A Highly Efficient Extraction and Preservation Method for Serum BICC1 Natural Protein

Abstract

This invention relates to the field of protein extraction technology, and in particular to a highly efficient method for extracting and preserving natural BICC1 protein from serum, comprising the following steps: 1) preparation of aminated magnetic Fe3O4 nanoparticles; 2) preparation of Fe3O4@Au nanoparticles; 3) preparation of BICC1 template protein; 4) binding of BICC1 template protein; 5) immobilization of BICC1 template protein; 6) elution of BICC1 template protein to prepare BICC1 magnetic molecularly imprinted polymer microspheres; 7) purification of natural BICC1 protein. This invention features a simple method with mild reaction conditions. The colloidal gold layer of the magnetic molecularly imprinted polymer achieves highly efficient adsorption of template molecules, and the outermost polymer immobilization layer, composed of neutral epoxy groups, reduces non-specific adsorption, enabling efficient and specific extraction of natural BICC1 protein directly from serum.

Description

Technical Field

[0001] This invention relates to the field of protein extraction technology, specifically to a method for the efficient extraction and preservation of serum BICC1 natural protein. Background Technology

[0002] In 1995, researchers discovered a gene in fruit flies (called the bitail-C gene) whose loss of function leads to the absence of a head and the formation of a bitail structure in fruit flies (Michele M, Emma ES, Paul FL. Localized Bicaudal –C RNA encodes a protein containing a KH domain, the RNA binding motif of FMR1). EMBO According to sequence analysis, the protein (BICC1) translated from this gene contains several N-terminal KH domains and C-terminal SAM domains (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 the double-tailed-C gene exist in many species, including humans. The human-derived BICC1 gene is a gene that encodes RNA-binding proteins and is widely expressed in the brain and surrounding tissues. It is not only related to the pathogenesis of depression, but also closely related to cancer and pancreatic cancer. Multiple studies have reported that the human double-tailed-C gene (called BICC1 gene) is involved in the pathogenesis 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 serum BICC1 level in normal individuals is 533.24±43.08 pg / mL, while the level in patients with unipolar depression (MDD) is 636.30±41.33 pg / mL, and the level in patients with bipolar depression (MDD) is 784.44±36.54 pg / mL. Therefore, BICC1 can be used as a biomarker to differentiate 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 and has a molecular weight of 104.8 kDa. Structural prediction indicated that the N-terminus contains three similar KH domains, the C-terminus has a SAM domain, and the remaining sequence corresponds to a disordered structure (e.g., ...). Figure 1(As shown). Both the KH and SAM domains are key domains for the biological function of this protein. 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 via 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 detection and analysis, the accurate determination of BICC1 levels in human serum is of great significance for the diagnosis, treatment monitoring, and biomedical research of unipolar (MDD) and bipolar (BD) depression. Assay kits require a series of low-concentration BICC1 proteins as calibrators and quality control samples. However, the BICC1 protein has highly unique characteristics, with a significant proportion of disordered structures in its amino acid sequence, which makes soluble recombinant expression of the full-length protein difficult. Therefore, the industry standard practice is to express a partial fragment of BICC1 using recombinant technology to meet calibration and quality control requirements. However, the problem is that the recombinant protein fragments differ from the native BICC1 protein in spatial structure and immunoeptopes, altering key antigenic determinants, which can easily lead to measurement bias and inaccuracies, severely affecting the reliability of the test results. To overcome this challenge, directly isolating and purifying native BICC1 protein from human serum is undoubtedly the most ideal solution.

[0005] BICC1, as an RNA-binding protein, possesses the ability to interact and bind with a variety of proteins. Its highly disordered structure makes the natural BICC1 protein extremely fragile. Researchers have attempted many traditional methods to improve its stability, such as adding buffer solutions to the protein storage solution to adjust the pH and maintain a stable ionic environment; adding NaCl to utilize ionic strength balance to reinforce the protein structure; incorporating sucrose and glycerol to leverage their osmotic pressure regulation properties and resist adverse external environmental interference; and even combining it with commonly used surfactants such as Tween 20 and Triton X100 to reduce the surface tension of the protein solution and decrease protein aggregation. Unfortunately, these conventional methods have failed to achieve the desired effect of improving the stability of the natural BICC1 protein.

[0006] Molecularly imprinted polymer technology has the characteristics of good mechanical and chemical stability, high selectivity and long lifespan, 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 iron oxide nanoparticles. Simultaneously, the core-shell molecularly imprinted polymer material is prepared by imprinting with a virtual template, betamethasone. CN107899557B discloses a magnetic molecularly imprinted polymer microsphere and its preparation method and application, which consists of the following components: (1) Fe3O4 nanoparticles; (2) functional polymer, which is a copolymer of carboxylic acid containing at least one olefinic unsaturated double bond and styrene; (3) 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 with double bonds on their surface are prepared by reacting 3(methacryloyloxy)propyltrimethoxysilane with Fe3O4. The magnetic nanoparticles with double bonds, 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 on the surface of the magnetic nanoparticles using ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) as initiators. Finally, the template molecule lysozyme is completely removed by dialysis in water to obtain the magnetic molecularly imprinted polymer for separating lysozyme protein. These patent documents all involve first preparing magnetic Fe3O4 nanoparticles, then directly coating the surface of the magnetic Fe3O4 nanoparticles with a layer of silica, or first modifying the surface with double bonds and then coating it with a polymer shell such as polystyrene. Template molecules and functional monomers are then simultaneously added to the magnetic polymer microspheres. Polymerization encapsulates the template molecules within the magnetic polymer microspheres, followed by elution steps to remove the template molecules. This process is complex and involves numerous steps, and the efficiency of encapsulating template molecules through polymerization is low.

[0007] Therefore, it is extremely important to develop a highly efficient method for extracting and preserving serum BICC1 natural protein. Summary of the Invention

[0008] The purpose of this invention is to provide a highly efficient method for extracting and preserving serum BICC1 natural protein.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A highly efficient method for extracting and preserving serum BICC1 natural protein, comprising the following steps:

[0011] 1) Preparation of aminated magnetic Fe3O4 nanoparticles: Aminated magnetic Fe3O4 nanoparticles were prepared by a solvothermal method.

[0012] 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;

[0013] 3) Preparation of BICC1 template protein: Using the key structural domains of the BICC1 protein, a specific immunogen was designed and prepared to generate a polyclonal antibody against the BICC1 protein, which served as the template protein;

[0014] 4) Binding to BICCI template protein: Fe3O4@Au nanoparticles bind to the BICC1 template protein via adsorption.

[0015] 5) Immobilization of BICC1 template protein: Magnetic gold nanoparticles bound to the template protein were dispersed in pure water, and cross-linking agent, glycidyl methacrylate and initiator were added. The mixture was reacted at 45°C for 6 hours.

[0016] 6) Preparation of BICC1 magnetic molecularly imprinted polymer microspheres by eluting BICC1 template protein: The template protein is removed by acid washing to form BICC1 magnetic molecularly imprinted polymer microspheres;

[0017] 7) Purification of native BICC1 protein: Human serum samples are adsorbed onto BICC1 magnetic molecularly imprinted polymer microspheres, followed by an elution step to effectively separate the native BICC1 protein.

[0018] Specifically, step (1) involves adding FeCl3∙6H2O, ethylene glycol, and 1,6-hexanediamine to a beaker, stirring until dissolved, then adding anhydrous sodium acetate, stirring for 2 hours until fully dissolved, and finally pouring the mixture into a polytetrafluoroethylene reactor and reacting at 180-280℃ for 8-16 hours. 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.

[0019] Specifically, step (2) involves dispersing an appropriate amount of chloroauric acid with a mass fraction of 1% in ultrapure water, adding sodium citrate with a mass fraction of 1% as a reducing agent at 85-95℃, and continuing the reaction for 0.5-1h; then adding aminated 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.

[0020] In step (3), the BICC1 protein domains include either the BICC1 N-terminal KH domain or the BICC1 C-terminal SAM domain; preferably, the BICC1 N-terminal KH domain; the antigen contains a solubilizing tag; specifically, the solubilizing tag includes any one of GST, MBP, and SUMO; preferably, the GST tag; the immunized animal includes any one of rabbits and sheep; preferably, sheep. The BICC1 polyclonal antibody is a serum-extracted antibody, 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.

[0021] In step (4), the adsorption of colloidal gold on the surface binds to the BICC1 template protein. Specifically, 50 mg of Fe3O4@Au nanoparticles are placed in a beaker, 50 mL of labeling buffer is added, magnetic separation is performed, the supernatant is removed, another 50 mL of labeling buffer is added, ultrasonic dispersion is performed, the process is repeated three times, and finally the product is stored in 50 mL of labeling buffer. 25 mg of template protein is added to the above beaker, quickly mixed, and incubated at room temperature for 2 h. 5 mL of 10% BSA blocking solution is added, and incubated at room temperature for 30 min. Magnetic separation is performed, the supernatant is removed, 50 mL of preservation solution is added, ultrasonic dispersion is performed, the process is repeated three times, and finally the product is stored in 50 mL of preservation solution.

[0022] In step (5), magnetic microparticles bound to the BICC1 template protein, glycidyl methacrylate, N,N'-methylenebisacrylamide, and sodium dodecyl sulfate are purged with nitrogen for 20 minutes, and the temperature is set to 45°C. When the temperature reaches 45°C, ammonium persulfate is added as an initiator, and the reaction continues for 6 hours. The molar ratio of the functional monomer to the crosslinking agent is 1:0.5-2.

[0023] In step (6), the protein is eluted by acid washing, specifically by eluting with elution buffer and neutralizing with neutralization buffer.

[0024] In step (7), the supernatant of the magnetic molecularly imprinted polymer microspheres is removed by magnetic separation, serum sample neutralized by PBS buffer is added, incubated at room temperature for 24 hours, magnetic separation is performed to remove unbound proteins, elution buffer is added for elution, neutralization buffer is used for neutralization, and finally the component containing natural BICC1 is obtained.

[0025] Furthermore, the elution buffer is 0.1 M Glycine, pH 3.0; and the neutralization buffer is 1 mM Tris-HCl, pH 8.0.

[0026] The purified BICC1 protein was diluted to 200 pg / mL with a diluent, and the diluted solution was stored at 2-8℃. The diluent was 20 mM MES, pH 6.5, containing 0.05% AES, 2% sucrose, 0.1 mM hypoxanthine, 1M NaCl, and 0.02% Proclin 300.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] This application prepares a magnetic molecularly imprinted polymer and uses molecular imprinting technology to efficiently purify the natural BICC1 protein. First, magnetic Fe3O4 nanoparticles are prepared, and then colloidal gold particles are combined with them by citric acid reduction. The colloidal gold particles are combined with the BICC1 template protein through electrostatic adsorption. Then, the template protein is fixed by polymerization of epoxy monomers. Finally, the template protein is eluted and preserved.

[0029] The extraction and preservation method of this invention is simple, the reaction conditions are mild, and the colloidal gold layer of the magnetic molecularly imprinted polymer achieves efficient adsorption of template molecules. The outermost polymer immobilization layer is composed of neutral epoxy groups, which reduces non-specific adsorption. This enables efficient and specific extraction of natural BICC1 protein directly from serum, ensuring that the structure of BICC1 protein is close to its natural state. Moreover, the magnetic molecularly imprinted polymer is recyclable and reusable, reducing research and development and production costs and improving separation efficiency.

[0030] In addition, the protein diluent provided in this application significantly improves the long-term stability of BICC1 protein, enabling the protein to maintain its biological activity during storage, which is of great significance for the preservation and application of the protein. Attached Figure Description

[0031] Figure 1 The diagram shows the structural prediction results for BICC1.

[0032] Figure 2 The results are SDS-PAGE of purified natural BICC1 from serum.

[0033] Figure 3 The image shows the SEC pattern of purified natural BICC1 from serum.

[0034] Figure 4 The results are from the BICC1 natural protein ELISA test.

[0035] Figure 5 To assess the long-term stability of low-concentration BICC1 protein at 2-8°C. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1: Aminated Magnetic Fe3O4 Nanoparticles

[0038] Add 30 mL of ethylene glycol, 1 g of FeCl3∙6H2O, and 3 g of 1,6-hexanediamine to a beaker, stir to dissolve, then add 9 g of anhydrous sodium acetate and stir at high speed for 2 hours. Pour the mixture into a polytetrafluoroethylene reactor and react at 200°C for 13 hours. Wash the mixture three times with ethanol and three times with water, then store it in pure water for later use.

[0039] Example 2 Fe3O4@Au nanoparticles

[0040] 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 at 90°C. Once the temperature reaches 90°C, add 5 mL of 1% sodium citrate. After reacting for 30 min, add 100 mg of the magnetic Fe3O4 prepared in Example 1, and continue stirring for another 30 min. Wash three times with water and finally store in pure water.

[0041] Example 3: Preparation of BICC1 template protein

[0042] Because the N-terminal KH domain has a molecular weight of approximately 9.5 kDa, it can cause immune escape and is unsuitable as an antigen for immunization. Therefore, an additional solubilizing tag, GST, is needed. The plasmid encoding (His tag)-GST-(BICC1 KH domain) (sequence shown in SEQ ID NO.1), obtained through total gene synthesis (completed by Shanghai Sangon Biotech Co., Ltd.), was transformed into... E. coli BL21In (DE3), expression was induced for 18 h at 20 °C using 0.2 mM IPTG, and the bacteria were harvested using a high-speed centrifuge. The collected bacteria were resuspended in 30 mL of buffer and sonicated to lyse 1 L of the bacterial culture (buffer: 10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1 mM PMSF, 10% glycerol). The lysate was centrifuged at 12,000 rpm, and the supernatant was filtered through a 0.45 μm filter. The supernatant was then loaded onto a 5 mL nickel column, equilibrated with 10% buffer B, and eluted with 10%–100% buffer B for 5 column volumes. The eluted sample was collected as 6His-GST-(BICC1 KH domain). Buffer B consisted of 10 mM Tris-HCl, 100 mM NaCl, and 500 mM imidazole.

[0043] To purify the natural BICC1 protein, this application employs an antigen-antibody interaction method. BICC1-specific antibodies need to be prepared; therefore, this invention selects to prepare sheep polyclonal antibodies. This step was outsourced to Nanjing Huibiao Biotechnology Co., Ltd., which provided sheep serum containing the N-terminal KH domain. 50 mL of sheep antiserum was diluted with an equal volume of 1×PBS (pH 7.4) and loaded into a pre-packed column (Sanyi Technology Co., Ltd., Protein GSarose 4FF) filled with Protein G packing material. The column was equilibrated with 50 mL of PBS, then eluted with 50 mL of 0.1 M Glycine (pH 3.0), followed by neutralization 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 for later use.

[0044] Example 4: BICC1 template protein binding

[0045] Take 50 mg of Fe3O4@Au nanoparticles from Example 2 into a beaker, add 50 mL of labeling buffer (50 mM PBS, pH 6.5), magnetically separate, remove the supernatant, add another 50 mL of labeling buffer, sonicate, 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, mix quickly, and incubate at room temperature for 2 h; add 5 mL of 10% BSA blocking solution, mix at room temperature and incubate for 30 min; magnetically separate, remove the supernatant, add 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), sonicate, repeat three times, and finally store in 50 mL of preservation solution.

[0046] Example 5: Immobilization of BICC1 template protein

[0047] 50 mL of the 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 crosslinking agent N,N'-methylenebisacrylamide, and 10 mg of the surfactant sodium dodecyl sulfate were added. After purging with nitrogen for 20 min, the temperature was set to 45 °C. When the temperature reached 45 °C, 30 mg of the initiator ammonium persulfate was added, and the reaction was continued for 6 h.

[0048] Example 6 Preparation of magnetic molecularly imprinted polymer microspheres by eluting BICC1 template protein

[0049] Elution was performed using 50 mL of elution buffer (0.1 M Glycine, pH 3.0), followed by neutralization using 12.5 mL of neutralization buffer (1 mM Tris-HCl, pH 8.0). Finally, the solution was stored in 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).

[0050] Example 7 Purification of BICC1 native protein

[0051] 20 mL of serum sample was neutralized with 50 mL of 1×PBS buffer. The supernatant of the magnetic molecularly imprinted polymer microspheres from Example 6 was removed by magnetic separation. Then, 50 mL of serum sample neutralized with PBS buffer was added, and the mixture was incubated at room temperature for 24 h. After magnetic separation, unbound proteins were removed. Then, 5 mL of elution buffer (0.1 M Glycine, pH 3.0) was added for elution. The mixture was then neutralized with 5 mL of neutralization buffer (1 mM Tris-HCl, pH 8.0) to finally obtain the fraction containing natural BICC1.

[0052] Example 8

[0053] The concentration of the purified natural protein in Example 7 was determined using the BCA method and was 0.2 mg / mL.

[0054] Example 9

[0055] Protein purity was analyzed using SDS-PAGE electrophoresis and SEC, respectively. Results are as follows: Figure 2-3 As shown.

[0056] Example 10: BICC1 Natural Protein Immunotiter Test

[0057] The titer of the natural BICC1 protein was tested using an ELISA method. The ELISA kit used was purchased from Hermes Criterion Biotechnology [HCB], Vancouver, BC, Canada. The BICC1 protein extracted in Example 7 was diluted a series of gradients with 1xPBS, using 1xPBS without protein as a control. The titer was determined according to the ELISA kit instructions. Results are as follows: Figure 4 As shown.

[0058] Example 11: Long-term stability test of BICC1 natural protein

[0059] Protein stabilizers used as calibrators and quality control materials for diagnostic reagents typically have a shelf life of one year. This study therefore evaluated the long-term stability of BICC1 protein at 2–8°C. In the experiments, BICC1 protein was diluted to 200 pg / mL using both conventional diluents (1xPBS, pH 7.4, containing 0.5% BSA, 0.1% Tween 20, 0.02% Proclin 300) and the protein diluent of this 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 solutions were stored at 2–8°C and retrieved at 0, 1, 3, 6, 9, 12, and 15 months for titer determination using an ELISA kit. Results are as follows: Figure 5 As shown.

[0060] To highlight the beneficial effects of the present invention, the following comparative experiments are provided.

[0061] Comparative Example 1

[0062] Take 50 mg of Fe3O4@Au nanoparticles from Example 2, 25 mg of template protein, 0.3 mmol of glycidyl methacrylate functional monomer, 0.3 mmol of N,N'-methylenebisacrylamide crosslinking agent, and 10 mg of sodium dodecyl sulfate surfactant. After purging with nitrogen for 20 min, set the temperature to 45°C. When the temperature reaches 45°C, add 30 mg of ammonium persulfate initiator and continue the reaction for 6 h.

[0063] Comparative Example 2

[0064] The template protein coated with 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 NaCl, 0.05% Tween-80, 1% BSA, 5% sucrose, 0.1% ProClin 300).

[0065] Comparative Example 3

[0066] 20 mL of serum sample was neutralized with 50 mL of 1×PBS buffer. The supernatant of the magnetic molecularly imprinted polymer microspheres from Comparative Example 2 was removed by magnetic separation. Then, 50 mL of serum sample neutralized with PBS buffer was added, and the mixture was incubated at room temperature for 24 h. After magnetic separation, unbound proteins were removed. Then, 5 mL of elution buffer (0.1 M Glycine, pH 3.0) was added for elution. The mixture was then neutralized with 5 mL of neutralization buffer (1 mM Tris-HCl, pH 8.0) to obtain the fraction containing natural BICC1.

[0067] Comparative Example 4

[0068] The concentration of purified natural protein in Comparative Example 3 was determined using the BCA method and was 0.12 mg / mL.

[0069] The experimental results above show that, in Example 8, the concentration of the purified natural protein in Example 7 was determined to be 0.2 mg / mL using the BCA method, while in Comparative Example 4, the concentration of the purified natural protein in Comparative Example 3 was determined to be 0.12 mg / mL using the BCA method. In the examples, the molecularly imprinted polymer was prepared by first adsorbing the template protein with colloidal gold and then immobilizing it; whereas in the comparative examples, the template molecule, functional monomers, and cross-linking agents were used to encapsulate the template protein during monomer polymerization. Data comparison shows that the method of the present invention, which involves first adsorbing the template protein and then immobilizing it, has high efficiency in binding the template protein and high protein purification efficiency.

[0070] Figure 2 and Figure 3 The protein purity of the purified protein was analyzed by SDS-PAGE electrophoresis and SEC method, respectively. SDS-PAGE showed a protein purity of ≥85%, and SEC analysis showed a protein purity of 88.2%, indicating that the purified BICC1 native protein of the present invention has high purity. Figure 4 The results of the ELISA method for testing the titer of BICC1 natural protein are presented. The sensitivity of the test can reach 15 pg / mL, indicating that the BICC1 natural protein purified from serum in this invention has a high titer. Figure 5 The figure shows the stability results of the BICC1 natural protein in a low-concentration preservation solution, indicating that the BICC1 protein can maintain its stability for up to 15 months in this preservation solution at 2-8℃.

[0071] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A highly efficient method for extracting and preserving serum BICC1 natural protein, characterized in that, Includes the following steps: 1) Preparation of aminated magnetic Fe3O4 nanoparticles: Aminated magnetic Fe3O4 nanoparticles were prepared by a 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: Using the key structural domains of the BICC1 protein, a specific immunogen was designed and prepared to generate a polyclonal antibody against the BICC1 protein, which served as the template protein; 4) Binding to BICCI template protein: Fe3O4@Au nanoparticles bind to the BICC1 template protein via adsorption. 5) Immobilization of BICC1 template protein: Magnetic gold nanoparticles bound to the template protein were dispersed in pure water, and cross-linking agent, glycidyl methacrylate and initiator were added. The mixture was reacted at 45°C for 6 hours. 6) Preparation of BICC1 magnetic molecularly imprinted polymer microspheres by eluting BICC1 template protein: 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 are adsorbed onto BICC1 magnetic molecularly imprinted polymer microspheres, followed by an elution step to effectively separate the native BICC1 protein.

2. The method for efficient extraction and preservation of serum BICC1 natural protein according to claim 1, characterized in that: Specifically, step (1) involves adding FeCl3∙6H2O, ethylene glycol, and 1,6-hexanediamine to a beaker, stirring until dissolved, then adding anhydrous sodium acetate, stirring for 2 hours until fully dissolved, and finally pouring the mixture into a polytetrafluoroethylene reactor. The reaction is carried out at 180-280℃ for 8-16 hours. 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 efficient extraction and preservation of serum BICC1 natural protein according to claim 1, characterized in that: Step (2) specifically involves dispersing an appropriate amount of chloroauric acid with a mass fraction of 1% in ultrapure water, adding sodium citrate with a mass fraction of 1% as a reducing agent at 85-95℃, and continuing the reaction for 0.5-1h; then adding aminated 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 efficient extraction and preservation of serum BICC1 natural protein according to claim 1, characterized in that: In step (3), the domains of the BICC1 protein include the BICC1 N-terminal KH domain or the BICC1 C-terminal SAM domain; the antigen contains a solubilizing tag, which includes any one of GST, MBP, and SUMO; the immunized animal includes any one of rabbit and sheep; the BICC1 polyclonal antibody is an antibody extracted from serum, including any one of N-terminal KH domain polyclonal antibody and BICC1 C-terminal SAM domain polyclonal antibody.

5. The method for efficient extraction and preservation of serum BICC1 natural protein according to claim 4, characterized in that: The BICC1 protein has an N-terminal KH domain; the solubilizing tag is a GST tag; the immunized animal type is sheep; and the BICC1 polyclonal antibody is an N-terminal KH domain polyclonal antibody.

6. The method for efficient extraction and preservation of serum BICC1 natural protein according to claim 1, characterized in that: In step (4), the adsorption of colloidal gold on the surface binds to the BICC1 template protein. Specifically, 50 mg of Fe3O4@Au nanoparticles are placed in a beaker, 50 mL of labeling buffer is added, magnetic separation is performed, the supernatant is removed, another 50 mL of labeling buffer is added, ultrasonic dispersion is performed, the process is repeated three times, and finally the product is stored in 50 mL of labeling buffer. 25 mg of template protein is added to the above beaker, quickly mixed, and incubated at room temperature for 2 h. 5 mL of 10% BSA blocking solution is added, and incubated at room temperature for 30 min. Magnetic separation is performed, the supernatant is removed, 50 mL of preservation solution is added, ultrasonic dispersion is performed, the process is repeated three times, and finally the product is stored in 50 mL of preservation solution.

7. The method for efficient extraction and preservation of serum BICC1 natural protein according to claim 1, characterized in that: In step (5), magnetic microparticles bound to the BICC1 template protein, glycidyl methacrylate, N,N'-methylenebisacrylamide, and sodium dodecyl sulfate are purged with nitrogen for 20 minutes, and the temperature is set to 45°C. When the temperature reaches 45°C, ammonium persulfate is added as an initiator, and the reaction continues for 6 hours. The molar ratio of the functional monomer to the crosslinking agent is 1:0.5-2.

8. The method for efficient extraction and preservation of serum BICC1 natural protein according to claim 1, characterized in that: In step (6), the protein is eluted by acid washing, specifically by eluting with elution buffer and neutralizing with 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 efficient extraction and preservation of serum BICC1 natural protein according to claim 1, characterized in that: In step (7), the supernatant of the magnetic molecularly imprinted polymer microspheres is removed by magnetic separation, serum sample neutralized with PBS buffer is added, incubated at room temperature for 24 h, magnetic separation is performed to remove unbound proteins, then elution buffer is added, and neutralization buffer is used to obtain the component containing natural BICC1; wherein, the elution buffer is 0.1 M Glycine, pH 3.0; the neutralization buffer is 1 mM Tris-HCl, pH 8.

0.

10. The method for efficient extraction and preservation of serum BICC1 natural protein according to claim 1, characterized in that: The purified BICC1 protein was diluted to 200 pg / mL with protein dilution buffer, and the diluted solution was stored at 2-8℃. The protein dilution buffer 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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