relationship between mycoplasma protein M and antibodies;

[0063] Figure 7 This is the analysis diagram of MNiNPs stability and loading capacity;

[0064] Figure 8 Non-reducing SDS-PAGE of mycoplasma protein M eluted from NiNP and MALDI-TOF MS (mass spectrometry) analysis of mycoplasma protein M eluted fractions;

[0065] Fig. 9 This is a picture of the binding of magnetic beads to mycoplasma protein M;

[0066] Fig.10 This is a picture of magnetic beads and mycoplasma protein M after elution;

[0067] Fig.11 is the MALDI-TOF mass spectrum;

[0068] Fig.12 is the adsorption capacity of MNiNP for IgGs and Fabs;

[0069] Fig.13 This is a statistical diagram of the binding ability of MNiNP and scFv. DETAILED DESCRIPTION

[0070] The following will describe the implementation methods of the present application in detail with reference to the accompanying drawings and embodiments, so that the implementation process of how the present application applies technical means to solve technical problems and achieve corresponding technical effects can be fully understood and implemented accordingly. The embodiments of the present application and the various features in the embodiments can be combined with each other without conflict, and the technical solutions formed are all within the protection scope of the present application.

[0071] It should be clear that the embodiments described below are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by technicians in the relevant field without making creative work are within the scope of protection of the present application. Example 1

[0072] This embodiment discloses a magnetic nanomaterial for antibody purification and a preparation method thereof, specifically:

[0073] 1. Reagents.

[0074] Ferric chloride hexahydrate (FeCl3·6H2O, ≥99.0%), ferrous chloride tetrahydrate (FeCl2·4H2O, ≥99.5%), ammonia (NH3·H2O, 25.0–28.0%), 3-aminopropyltrimethoxysilane (APTMS, 99.0%), tetraethyl silicate (TEOS, ≥99.0%), and sodium chloroacetate (≥99.0%) were purchased from Shanghai MacLean Biochemical Co., Ltd. (Shanghai, China). Nickel sulfate hexahydrate (NiSO4·6H2O) and isopropyl-β-D-thiogalactopyranoside (IPTG, ≥99.0%) were purchased from Aladdin Co., Ltd. (Shanghai, China). All the above organic solvents were of analytical grade and used without further purification.

[0075] 2. Synthesis of Fe3O4 nanoparticles.

[0076] Fe3O4 nanoparticles were synthesized by coprecipitation method: 5.4 g FeCl3·6H2O and 2.98 g FeCl2·4H2O were dissolved in 100 mL deionized water in a 250 mL three-necked flask. Under argon protection, 25 mL ammonia water was slowly added to the solution, stirred at 250 rpm for 12 h at 60°C, and Fe3O4 nanoparticles were collected by external magnet, washed with deionized water until neutral, and washed with 50% ethanol solution for 10-15 times. Stored in 50% ethanol solution, vacuum dried at 60°C for 12 h, and used for later use.

[0077] 3. Synthesis of Fe3O4@SiO2.

[0078] Fe3O4@SiO2 was prepared by sol-gel method: 0.5 g Fe3O4 nanoparticles were dispersed in 50 mL ethanol in a 100 mL three-necked flask, 10 mL deionized water and 2.5 mL ammonia were added, 1.5 mL tetraethyl silicate was added dropwise under ultrasonic conditions at room temperature for 30 min, and then ultrasonicated for 15 min. Finally, the obtained mixture was stirred at 300 rpm at 25 °C for 12 h. Fe3O4@SiO2 was magnetically separated and washed with deionized water for 10-15 times. Fe3O4@SiO2 was stored in deionized water, dried under vacuum at 60 °C for 12 h, and then used.

[0079] 4. Synthesis of Fe3O4@SiO2–APTMS.

[0080] The preparation method of Fe3O4@SiO2-APTMS is as follows: 0.5 g of Fe3O4@SiO2 is dispersed in 16 mL of toluene in a 50 mL three-necked flask, 0.85 mL of 3-aminopropyltrimethoxysilane (APTMS) is added dropwise, and the mixture is refluxed at 95°C for 12 h under argon protection to obtain Fe3O4@SiO2-APTMS. After magnetic separation, Fe3O4@SiO2-APTMS is washed with pure methanol for 3-5 times. Fe3O4@SiO2-APTMS is stored in pure methanol, dried under vacuum at 60°C for 12 h, and then used.

[0081] 5. Fe3O4@SiO2–PIDA–Ni 2+ synthesis.

[0082] Fe3O4@SiO2-PIDA was synthesized by dispersion method: 0.5 g Fe3O4@SiO2-APTMS was dispersed in 15 mL ethanol and 10 mL triethylamine solution containing 1.28 g sodium chloroacetate in a 50 mL three-necked flask. Fe3O4@SiO2-PIDA was obtained by reflux at 80 °C for 12 h under argon protection, and washed with pure methanol and deionized water for 5-10 times, respectively. 0.5 g Fe3O4@SiO2-PIDA was dispersed in 20 mL 0.1 M NiSO4·6H2O solution and shaken at 25 °C for 12 h to obtain Fe3O4@SiO2–PIDA–Ni 2 + Fe3O4@SiO2–PIDA–Ni 2+ Wash with deionized water for 3-5 times, store in 20% ethanol as a suspension (0.1 g / mL) for later use. 2+ In this paper, it is represented by NiNP. Example 2

[0083] This example discloses the preparation of immobilized mycoplasma protein M.

[0084] 1. Expression and purification of mycoplasma protein M.

[0085] Expression and purification of mycoplasma protein M mutant: Transform the pET28a-M-8his plasmid into E. coli BL21 (DE3). Spread the colonies on LB plates containing 50 μg / mL kanamycin and incubate at 37°C overnight. Take a single colony and transfer it to 5 mL of culture medium (LB medium) containing 50 μg / mL kanamycin and incubate it at 37°C, 200 rpm overnight.

[0086] Then, the resulting culture was transferred to 400 mL of 2YT medium (composition: 5.0 g / L sodium chloride, 10 g / L yeast extract, 16 g / L peptidone). The above culture medium was divided into two 500 mL flasks and incubated until OD 600 At 0.5-0.6, IPTG (62.5 μL, 0.8 M) was added to promote induction and incubated at 20°C, 200 rpm for 16-18 h. During the incubation period, the culture was centrifuged at 8000 rpm, 4°C for 10 min and resuspended in binding buffer (5 mM imidazole buffer, pH 7.7) to ensure that the culture was evenly distributed in the medium.

[0087] After the culture was completed, the cells were lysed with a high-pressure homogenizer at 600 bar for 5-7 min. The cell lysate was centrifuged at 8000 rpm and 4°C for 1 h. After the treatment, the supernatant was collected and filtered using a 0.22 μm filter. The Ni-NTA-agarose column was pre-calibrated with 2 column volumes of binding buffer. The protein was washed with 5 column volumes of washing buffer (20 mM imidazole buffer, pH 7.7) and finally eluted with elution buffer (250 mM imidazole buffer, pH 7.7). The purity was determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the concentration was determined by Bradford method (Coomassie Brilliant Blue method).

[0088] 2. Immobilization and elution of mycoplasma protein M on NiNPs.

[0089] In order to immobilize the mycoplasma protein M mutant on NiNP, NiNP was washed several times with deionized water, mycoplasma protein M binding buffer, and 5 mM imidazole buffer (pH 7.7).

[0090] 500 μL of mycoplasma protein M cell lysate from 8 samples and 5 mg of NiNP were ultrasonicated for 5-40 min at 4°C to obtain immobilized mycoplasma protein M at different times, which was recorded as MNiNP. MNiNP was separated by an external magnet, washed several times with washing buffer, and stored in phosphate buffered saline.

[0091] MNiNPs were separated by an external magnet, washed with deionized water and 20% ethanol solution, and stored as a suspension (0.1 mg / mL) in 20% ethanol solution. The purity of the eluate was determined by SDS-PAGE, and its concentration was determined by Bradford method. The purpose of the above test was to test the loading rate and reusability of NiNP for mycoplasma protein M. Example 3

[0092] 1. Purification of trastuzumab scFv

[0093] Transform the pET28a-trastuzumab scFv plasmid into E. coli BL21 (DE3). Spread the colonies on LB plates containing 50 μg / mL kanamycin and incubate at 37°C overnight. Take a colony and transfer it to 5 mL LB containing 50 μg / mL kanamycin and incubate it at 37°C, 200 rpm overnight. Then, transfer the culture to 400 mL LB medium and divide it into two 500 mL bottles to measure the OD 600 value.

[0094] When the culture concentration reached 0.5-0.6, add isopropyl-β-D-thiogalactoside (IPTG, 50 μL, 0.8 M) to promote induction, incubate at 25°C, 200 rpm for 14-16 h, centrifuge at 8000 rpm, 4°C for 10 min, and resuspend in binding buffer (5 mM imidazole buffer, pH 7.7).

[0095] After the culture was completed, the cells were homogenized at 300 bar for 5 min, and the cell lysate was centrifuged at 8000 rpm for 1 h at 4°C to collect the supernatant and filter it with a filter with a pore size of 0.22 μm. In a 1.5 mL centrifuge tube, 1 mL of filtered cell lysate and 5 mg of MNiNP were added and incubated at 4°C for different times (5-40 min) with regular shaking (once every 5-7 min).

[0096] The immobilized trastuzumab scFv was magnetically separated and eluted with 0.8 mL of citric acid buffer (0.1 M, pH 3.5-6) ​​at room temperature for 5 min with gentle shaking. Subsequently, the MNiNPs were separated with an external magnet, and the pH value of the elution solution was calibrated to 7.4-7.6 by adding an appropriate amount of Tris-HCl buffer solution (1 M, pH 8.5).

[0097] 2. Purification of S2P6 and 76E1

[0098] The purification process of S2P6 IgG, Fab, 76E1 IgG and Fab was as follows: 300 μL of cell lysate was mixed with 5 mg of MNiNP in a 1.5 mL centrifuge tube. The suspension was incubated at 4 °C for different times (20-80 min) with regular shaking (every 5-7 min). The cell lysate was then separated and the MNiNP containing the immobilized antibody was washed 3-4 times with 1 mL of PBS (pH 7.2-7.4). The washing step was performed because IgGs and Fabs contained significant concentrations of bovine serum albumin in the cell lysate. The antibody was then eluted with 200 μL of citrate buffer (0.1 M, pH 4.5) with gentle shaking at room temperature for 5 min. The MNiNP was separated with an external magnet and the pH of the elution solution was calibrated to 7.4-7.6 by adding 45 μL of Tris-HCl buffer (1 M, pH 8.5). Example 4

[0099] In this example, the antibody loading capacity of MNiNP was calculated by formula 1:

[0100] (1)

[0101] Where Qe (mg g −1 ) represents the loading capacity of MNiNPs, V (L) represents the total volume of elution, Co-Ct represents the elution concentration of mycoplasma protein M or antibody in mg / L, and m (g) represents the mass of MNiNPs. Example 5

[0102] This example tests the stability of MNiNPs.

[0103] A 0.1 g / mL MNiNP suspension was stored in PBS buffer solution (50 mM, pH 7.4) for 15 days. Every 3 days, 5 mg of MNiNP suspension was taken, washed with deionized water and trastuzumab scFv binding buffer, and dispersed in 1 mL of trastuzumab scFv cell lysate. The suspension was incubated at 4 °C for 30 min with regular shaking (every 5-7 min). The immobilized trastuzumab scFv was magnetically separated and gently eluted with 0.8 mL of citric acid buffer (0.1 M, pH 4.5) for 5 min at room temperature. The MNiNPs were then separated with an external magnet, and the pH of the eluted solution was calibrated to 7.4 by adding an appropriate amount of Tris-HCl buffer solution (1 M, pH 8.5). The MNiNPs were washed three times with PBS buffer solution (50 mM, pH 7.4) and stored in the same buffer. The antibody concentration was determined by Bradford method. Example 6

[0104] This example tests the reusability of MNiNPs. The MNiNP reusability test was performed using trastuzumab purification.

[0105] 5 mg of MNiNPs were washed with deionized water and trastuzumab scFv binding buffer and dispersed in 1 mL of trastuzumab scFv cell lysate. The suspension was incubated at 4 °C for 30 min with regular shaking (every 5-7 min). The immobilized trastuzumab scFv was magnetically separated and gently eluted with 0.8 mL of citric acid buffer (0.1 M, pH 4.5) at room temperature for 5 min. The MNiNPs were then separated with an external magnet, and the pH of the elution solution was calibrated to 7.4 by adding an appropriate amount of 1M Tris-HCl buffer solution (pH 8.5). The MNiNPs were washed three times with PBS buffer solution (50 mM, pH 7.4), and the above experiment was repeated 10 times using the same MNiNPs. Example 7

[0106] This example optimizes the loading capacity of mycoplasma protein M on NiNP.

[0107] Mycoplasma protein M cell lysate was diluted with Mycoplasma protein M binding buffer with dilution factors of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5. Eight different dilutions of Mycoplasma protein M cell lysate (1 mL) were pre-calibrated with deionized water and Mycoplasma protein M binding buffer, and 10 mg of NiNPs were immobilized under sonication at 4°C for 25 min. Subsequently, MNiNPs were separated with an external magnet, washed several times with washing buffer, and stored in phosphate buffered saline (pH 7.7). To test the purity and Mycoplasma protein M loading capacity, MNiNPs of each sample were eluted with elution buffer under sonication at 4°C for 30 min. 1 mL of trastuzumab scFv cell lysate was pre-calibrated with deionized water and binding buffer, and MNiNP (5 mg) samples were added to 8 1.5 mL centrifuge tubes. The suspension prepared above was incubated at 4°C for 30 min with regular shaking (once every 5-7 min).

[0108] The immobilized trastuzumab scFv was magnetically separated and gently eluted with 0.8 mL of citric acid buffer (0.1 M, pH 4.5) at room temperature for 5 min. Subsequently, the MNiNPs were separated with an external magnet, and an appropriate amount of Tris-HCl buffer solution (1 M, pH 8.5) was added to calibrate the pH value of the elution solution to 7.4-7.6. Finally, the optimal mycoplasma protein M loading amount was determined by the concentration of the trastuzumab scFv eluate. Example 8

[0109] This example analyzes the loading and elution effects of immobilized Mycoplasma protein M by CLSM (confocal laser scanning microscopy).

[0110] 0.5 mL of MNiNP suspension (0.1 g / mL) in PBS buffer solution (pH 7.4) was placed in a 1.5 mL centrifuge tube and sonicated for 3 min, and then 0.18 mL of the sample was transferred to a new 1.5 mL centrifuge tube.

[0111] By using a pipette tip, a soybean-sized sample was picked up and placed on a glass slide. 20 μL of 0.02% FITC (fluorescein isothiocyanate, purchased from Shanghai MacLean Biochemical Co., Ltd.) was added and stored in the dark at room temperature for 30 minutes. Subsequently, 20 μL of the stained sample was placed on a slide and observed at a magnification of 3000 times on a confocal microscope (Olympus FV3000). The Ex / Em (fluorescence excitation spectrum / emission spectrum) of FIT (fluorescein isothiocyanate) is 488 / 510-530 nm (green).

[0112] Results and Discussion of Examples 3-8

[0113] 1. Synthesis and modification of magnetic beads. Fe3O4 was synthesized by coprecipitation. A silica layer Fe3O4@SiO2 was added to its surface by sol-gel process, and an amine coating was formed by APTMS. Sodium chloroacetate was used to replace the amine group to convert it into a PIDA structure Fe3O4@SiO2-PIDA with chelating ability, and Ni was further combined. 2+ Fe3O4@SiO2-PIDA-Ni was obtained by activation 2+ , stored in a 20% ethanol solution in the form of a suspension for later use. 2+ The synthesis flow chart is shown in Figure 1 .

[0114] 2. Representation. Figure 2 TEM image of Fe3O4 superparamagnetic nanoparticles is shown. The nanoparticle core is spherical, almost monodisperse, and has an average diameter of 15.9 nm.

[0115] Fe3O 4， Fe3O4@SiO 2， Fe3O4@SiO2-APTMS, Fe3O4@SiO2-PIDA and Fe3O4@SiO2-PIDA-Ni 2+ The crystal structure and purity of Figure 3 shown.

[0116] Interpretation: The Fe3O4 XRD pattern is very consistent with the standard cubic structure of Fe3O4, see the figure in PDF # 88-0866. The peaks at 2θ = 18.4°, 30.1°, 35.5°, 37.0°, 43.2°, 53.4°, 57.0° and 62.7° are (111), (220), (311), (222), (400), (422), (511) and (440), respectively. The presence of amorphous silica in the structure is confirmed by the obvious diffuse peaks at 2θ = 15-25° for Fe3O4@SiO2, Fe3O4@SiO2-APTMS and Fe3O4@SiO2-PIDA. The patterns of Fe3O4@SiO2-APTMS and Fe3O4@SiO2-PIDA are very similar to those of Fe3O4@SiO2, so it can be concluded that the modification of APTMS and PIDA does not lead to the loss of crystallinity of the core structure.

[0117] Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2-APTMS, Fe3O4@SiO2-PIDA and Fe3O4@SiO2-PIDA-Ni 2+The decomposition rate of is measured by TGA, such as Figure 4 shown.

[0118] Interpretation: Fe3O4 only lost 2.41% of its total weight due to the removal of adsorbed water molecules. After SiO2 modification, the overall weight loss increased to 3.87%, APTMS, PIDA and Ni 2+ The weight of the derivatives decreased more significantly at temperatures above 200 °C.

[0119] 3. The loading capacity of MNiNP was evaluated and compared with the commercial Ni-NTA agarose resin chromatography filler. MNiNP had a higher maximum loading capacity of 74.41 mg / g at a fixed time of 25 min. Although the results of Ni-NTA agarose resin were more stable, the loading capacity was lower, with a maximum loading capacity of 49.01 mg / g. MNiNP showed very high positional specificity for the mycoplasma protein M and no washing was required before the elution step. Ni-NTA agarose resin required extensive washing (3-5 CV with washing buffer). Results are shown in Figure 5 shown.

[0120] 4. The reproducibility and storage stability of MNiNP are important parameters. MNiNP showed high reusability. After 4 consecutive cycles, the binding capacity of MNiNP and scFv was 90.5% of the initial capacity in the first cycle. Figure 6 This may once again prove the stability of mycoplasma protein M on NiNP and the effectiveness of active sites. Figure 7 The storage stability analysis of MNiNPs over 15 days is shown. MNiNPs were stored in PBS solution (50 mM, pH 7.4) at 4°C. After 15 days of storage, the ability of MNiNPs to capture scFv from cell lysate was still equal to 76.1% of the initial capacity at day 0.

[0121] 5. Immobilization of mycoplasma protein M and purification of antibodies. Mycoplasma protein M is immobilized by his-tag and Ni 2+ The non-covalent interaction between them was immobilized from the cell lysate. NiNPs were detected by SDS-PAGE (polyacrylamide gel electrophoresis) and the mycoplasma protein M was eluted after immobilization with 250 mM imidazole buffer. The non-reducing SDS-PAGE (M: molecular weight marker, L: cell lysate, E: eluate) of mycoplasma protein M eluted from NiNPs and the MALDI-TOF MS (mass spectrometry) analysis of the mycoplasma protein M elution fractions are shown in Figure 2. Figure 8 shown.

[0122] Reference Figure 8As shown, a clear band was observed around 46 kDa. The MALDI-TOF mass spectrometry analysis showed a peak at 48441, which corresponds to 48289 Da, corresponding to the amino acid sequence analysis of mycoplasma protein M. The slight difference between MALDI-TOF MS and SDS-PAGE may be due to the presence of more negatively charged residues, thus migrating faster than expected on SDS-PAGE.

[0123] To further confirm the presence of mycoplasma protein M on MNiNPs, CLSM analysis was performed again. FITC staining was performed on the magnetic beads before and after mycoplasma protein M was eluted. Fig. 9 The expected presence of mycoplasma protein M on the beads was shown. Based on the CLSM results, the complete elution of mycoplasma protein M was confirmed. Fig.10 shown.

[0124] 6. To confirm the ability of immobilized mycoplasma protein M to capture antibodies, it was first tested on trastuzumab scFv. Trastuzumab scFv was rapidly purified in cell lysate. As expected, the PelB signal peptide was cleaved, which reduced the final molecular weight. The elution fraction was pure and the expected molecular weight was determined to be approximately 26 kDa. The elution fraction was purified by MALDI-TOF mass spectrometry (see Fig.11 ) describes the exact molecular weight, and the results show a distinct peak at 26,260 Da, which is highly correlated with the expected molecular weight of 26,290 Da.

[0125] 7. To test the effect of immobilized mycoplasma protein M in capturing IgG and Fab, S2P6 IgG and Fab, and 76E1 IgG and Fab were used. Fig.12 The adsorption capacity of MNiNP for IgGs and Fabs is shown. Compared with scFv, IgGs and Fabs require longer incubation time to reach the maximum loading capacity. The maximum loading capacity is only achieved after incubation at 4°C for 60-80 min. Compared with the scFv results, the overall results are significantly lower, with the detection results for S2P6 Fab, S2P6 IgG, 76E1 Fab and 76E1IgG being 22.58, 17.32, 12.65 and 10.21 mg / g, respectively.

[0126] The reproducibility and storage stability of MNiNPs are important parameters that have a significant impact on further applications. MNiNPs showed high reusability. After 4 consecutive cycles, the binding capacity of MNiNPs to scFv was 90.5% of the initial capacity in the first cycle (see Fig.13 ). This once again demonstrated the stability of mycoplasma protein M on NiNP and the effectiveness of the active sites.

[0127] Conclusion: The antibody purification method described in this paper is effectively applicable to a wide range of antibodies, including IgG, Fab, and scFv. A new antibody purification carrier and method were developed and optimized based on the newly discovered Mycoplasma protein M. The antibody loading on MNiNP varied from 10.21 to 52.01 mg / g depending on the antibody type. The optimal Mycoplasma protein M loading was found to be between 40 and 60 mg / g. In addition, the immobilized Mycoplasma protein M showed excellent stability and recyclability, maintaining 76.1% of the initial activity after storage at 4°C for 15 days and 90.5% after four consecutive application cycles. The results of this application are conducive to further research on Mycoplasma protein M and its application in industry. This technology can simplify antibody production in non-professional laboratories and promote the research of biopharmaceuticals.

Claims

1. A method for broad-spectrum separation of antibodies using magnetic nano-mycoplasma protein, characterized in that: The steps include: S01, Magnetic Nanomaterials Fe3O4@SiO2–PIDA–Ni 2+ The synthesis is denoted as NiNP; The synthesis steps of NiNPs include: S01.

1. Synthesis of Fe3O4 nanoparticles Dissolve FeCl3·6H2O and FeCl2·4H2O in deionized water to obtain a mixed solution; Under argon protection, ammonia water was added dropwise to the mixed solution, and stirred at 250 rpm for 10-12 h at 60-65 °C to obtain crude Fe3O4 nanoparticles; The crude Fe3O4 nanoparticles were collected by an external magnet, washed with deionized water until neutral, and then washed with a 50% ethanol solution for 10-15 times, and vacuum dried at 60°C for 12-24 h to obtain the Fe3O4 nanoparticles; S01.

2. Synthesis of Fe3O4@SiO2 The Fe3O4 nanoparticles were dispersed in ethanol, and deionized water and ammonia were added. 1.5 mL of tetraethyl silicate was added dropwise during 30 min of ultrasonication at room temperature, and then ultrasonication was continued for 15 min. The mixture obtained above was stirred at 300 rpm at 25 °C for 12 h to obtain a crude Fe3O4@SiO2 product; The crude Fe3O4@SiO2 was collected by an external magnet, washed with deionized water for 10-15 times, and vacuum dried at 60°C for 12-24h to obtain Fe3O4@SiO2; S01.

3. Synthesis of Fe3O4@SiO2–APTMS Fe3O4@SiO2 was dispersed in toluene, 3-aminopropyltrimethoxysilane was added dropwise, and the mixture was refluxed at 95 °C for 12 h under argon protection to obtain a crude Fe3O4@SiO2–APTMS product; The crude Fe3O4@SiO2–APTMS was collected by an external magnet, washed with pure methanol for 3–5 times, and dried under vacuum at 60 °C for 12 h to obtain Fe3O4@SiO2–APTMS; S01.4, Fe3O4@SiO2–PIDA–Ni 2+ Synthesis The obtained Fe3O4@SiO2-APTMS was dispersed in a mixed solution of ethanol and triethylamine in which sodium chloroacetate was dissolved, and refluxed at 80°C for 12 h under argon protection to obtain a crude Fe3O4@SiO2-PIDA product; The crude Fe3O4@SiO2-PIDA was washed with pure methanol and deionized water for 5-10 times to obtain pure Fe3O4@SiO2-PIDA; Pure Fe3O4@SiO2-PIDA was dispersed in NiSO4·6H2O solution and shaken at 25℃ for 12 h to obtain Fe3O4@SiO2–PIDA–Ni 2+ crude product; Fe3O4@SiO2–PIDA–Ni 2+ The crude product was washed with deionized water for 3-5 times to obtain pure Fe3O4@SiO2–PIDA–Ni 2+ ; S02, expression and purification of mycoplasma protein M-8his; S02.

1. Transform the pET28a-M-8his plasmid into Escherichia coli cells, spread the Escherichia coli cells on an LB plate containing 50 μg / mL kanamycin, and incubate at 37°C overnight to obtain Escherichia coli colonies; S02.

2. Take a single E. coli colony and transfer it to 5 mL of culture medium containing 50 μg / mL kanamycin. Incubate overnight at 37°C and 200 rpm. S02.

3. Transfer the culture obtained above into 2YT medium and incubate until OD 600 At 0.5-0.6, isopropyl-β-D-thiogalactoside was added to promote induction, and the mixture was incubated at 20°C and 200 rpm for 16-18 h to obtain Escherichia coli colonies expressing the target protein; S02.4, collect the above-mentioned E. coli cells, centrifuge at 4°C and 8000 rpm for 10 min, resuspend with buffer solution, and lyse by high-pressure homogenization to obtain cell lysate; S02.5, the cell lysate was centrifuged at 4°C and 8000 rpm for 1 h, and the supernatant was filtered with a 0.22 μm filter membrane to obtain a solution containing the mycoplasma protein M-8his; S03, Mycoplasma protein M-8his in magnetic nanomaterials Fe3O4@SiO2–PIDA–Ni 2+ The immobilization on is denoted as MNiNP; S04, separating the MNiNP adsorbed with the target antibody by an external magnet, eluting, and obtaining the purified target antibody; the MNiNP can be reused after elution; The amino acid sequence of the mycoplasma protein M-8his is shown in SEQ ID NO:

1.

2. A method for separating antibodies using magnetic nano-mycoplasma protein broad spectrum according to claim 1, characterized in that: In step S02.3, 2YT medium is prepared from the following components: 1000 mL of distilled water, 5.0 g of sodium chloride, 10 g of yeast extract, and 16 g of peptone.

3. The method for separating antibodies using magnetic nano-mycoplasma protein broad spectrum according to claim 1, characterized in that: In step S02.4, the centrifugation conditions for the E. coli colonies are: rotation speed 8000 rpm, temperature 4°C, centrifugation time 10 min; the buffer solution is 5 mM imidazole buffer.

4. The method for separating antibodies using magnetic nano-mycoplasma protein broad spectrum according to claim 1, characterized in that: In step S02.5, the centrifugation conditions of the cell lysate are 8000 rpm, 4°C, and 1 h; The elution conditions of the cell lysate were as follows: the Ni-NTA-agarose column was precalibrated with 2 column volumes of binding buffer, then washed with 5 column volumes of wash buffer, and finally the protein was eluted with elution buffer; The washing buffer was 20 mM imidazole buffer, and the elution buffer was 250 mM imidazole buffer.

5. The method for broad-spectrum separation of antibodies using magnetic nano-mycoplasma protein according to claim 1, characterized in that: Step S03 is specifically as follows: S03.

1. Put Fe3O4@SiO2–PIDA–Ni 2+ Wash 1-2 times with deionized water and 5 mM imidazole buffer, pH 7.7; S03.2, 1% Fe3O4@SiO2–PIDA–Ni was added to the mycoplasma protein M-8his solution 2+ Ultrasonication was performed at 4°C for 5-40 min and external magnetic separation was performed to obtain immobilized mycoplasma protein M, which was recorded as MNiNP.

6. The method for broad-spectrum separation of antibodies using magnetic nano-mycoplasma protein according to claim 1, characterized in that: Step S04 is specifically as follows: S04.1 Disperse the E. coli containing the target antibody to be purified into the binding buffer, lyse the cells and release the target antibody by high-pressure homogenization, add 1-2% MNiNP by mass ratio, shake at 4°C, and after 5-40 minutes, separate the MNiNP by an external magnet, and separate the target antibody from the cell lysate; S04.2 The separated MNiNPs are redispersed in the elution buffer, and the MNiNP concentration is maintained at 2-4%. After shaking at room temperature for 5 min, the target antibody is released from the MNiNPs, and the MNiNPs are separated by an external magnet to obtain the purified target antibody; MNiNPs are separated and reused; The binding buffer was 5 mM imidazole buffer at pH 7.7, and the elution buffer was 0.1 M citrate buffer.

7. The method for broad-spectrum separation of antibodies using magnetic nano-mycoplasma protein according to claim 6, characterized in that: In step S04.1, the antibodies used for separation and purification of magnetic nanomaterials include: antibody S2P6, antibody 76E1 and trastuzumab scFv.

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