Efficient extraction and preservation method of serum BICC1 natural protein

Abstract

The invention relates to the technical field of protein extraction, in particular to an efficient extraction and preservation method of serum BICC1 natural protein, which comprises the following steps: 1) preparing aminated magnetic Fe3O4 nanoparticles; (2) preparing Fe3O4 (at) Au nanoparticles; (3) preparing a BICC1 template protein; (4) binding the BICC1 template protein; (5) fixing a BICC1 template protein; (6) eluting the BICC1 template protein to prepare a BICC1 magnetic molecularly imprinted polymer microsphere; the method is simple, reaction conditions are mild, efficient adsorption of template molecules is achieved through the colloidal gold layer of the magnetic molecularly imprinted polymer, the polymer fixing layer on the outermost layer is a neutral epoxy group, non-specific adsorption is reduced, and the natural BICC1 natural protein is directly, efficiently and specifically extracted from serum.

Description

Technical Field

[0001] The present invention relates to the technical field of protein extraction, and specifically relates to a method for efficiently extracting and preserving serum BICC1 natural protein. Background Art

[0002] In 1995, researchers discovered a gene in Drosophila (called the Bicaudal-C gene), the loss of which leads to the loss of the Drosophila head and the formation of a double-tail structure (Michele M, Emma ES, Paul FL. Localized Bicaudal –C RNA encodes a protein containing a KH domain, the RNA binding motif of FMR1. EMBO , 1995, 14(9): 2043-2055). According to sequence analysis, the protein (BICC1) translated from this gene contains several KH domains at the N-terminus and a SAM domain at the C-terminus (Leettola CN, Knight MJ,Cascio D, et al. Characterization of the SAM domain of the PKD-related protein ANKS6 and its interaction with ANKS3. BMC structural biology, 2014,14(1): 1-15; Rothé B, Leettola CN, Leal-Esteban L, et al. Crystal structureof Bicc1 SAM polymer and mapping of interactions between the ciliopathy-associated proteins Bicc1, ANKS3, and ANKS6. Structure, 2018, 26(2): 209-224.e6). According to sequence alignment, homologous genes of BICC1 are found in many species, including humans. The human BICC1 gene is a gene encoding RNA-binding proteins that is widely expressed in the brain and peripheral tissues. It is not only related to the onset of depression, but also closely related to cancer and pancreatic cancer. Many papers have reported that the human BICC1 gene (called BICC1 gene) is involved in the onset of depression (Ota KT, Andres W, Lewis DA, Stockmeier CA, Duman RS. BICC1 expression is elevated in depressed subjects and contributes to depressive behavior in rodents. Neuropsychopharmacology. 2015. 40(3): 711-718). The BICC1 level in normal human serum is 533.24±43.08 pg / mL, while that in patients with unipolar depression (MDD) is 636.30±41.33 pg / mL and in patients with bipolar depression (MDD) is 784.44±36.54 pg / mL. Therefore, BICC1 can be used as a marker to distinguish between unipolar (MDD) and bipolar (BD) depression (Chen S, Jiang H, Xu Z, et al. Serum BICC1 levels are significantly different in various mood disorders. Neuropsychiatric Disease and Treatment , 2019: 259-265; Ota KT, Andres W, Lewis DA, et al. BICC1 expression is elevated in depressed subjects andcontributes to depressive behavior in rodents[J]. Neuropsychopharmacology, 2015, 40(3): 711-718).

[0003] Bioinformatics analysis showed that the full-length BICC1 protein contains 974 amino acids with a molecular weight of 104.8 kDa. Structural prediction showed that the N-terminus contains three similar KH domains, the C-terminus has a SAM domain, and the rest of the sequence corresponds to a disordered structure (such as Figure 1Both the KH and SAM domains are key to the protein's biological function. Studies have shown that BICC1 is an RNA-binding protein capable of binding to a variety of RNA molecules. BICC1 can also bind to a variety of proteins. For example, BICC1 can bind to the ANKS6 protein through its KH domain (Yakulov TA, Yasunaga T., Ramachandran H., Engel C., Mueller B., Hoff S., Dengjel J., Lienkamp SS, Walz G. Anks3 interacts with nephronophthisis proteins and is required for normal renal development). Kidney Int. 87:1191-1200 (2015)).

[0004] In the field of biochemical testing and analysis, accurate measurement of BICC1 levels in human serum is crucial for differentiating between monopolar (MDD) and bipolar (BD) depression, diagnosis, treatment monitoring, and biomedical research. Assay kits require a series of low-concentration BICC1 proteins as calibrators and quality controls. However, BICC1 protein has unique characteristics, with a high proportion of disordered structures in its amino acid sequence, which makes soluble recombinant expression of the full-length protein challenging. Consequently, the industry's standard approach is to recombinantly express fragments of BICC1 to meet calibration and quality control requirements. However, these recombinant protein fragments differ from native BICC1 in spatial structure and immunogenic epitopes, altering key antigenic determinants. This can easily lead to bias and inaccuracy in measurement results, seriously compromising the reliability of test results. To overcome this challenge, direct isolation and purification of native BICC1 protein from human serum is undoubtedly the ideal solution.

[0005] As an RNA-binding protein, BICC1 has the ability to interact and bind to a variety of proteins. Its highly disordered structure makes the stability of native BICC1 proteins very fragile. Technicians have tried many traditional methods to improve its stability, such as adding buffers to the protein storage solution to adjust the pH and maintain a stable ionic environment; adding NaCl to strengthen the protein structure by balancing ionic strength; incorporating sucrose and glycerol to protect against adverse environmental interference through their osmotic pressure-regulating properties; and even adding commonly used surfactants such as Tween 20 and Triton X100 to reduce the surface tension of the protein solution and minimize protein aggregation. Unfortunately, these conventional methods have failed to achieve the desired effect of improving the stability of native BICC1 proteins.

[0006] Molecularly imprinted polymer technology has the characteristics of good mechanical and chemical stability, high selectivity and long life, and can be widely used in chemical sensors, drug delivery, separation and artificial antibodies. CN117531486B discloses a core-shell magnetic molecularly imprinted polymer material and its preparation method and application. Based on the polymerization reaction of 3,3-dihydroxybenzidine and p-benzoquinone, a polymer layer is formed by precipitation polymerization on the surface of silica-modified magnetic ferroferric oxide nanoparticles, and a virtual template betamethasone is used for imprinting to obtain a core-shell molecularly imprinted polymer material. CN107899557B discloses a magnetic molecularly imprinted polymer microsphere and its preparation method and application. It consists of the following components: (1) Fe3O4 nanoparticles; (2) a functional polymer, which is a copolymer of carboxylic acid and styrene containing at least one ethylenically unsaturated double bond; (3) a template molecule, which is a compound that can form hydrogen bonds with the functional polymer. CN110330607A discloses a method for preparing a magnetic molecularly imprinted polymer for separating lysozyme protein and its application. First, magnetic nanoparticles containing double bonds on the surface are prepared by reacting 3 (methacryloyloxy) propyltrimethoxysilane with Fe3O4. The magnetic nanoparticles containing double bonds on the surface, N (3-dimethylaminopropyl) methacrylamide, N,N'-methylenebisacrylamide, methacrylic acid, and lysozyme are placed in a reaction vessel. The magnetic molecularly imprinted polymer is prepared by free radical cross-linking copolymerization of initiators ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) on the surface of the magnetic nanoparticles. The template molecule lysozyme is then completely removed by dialysis in water to finally prepare the magnetic molecularly imprinted polymer for separating lysozyme protein. These patents all involve first preparing magnetic Fe3O4 nanoparticles, then directly coating the surface of the magnetic Fe3O4 nanoparticles with a layer of silica or first performing double-bond modification and then coating them with a polymer shell such as polystyrene. Template molecules and functional monomers are then simultaneously added to the magnetic polymer microspheres, polymerized to encapsulate the template molecules within the magnetic polymer microspheres, and then eluted by performing other steps. The steps are complex and inefficient, and the efficiency of encapsulating the template molecules through polymerization is low.

[0007] Therefore, it is extremely important to develop an efficient extraction and preservation method for serum BICC1 natural protein. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for efficiently extracting and preserving serum BICC1 natural protein.

[0009] To achieve the above object, the present invention provides the following technical solutions: A method for efficiently extracting and preserving serum BICC1 native protein comprises the following steps: 1) Preparation of amino-modified magnetic Fe3O4 nanoparticles: Surface amino-modified magnetic Fe3O4 nanoparticles were prepared by solvothermal method; 2) Preparation of Fe3O4@Au nanoparticles: Gold nanoparticles were prepared by citric acid reduction and distributed on the surface of magnetic Fe3O4 nanoparticles through electrostatic adsorption and covalent bonding to form Fe3O4@Au nanoparticles; 3) Preparation of BICC1 template protein: Design and prepare specific immunogens using key structural domains of the BICC1 protein to generate polyclonal antibodies against the BICC1 protein as template protein; 4) BICCI template protein binding: Fe3O4@Au nanoparticles bind to BICC1 template protein by adsorption; 5) Immobilization of the BICC1 template protein: Disperse the template protein-bound magnetic gold nanoparticles in pure water, add a crosslinker, glycidyl methacrylate, and an initiator, and react at 45°C for 6 h. 6) Elution of BICC1 template protein to prepare BICC1 magnetic molecularly imprinted polymer microspheres: The template protein is removed by acid washing to form BICC1 magnetic molecularly imprinted polymer microspheres; 7) Purification of native BICC1 protein: Human serum samples were adsorbed onto BICC1 magnetic molecularly imprinted polymer microspheres, followed by an elution step to effectively isolate the native BICC1 protein.

[0010] Specifically, step (1) comprises adding FeCl3∙6H2O, ethylene glycol, and 1,6-hexanediamine into a beaker, stirring and dissolving the mixture, adding anhydrous sodium acetate, stirring for 2 hours to fully dissolve the mixture, and then pouring the mixture into a polytetrafluoroethylene reactor and reacting the mixture at 180-280°C for 8-16 hours; wherein the mass ratio of FeCl3∙6H2O to 1,6-hexanediamine is 1:1-6, and the mass ratio of FeCl3∙6H2O to anhydrous sodium acetate is 1:5-10.

[0011] Specifically, step (2) comprises dispersing an appropriate amount of 1% chloroauric acid in ultrapure water, adding 1% sodium citrate as a reducing agent at 85-95° C., and continuing the reaction for 0.5-1 h; then adding amino-modified magnetic Fe3O4 nanoparticles, and continuing the reaction for 0.5-1 h; wherein the mass fraction of chloroauric acid in the entire system is 0.005%-0.01%, and the mass ratio of chloroauric acid to sodium citrate is 1:5-20.

[0012] Wherein, in said step (3), the structural domain of the BICC1 protein includes the BICC1 N-terminal KH domain or the BICC1 C-terminal SAM domain; preferably the BICC1 N-terminal KH domain; the antigen contains a solubility tag; specifically, the solubility tag includes any one of GST, MBP, and SUMO; preferably a GST tag; the immunized animal includes any one of rabbit and sheep; preferably sheep. Wherein, the BICC1 polyclonal antibody is an antibody extracted from serum, including any one of an N-terminal KH domain polyclonal antibody and a BICC1 C-terminal SAM domain polyclonal antibody; preferably an N-terminal KH domain polyclonal antibody.

[0013] Wherein, in said step (4), the surface colloidal gold is combined with the BICC1 template protein through the adsorption effect, specifically: take 50 mg of Fe3O4@Au nanoparticles in a beaker, then add 50 mL of labeling buffer, magnetically separate, remove the supernatant, then add 50 mL of labeling buffer, ultrasonically disperse, repeat three times, and finally store in 50 mL of labeling buffer; take 25 mg of template protein and add it to the above beaker, quickly mix, and incubate at room temperature for 2 h; add 5 mL of 10% BSA blocking solution, mix and incubate at room temperature for 30 min; magnetically separate, remove the supernatant, add 50 mL of preservation solution and ultrasonically disperse, repeat three times, and finally store in 50 mL of preservation solution.

[0014] In step (5), the magnetic particles bound to the BICC1 template protein, the functional monomer glycidyl methacrylate, the cross-linking agent N,N'-methylenebisacrylamide, and the surfactant sodium dodecyl sulfate are purged with nitrogen for 20 minutes and then the temperature is set to 45°C; when the temperature rises to 45°C, the initiator ammonium persulfate is added and the reaction is continued for 6 hours; wherein the molar ratio of the functional monomer to the cross-linking agent is 1:0.5-2.

[0015] Wherein, in said step (6), the protein is eluted by an acid washing method, specifically: eluted using an elution buffer and neutralized using a neutralization buffer.

[0016] In step (7), the supernatant is removed by magnetic separation of the magnetic molecularly imprinted polymer microspheres, a serum sample neutralized with PBS buffer is added, and the mixture is incubated at room temperature for 24 hours, magnetically separated to remove unbound proteins, and then eluted with elution buffer and neutralized with neutralization buffer to finally obtain a component containing natural BICC1.

[0017] Furthermore, the elution buffer is 0.1 M Glycine, pH 3.0; and the neutralization buffer is 1 mMTris-HCl, pH 8.0.

[0018] The purified BICC1 protein was diluted to 200 pg / mL with a diluent, and the diluted solution was stored at 2-8°C; wherein the diluent was 20 mM MES, pH 6.5, containing 0.05% AES, 2% sucrose, 0.1 mM hypoxanthine, 1 M NaCl, and 0.02% Proclin 300.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This application prepares a magnetic molecular imprinted polymer, which uses molecular imprinting technology to efficiently purify BICC1 natural protein. First, magnetic Fe3O4 nanoparticles are prepared, and then colloidal gold particles are combined through citric acid reduction method. The colloidal gold particles are combined with the BICC1 template protein through electrostatic adsorption. The template protein is then fixed by polymerization using epoxy monomers. Finally, the template protein is eluted and preserved.

[0020] The extraction and preservation method of the present invention is simple and has mild reaction conditions. The colloidal gold layer of the magnetic molecularly imprinted polymer achieves efficient adsorption of template molecules. The outermost polymer fixed layer is a neutral epoxy group, which reduces nonspecific adsorption, thereby achieving efficient and specific extraction of natural BICC1 protein directly from serum, ensuring that the BICC1 protein structure is close to its natural state. In addition, the magnetic molecularly imprinted polymer is recyclable and reusable, reducing R&D and production costs and improving separation efficiency.

[0021] In addition, the protein diluent provided in the present application significantly improves the long-term stability of the BICC1 protein, allowing the protein to maintain its biological activity during storage, which is of great significance for the preservation and application of the protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the structural prediction result of BICC1.

[0023] Figure 2 The SDS-PAGE results of purified natural BICC1 from serum.

[0024] Figure 3 This is the SEC profile of purified native BICC1 from serum.

[0025] Figure 4 The results of ELISA test of BICC1 native protein are shown.

[0026] Figure 5 The long-term stability of low-concentration BICC1 protein at 2-8°C. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts are within the scope of protection of the present invention.

[0028] Example 1 Aminated Magnetic Fe3O4 Nanoparticles Add 30 mL of ethylene glycol, 1 g of FeCl₃∙6H₂O, and 3 g of 1,6-hexanediamine to a beaker and stir to dissolve. Afterwards, add 9 g of anhydrous sodium acetate and stir at high speed for 2 h. Pour the mixture into a polytetrafluoroethylene reactor and react at 200°C for 13 h. Rinse the mixture three times with ethanol and three times with water, then store in pure water until use.

[0029] Example 2 Fe3O4@Au nanoparticles Disperse 0.5 mL of 1% chloroauric acid in 49.5 mL of ultrapure water. Place the reaction flask in a constant-temperature water bath set to 90°C. Once the temperature reaches 90°C, add 5 mL of 1% sodium citrate. After reacting for 30 minutes, add 100 mg of the magnetic Fe₃O₄ prepared in Example 1 and continue stirring for another 30 minutes. Rinse the flask three times with water and store in pure water.

[0030] Example 3 Preparation of BICC1 template protein Because the molecular weight of the N-terminal KH domain is approximately 9.5 kDa, it can cause immune escape and is not suitable as an antigen for immunization. Therefore, a solubility-enhancing tag GST needs to be added. The plasmid encoding (His tag)-GST-(BICC1 KH domain) (sequence shown in SEQ ID NO.1) obtained by gene synthesis (completed by Shanghai Sangon Biotechnology Co., Ltd.) was transformed into E. coli BL21In DE3, expression was induced with 0.2 mM IPTG at 20°C for 18 hours, and the cells were harvested by high-speed centrifugation. The harvested cells were resuspended in 30 mL of 10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1 mM PMSF, and 10% glycerol, and lysed by sonication. The lysate was centrifuged at 12,000 rpm, and the supernatant was filtered through a 0.45 μm filter. The sample was then loaded onto a 5 mL nickel column. After equilibration with 10% buffer B, the column was eluted with 10%-100% buffer B for five column volumes. The eluted sample was collected as 6-His-GST-(BICC1 KH domain). Buffer B consisted of 10 mM Tris-HCl, 100 mM NaCl, and 500 mM imidazole.

[0031] To purify the native BICC1 protein, the method used in this application is an antigen-antibody interaction method. BICC1-specific antibodies need to be prepared, and the present invention chose to prepare sheep polyclonal antibodies. This step was commissioned to Nanjing Huibiao Biotechnology Co., Ltd., which provided sheep serum against the N-terminal KH domain. 50 mL of sheep antiserum was diluted with an equal volume of 1× PBS (pH 7.4) and loaded onto a prepacked column packed with Protein G (Sanyi Technology Co., Ltd., Protein GS Anarose 4FF). The column was equilibrated with 50 mL of PBS, then eluted with 50 mL of 0.1 M Glycine, pH 3.0, and neutralized with 5 mL of 1 mM Tris-HCl, pH 8.0. The eluted polyclonal antibody was diluted with 1× PBS (pH 7.4) to a final concentration of 20 mg / mL and stored at -20°C until use.

[0032] Example 4 BICC1 template protein binding 50 mg of the Fe3O4@Au nanoparticles in Example 2 were placed in a beaker, and 50 mL of labeling buffer (50 mM MES, pH 6.5) was added. The mixture was subjected to magnetic separation, the supernatant was removed, and 50 mL of labeling buffer was added. The mixture was ultrasonically dispersed, repeated three times, and finally stored in 50 mL of labeling buffer. 25 mg of the template protein was added to the above beaker, quickly mixed, and incubated at room temperature for 2 h. 5 mL of 10% BSA blocking solution was added, and the mixture was mixed and incubated at room temperature for 30 min. The mixture was magnetically separated, the supernatant was removed, and 50 mL of preservation solution (10 mM PBS (pH 7.4±0.05), 10 mM NaCl, 0.05% Tween-80, 1% BSA, 5% sucrose, 0.1% ProClin 300) was added and ultrasonically dispersed. The mixture was repeated three times, and finally stored in 50 mL of preservation solution.

[0033] Example 5 Immobilization of BICC1 Template Protein 50 mL of magnetic microparticles bound to the BICC1 template protein from Example 4, 0.3 mmol of the functional monomer glycidyl methacrylate, 0.3 mmol of the cross-linker N,N'-methylenebisacrylamide, and 10 mg of the surfactant sodium dodecyl sulfate were added to the mixture. After nitrogen flow for 20 minutes, the temperature was set to 45°C. Once the temperature reached 45°C, 30 mg of the initiator ammonium persulfate was added, and the reaction was continued for 6 hours.

[0034] Example 6: Elution of BICC1 Template Protein to Prepare Magnetic Molecularly Imprinted Polymer Microspheres Elution was performed with 50 mL of elution buffer (0.1 M Glycine, pH 3.0), neutralization was performed with 12.5 mL of neutralization buffer (1 mM Tris-HCl, pH 8.0), and finally storage was performed in 50 mL of storage solution (10 mM PBS (pH 7.4 ± 0.05), 10 mM NaCI, 0.05% Tween-80, 1% BSA, 5% sucrose, 0.1% ProClin 300).

[0035] Example 7 Purification of BICC1 native protein A 20 mL serum sample was neutralized with 50 mL of 1× PBS buffer, and the supernatant was removed by magnetic separation using the magnetic molecularly imprinted polymer microspheres described in Example 6. Then, 50 mL of serum sample neutralized with PBS buffer was added, and the mixture was incubated at room temperature for 24 h. Unbound proteins were removed by magnetic separation, and then 5 mL of elution buffer (0.1 M Glycine, pH 3.0) was added for elution. The mixture was neutralized with 5 mL of neutralization buffer (1 mM Tris-HCl, pH 8.0) to obtain a fraction containing native BICC1.

[0036] Example 8

[0037] The concentration of the purified native protein in Example 7 was determined by the BCA method and was 0.2 mg / mL.

[0038] Example 9

[0039] The protein purity was analyzed by SDS-PAGE electrophoresis and SEC. Figure 2-3 shown.

[0040] Example 10 BICC1 native protein immune titer test The titer of native BICC1 protein was tested using an ELISA kit purchased from Hermes Criterion Biotechnology [HCB], Vancouver, BC, Canada. The BICC1 protein extracted in Example 7 was diluted in a series of 1xPBS dilutions. Protein-free 1xPBS was used as a control and assayed according to the ELISA kit instructions. The results are shown in Table 1. Figure 4 shown.

[0041] Example 11 Long-term stability test of BICC1 native protein Protein stabilization solutions used as diagnostic reagent calibrators and quality control products usually have a shelf life of one year. Therefore, this study evaluated the long-term stability of BICC1 protein at 2-8°C. In the experiment, BICC1 protein was diluted to 200 pg / mL using conventional diluent (1xPBS, pH 7.4, containing 0.5% BSA, 0.1% Tween 20, 0.02% Proclin 300) and the protein diluent of the present invention (20 mM MES, pH 6.5, containing 0.05% AES, 2% sucrose, 0.1 mM hypoxanthine, 1 M NaCl, 0.02% Proclin 300). The diluted solution was stored at 2-8°C and taken out at 0, 1, 3, 6, 9, 12 and 15 months, and the titer was determined using an ELISA kit. The results are shown in Figure 2. Figure 5 shown.

[0042] In order to highlight the beneficial effects of the present invention, the following comparative example experiments are given.

[0043] Comparative Example 1 A mixture of 50 mg of the Fe₃O₄@Au nanoparticles from Example 2, 25 mg of the template protein, 0.3 mmol of the functional monomer glycidyl methacrylate, 0.3 mmol of the crosslinker N,N'-methylenebisacrylamide, and 10 mg of the surfactant sodium dodecyl sulfate was prepared. After nitrogen flow for 20 minutes, the temperature was set to 45°C. Once the temperature reached 45°C, 30 mg of the initiator ammonium persulfate was added, and the reaction was continued for 6 hours.

[0044] Comparative Example 2 The template protein coated with the polymer microspheres in Comparative Example 1 was eluted with 50 mL of elution buffer (0.1 M Glycine, pH 3.0), neutralized with 12.5 mL of neutralization buffer (1 mM Tris-HCl, pH 8.0), and finally stored in 50 mL of preservation solution (10 mM PBS (pH 7.4 ± 0.05), 10 mM NaCI, 0.05% Tween-80, 1% BSA, 5% sucrose, 0.1% ProClin 300).

[0045] Comparative Example 3 20 mL of serum sample was neutralized with 50 mL of 1× PBS buffer, and the supernatant was removed by magnetic separation using the magnetic molecularly imprinted polymer microspheres in Comparative Example 2. 50 mL of serum sample neutralized with PBS buffer was then added, and the mixture was incubated at room temperature for 24 h. Unbound proteins were removed by magnetic separation, and then 5 mL of elution buffer (0.1 M Glycine, pH 3.0) was added for elution. The mixture was neutralized with 5 mL of neutralization buffer (1 mM Tris-HCl, pH 8.0) to obtain a fraction containing native BICC1.

[0046] Comparative Example 4 The concentration of the purified natural protein in Comparative Example 3 was determined by the BCA method and was 0.12 mg / mL.

[0047] The above experimental results show that Example 8 is the concentration of the purified natural protein in Example 7 determined by the BCA method, which is 0.2 mg / mL. Comparative Example 4 is the concentration of the purified natural protein in Comparative Example 3 determined by the BCA method, which is 0.12 mg / mL. The molecularly imprinted polymer in the embodiment is prepared by first adsorbing the template protein with colloidal gold and then immobilizing it; while in the comparative example, the template protein is encapsulated by the template molecule, functional monomers, cross-linking agents, etc. during the monomer polymerization process. From the data comparison, it can be seen that the method of first adsorbing the template protein and then immobilizing the protein in the method of the present invention has high efficiency in binding to the template protein and high protein purification efficiency.

[0048] Figure 2 and Figure 3The protein purity of the protein purified by the embodiment technology was analyzed by SDS-PAGE electrophoresis and SEC, respectively. SDS-PAGE showed that the protein purity was ≥85%, and SEC analysis showed that the protein purity was 88.2%, indicating that the BICC1 natural protein purified by the present invention had a high purity. Figure 4 The results of the ELISA test on the potency of the BICC1 native protein are given. The sensitivity of the test can reach 15 pg / mL, indicating that the BICC1 native protein purified from serum in the present invention has a high potency. Figure 5 The stability results of BICC1 native protein in low-concentration storage solution are shown in the figure, indicating that BICC1 protein can maintain stability for up to 15 months in this storage solution at 2-8°C.

[0049] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for efficiently extracting and preserving serum BICC1 natural protein, characterized in that: The following steps are involved: 1) Preparation of amino-modified magnetic Fe3O4 nanoparticles: Surface amino-modified magnetic Fe3O4 nanoparticles were prepared by solvothermal method; 2) Preparation of Fe3O4@Au nanoparticles: Gold nanoparticles were prepared by citric acid reduction and distributed on the surface of magnetic Fe3O4 nanoparticles through electrostatic adsorption and covalent bonding to form Fe3O4@Au nanoparticles; 3) Preparation of BICC1 template protein: Design and prepare specific immunogens using key structural domains of the BICC1 protein to generate polyclonal antibodies against the BICC1 protein as template protein; 4) BICCI template protein binding: Fe3O4@Au nanoparticles bind to BICC1 template protein by adsorption; 5) Immobilization of the BICC1 template protein: Disperse the template protein-bound magnetic gold nanoparticles in pure water, add a crosslinker, glycidyl methacrylate, and an initiator, and react at 45°C for 6 h. 6) Elution of BICC1 template protein to prepare BICC1 magnetic molecularly imprinted polymer microspheres: The template protein is removed by acid washing to form BICC1 magnetic molecularly imprinted polymer microspheres; 7) Purification of native BICC1 protein: Human serum samples were adsorbed onto BICC1 magnetic molecularly imprinted polymer microspheres, followed by an elution step to effectively isolate the native BICC1 protein.

2. The method for efficiently extracting and preserving serum BICC1 native protein according to claim 1, characterized in that: The step (1) specifically comprises adding FeCl3∙6H2O, ethylene glycol, and 1,6-hexanediamine into a beaker, stirring to dissolve, adding anhydrous sodium acetate, stirring for 2 hours to fully dissolve, pouring into a polytetrafluoroethylene reactor, and reacting at 180-280°C for 8-16 hours; wherein the mass ratio of FeCl3∙6H2O to 1,6-hexanediamine is 1:1-6, and the mass ratio of FeCl3∙6H2O to anhydrous sodium acetate is 1:5-10.

3. The method for efficiently extracting and preserving serum BICC1 native protein according to claim 1, characterized in that: The step (2) is specifically as follows: taking an appropriate amount of 1% chloroauric acid and dispersing it in ultrapure water; adding 1% sodium citrate as a reducing agent at 85-95°C; and continuing the reaction for 0.5-1h; then adding amino-modified magnetic Fe3O4 nanoparticles; and continuing the reaction for 0.5-1h; wherein the mass fraction of chloroauric acid in the entire system is 0.005%-0.01%, and the mass ratio of chloroauric acid to sodium citrate is 1:5-20.

4. The method for efficiently extracting and preserving serum BICC1 native protein according to claim 1, characterized in that: In the step (3), the structural domain of the BICC1 protein includes the BICC1 N-terminal KH domain or the BICC1 C-terminal SAM domain; the antigen contains a solubility tag, and the solubility tag includes any one of GST, MBP, and SUMO; the immunized animal includes any one of a rabbit and a sheep; and the BICC1 polyclonal antibody is an antibody extracted from serum, including any one of an N-terminal KH domain polyclonal antibody and a BICC1 C-terminal SAM domain polyclonal antibody.

5. The method for efficiently extracting and preserving serum BICC1 native protein according to claim 4, characterized in that: The structural domain of the BICC1 protein is the BICC1 N-terminal KH domain; the solubility tag is a GST tag; the immunized animal is a sheep, and the BICC1 polyclonal antibody is an N-terminal KH domain polyclonal antibody.

6. The method for efficiently extracting and preserving serum BICC1 native protein according to claim 1, characterized in that: In the step (4), the surface colloidal gold is combined with the BICC1 template protein through the adsorption effect, specifically: take 50 mg of Fe3O4@Au nanoparticles in a beaker, then add 50 mL of labeling buffer, magnetically separate, remove the supernatant, then add 50 mL of labeling buffer, ultrasonically disperse, repeat three times, and finally store in 50 mL of labeling buffer; take 25 mg of template protein and add it to the above beaker, quickly mix, and incubate at room temperature for 2 h; add 5 mL of 10% BSA blocking solution, mix and incubate at room temperature for 30 min; magnetically separate, remove the supernatant, add 50 mL of preservation solution and ultrasonically disperse, repeat three times, and finally store in 50 mL of preservation solution.

7. The method for efficiently extracting and preserving serum BICC1 native protein according to claim 1, characterized in that: In the step (5), the magnetic particles bound to the BICC1 template protein, the functional monomer glycidyl methacrylate, the cross-linking agent N,N'-methylenebisacrylamide, and the surfactant sodium dodecyl sulfate are purged with nitrogen for 20 minutes and then the temperature is set to 45°C. When the temperature rises to 45°C, the initiator ammonium persulfate is added and the reaction is continued for 6 hours. The molar ratio of the functional monomer to the cross-linking agent is 1:0.5-2.

8. The method for efficiently extracting and preserving serum BICC1 native protein according to claim 1, characterized in that: In the step (6), the protein is eluted by an acid washing method, specifically: elution is performed using an elution buffer and neutralization is performed using a neutralization buffer; wherein the elution buffer is 0.1 M Glycine, pH 3.0; and the neutralization buffer is 1 mM Tris-HCl, pH 8.

0.

9. The method for efficiently extracting and preserving serum BICC1 native protein according to claim 1, characterized in that: In the step (7), the supernatant is removed by magnetic separation of the magnetic molecularly imprinted polymer microspheres, and a serum sample neutralized with PBS buffer is added, incubated at room temperature for 24 hours, and magnetically separated to remove unbound proteins. Then, an elution buffer is added for elution, and neutralized with a neutralization buffer to finally obtain a component containing natural BICC1; wherein the elution buffer is 0.1 M Glycine, pH 3.0; and the neutralization buffer is 1 mM Tris-HCl, pH 8.

0.

10. The method for efficiently extracting and preserving serum BICC1 native protein according to claim 1, characterized in that: The purified BICC1 protein was diluted to 200 pg / mL with protein diluent, and the diluted solution was stored at 2-8°C; wherein the protein diluent was 20 mM MES, pH 6.5, containing 0.05% AES, 2% sucrose, 0.1 mM hypoxanthine, 1 M NaCl, and 0.02% Proclin 300.

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