Magnetic-mediated spina gleditsiae-shaped nano nickel protein purifying agent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nano magnetic materials and biology, and discloses a magnetic-mediated spina gleditsiae-shaped nano nickel protein purifying agent as well as a synthesis method and application of the magnetic-mediated spina gleditsiae-shaped nano nickel protein purifying agent. The preparation method comprises the following steps: dissolving nickel chloride in ethylene glycol, adjusting the pH value to be alkaline, and carrying out high-pressure hydrothermal reaction to obtain the magnetic-mediated spina gleditsiae-shaped nano nickel protein purifying agent. The synthesis process is simple and easy to operate, the synthesized spina gleditsiae-shaped simple substance nickel has magnetism and contains a large number of active sites, His-tagged fusion protein can be directly captured in a cell lysis solution without surface modification, the saturation adsorption capacity on target protein reaches 80.36 mg / g, and the spina gleditsiae-shaped simple substance nickel can be used as a target substance. The effect of the method is far better than the purification efficiency of imported commercially available commodities in contrast experiments. And the synthesized Ni nano adsorbent has excellent specific capture capability on His-tagged protein, good universality and recycling performance, and has strong market competitiveness and application prospects.

Description

Technical Field

[0001] The invention belongs to the field of nano magnetic materials and biotechnology, and specifically refers to a magnetic-mediated nano nickel protein purifying agent. Background Art

[0002] Histidine tags have been widely used in protein purification and separation processes. Agarose gels that coordinate nickel ions can quickly and highly selectively bind to target proteins with histidine tags, which improves the speed of protein separation and purification and reduces the cost of protein purification. Multifunctional proteins have been commercialized and applied using this production process. In the purification process of these functional proteins, agarose gel nickel columns require complex pretreatment and low-temperature storage. The operation is complex and energy-intensive. Therefore, the development of fast, efficient, and low-cost protein separation and purification methods has always been a bottleneck in the development of the protein industry.

[0003] Magnetic nanoparticles have unique advantages in the field of protein separation and purification due to their large specific surface area, easy surface modification, and ability to achieve rapid separation under the action of an external magnetic field. With the development of magnetic nanotechnology, magnetic nanoparticles are gradually combined with gold nanoparticles (CN108371942A), metal ion probes or organic polymers to obtain composite microspheres, which are used to improve separation and purification capabilities. For example, the application with publication number CN115504515A discloses a method based on magnetic nano γ-Fe 2 O 3 @Al 2 O 3 The magnetically responsive nanomaterial, its preparation method and application are disclosed, but its preparation steps are cumbersome and require multi-step surface functionalization. When it is used for adsorption and enrichment of His-tagged fusion protein, its enrichment capacity is only 45.03 mg / g.

[0004] Elemental Ni nanoparticles (Ni NPs) have excellent ferromagnetic properties, such as magnetocrystalline anisotropy, high coercivity, and good chemical stability, and have been widely used in catalysis, battery manufacturing, coating production, electrospinning fiber manufacturing, capacitor enhancement, new ink printing, optical switch manufacturing, etc. 2+ There is a strong chemical interaction between the His-tagged fusion protein molecule and Ni 2+Separation of His-tagged fusion protein molecules. For example, publication number CN104607143A discloses an iron-nickel magnetic microsphere, which has a separation and purification ability that is 5 times that of the microsphere and is equivalent to the separation purity of agarose gel combined with nickel particles, and has a low separation and purification ability. There is currently no research on the separation and purification of His-tagged fusion proteins using single-substance Ni nanoparticles; moreover, the morphology of the prepared nano-nickel is spherical, and the adsorption and enrichment effect of the prepared nano-nickel with other morphologies on His-tagged fusion protein molecules is unknown. Therefore, it is crucial to regulate the morphology of single-substance Ni nanoparticles to further improve their enrichment and purification capabilities. Summary of the invention

[0005] In view of the above technical problems, the present invention proposes a magnetically mediated sapodilla thorn-shaped nano-nickel protein purification agent and a preparation method and application thereof.

[0006] To achieve the above object, the technical solution of the present invention is implemented as follows:

[0007] A method for preparing a magnetically mediated sapodilla thorn-shaped nano-nickel protein purifier comprises the following steps: dissolving nickel chloride in ethylene glycol, adjusting the pH to alkaline, and subjecting the mixture to a high-pressure hydrothermal reaction to obtain a magnetically mediated sapodilla thorn-shaped nano-nickel protein purifier, also known as Ni NPs nano-purifier or Ni NPs.

[0008] Furthermore, the concentration of the nickel chloride is 22.23-44.45 mM.

[0009] Furthermore, the pH is adjusted to 8-12.

[0010] Furthermore, the high pressure hydrothermal reaction temperature is 140-220° C., and the reaction time is 8-24 hours.

[0011] The magnetically mediated soapberry thorn-shaped nano-nickel protein purifier prepared by the above-mentioned preparation method is in the shape of a soapberry thorn, and the magnetic saturation intensity can be as high as 165.03emu / g.

[0012] The application of the magnetically mediated sapodilla thorn-shaped nano-nickel protein purifier in separating and purifying target proteins.

[0013] Furthermore, the above-mentioned target protein is a His-tagged fusion protein.

[0014] The above separation and purification method is: adding the magnetically mediated sapodilla thorn-shaped nano-nickel protein purifier to the His-tagged fusion protein solution, incubating at low temperature, magnetic separation, and separating the composite precipitate of the magnetically mediated sapodilla thorn-shaped nano-nickel protein purifier with the target protein; washing the precipitate with TBS or PBS buffer, and then soaking and eluting with an imidazole solution.

[0015] Furthermore, the mass ratio of the magnetically mediated sapodilla thorn-shaped nano-nickel protein purification agent to the His-tagged fusion protein is 1:(0.03-0.11); and the low-temperature incubation time is 5-120 min.

[0016] The beneficial effects produced by the present invention are:

[0017] (1) The present invention uses a one-step method to propose a method for synthesizing a magnetically mediated sapodilla thorn-like nano-nickel protein purifier. Using ethylene glycol as a solvent and a reducing agent and nickel chloride as a nickel source, a high-pressure hydrothermal method is used to synthesize magnetic single-substance nickel nanoparticles. By controlling the synthesis conditions (pH value, hydrothermal temperature and hydrothermal time), Ni NPs nanoadsorbents with relatively uniform size (excluding thorn length, particle size 250-640nm) and sapodilla thorn-like structure (thorn length 159-278nm) are prepared. The spike structure provides more contact area and active sites due to its unique geometric shape, which is conducive to improving the adsorption efficiency and adsorption amount. In addition, due to its anisotropy, the spike-structured nanomaterial can provide more binding sites during the adsorption process, while also reducing the steric hindrance of the target protein capture, thereby greatly enhancing its saturated adsorption capacity; in addition, the magnetic saturation intensity of the prepared Ni NPs is as high as 165.03emu / g; it can be evenly dispersed in water and can be quickly recovered (within 3s) under the action of an external magnetic field.

[0018] (2) The magnetically mediated sapodilla thorn-shaped nano-nickel protein purifier prepared by the present invention is in the shape of sapodilla thorns and contains a large number of nickel binding sites. It can directly capture His-tagged fusion proteins from cell lysates containing His-tagged proteins without surface modification. The saturated adsorption capacity of the target protein reaches 80.36 mg / g, which is much better than the purification efficiency of imported commercial products in the comparative experiment (1.80 mg / g); this is due to the special sapodilla thorn-shaped structure and high-density active Ni sites on the surface of the magnetically mediated sapodilla thorn-shaped nano-nickel protein purifier. At the same time, the synthesized nano-nickel protein purifier has excellent specific capture ability, good universality and recycling performance for His-tagged fusion proteins. After 5 cycles of separation and purification of proteins, its separation efficiency is still higher than 80%. The nano-protein purifier has completed small-scale experiments and is undergoing pilot-scale expansion. It has strong market competitiveness and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is the SEM image of Ni NPs prepared in Example 1.

[0021] Figure 2 This is the SEM image of Ni NPs prepared in Example 2.

[0022] Figure 3 This is the SEM image of Ni NPs prepared in Example 3.

[0023] Figure 4 TEM images (a, b) of Ni NPs prepared in the present invention and (c) Ni element and (d) O element surface distribution maps.

[0024] Figure 5 XRD spectrum of Ni NPs (a), FTIR spectrum of the solvent before and after the reaction (b), and TG curve of elemental Ni (c).

[0025] Figure 6 N is the Ni NPs prepared in the present invention. 2 Adsorption–desorption curves (a) and hysteresis loops (b).

[0026] Figure 7 Schematic diagram of the preparation of Ni NPs nanoadsorbents and the separation process of His-tagged proteins.

[0027] Figure 8 The adsorption performance and adsorption kinetic model of Ni NPs nanoadsorbent for His-tagged GFP protein; (a) is the equilibrium adsorption amount-protein concentration bar graph; (b) is the adsorption amount-time curve; (c) is the simulation analysis result of the pseudo-first-order kinetic model; (d) is the simulation analysis result of the pseudo-second-order kinetic model.

[0028] Fig. 9 Laser confocal microscopy images of Ni NPs nanoadsorbents; (a, d) are Ni NPs after incubation with TBS; (b, e) are Ni NPs washed four times with TBS after incubation with His-tagged GFP protein; (c, f) are Ni NPs washed with imidazole solution after incubation with His-tagged GFP protein.

[0029] Fig.10 The SDS-PAGE images (ac) and corresponding Western blot images (df) of the complexes obtained by separating different His-tagged proteins by Ni NPs nanoadsorbents; wherein, (a) lane 1, marker; lane 2, protein purified by commercial gel nickel column; lane 3, protein purified by Ni NPs; lane 4, His-tagged GFP Escherichia coli lysate; (b) lane 1, marker; lane 2, protein purified by commercial gel nickel column; lane 3, protein purified by Ni NPs; lane 4, His-tagged PYL3 Escherichia coli lysate; (c) lane 1, marker; lane 2, protein purified by commercial gel nickel column; lane 3, protein purified by Ni NPs; lane 4, His-tagged KAI2 Escherichia coli lysate; (df) are Western blot images corresponding to the SDS-PAGE images of (ac), respectively.

[0030] Fig.11 The figure shows the recycling performance of Ni NPs nanoadsorbent; wherein, (a) is the SDS-PAGE diagram of the complex obtained by repeatedly using Ni NPs nanoadsorbent to separate His-tagged GFP protein, wherein, lane 1, marker; lane 2, first use; lane 3, second use; lane 4, third use; lane 5, fourth use; lane 6, fifth use; lane 7, protein eluted from a commercial gel nickel column; lane 8, Escherichia coli lysate containing His-tagged GFP; (b) purification ability of Ni NPs nanoadsorbent when repeatedly separating His-tagged GFP protein. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Experimental Materials:

[0033] Nickel chloride hexahydrate: purity (mass fraction) is 98%, purchased from Tianjin Komiou Chemical Reagent Co., Ltd.;

[0034] Ethylene glycol: purity (mass fraction) is 98%, Tianjin Fuyu Fine Chemical Co., Ltd.;

[0035] NaOH: purity (mass fraction) is 98%, Tianjin Komiou Chemical Reagent Co., Ltd.;

[0036] Commercial Ni Smart Beads 6FF (commercial gel nickel column): purchased from Changzhou Tiandirenhe Biotechnology Co., Ltd.

[0037] Example 1

[0038] The preparation method of the magnetically mediated sapodilla thorn-shaped nano-nickel protein purification agent of this embodiment comprises the following steps:

[0039] Weigh 22.23 mM NiCl 2 6H 2 An appropriate amount of O was placed in a beaker, 60 mL of ethylene glycol was added, and magnetic stirring was performed at room temperature for 30 min to fully dissolve it. Then, NaOH solution was added to adjust the pH to 12. After stirring and mixing, the mixed solution was transferred into a 100 mL polytetrafluoroethylene high-pressure hydrothermal reactor and reacted at 180 ° C for 12 h. After the reaction, magnetic separation was performed, and the precipitate was washed 3 times with deionized water and ethanol respectively, and then freeze-dried for 24 h to obtain Ni NPs. Figure 1 The figure shows a scanning electron microscope (SEM) image of the synthesized Ni NPs. It can be seen that the synthesized Ni NPs nanoadsorbent is in the shape of a soapberry thorn, with good dispersibility, an average particle size (excluding the thorn length) of 640nm, and a thorn length of 214nm. Due to its anisotropy, the nanomaterial with a soapberry thorn structure greatly reduces the steric hindrance when capturing the target protein, and provides more active specific binding sites, effectively enhancing the saturated adsorption capacity of the target protein.

[0040] Example 2

[0041] The preparation method of the magnetically mediated sapodilla thorn-shaped nano-nickel protein purification agent of this embodiment comprises the following steps:

[0042] Weigh 44.45 mM NiCl 2 6H 2 An appropriate amount of O was placed in a beaker, 60 mL of ethylene glycol was added, and magnetic stirring was performed at room temperature for 30 min to fully dissolve it. Then, NaOH solution was added to adjust the pH to 10. After stirring and mixing, the mixed solution was transferred into a 100 mL polytetrafluoroethylene high-pressure hydrothermal reactor and reacted at 160 ° C for 8 h. After the reaction, magnetic separation was performed, and the precipitate was washed 3 times with deionized water and ethanol respectively, and then freeze-dried for 24 h to obtain Ni NPs. Figure 2 The SEM image of the synthesized Ni NPs shows that the synthesized Ni NPs nanoadsorbent is in the shape of a soapberry thorn, has good dispersibility, an average particle size (excluding the thorn length) of 257nm, and a thorn length of 278nm.

[0043] Example 3

[0044] The preparation method of the magnetically mediated sapodilla thorn-shaped nano-nickel protein purification agent of this embodiment comprises the following steps:

[0045] Weigh 30.20 mM NiCl 2 6H 2 An appropriate amount of NiO was placed in a beaker, 60 mL of ethylene glycol was added, and magnetic stirring was performed at room temperature for 30 min to fully dissolve it. Then, NaOH solution was added to adjust the pH to 8. After stirring and mixing, the mixed solution was transferred into a 100 mL polytetrafluoroethylene high-pressure hydrothermal reactor and reacted at 140 ° C for 24 h. After the reaction, magnetic separation was performed, and the precipitate was washed 3 times with deionized water and ethanol respectively, and then freeze-dried for 24 h to obtain single Ni NPs. Figure 3 The SEM image of the synthesized Ni NPs shows that the synthesized Ni NPs nanoadsorbent is in the shape of a soapberry thorn, has good dispersibility, an average particle size (excluding the thorn length) of 265nm, and a thorn length of 159nm.

[0046] Example 4

[0047] The preparation method of the magnetically mediated sapodilla thorn-shaped nano-nickel protein purification agent of this embodiment comprises the following steps:

[0048] Weigh 30.20 mM NiCl 2 6H 2 An appropriate amount of NiO was placed in a beaker, 60 mL of ethylene glycol was added, and magnetic stirring was performed at room temperature for 30 min to fully dissolve it. Then, NaOH solution was added to adjust the pH to 8. After stirring and mixing, the mixed solution was transferred into a 100 mL polytetrafluoroethylene high-pressure hydrothermal reactor and reacted at 220 ° C for 8 h. After the reaction, magnetic separation was performed, and the precipitate was washed 3 times with deionized water and ethanol respectively, and then freeze-dried for 24 h to obtain single Ni NPs.

[0049] Implementation effect example

[0050] The magnetically mediated sapodilla thorn-shaped nano-nickel protein purifier (Ni NPs nanoadsorbent or Ni NPs) prepared in the above embodiment was tested and analyzed for its TEM image, crystal structure, functional group, thermal stability and adsorption performance for His-tagged fusion protein, and the results are as follows.

[0051] (1) Morphology and structure

[0052] Figure 4 The figure shows the transmission electron microscope (TEM) image and element surface distribution diagram of Ni NPs prepared by the present invention. It can be seen that the prepared Ni NPs have obvious thorn-like morphology, and the surface distribution of Ni and O elements corresponds to its morphology. This indicates that part of the metal Ni on the surface of the Ni NPs sample can be oxidized by oxygen in the air and converted into NiO.

[0053] Figure 5 Shown are the X-ray diffraction (XRD) pattern of Ni NPs synthesized by the present invention, the Fourier transform infrared spectrum (FTIR) of the solvent before and after the reaction, and the thermogravimetric analysis (TG) curve of Ni NPs. Figure 5 As can be seen in a, the synthesized Ni NPs show strong diffraction peaks at 2θ = 44.5°, 51.8°, and 76.4°, respectively, which are attributed to the diffraction of the (111), (200), and (220) crystal planes of Ni NPs (PDF 04-0850). This shows that the product synthesized by high-pressure hydrothermal reaction is indeed Ni NPs. Liquid infrared spectroscopy tests were performed on the solvents before and after the reaction. The results showed that the solvent after high-pressure hydrothermal reaction had a peak at 1602 cm -1 There is an obvious absorption peak ( Figure 5 b); Combined with the corresponding XRD analysis results, it can be inferred that Ni 2+ is reduced to elemental Ni, while ethylene glycol is oxidized. In the entire reaction system, ethylene glycol acts as both a reaction solvent and a reducing agent. From the TG curve of the synthesized Ni NPs ( Figure 5 c) It can be seen that the weight loss from room temperature to 300°C is only 1.2%; this weight loss may be due to the evaporation of water.

[0054] Figure 6 The N of Ni NPs prepared by the present invention is shown as 2 Adsorption-desorption curve and hysteresis loop. It can be seen that its N 2 The adsorption-desorption curve is consistent with the IV type adsorption-desorption curve ( Figure 6 a). From the corresponding hysteresis loop, it can be seen that the magnetic saturation intensity of NiNPs prepared in the present invention is 165.03emu / g ( Figure 6 b), indicating that it has good magnetic responsiveness. Figure 6 As shown in the inset in b, the Ni NPs prepared in the present invention can be uniformly dispersed in water and can be quickly recovered (within 3 s) under the action of an external magnetic field.

[0055] (2) Adsorption performance

[0056] When synthesizing Ni NPs nanoadsorbents by high pressure hydrothermal method, the target product with a sapodilla thorn-like structure can be obtained by regulating the pH value of the reaction system, the reaction temperature and the reaction time. The obtained Ni NPs nanoadsorbent itself has ferromagnetism and can be partially oxidized into Ni by oxygen in the air. 2+ ; Because of Ni 2+Ni NPs nanoadsorbents have specific recognition ability and excellent adsorption and purification ability for specific fusion proteins (such as His-tagged fusion proteins). Therefore, they can specifically recognize and capture His-tagged fusion proteins, thereby achieving rapid separation of His-tagged fusion proteins. Figure 7 As shown, the Ni NPs nanoadsorbent prepared in the present invention can specifically enrich and purify His-tagged fusion proteins from a mixed protein sample solution.

[0057] Different concentrations of His-tagged GFP were selected as an example for adsorption experiments to evaluate the adsorption capacity of Ni NPs nanoadsorbents for His-tagged fusion proteins. The specific test process is as follows: 6 portions of 0.02g Ni NPs were weighed and placed in 2mL microcentrifuge tubes (EP tubes), washed three times with Tris-HCl buffered saline (TBS buffer), and 1.50mL of His-tagged GFP protein solutions with concentrations of 0.40mg / mL, 0.60mg / mL, 0.80mg / mL, 1.00mg / mL, 1.20mg / mL and 1.40mg / mL were added respectively; the mixed solution was placed in a silent reverberator and incubated at 4℃ for 1.5h; after the incubation, it was placed on a magnetic stand for magnetic separation, the supernatant was aspirated, and the concentration of the remaining His-tagged GFP protein in the supernatant after incubation was measured with a micro-spectrophotometer (wavelength 280nm). According to the measured concentration of the target protein, the adsorption performance of the Ni NPs nanoadsorbent for the target protein was calculated using formula (1):

[0058]

[0059] Where: C e (mg / mL) is the concentration of His-tagged GFP protein remaining in the EP tube after incubation of Ni NPs nanoadsorbent and different concentrations of His-tagged fusion protein; C 0 (mg / mL) is the concentration of His-tagged GFP protein in the EP tube before incubation with Ni NPs nanoadsorbent; Q e (mg / g) is the equilibrium adsorption capacity of Ni NPs nanoadsorbent for His-tagged GFP protein; M (mg / g) is the mass of Ni NPs nanoadsorbent added. Figure 8 As shown in a, the adsorption amount of His-tagged GFP protein by Ni NPs nanoadsorbent first increased and then decreased with the increase of protein concentration; when the protein concentration increased to 1.2 mg / mL, its adsorption reached saturation, and its maximum adsorption amount was 80.36 mg / g.

[0060] In order to study the adsorption kinetics of Ni NPs on His-tagged proteins at different adsorption times, pure His-tagged GFP protein was selected for adsorption experiments. The specific steps are as follows: weigh 12 portions of 0.02g Ni NPs and place them in 2mL EP tubes, wash them with TBS solution three times; add 1.50mL of His-tagged GFP protein solution with a concentration of 1.20mg / mL to each; place the mixed solution in a silent reverberator and incubate at 4℃ for different times (5min, 10min, 15min, 20min, 25min, 30min, 40min, 50min, 60min, 70min, 80min and 90min). After the incubation, place the mixed solution on a magnetic stand for magnetic separation, aspirate the supernatant, and measure the concentration of His-tagged GFP protein remaining in the supernatant after incubation with a micro-spectrophotometer (wavelength 280nm). And the adsorption amount-time ( Figure 8 b) and fitting analysis of adsorption kinetics (including pseudo-first-order kinetic model and pseudo-second-order kinetic model). The adsorbent was subjected to kinetic study to determine the time required for the adsorption of protein to reach adsorption equilibrium. The pseudo-first-order kinetic equation (Equation 2) and the pseudo-second-order kinetic equation (Equation 3) are as follows:

[0061] ln(Q e -Q t )=-k 1 t+lnQ e Equation 2

[0062]

[0063] Where: Q e (mg / g) and Q t (mg / g) is the equilibrium adsorption capacity and the adsorption capacity at time t of Ni NPs nanoadsorbent for His-tagged GFP protein; k 1 (min -1 ) and k 2 (g·mg -1 ·min -1 ) are pseudo-first-order kinetic constants and pseudo-second-order kinetic constants, respectively. The adsorption kinetics of Ni NPs nanoadsorbents on His-tagged GFP protein were analyzed ( Figure 8 c and 8d), the corresponding adsorption data are more consistent with the pseudo-second-order kinetic model; R 2 =0.999, which is higher than R based on pseudo-first-order kinetic equation 2 =0.988.

[0064] (3) Fluorescence performance test

[0065] Taking green fluorescent protein His-tagged GFP as an example, the binding effect between Ni NPs nanoadsorbent and His-tagged fusion protein was analyzed and verified (under visible light irradiation, GFP protein emits green fluorescence at an excitation wavelength of 488nm, and the fluorescence position and intensity remain unchanged after the nanomaterial binds to His-tagged GFP). Weigh 3 portions of 0.02g of Ni NPs nanoadsorbent and place them in 2mL EP tubes. Add 1.50mL of Escherichia coli lysate to each of the two tubes, and add 1.5mL of TBS to the other tube as a blank control; place the mixed solution in a silent reverberator and incubate at 4℃ for 1.5h; after the incubation, place it on a magnetic stand for magnetic separation and discard the supernatant. Wash the Ni NPs / His-tagged PYL3 complex precipitate 4 times with TBS buffer. One EP tube with cell lysate was soaked and eluted with imidazole solution (concentration of 500mM), and the precipitate was collected after elution; the other EP tube with cell lysate was not soaked and eluted with imidazole solution, and the precipitate was collected directly after magnetic separation; the blank control sample was directly magnetically separated under the same conditions, and the precipitate was collected for testing. The precipitates taken from the three EP tubes were observed with a laser confocal microscope to observe whether they emitted green fluorescence.

[0066] Fig. 9 The laser confocal microscopy image of the complex after incubation of Ni NPs and His-tagged GFP protein is shown. It can be seen that Ni NPs incubated with TBS have no green fluorescence under the irradiation of the laser confocal light source ( Fig. 9 a, d); After incubation of Ni NPs with His-tagged GFP protein, the resulting complex was washed four times with TBS and emitted obvious green fluorescence under laser confocal light source ( Fig. 9 b, e); and the distribution image of its green fluorescence corresponds to the bright field morphology of Ni NPs. This indicates that the His-tagged GFP protein is evenly distributed on the surface of Ni NPs. However, when the complex obtained after the incubation of Ni NPs with the His-tagged GFP protein is soaked and eluted in imidazole solution, it emits almost no green fluorescence ( Fig. 9 c, f). This is because the strong chemical binding between Ni NPs nanoadsorbent and His-tagged GFP protein was destroyed and His-tagged GFP protein fell off from the surface of Ni NPs due to immersion and elution in imidazole solution. This indicates that the binding between Ni NPs nanoadsorbent and His-tagged GFP protein is a specific chemical binding, rather than a simple physical adsorption.

[0067] Application Examples

[0068] In order to verify the universality of Ni NPs for His-tagged proteins and its application in the actual separation and purification of mixed proteins, three His-tagged fusion proteins (His-tagged PYL3 fusion protein, His-tagged KAI2 fusion protein, His-tagged GFP fusion protein) were selected and expressed and broken in Escherichia coli to obtain the target proteins; the crude protein extract was taken for testing, and the captured target proteins were subjected to immunoblotting experiments (Western blot analysis).

[0069] The specific steps of the universality investigation are as follows: three His-tagged fusion protein expression strains were added to the culture medium to induce amplification and collect the cells. TBS solution (Tris-HCl buffered saline) was added to the collected cells for re-suspending by shaking. After re-suspending, the cells were subjected to high pressure and ultrasonic disruption to obtain E. coli cell lysate containing His-tagged fusion proteins. Adsorption and elution experiments were performed to investigate its ability to separate and purify His-tagged proteins from actual complex biological samples. The process of adsorption and elution experiments is as follows: 3 portions of 0.02g Ni NPs nanoadsorbent were weighed in parallel and placed in 3 EP tubes, and 1.50mL of Escherichia coli lysate containing His-tagged PYL3 fusion protein, His-tagged KAI2 fusion protein and His-tagged GFP fusion protein were added to each tube; each mixed solution was placed in a silent reverberator and incubated at 4°C for 1.5h; after the incubation, the mixed solution was placed on a magnetic stand for magnetic separation, the precipitate was collected and retained, and the precipitate was washed 4 times with TBS buffer to obtain Ni NPs / His-tagged PYL3, Ni NPs / His-tagged KAI2 and Ni NPs / His-tagged GFP complexes, respectively. The obtained complexes were soaked in 600μL imidazole solution (500mmol / L) to elute the captured His-tagged fusion protein, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments were performed respectively.

[0070] In addition, the target protein purified and captured by Ni NPs nanoadsorbent was determined by immunoblotting (Western blot method) to study whether Ni NPs nanoadsorbent has specific recognition and purification ability for His-tagged proteins. The target protein obtained after purification and elution by Ni NPs nanoadsorbent was used for SDS-PAGE experiment; after the electrophoresis experiment, the membrane was transferred. The steps of membrane transfer are: select a suitable polyvinylidene fluoride membrane (PVDF membrane), place the protein gel on the PVDF membrane, and transfer the membrane at 180mA for 90min. Then put the PVDF membrane in a culture dish, add 15mL skim milk powder solution (0.1g / mL) to block for 4h; after blocking, wash it 5 times with TBS+Tween-20 (referred to as TBST). After washing, add 15mL skim milk powder solution (0.01g / mL) and 1.5μL primary antibody to the mixed solution and incubate for 2h; after incubation, wash 5 times with TBST to remove the unadsorbed primary antibody; then add 15mL skim milk powder solution (0.01g / mL) and 1.5μL secondary antibody to it and incubate for 1h; after incubation, wash 5 times with TBST to ensure that the unbound secondary antibody is thoroughly washed. After washing, the PVDF membrane is exposed.

[0071] Fig.10 The SDS-PAGE and Western blot images of the complexes obtained by separating different His-tagged proteins using Ni NPs nanoadsorbents are shown. Fig.10 ac), except for the electrophoretic bands corresponding to the target protein, there are almost no bands of other impurity proteins. The three purified fusion proteins were subjected to electrophoretic transfer experiments, and it was found that the exposed bands of the PVDF membrane after transfer corresponded to the bands in the corresponding gel electrophoresis diagram ( Fig.10 df). This indicates that the purified target protein is a His-tagged fusion protein. It can be seen that the synthesized Ni NPs nanoadsorbent has a good separation and purification ability for His-tagged fusion proteins and has a good application prospect in the separation and purification of actual mixed protein sample solutions.

[0072] In order to verify the cyclic performance of the prepared Ni NPs for His-tagged GFP protein adsorption, a cyclic adsorption test was performed. The specific steps are as follows: 0.02g of Ni NPs were weighed and placed in a 2mL EP tube, washed three times with TBS solution, and 1.50mL of His-tagged GFP protein solution with a concentration of 1.00mg / mL was added to it; the mixed solution was placed in a silent reverberator and incubated at 4°C for 1.5h. After the incubation, the mixture was placed on a magnetic stand for magnetic separation, the supernatant was aspirated, and the concentration of the remaining target protein in the supernatant after the incubation was measured with a micro-spectrophotometer (wavelength 280nm); the precipitate was washed with imidazole solution and TBS buffer to remove the adsorbed protein, and 1.5mL of His-tagged GFP protein solution with a concentration of 1.00mg / mL was added after the washing; the mixed solution was placed in a silent reverberator and incubated at 4°C for 1.5h. After the incubation, the mixture was placed on a magnetic stand for magnetic separation, the supernatant was aspirated, and the concentration of the remaining protein in the supernatant after the incubation was measured with a micro-spectrophotometer (280nm). The above steps were repeated 4 times to evaluate the recycling performance of the Ni NPs nanoadsorbent, and the results are shown in Figure 2. Fig.11 shown.

[0073] Fig.11 a is the SDS-PAGE image of the complex obtained after Ni NPs nanoadsorbent was repeatedly used to purify His-tagged GFP protein. It can be seen that after 5 cycles of use, Ni NPs nanoadsorbent still has a good separation effect and strong specific recognition ability for the target protein. Its separation ability for the target protein after 5 cycles is still higher than 80% ( Fig.11 b). This indicates that Ni NPs nanoadsorbents have strong binding and specific recognition capabilities for target proteins, and have good application prospects in the field of protein separation and purification.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a magnetically mediated sapodilla thorn-shaped nano-nickel protein purifier, characterized in that: The steps are as follows: nickel chloride is dissolved in ethylene glycol, the pH is adjusted to alkaline, and then high-pressure hydrothermal reaction is performed to obtain the product.

2. The method for preparing the magnetically mediated sapodilla thorn-shaped nano-nickel protein purifying agent according to claim 1, characterized in that: The concentration of nickel chloride is 22.23-44.45 mM.

3. The method for preparing the magnetic-mediated sapodilla thorn-shaped nano-nickel protein purifying agent according to claim 2, characterized in that: The pH is adjusted to 8-12.

4. The method for preparing the magnetically mediated sapodilla thorn-shaped nano-nickel protein purifying agent according to claim 3, characterized in that: The high pressure hydrothermal reaction is carried out at a temperature of 140-220° C. and for a time of 8-24 h.

5. A magnetically mediated sapodilla thorn-shaped nano-nickel protein purifying agent prepared by the preparation method according to any one of claims 1 to 4, characterized in that: The magnetically mediated soapberry thorn-shaped nano-nickel protein purifier is in the shape of a soapberry thorn, and the magnetic saturation intensity is as high as 165.03 emu / g.

6. Use of the magnetically mediated sapodilla thorn-shaped nano-nickel protein purifier according to claim 5 in separating and purifying target proteins.

7. The use according to claim 6, characterized in that: The target protein is a His-tagged fusion protein.

8. The use according to claim 7, characterized in that: The separation and purification method comprises the following steps: adding a magnetically mediated sapodilla thorn-shaped nano-nickel protein purifier to a His-tagged fusion protein solution, incubating at low temperature, and magnetically separating to separate a composite precipitate of the magnetically mediated sapodilla thorn-shaped nano-nickel protein purifier carrying the target protein; washing the composite precipitate with a buffer solution and then eluting with an imidazole solution.

9. The use according to claim 8, characterized in that: The buffer for washing the composite precipitate is TBS or PBS buffer solution.

10. The use according to claim 9, characterized in that: The mass ratio of the magnetic-mediated sapodilla thorn-shaped nano-nickel protein purifier to the His-tagged fusion protein is 1:(0.03-0.11); the low-temperature incubation time is 5-120 min.

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