WO2017128888A1 - 一种基于蛋白纳米线的3d探针-磁性微珠复合物及其应用 - Google Patents

一种基于蛋白纳米线的3d探针-磁性微珠复合物及其应用 Download PDF

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WO2017128888A1
WO2017128888A1 PCT/CN2016/111261 CN2016111261W WO2017128888A1 WO 2017128888 A1 WO2017128888 A1 WO 2017128888A1 CN 2016111261 W CN2016111261 W CN 2016111261W WO 2017128888 A1 WO2017128888 A1 WO 2017128888A1
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protein
magnetic
nanowire
probe
nanowires
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PCT/CN2016/111261
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English (en)
French (fr)
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门冬
张先恩
周娟
张治平
李唯
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中国科学院武汉病毒研究所
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the invention relates to the field of immunoassay, in particular to a protein nanowire-based 3D probe-magnetic microbead composite and its application in immunodetection.
  • the affinity-mediated immobilization method is to reduce the fixed randomness of the probe by the avidin-biotin system, thereby increasing the effective probe density;
  • the silicon nanowire-mediated immobilization method is on the surface modification of the silicon nanowire.
  • An aldehyde group which exhibits a high-density fixation by forming a chemical bond between an amino group and an aldehyde group to form a chemical bond;
  • a virus nanoparticle-mediated immobilization method is to fuse SPA on a viral capsid protein subunit through SPA and
  • the antibody acts to display the antibody, and the His-tag on the viral capsid protein interacts with the nickel nano-array to achieve high-density fixation;
  • the giant magnetoresistance-mediated immobilization method uses the avidin-biotin system to bind the antibody.
  • the magnetic particles are fixed at the interface by the giant magnetoresistance effect, thereby achieving high-density display of the antibody probe.
  • increasing the fixed density of the protein probe is mainly achieved by two ways: one, instead of random physical adsorption by specific fixation; and two, increasing the number of immobilized protein probes.
  • the former increases the number of probes.
  • the affinity-mediated immobilization can only solve the problem of random orientation of the probe to a certain extent, and can not improve the real probe density.
  • the virus nanoparticle-mediated immobilization method is limited by the virus.
  • the size of the particles, the number of immobilized probes is limited, and the effect of His-tag and nickel is unstable.
  • the latter requires a complicated chemical modification process, or requires complicated instruments and operating steps, and is costly.
  • nanomaterials can increase the specific surface area, the modification method is random and complicated, and the protein probe is damaged during the modification process. Biological activity, resulting in reduced capture capacity.
  • the technical problem to be solved by the present invention is to provide a rapid and highly sensitive immunodetection product and method for realizing efficient and high-density immunodetection in a three-dimensional (3D) space.
  • the present invention adopts the following technical solutions:
  • the present invention provides a protein nanowire-based 3D probe-magnetic microbead composite comprising magnetic microbeads and protein nanowires formed by self-assembly of a blunt protein, and the protein nanowires
  • the surface comprises at least one functional ligand and at least one linking ligand, the protein nanowires being attached to the surface of the magnetic microbeads by a linking ligand.
  • the magnetic microbead refers to a nano or micro-scale microsphere having a certain magnetic and special surface structure, which can be composed of an inorganic magnetic substance and various materials containing active functional groups.
  • the inorganic magnetic material comprises a magnetic nano/micro material or a magnetic nano/micro composite, preferably, the magnetic nano/micro material comprises a magnetic element (such as iron, cobalt, nickel, ruthenium, boron, zirconium, chromium, etc.)
  • Doped nano/micro materials for example, iron oxide ( ⁇ -Fe 2 O 3 , ⁇ -Fe 3 O 4 ), ferrite (CoFe 2 O 4 , BaFe 12 O 19 ), chromium oxide (CrO 2 ) Zirconium oxide (ZrO 2 ), iron nitride (Fe 4 N), metal alloy (Fe, Co, Ni, Al), and the like.
  • the active functional group-containing material includes synthetic polymer materials such as polyethylene glycol, polyvinyl alcohol, polyglycolic acid, polyacrylic acid, silane derivatives, or cellulose and its derivatives, agarose, gelatin, and dextran. , chitosan and its derivatives, hyaluronic acid, alginic acid and other natural polymer materials.
  • the magnetic microbeads can be rapidly positioned, guided and separated under the action of an external magnetic field, and on the other hand, various active functional groups such as hydroxyl groups can be imparted to the surface of the magnetic microbead by surface modification or chemical polymerization.
  • a carboxyl group, an aldehyde group, an amino group or the like, and a magnetic microbead may also bind a bioactive substance such as an antibody, a cell or a DNA by a covalent bond.
  • the line-forming protein is a protein containing a self-assembling domain capable of being assembled into a linear nanostructure, and may be, for example, yeast prion protein Sup35, amyloid Ure2, silk fibroin, or the like.
  • the troponin is a yeast prion protein, and the amino acids 1-61 of Sup35 are self-assembling domains.
  • the functional ligand and the linking ligand may be the same or different, and are independently selected from one or more of the group consisting of an antigen, a functionalized antibody having specific binding ability, Protein A (SPA), Protein G (Protein G, SPG), SPL protein, protein of this week (BJP), ⁇ 2-macroglobulin ( ⁇ 2m), calcitonin (CT), human chorionic gonadotropin ( hCG), adrenocorticotropic hormone (ACTG), thyroid hormone (PHT), fluorescent protein, biotin and avidin.
  • an antigen Protein A
  • Protein G Protein G
  • SPG Protein G
  • SPL protein protein of this week
  • BJP ⁇ 2-macroglobulin
  • CT calcitonin
  • hCG human chorionic gonadotropin
  • ACTG adrenocorticotropic hormone
  • PHT thyroid hormone
  • the antigen includes an antigen, a viral antigen or a microbial antigen associated with an autoimmune defect, a malignant cell or a cancer, including but not limited to any viral peptide, microbial peptide, polypeptide protein, carbohydrate, which is capable of eliciting an immune response.
  • Polysaccharide lipid molecules such as HIV-p24, HIV-gp41, HIV-gp120, HIV-gp160, HIV-nef, HA1, HAV, HBV, HCV, HDV, HEV, HBsAg, HBcAg, Ebola, EV71, SV40, HTLV- I, CBV, EB, SARS, CEA, AFP, PSA, POA, PSCA, PSMA, CA125, CA19-9, CA15-3, CA50, CA242, TNF- ⁇ , RSV-F, CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD27, CD28, CD30, CD33, CD37, CD38, CD40, CD56, CD70, CD79, CD79b, CD90, CD125, CD134, CD147, CD152/CTLA-4, and the like.
  • the protein nanowires can be connected to the magnetic microbeads by a linking ligand, and the protein nanowires combine with the large specific surface area of the magnetic microbeads to form a 3D structure, thereby increasing the fixed density; by fusion on the protein nanowires
  • the functional ligand achieves specific binding to a target molecule, and enhances the fixed orientation of an antigen or an antibody, thereby performing antibody screening or pathogen or disease detection.
  • the linking ligand is biotin.
  • the functional ligand is selected from the group consisting of an antigen, SPA, SPG or SPL, etc., preferably from one or more of the following groups: HIV-p24, HIV-gp41, HIV-gp120, HIV-gp160, HIV-nef , HA1, HAV, HBV, HCV, HDV, HEV, HBsAg, HBcAg, Ebola, EV71, SV40, HTLV-I, CBV, EB, SARS, CEA, AFP, PSA, POA, PSCA, PSMA, CA125, CA19-9 , CA15-3, CA50, CA242, SPA, SPG, etc.
  • HIV-p24 HIV-gp41, HIV-gp120, HIV-gp160, HIV-nef , HA1, HAV, HBV, HCV, HDV, HEV, HBsAg, HBcAg, Ebola, EV71, SV40, HTLV-I, CBV,
  • the protein nanowires are covalently cross-linked by biotin-avidin interaction, chemical covalent crosslinking (for example, surface carboxyl functionalized magnetic microbeads and amino groups on protein nanowires)
  • transition metal ions such as Co, Ni, Cu, Zn and other specific amino acid side chains, such as natural histidine tag HAT, polyhistidine tag His tag and NTA-Ni (nitrosotriacetic acid - Interaction between nickel) or Co-CMA (cobalt-carboxymethylaspartate)
  • GST glutthione thiotransferase
  • protein cellulose domain and fiber Interacting interactions or other interacting polypeptides
  • specific DNA-protein interactions eg surface-specific DNA functionalized magnetic microbeads interact with specific protein-fused protein nanowires
  • a specific binding mode is attached to the surface of the magnetic microbeads.
  • the protein nanowires are attached to the surface of the magnetic microbeads by a biotin-avidin interaction.
  • Another aspect of the present invention provides a method for preparing a protein nanowire-based 3D probe-magnetic microbead composite of the present invention, comprising the steps of:
  • the fusion protein gene LP-L was formed by fusion of the self-assembled domain of Linear Protein (LP) with at least one functional ligand (Ligand, L) by molecular cloning, and the fusion protein LP was obtained after purification.
  • LP Linear Protein
  • Ligand L
  • the self-assembling domain of the mitochondrial LP is fused with at least one functional ligand L by molecular cloning, and then fused with at least one linking ligand CL to form a fusion protein gene LP-L-CL (LP-CL for short). After purification by expression, the fusion protein LP-CL is obtained;
  • the protein nanowire-based 3D probe-magnetic microbead complex obtained by the preparation method of the invention enables high-density display of functional ligands such as antigen molecules or specific binding proteins on the complex, and the complex is made by multivalent effects.
  • the object can quickly bind to the target molecule to achieve a highly sensitive and rapid detection of the target molecule.
  • protein fusion when a fusion protein gene is formed by molecular cloning, protein fusion can be performed after fusion of a flexible polypeptide between two protein genes, thereby reducing the influence of possible steric hindrance.
  • the expression and purification of the fusion protein gene can be carried out by a method conventionally used in the art for purification of protein expression, for example, the fusion protein gene is cloned into an expression vector, and the expression vector and/or the co-expression vector are transferred into an expression host for cultivation. After activation to the logarithmic growth phase, the induced albumin is added, and after being crushed and purified, the fusion protein is obtained.
  • the present invention does not limit the type and class of the expression vector, the co-expression vector, and the expression host, and the vector and the host conventionally used for genetic modification in the art may be selected.
  • the expression vector may be pET-28, pET-32,
  • the co-expression vector of pET-15 or pET-11 may be pCDFDuet-1 or the like; the expression host may be selected from Escherichia coli, Bacillus subtilis, Bacillus megaterium, Corynebacterium, Saccharomyces cerevisiae, Pichia or mammalian cells.
  • cloning can be accomplished, for example, by chain enzyme polymerization (PCR).
  • PCR chain enzyme polymerization
  • the line-forming protein is a protein containing a self-assembling domain capable of being assembled into a linear nanostructure, and may be, for example, yeast prion protein (Sup35), amyloid Ure2, silk fibroin, or the like.
  • the troponin is a yeast prion protein, and the amino acids 1-61 of Sup35 are self-assembling domains.
  • At least one functional ligand and at least one linking ligand are as defined in the foregoing functional ligands and linking ligands of the present invention, and are not described herein.
  • the linking ligand is biotin.
  • the step (2) has The body includes the following steps:
  • the fusion protein gene LP-BAP is formed by fusion of the self-assembling domain of the mitochondrial LP with the biotin-receiving peptide (BAP) by molecular cloning; or the self-assembling domain of the lignin LP is at least a functional ligand L is fused, and then fused with the biological acceptor polypeptide BAP to form a fusion protein gene LP-L-BAP (abbreviated as LP-BAP);
  • the fusion protein gene LP-BAP was cloned into an expression vector, and Biotin-protein ligase (BirA) was cloned into a co-expression vector;
  • biotinylated fusion protein LP-BAP was prepared.
  • the cloning of the biotin ligase (BirA) into the co-expression vector specifically comprises: obtaining the nucleotide sequence of the BirA gene, designing primers, and adding restriction endonuclease in the upstream and downstream primers, respectively.
  • the enzyme cleavage sites of NcoI and SalI were used to amplify the BirA gene by PCR; the PCR product BirA and the expression vector pCDFDuet-1 were double-digested to collect the digested products; the collected digested products BirA and pCDFDuet-1 were collected.
  • the carrier was subjected to a ligation reaction at a ratio of 6:1 to give BirA-pCDFDuet-1.
  • the expression vector and the co-expression vector are transferred into an expression host, and activation to the logarithmic growth phase after the addition of the induced albumin and biotin specifically comprises: adding the expression vector and the co-expression vector to the inclusion
  • the medium in which the host and the antibiotic were expressed was cultured overnight until activation to the logarithmic growth phase, and IPTG was added to induce albumin and biotin overnight, and cultured and expressed.
  • the functional ligand is selected from one or more of the group consisting of HIV-p24, HIV-gp41, HIV-gp120, HIV-gp160, HIV-nef, HA1, HAV, HBV, HCV, HDV, HEV, HBsAg, HBcAg, Ebola, EV71, SV40, HTLV-I, CBV, EB, SARS, CEA, AFP, PSA, POA, PSCA, PSMA, CA125, CA19-9, CA15-3, CA50, CA242, SPA, SPG, CT, hCG, etc.
  • the seed is attached to the surface of the magnetic microbead through biotin-avidin interaction, chemical covalent crosslinking (for example, surface carboxyl functionalization) Magnetic microbeads covalently bind to amino groups on protein nanowires), specific DNA-protein interactions (eg surface-specific DNA functionalized magnetic microbeads interact with specific protein-fused protein nanowires) Or other high affinity and strong specific binding means.
  • biotin-avidin interaction for example, chemical covalent crosslinking (for example, surface carboxyl functionalization)
  • Magnetic microbeads covalently bind to amino groups on protein nanowires
  • specific DNA-protein interactions eg surface-specific DNA functionalized magnetic microbeads interact with specific protein-fused protein nanowires
  • other high affinity and strong specific binding means eg surface-specific DNA functionalized magnetic microbeads interact with specific protein-fused protein nanowires
  • the seed is attached to the surface of the magnetic microbeads by a biotin-avidin interaction, wherein biotin acts as a linking ligand and the avidin is modified on the surface of the magnetic microbeads.
  • the step (3) specifically includes the following steps:
  • biotinylated fusion protein LP-BAP was incubated at 4 ° C for one week to form nanowires, and the nanowires were broken into nanowire fragments by ultrasound to prepare biotinylated LP-BAP seeds; avidin modified
  • the magnetic microbeads were incubated with excess biotinylated seed LP-BAP at 37 ° C and washed by magnetic field separation to obtain a seed-magnetic microbead complex.
  • the step (4) specifically includes the following steps:
  • the seed-magnetic microbead complex obtained in the step (3) is subjected to seed-induced self-assembly, and the fusion protein LP-L containing the functional ligand is added, and the protein nanowire is grown on the surface of the magnetic microbead at room temperature.
  • the protein nanowire-based 3D probe-magnetic microbead composite of the present invention is obtained by magnetic field separation and washing.
  • the method for modifying the avidin on the magnetic microbeads in the present invention is well known to those skilled in the art, and commercially available avidin-modified magnetic microbeads may also be used, and no further details are provided herein.
  • the invention adopts the seed-induced self-assembly method, and the seed can rapidly induce the fusion protein with the self-assembled domain to be assembled at the end thereof, and control the reaction time to control the assembly ratio of the seed and the fusion protein, thereby realizing the control of the length of the nanowire.
  • a fusion protein containing a functional ligand can be controlled to be assembled to the end of the protein nanowire, and further, by controlling the proportion of the functional ligand in the protein nanowire, A product for immunoassay with high sensitivity and low non-specific adsorption is obtained.
  • the present invention provides a product for immunoassay comprising the protein nanowire-based 3D probe-magnetic microbead complex of the present invention.
  • the product may be in the form of a probe (sensor), a test strip, a chip, a kit, etc., and when used, the protein nanowire-magnetic bead composite of the present invention and other existing commercial reagents (such as Mixing enzyme-labeled antibodies, fluorescently labeled antibodies, chromogenic reagents, substrates, etc., can be used for various forms of immunoassay, such as antibody detection, antibody screening, antigen detection, pathogen detection, protein detection, protein interaction screening, high Flux target protein detection, protein-nucleic acid interaction analysis, drug screening, and the like.
  • immunoassay such as antibody detection, antibody screening, antigen detection, pathogen detection, protein detection, protein interaction screening, high Flux target protein detection, protein-nucleic acid interaction analysis, drug screening, and the like.
  • the probe When the product is in the form of a probe (sensor), the probe can be used for efficient, high-density solids
  • the antigen or antibody is determined for highly sensitive detection of the target molecule in three dimensions.
  • the composite of the present invention can be placed on a detection line for capturing target molecules, and detection is performed using gold-labeled nanoparticles; further, when the complex is fused with different functional ligands, Simultaneous detection of multiple target molecules.
  • the complex of the present invention can be placed in a chip for point-of-care (POC); further, when the complex is fused with different functional ligands, it can be Separately placed in different channels of the chip, high-throughput detection of multiple target molecules can be achieved simultaneously.
  • POC point-of-care
  • the kit may further include a buffer, a washing solution, a diluent or a color developing agent.
  • the present invention also provides the use of the protein nanowire-based 3D probe-magnetic microbead complex of the present invention in immunoassay.
  • the immunoassay can be indirect immunization, sandwich immunization, etc., and can be used for various forms of immunoassay, such as antibody detection, antibody screening, antigen detection, pathogen detection, protein detection, protein interaction screening, high throughput Target protein detection, protein-nucleic acid interaction analysis, drug screening, and the like.
  • the present invention is based on a protein nanowire-based 3D probe-magnetic microbead complex for solution phase immunoassay.
  • protein nanowire-based 3D probe-magnetic microbead complex of the present invention can be applied to the detection for the purpose of treatment or non-treatment.
  • the 3D probe-magnetic microbead complex based on the protein nanowire can capture target molecules efficiently and at high density, and achieve rapid and highly sensitive detection, which can be improved by more than 100 times compared with the conventional ELISA.
  • the 3D probe-magnetic microbead composite based on the protein nanowire of the invention is simple and easy to prepare, and can be applied to different immunoassay modes, such as indirect ELISA, sandwich ELISA, etc., especially suitable for liquid phase.
  • the immunoassay in the middle is only to replace one of the original detection methods, without changing the original operation steps, and no additional equipment and instruments are needed.
  • the present invention utilizes specific biological interaction to grow multifunctional protein nanowires in situ on the surface of magnetic microbeads, which not only has a simple operation process, but also has a short reaction time, and a high affinity biological action can improve the immobilization ability and stability of functional molecules.
  • the detection system constructed by the invention is very flexible, and the functional protein nanowires suitable for detecting various desired target molecules can be grown in situ by simply replacing the functional fusion protein unit.
  • the present invention utilizes the 3D protein display capability of protein nanowires, and utilizes high-density display to bring about large specific surface area and multivalent state effects, thereby improving the ability to capture target molecules in a sample (currently the conventional ELISA is on a two-dimensional plane)
  • the protein probe is immobilized by physical adsorption, the density of the immobilized protein probe is limited, which results in lower efficiency of capturing the target molecule, and thus the sensitivity is low.
  • the magnetic microbead can be used for rapid separation and the 3D display nanowire Used in combination with magnetic beads, the detection time is greatly reduced.
  • Fig. 1 is a schematic view showing the preparation of a 3D probe-magnetic microbead composite based on protein nanowires of the present invention.
  • Fig. 2 is a schematic diagram showing the principle of 3D high-sensitivity indirect immunoassay based on protein nanowire-based 3D probe-magnetic microbead complex.
  • Figure 3 is a graph showing the results of 3D high-sensitivity indirect immunodetection based on protein nanowire-based 3D probe-magnetic microbead complex.
  • ⁇ is a p24 protein nanowire-magnetic microbead (denoted as p24-NW-MB)
  • the present invention is based on a protein nanowire-based 3D probe-magnetic microbead complex
  • ⁇ is a p24-magnetic microbead (denoted as p24) -MB)
  • biotinylated p24 pathogenic protein was directly displayed on magnetic beads
  • 15 ⁇ was a commercial ELISA reagent, compared to conventional methods.
  • Fig. 4 is a schematic diagram showing the principle of 3D high-sensitivity sandwich immunoassay based on protein nanowire-based 3D probe-magnetic microbead complex.
  • Figure 5 is a graph showing the results of 3D high-sensitivity sandwich immunoassay based on protein nanowire-based 3D probe-magnetic microbead complex.
  • “genetic modification” refers to genetic modification of the genome of an organism by molecular biological techniques, and the resulting gene composition and trait changes.
  • “Avidin” includes, but is not limited to, Avidin, Streptomyces Avidin.
  • Biotin Accepted Peptide” (BAP) is a polypeptide capable of being fused to the N-terminus of ferritin and capable of linking biotin.
  • Biotin-protein Ligase (BirA) refers to an enzyme that is capable of activating biotin and attaching biotin to a biotin receptor protein.
  • Linear Protein refers to a protein containing a self-assembling domain capable of being assembled into a linear nanostructure.
  • LP-L-BAP and “LP-BAP” are used interchangeably, and can represent LP-BAP of modified/unmodified biotin, and the specific meanings are understood according to the context. .
  • the present invention provides a 3D probe-magnetic microbead composite based on protein nanowires, comprising magnetic microbeads and protein nanowires, which are formed by self-assembly of line-forming proteins.
  • the protein nanowire surface comprises at least one functional ligand and at least one linking ligand, the protein nanowires being attached to the surface of the magnetic microbeads by a linking ligand.
  • the present invention provides a method for preparing a 3D probe-magnetic microbead composite based on protein nanowires, comprising the following steps:
  • the fusion protein gene LP-L is formed by fusion of the self-assembling domain of the mitochondrial LP with at least one functional ligand L by molecular cloning, and the fusion protein LP-L is obtained after purification;
  • the self-assembling domain of the mitochondrial LP is fused with at least one functional ligand L by molecular cloning, and then fused with at least one linking ligand CL to form a fusion protein gene LP-L-CL (LP-CL for short). After purification by expression, the fusion protein LP-CL is obtained;
  • the invention provides a product for immunoassay comprising a protein nanowire-based 3D probe-magnetic microbead complex of the invention.
  • the product may be in the form of a probe (sensor), a test strip, a chip, a kit, etc., and when used, the protein nanowire-magnetic bead composite of the present invention and other existing commercial reagents (such as Mixing enzyme-labeled antibodies, fluorescently labeled antibodies, chromogenic reagents, substrates, etc., can be used for various forms of immunoassay, such as antibody detection, antibody screening, antigen detection, pathogen detection, protein detection, protein interaction screening, high Flux target protein assay Measurement, protein-nucleic acid interaction analysis, drug screening, and the like.
  • the invention provides a protein nanowire-based 3D probe-magnetic microbead complex for use in immunoassays.
  • the Sup35-p24/Sup35-BAP protein nanowire-based 3D probe-magnetic microbead composite (hereinafter referred to as p24 protein nanowire-magnetic microbead composite) is taken as an example to the present invention in combination with specific examples 1-2.
  • p24 protein nanowire-magnetic microbead composite The preparation of 3D probe-magnetic microbead complex based on protein nanowires and its application in immunoassay are further elaborated.
  • IPTG was added to the culture at a final concentration of 1 mM, and the protein expression was induced by shaking culture at 25 ° C and 120 rpm for 8 hours.
  • the cells were collected by centrifugation at 8000 rpm for 5 minutes, and the cells were sonicated, centrifuged at 10,000 ⁇ g for 30 minutes to remove cell debris, and the supernatant was purified by Ni affinity chromatography to obtain a purified functional fusion protein Sup35-p24.
  • the positive clone E.coli BL21 (Sup35-BAP/pCDFDuet-BirA) was secondarily activated into kanamycin and streptomycin double-antibody LB medium, and cultured at 37 ° C, shaking at 200 rpm to logarithmic growth phase (OD). The value is approximately 0.5).
  • IPTG at a final concentration of 1 mM and biotin at a working concentration of 50 ⁇ M were added to the culture, and protein expression was induced by shaking culture at 25 ° C and 120 rpm for 8 hours.
  • the cells were collected by centrifugation at 8000 rpm for 5 minutes, and the cells were sonicated, centrifuged at 10,000 ⁇ g for 30 minutes to remove cell debris, and the supernatant protein was purified by affinity chromatography to obtain a purified biotinylated fusion protein Sup35-BAP.
  • the cloning is accomplished by chain enzyme polymerization (PCR).
  • Cloning of BirA into pCDFDuet-1 specifically includes: obtaining the nucleotide sequence of BirA gene, designing primers, adding restriction endonucleases of restriction enzymes NcoI and SalI to the upstream and downstream primers, and amplifying BirA gene by PCR.
  • the PCR product BirA and the expression vector pCDFDuet-1 were double-digested to collect the digested product; the collected digested products BirA and pCDFDuet-1 vectors were ligated in a ratio of 6:1 to obtain BirA.
  • -pCDFDuet-1 The PCR product BirA and the expression vector pCDFDuet-1 were double-digested to collect the digested product; the collected digested products BirA and pCDFDuet-1 vectors were ligated in a ratio of 6:1 to obtain BirA. -pCDFDuet-1.
  • the expression vector can be a plasmid vector, including but not limited to pET-28, pET-32, pET-15 or pET-11 plasmid vector, etc.; the expression host can also be Bacillus subtilis, Bacillus megaterium A host capable of protein expression, such as Corynebacterium, Saccharomyces cerevisiae, Pichia pastoris or mammalian cells.
  • the p24 protein nanowire-magnetic microbead is applied to a highly sensitive 3D immunoassay for rapid and highly sensitive detection of the p24 antibody.
  • the principle of antibody detection is as shown in Figure 2. Since the protein nanowires have a large specific surface area, the affinity molecules on them can be more fully combined with the target molecules in the solution, thereby improving the capture efficiency of the target molecules; The ability of the beads to be rapidly separated allows for fast, highly sensitive detection of target molecules in the sample.
  • the present invention also uses conventional methods (p24-magnetic microbeads and commercial ELISA reagents) for p24 antibody detection, replacing the p24 protein nanowire-magnetic microbead complex of the present invention with p24 antigen-magnetic microbead complex and commercial ELISA reagent The remaining steps and parameters are similar to the present invention.
  • the detection result is shown in FIG. 3.
  • the detection sensitivity of the method for detecting a protein nanowire-based 3D probe-magnetic microbead according to the present invention is improved by more than 100 times compared with the conventional ELISA method. And the entire inspection time is greatly reduced to less than half an hour.
  • the detection sensitivity can be increased to a higher level.
  • the p24 protein nanowire-magnetic microbead complex of the present invention can replace the functional ligand with other capture antigen according to actual needs, and can realize the capture and detection of different target molecules (antibodies).
  • the functional ligand can be an antigen of an infectious pathogen (such as HIV, HBV, HCV, Ebola, EV71, various viruses, bacteria and even parasites), and can detect antibodies in the blood to know whether the human body is infected or infected. This type of disease.
  • SPG protein nanowire-magnetic microbead composite SPG protein nanowire-magnetic microbead composite
  • BAP was fused at the C-terminus of the linear protein Sup35 to form a biotinylated fusion protein Sup35-BAP.
  • the BAP tag in the fusion protein can be used in Escherichia coli biotin.
  • the ligase Biotin-protein ligase (EC 6.3.4.15), BirA) was biotinylated.
  • IPTG was added to the culture at a final concentration of 1 mM, and the protein expression was induced by shaking culture at 25 ° C and 120 rpm for 8 hours.
  • the cells were collected by centrifugation at 8000 rpm for 5 minutes, and the cells were sonicated, centrifuged at 10,000 ⁇ g for 30 minutes to remove cell debris, and the supernatant was subjected to Ni affinity chromatography to purify the target protein, thereby obtaining a purified functional fusion protein Sup35-SPG.
  • the positive clone E.coli BL21 (Sup35-BAP/pCDFDuet-BirA) was secondarily activated into kanamycin and streptomycin double-antibody LB medium, and cultured at 37 ° C, shaking at 200 rpm to logarithmic growth phase (OD). The value is approximately 0.5).
  • IPTG at a final concentration of 1 mM and biotin at a working concentration of 50 ⁇ M were added to the culture, and protein expression was induced by shaking culture at 25 ° C and 120 rpm for 8 hours. The cells were collected by centrifugation at 8000 rpm for 5 minutes.
  • the cells were sonicated, centrifuged at 10000 ⁇ g for 30 minutes to remove cell debris, and the supernatant was subjected to Ni affinity chromatography to purify the target protein to obtain a purified biotinylated fusion protein Sup35-BAP.
  • the cloning is accomplished by chain enzyme polymerization (PCR).
  • Cloning of BirA into pCDFDuet-1 specifically includes: obtaining the nucleotide sequence of BirA gene, designing primers, adding restriction endonucleases of restriction enzymes NcoI and SalI to the upstream and downstream primers, and amplifying BirA gene by PCR.
  • the PCR product BirA and the expression vector pCDFDuet-1 were double-digested to collect the digested product; the collected digested products BirA and pCDFDuet-1 vectors were ligated in a ratio of 6:1 to obtain BirA.
  • -pCDFDuet-1 The PCR product BirA and the expression vector pCDFDuet-1 were double-digested to collect the digested product; the collected digested products BirA and pCDFDuet-1 vectors were ligated in a ratio of 6:1 to obtain BirA. -pCDFDuet-1.
  • the expression vector can be a plasmid vector, including but not limited to pET-28, pET-32, pET-15 or pET-11 plasmid vector, etc.; the expression host can also be Bacillus subtilis, Bacillus megaterium A host capable of protein expression, such as Corynebacterium, Saccharomyces cerevisiae, Pichia pastoris or mammalian cells.
  • the SPG protein nanowire-magnetic microbead is applied to a highly sensitive 3D sandwich immunoassay for rapid and highly sensitive detection of the target antigen (p24).
  • the principle of antibody detection is as shown in Fig. 4. Since the protein nanowire has a large specific surface area, the affinity molecule SPG displayed thereon can be more fully combined with the immobilized antibody in the solution, thereby improving the capture efficiency of the target molecule p24;
  • the magnetic microbeads are capable of rapid separation and enable rapid and highly sensitive detection of target molecules in the sample.
  • the detection concentration of the complex of the present invention for the target molecule p24 can be as low as 0.01 ng/mL.
  • the functional ligand SPG can also adopt other proteins having specific binding ability to antibodies such as SPA, SPL, etc., and adopt a sandwich immunological method, and when combined with different capture antibodies, can be specific to the capture antibody.
  • sexually bound corresponding target molecules eg, macroglobulin, calcitonin, cancer-associated antigen, etc.

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Abstract

一种基于蛋白纳米线的3D探针-磁性微珠复合物，包括磁性微珠和蛋白纳米线，蛋白纳米线表面包括至少一种功能配体和至少一种连接配体，蛋白纳米线通过连接配体连接在磁性微珠表面。该结构利用纳米线较大的比表面积，展示在其上的配体形成3D高密度固定，一方面提高配体数量，利用高密度展示形成多价效应，提高特异性配体的结合力；另一方面通过自组装的方式实现抗原或抗体等分子的定向固定，最大程度的保留功能分子活性，减少常规化学交联的损伤，从而提高对样品中待测目标的捕获效率。复合物通过连接配体将蛋白纳米线与磁性微珠相连，利用磁性微珠快速分离的性质，减少检测时间，进而实现快速高通量的病原筛查或疾病检测。

Description

一种基于蛋白纳米线的3D探针-磁性微珠复合物及其应用 技术领域
本发明涉及免疫检测领域，特别涉及一种基于蛋白纳米线的3D探针-磁性微珠复合物及其在免疫检测中的应用。
背景技术
各种传染性疾病大范围的流行传播，对我国的国家安全和人口健康造成严重威胁。发展灵敏、准确的病原分析方法和检测技术对于相关疾病的快速诊断和及时治疗、生物恐怖和突发性公共卫生事件的有效防范和快速处置具有重要意义。经典的微生物分离鉴定麻烦费时，难以应用于致病微生物的现场快速检测；基于病原核酸的检测方法大多具有较高的检测灵敏度，但需要复杂的核酸抽提过程，而且容易出现假阳性；免疫学方法因基于抗体对病原的特异性识别作用而具有较好的特异性，同时操作简单，因此，广泛应用于临床和基础研究的各个领域。
常规的免疫分析方法，如ELISA，其过程主要依赖于抗体的特异性捕获和固相亲和分离等过程，因此有着特异性好，背景信号低，灵敏度高等特点。但随着社会的不断发展，在海关、口岸和CDC等进出口人员、货物密集交通的地方，对现场、快速、高灵敏的病原检测有着非常高的要求，也是国家疾病防控的重要关口。目前，常规的ELISA检测中，固定蛋白探针是通过物理吸附作用，使得蛋白探针随机取向，且吸附作用是在二维平面上的，其固定密度较低，导致捕获检测物的效率降低而限制其灵敏度；在操作过程中，包被、封阻、结合、洗涤需要花费大量时间，限制了对样品的现场检测。而快速免疫分析方法，如试纸条等，虽然能够实现快速检测，但由于该方法通常通过肉眼识别纳米金聚集显色，其灵敏度更是低于ELISA。针对于此，我们亟需开发一种快速、高灵敏的免疫检测方法。
现有提高蛋白探针固定有效方向和密度而调高灵敏度的方法有：亲和作用(Analytical Chemistry，1999，71(17):3846-3852.)、硅纳米线介导(Nature biotechnology，2005，23(10):1294-1301.)、病毒纳米颗粒介导(Nature nanotechnology，2009，4(4):259-264.)、巨磁阻介导(Nature nanotechnology， 2011，6(5):314-320.)等。
亲和作用介导的固定法是通过亲和素-生物素系统，使得探针的固定随机性降低，从而提高有效探针密度；硅纳米线介导的固定法是在硅纳米线表面修饰上醛基，将抗体探针通过氨基与醛基形成化学键而在其表面展示，达到高密度的固定；病毒纳米颗粒介导的固定法是将SPA融合在病毒衣壳蛋白亚基上，通过SPA与抗体的作用而展示抗体，另外通过病毒衣壳蛋白上的His-tag与镍纳米阵列作用，达到高密度的固定；巨磁阻介导的固定法是利用亲和素-生物素系统将抗体结合在磁颗粒上，磁颗粒通过巨磁阻效应固定在界面上，从而达到抗体探针的高密度展示。
上述方法中，提高蛋白探针的固定密度主要通过两种途径来实现：一、通过特异性的固定代替随机的物理吸附；二、提高固定蛋白探针的数量。前者提高探针数量有限，如亲和作用介导的固定只能在一定程度上解决探针随机取向的问题，并不能提高真实探针密度；而病毒纳米颗粒介导的固定法受限于病毒颗粒的大小，固定的探针数量有限，且His-tag与镍的作用不稳定。后者需要通过复杂的化学修饰过程，或者需要复杂的仪器和操作步骤，成本高昂，如纳米材料虽然能够提高比表面积，但其修饰方法随机、复杂，且在修饰过程中会损伤蛋白探针的生物活性，导致捕获能力降低。
发明内容
本发明主要解决的技术问题是提供一种快速、高灵敏的免疫检测产品及方法，实现高效、高密度的在三维(3D)空间上的免疫检测。
为了实现本发明的目的，本发明采用如下技术方案：
本发明一方面提供一种基于蛋白纳米线的3D探针-磁性微珠复合物，包括磁性微珠和蛋白纳米线，所述蛋白纳米线通过成线蛋白自组装形成，且所述蛋白纳米线表面包括至少一种功能配体和至少一种连接配体，所述蛋白纳米线通过连接配体连接在磁性微珠表面。
本发明中，所述磁性微珠是指具有一定磁性及特殊表面结构的纳米或微米尺度的微球，其可由无机磁性物质及各种含活性功能基团的材料组合而成。其中，所述无机磁性材料包括磁性纳米/微米材料或磁性纳米/微米复合材料，优选的，所述磁性纳米/微米材料包括磁性元素(如铁、钴、镍、钕、硼、锆、铬等)掺 杂的纳米/微米材料，例如，氧化铁(γ-Fe2O3，γ-Fe3O4)、铁氧体(CoFe2O4，BaFe12O19)、氧化铬(CrO2)、氧化锆(ZrO2)、氮化铁(Fe4N)、金属合金(Fe、Co、Ni、Al)等。所述含活性功能基团的材料包括聚乙二醇、聚乙烯醇、聚乙醇酸、聚丙烯酸、硅烷衍生物等合成高分子材料或纤维素及其衍生物、琼脂糖、明胶、葡聚糖、壳聚糖及其衍生物、透明质酸、海藻酸等天然高分子材料。
本发明中，磁性微珠一方面可在外加磁场的作用下快速定位、导向和分离，另一方面可通过表面改性或化学聚合等赋予磁性微珠表面多种活性功能基团，如羟基、羧基、醛基、氨基等，此外，磁性微珠也可以通过共价键来结合抗体、细胞、DNA等生物活性物质。
在本发明一具体实施方式中，所述成线蛋白为含有能够组装成线性纳米结构自组装结构域的蛋白，例如可为酵母朊蛋白Sup35、淀粉样蛋白Ure2、丝素蛋白等。优选的，成线蛋白为酵母朊蛋白，Sup35的第1-61位氨基酸为自组装结构域。
在本发明一具体实施方式中，所述功能配体和连接配体可相同或不同，并独立的选自以下组中的一种或多种：抗原、具有特异性结合能力的功能化抗体、蛋白A(Protein A，SPA)、蛋白G(Protein G，SPG)、SPL蛋白、本周蛋白(BJP)、β2-巨球蛋白(β2m)、降钙素(CT)、人绒毛膜性腺激素(hCG)、促肾上腺皮质激素(ACTG)、甲状腺激素(PHT)、荧光蛋白、生物素和亲和素等。
本发明中，所述抗原包括与自身免疫缺陷、恶性细胞或癌症相关的抗原、病毒抗原或微生物抗原，包括但不限于，能够引起免疫应答的任何病毒肽、微生物肽、多肽蛋白、糖类、多糖脂质分子，如HIV-p24、HIV-gp41、HIV-gp120、HIV-gp160、HIV-nef、HA1、HAV、HBV、HCV、HDV、HEV、HBsAg、HBcAg、Ebola、EV71、SV40、HTLV-Ⅰ、CBV、EB、SARS、CEA、AFP、PSA、POA、PSCA、PSMA、CA125、CA19-9、CA15-3、CA50、CA242、TNF-α、RSV-F、CD2、CD3、CD4、CD8、CD19、CD20、CD22、CD27、CD28、CD30、CD33、CD37、CD38、CD40、CD56、CD70、CD79、CD79b、CD90、CD125、CD134、CD147、CD152/CTLA-4等。
本发明中，可通过连接配体将蛋白纳米线与磁性微珠相连，蛋白纳米线结合磁性微珠大的比表面积形成3D结构，提高固定密度；通过融合在蛋白纳米线上 的功能配体实现与目标分子的特异性结合，提高抗原或抗体等固定取向性，从而进行抗体筛选或病原或疾病检测。
优选的，所述连接配体为生物素。
优选的，所述功能配体选自抗原、SPA、SPG或SPL等，优选自以下组中的一种或多种：HIV-p24、HIV-gp41、HIV-gp120、HIV-gp160、HIV-nef、HA1、HAV、HBV、HCV、HDV、HEV、HBsAg、HBcAg、Ebola、EV71、SV40、HTLV-Ⅰ、CBV、EB、SARS、CEA、AFP、PSA、POA、PSCA、PSMA、CA125、CA19-9、CA15-3、CA50、CA242、SPA、SPG等。
在本发明一具体实施方式中，所述蛋白纳米线通过生物素-亲和素相互作用、化学共价交联(例如表面羧基功能化的磁性微珠与蛋白纳米线上的氨基共价交联，过渡金属离子如Co、Ni、Cu、Zn等离子与特定氨基酸侧链之间的相互作用，如天然组氨酸标签HAT、聚组氨酸标签His tag与NTA-Ni(亚硝基三乙酸-镍)或Co-CMA(钴-羧甲基天冬氨酸)之间的相互作用、GST(谷胱甘肽巯基转移酶)的结构域与蛋白之间的相互作用、纤维素结构域与纤维素之间的相互作用或者其它相互作用多肽)、特异性DNA-蛋白相互作用(例如表面特异性DNA功能化的磁性微珠与特异性蛋白融合的蛋白纳米线相互作用)或其它高亲和力和强特异性结合的方式连接在磁性微珠表面。
优选的，所述蛋白纳米线通过生物素-亲和素相互作用的方法连接在磁性微珠表面。
本发明另一方面提供一种本发明基于蛋白纳米线的3D探针-磁性微珠复合物的制备方法，包括以下步骤：
(1)通过分子克隆将成线蛋白(Linear Protein，LP)的自组装结构域与至少一种功能配体(Ligand，L)融合后形成融合蛋白基因LP-L，经表达纯化后得到融合蛋白LP-L；
(2)通过分子克隆将成线蛋白LP的自组装结构域与至少一种连接配体(Connection Ligand，CL)融合后形成融合蛋白基因LP-CL，经表达纯化后得到融合蛋白LP-CL；或者，
通过分子克隆将成线蛋白LP的自组装结构域与至少一种功能配体L融合后，再与至少一种连接配体CL融合，形成融合蛋白基因LP-L-CL(简称LP-CL)， 经表达纯化后得到融合蛋白LP-CL；
(3)将步骤(2)得到的融合蛋白LP-CL破碎作为种子，并将所述种子连接在磁性微珠表面，得到种子-磁性微珠复合物；
(4)将步骤(3)得到的种子-磁性微珠复合物表面进行种子诱导自组装，将步骤(1)得到的融合蛋白LP-L组装成蛋白纳米线，得到基于蛋白纳米线的3D探针-磁性微珠复合物。
采用本发明制备方法得到的基于蛋白纳米线的3D探针-磁性微珠复合物，使得功能配体如抗原分子或特异性结合蛋白等高密度的展示在复合物上，通过多价效应使得复合物能够快速的结合目标分子，而达到目标分子的高灵敏、快速的检测。
在本发明中，通过分子克隆形成融合蛋白基因时，可在两个蛋白基因之间融合柔性多肽之后再进行蛋白融合，减少可能存在的位阻的影响。
本发明中，融合蛋白基因的表达、纯化可采用本领域常规用于蛋白表达纯化的方法，例如将融合蛋白基因克隆入表达载体，将表达载体和/或共表达载体转入表达宿主中培养，活化至对数生长期后加入诱导表白蛋白，经破碎、纯化后得到融合蛋白。其中，本发明对表达载体、共表达载体、表达宿主的种类和类别不作限定，可选用本领域常规用于遗传修饰的载体和宿主，具体的，表达载体可为pET-28、pET-32、pET-15或pET-11的等，共表达载体可为pCDFDuet-1等；表达宿主可选自大肠杆菌、枯草芽孢杆菌、巨大芽孢杆菌、棒状杆菌、酿酒酵母、毕赤酵母或哺乳动物细胞。
本发明中，克隆可通过例如链式酶聚合反应(PCR)完成。
在本发明一具体实施方式中，所述成线蛋白为含有能够组装成线性纳米结构自组装结构域的蛋白，例如可为酵母朊蛋白(Sup35)、淀粉样蛋白Ure2、丝素蛋白等。优选的，成线蛋白为酵母朊蛋白，Sup35的第1-61位氨基酸为自组装结构域。
在本发明一具体实施方式中，至少一种功能配体和至少一种连接配体如本发明前述功能配体和连接配体所定义，在此不作赘述。
优选的，所述连接配体为生物素。
在本发明一具体实施方式中，当生物素作为连接配体时，所述步骤(2)具 体包括如下步骤：
通过分子克隆将成线蛋白LP的自组装结构域与生物素接受多肽(biotin accepted peptide，BAP)融合后形成融合蛋白基因LP-BAP；或者，通过分子克隆将成线蛋白LP的自组装结构域与至少一种功能配体L融合后，再与生物接受多肽BAP融合，形成融合蛋白基因LP-L-BAP(简称LP-BAP)；
将融合蛋白基因LP-BAP克隆入表达载体，将生物素蛋白连接酶(Biotin-protein ligase，BirA)克隆入共表达载体中；
将所述表达载体、共表达载体转入表达宿主中培养，活化至对数生长期后加入诱导表白蛋白和生物素；
经破碎、纯化后制得生物素化的融合蛋白LP-BAP。
更具体的，本发明中，将生物素蛋白连接酶(BirA)克隆入共表达载体中具体包括：获取BirA基因的核苷酸序列，设计引物，在上、下游引物中分别加入限制性内切酶NcoI和SalI的酶切位点，通过PCR扩增BirA基因；将PCR产物BirA和表达载体pCDFDuet-1进行双酶切反应，收集酶切产物；将收集到的酶切产物BirA和pCDFDuet-1载体按物质的量比以6：1进行连接反应，得到BirA-pCDFDuet-1。
更具体的，本发明中，将所述表达载体、共表达载体转入表达宿主中培养，活化至对数生长期后加入诱导表白蛋白和生物素具体包括：将表达载体和共表达载体加入含有表达宿主和抗生素的培养基中培养过夜，直至活化至对数生长期，加入IPTG诱导表白蛋白和生物素过夜，进行培养和表达。
优选的，所述功能配体选自以下组中的一种或多种：HIV-p24、HIV-gp41、HIV-gp120、HIV-gp160、HIV-nef、HA1、HAV、HBV、HCV、HDV、HEV、HBsAg、HBcAg、Ebola、EV71、SV40、HTLV-Ⅰ、CBV、EB、SARS、CEA、AFP、PSA、POA、PSCA、PSMA、CA125、CA19-9、CA15-3、CA50、CA242、SPA、SPG、CT、hCG等。
在本发明一具体实施方式中，所述步骤(3)中，将所述种子连接在磁性微珠表面可通过生物素-亲和素相互作用、化学共价交联(例如表面羧基功能化的磁性微珠与蛋白纳米线上的氨基共价结合)、特异性DNA-蛋白相互作用(例如表面特异性DNA功能化的磁性微珠与特异性蛋白融合的蛋白纳米线相互作用) 或其它高亲和力和强特异性结合的方式连接。
优选的，将所述种子连接在磁性微珠表面可通过生物素-亲和素相互作用的方式连接，其中生物素作为连接配体，亲和素修饰在磁性微珠表面。
在本发明一具体实施方式中，所述步骤(3)具体包括以下步骤：
将生物素化的融合蛋白LP-BAP置于4℃下孵育一周后，形成纳米线，通过超声将纳米线破碎为纳米线片段，制备生物素化的LP-BAP种子；将亲和素修饰的磁性微珠与过量生物素化的种子LP-BAP在37℃下孵育，通过磁场分离洗涤，得到种子-磁性微珠复合物。
在本发明一具体实施方式中，所述步骤(4)具体包括以下步骤：
将步骤(3)得到的种子-磁性微珠复合物表面进行种子诱导自组装，加入含功能配体的融合蛋白LP-L，在室温下孵育，使蛋白纳米线在磁性微珠表面进行生长，通过磁场分离洗涤，得到本发明所述基于蛋白纳米线的3D探针-磁性微珠复合物。
本发明中亲和素修饰在磁性微珠上的方法为本领域技术人员所公知，也可采用市售亲和素修饰的磁性微珠，在此不作赘述。
本发明采用种子诱导自组装方法，种子可以快速的诱发融合有自组装结构域的融合蛋白组装在其末端，通过控制反应时间来控制种子与融合蛋白的组装比，从而实现对纳米线长度的控制；此外，可通过控制组装的顺序，可控的将含有功能配体的融合蛋白组装至蛋白纳米线的端部，进一步的，可通过控制功能配体在蛋白纳米线中所占的比例，制备得到灵敏度高、非特异性吸附低的用于免疫分析的产品。
本发明还一方面提供一种用于免疫分析的产品，所述产品包括本发明所述的基于蛋白纳米线的3D探针-磁性微珠复合物。
其中，产品的形式可为探针(传感器)、试纸条、芯片、试剂盒等，在使用时，将本发明所述的蛋白纳米线-磁珠复合物与其它现有的商业试剂(如酶标抗体、荧光标记抗体、显色剂、底物等)混合，可用于各种形式的免疫分析，例如抗体检测、抗体筛选、抗原检测、病原检测、蛋白检测、蛋白相互作用筛查、高通量靶标蛋白检测、蛋白-核酸相互作用分析、药物筛选等。
产品以探针(传感器)的形式存在时，所述探针可用于高效、高密度的固 定抗原或抗体，从而用于目标分子在三维空间内的高灵敏检测。
产品以试纸条的形式存在时，可将本发明复合物置于检测线，用于捕获目标分子，采用金标纳米粒子等进行检测；进一步的，当复合物融合不同的功能配体时，可实现多种目标分子的同时检测。
产品以芯片的形式存在时，可将本发明复合物置于芯片中，用于现场即时检测(Point-of-Care，POC)；进一步的，当复合物融合不同的功能配体时，可将其分别置于芯片的不同通道，可同时实现多种目标分子的高通量检测。
产品以试剂盒的形式存在时，试剂盒中还可包括缓冲液、洗涤液、稀释液或显色剂等。
此外，本发明还提供一种本发明所述的基于蛋白纳米线的3D探针-磁性微珠复合物在免疫分析中的应用。
本发明中，免疫分析可为间接免疫、夹心免疫等方式，可用于各种形式的免疫分析，例如抗体检测、抗体筛选、抗原检测、病原检测、蛋白检测、蛋白相互作用筛查、高通量靶标蛋白检测、蛋白-核酸相互作用分析、药物筛选等。
优选的，本发明基于蛋白纳米线的3D探针-磁性微珠复合物用于溶液相免疫分析。
本领域技术人员知晓，本发明中基于蛋白纳米线的3D探针-磁性微珠复合物可以应用于以治疗为目的或非治疗为目的检测中。
本发明有益效果：
(1)本发明基于蛋白纳米线的3D探针-磁性微珠复合物可高效、高密度的捕获目标分子，达到快速、高灵敏的检测，相对于传统ELISA灵敏度可提高100倍以上。
(2)本发明基于蛋白纳米线的3D探针-磁性微珠复合物制备过程简单、易行，可适用于不同的免疫检测模式，如可用于间接ELISA、夹心ELISA等，尤其适用于液相中的免疫分析，仅仅是替换原有检测方法中的一种试剂，不改变原有操作步骤，不需要额外的设备和仪器。
(3)本发明利用特异性生物相互作用在磁性微珠表面原位生长多功能蛋白纳米线，不仅操作过程简单，反应时间短，而且高亲和力的生物作用可以提高功能分子固定能力和稳定性。
(4)本发明所构建检测系统十分灵活，仅通过简单更换功能融合蛋白单元，即可原位生长得到适用于检测各种预期目标分子的功能蛋白纳米线。
(5)本发明利用蛋白纳米线的3D蛋白展示能力，利用高密度展示带来大比表面积和多价态效应，提高对样品中目标分子的捕获能力(目前常规的ELISA是在二维平面上通过物理吸附作用固定蛋白探针，固定的蛋白探针密度有限，这导致捕获目标分子的效率较低，因而灵敏度较低)，此外，利用磁性微珠可以快速分离的性质，将3D展示纳米线与磁性微珠联用，使检测时间大大减小。
附图说明
图1本发明基于蛋白纳米线的3D探针-磁性微珠复合物的制备示意图。
图2基于蛋白纳米线的3D探针-磁性微珠复合物的3D高灵敏间接免疫检测原理示意图。
图3基于蛋白纳米线的3D探针-磁性微珠复合物的3D高灵敏间接免疫检测结果分析图。其中，■为p24蛋白纳米线-磁性微珠(记为p24-NW-MB)，本发明基于蛋白纳米线的3D探针-磁性微珠复合物；▲为p24-磁性微珠(记为p24-MB)，磁性微珠上直接展示生物素化的p24病原蛋白；15◆为商业ELISA试剂，传统方法对照。
图4基于蛋白纳米线的3D探针-磁性微珠复合物的3D高灵敏夹心免疫检测原理示意图。
图5基于蛋白纳米线的3D探针-磁性微珠复合物的3D高灵敏夹心免疫检测结果分析图。
具体实施方式
本发明具体实施方式、实施例中“遗传修饰”指的是通过分子生物学技术对生物体的基因组进行遗传修饰，所得到的基因组成和性状改变。“亲和素”包括但不限于亲和素(Avidin)、链酶亲和素(Streptomyces Avidin)。“生物素接受多肽”(Biotin Accepted Peptide，BAP)为能够与铁蛋白N端融合且能够连接生物素的多肽。“生物素蛋白连接酶”(Biotin-protein Ligase，BirA)是指能够活化生物素并将生物素连接到生物素受体蛋白上的酶。
本发明具体实施方式、实施例中“线性蛋白”(Linear Protein，LP)指的是含有能够组装成线性纳米结构自组装结构域的蛋白。
本发明具体实施方式、实施例中缩写“LP-L-BAP”与“LP-BAP”可互换使用，并可代表修饰/未修饰生物素的LP-BAP，具体所代表的含义依据上下文理解。
本发明具体实施方式、实施例中缩写“LP-L”、“LP-CL”、“LP-L-CL”、“LP-BAP”、“LP-L-BAP”不用于限定保护范围，仅用于区分相同或不同的融合蛋白基因和/或融合蛋白。
在本发明一具体实施方式中，本发明提供一种基于蛋白纳米线的3D探针-磁性微珠复合物，包括磁性微珠和蛋白纳米线，所述蛋白纳米线通过成线蛋白自组装形成，且所述蛋白纳米线表面包括至少一种功能配体和至少一种连接配体，所述蛋白纳米线通过连接配体连接在磁性微珠表面。
在本发明一具体实施方式中，本发明提供一种基于蛋白纳米线的3D探针-磁性微珠复合物的制备方法，包括以下步骤：
(1)通过分子克隆将成线蛋白LP的自组装结构域与至少一种功能配体L融合后形成融合蛋白基因LP-L，经表达纯化后得到融合蛋白LP-L；
(2)通过分子克隆将成线蛋白LP的自组装结构域与至少一种连接配体CL融合后形成融合蛋白基因LP-CL，经表达纯化后得到融合蛋白LP-CL；或者，
通过分子克隆将成线蛋白LP的自组装结构域与至少一种功能配体L融合后，再与至少一种连接配体CL融合，形成融合蛋白基因LP-L-CL(简称LP-CL)，经表达纯化后得到融合蛋白LP-CL；
(3)将步骤(2)得到的融合蛋白LP-CL破碎作为种子，并将所述种子连接在磁性微珠表面，得到种子-磁性微珠复合物；
(4)将步骤(3)得到的种子-磁性微珠复合物表面进行种子诱导自组装，将步骤(1)得到的融合蛋白LP-L组装成蛋白纳米线，得到基于蛋白纳米线的3D探针-磁性微珠复合物。
在本发明一具体实施方式中，本发明提供一种用于免疫分析的产品，所述产品包括本发明所述的基于蛋白纳米线的3D探针-磁性微珠复合物。其中，产品的形式可为探针(传感器)、试纸条、芯片、试剂盒等，在使用时，将本发明所述的蛋白纳米线-磁珠复合物与其它现有的商业试剂(如酶标抗体、荧光标记抗体、显色剂、底物等)混合，可用于各种形式的免疫分析，例如抗体检测、抗体筛选、抗原检测、病原检测、蛋白检测、蛋白相互作用筛查、高通量靶标蛋白检 测、蛋白-核酸相互作用分析、药物筛选等。
在本发明一具体实施方式中，本发明提供一种基于蛋白纳米线的3D探针-磁性微珠复合物在免疫分析中的应用。
下面结合具体实施例1-2，以Sup35-p24/Sup35-BAP基于蛋白纳米线的3D探针-磁性微珠复合物(以下简称p24蛋白纳米线-磁性微珠复合物)为例，对本发明基于蛋白纳米线的3D探针-磁性微珠复合物的制备及其在免疫分析中的应用作进一步阐述。
实施例1 p24蛋白纳米线-磁性微珠复合物的制备
A.功能化融合蛋白的制备
(1)功能化蛋白Sup35-p24的克隆：通过分子克隆将酵母朊蛋白Sup35自组装结构域(第1-61位氨基酸)与功能配体HIV-p24(简称p24)通过柔性连接多肽基因融合连接形成融合蛋白Sup35-p24。
(2)功能化蛋白Sup35-BAP的克隆：在融合蛋白Sup35-p24的基础上在其C末端融合生物素接受多肽(biotin accepted peptide，BAP)，形成可生物素化融合蛋白Sup35-p24-BAP(简称Sup35-BAP)，该融合蛋白中的BAP标签可以在大肠杆菌生物素连接酶(Biotin-protein ligase(EC 6.3.4.15)，BirA)的作用下被生物素化。
(3)功能蛋白Sup35-p24表达、纯化：融合蛋白Sup35-p24的基因被克隆入表达载体pET28(该蛋白可在多种表达载体和表达宿主中良好表达，这里仅描述在大肠杆菌中的表达、纯化)，将构建的功能蛋白表达载体转化到大肠杆菌BL21表达株中，卡那霉素、链霉素双抗平板挑取阳性克隆。将阳性克隆二次活化到卡那霉素、链霉素双抗LB培养基，37℃、200rpm振荡培养至对数生长期(OD值约为0.5)。向培养物中加入工作终浓度为1mM的IPTG，25℃、120rpm振荡培养诱导蛋白表达8小时。8000rpm离心收集菌体5分钟，超声破碎菌体，10000×g离心30分钟去除细胞碎片，取上清Ni亲和层析纯化目标蛋白，即获得纯化的功能性融合蛋白Sup35-p24。
(4)功能蛋白Sup35-BAP表达、纯化：融合蛋白Sup35-BAP的基因被克隆入表达载体pET28中，得到pET28-Sup35-BAP。同时克隆大肠杆菌生物素蛋白连接酶BirA到共表达载pCDFDuet中，得到pCDFDuet-BirA。将两个载体共 转化入大肠杆菌表达菌株BL21，卡那霉素、链霉素双抗平板挑取阳性克隆。将挑取的阳性克隆E.coli BL21(Sup35-BAP/pCDFDuet-BirA)二次活化到卡那霉素、链霉素双抗LB培养基，37℃、200rpm振荡培养至对数生长期(OD值约为0.5)。向培养物中加入工作终浓度为1mM的IPTG和工作中浓度为50μM的生物素，25℃、120rpm振荡培养诱导蛋白表达8小时。8000rpm离心收集菌体5分钟，超声破碎菌体，10000×g离心30分钟去除细胞碎片，取上清Ni亲和层析纯化目标蛋白，即获得纯化的生物素化的融合蛋白Sup35-BAP。
本领域技术人员知晓，本发明上述具体实施方式中蛋白的表达与纯化中具体参数(例如浓度、时间、温度等)数值并不用于限制本发明，本领域技术人员可以依据实际需求作调整。
在一个具体的实施方式中，克隆通过链式酶聚合反应(PCR)完成的。将BirA克隆入pCDFDuet-1具体包括：获取BirA基因的核苷酸序列，设计引物，在上、下游引物中分别加入限制性内切酶NcoI和SalI的酶切位点，通过PCR扩增BirA基因；将PCR产物BirA和表达载体pCDFDuet-1进行双酶切反应，收集酶切产物；将收集到的酶切产物BirA和pCDFDuet-1载体按物质的量比以6：1进行连接反应，得到BirA-pCDFDuet-1。
在一个具体的实施方式中，表达载体可为质粒载体，包括但不限于pET-28、pET-32、pET-15或pET-11质粒载体等；表达宿主还可为枯草芽孢杆菌、巨大芽孢杆菌、棒状杆菌、酿酒酵母、毕赤酵母或哺乳动物细胞等能够进行蛋白表达的宿主。
B.p24蛋白纳米线-磁性微珠复合物的制备
(1)生物素化种子的制备：将生物素化的融合蛋白Sup35-BAP单体置于4℃孵育一周后，通过超声所产生的剪切力将长的纳米线打断成为纳米线片段制备种子，该种子通过生物素结合在亲和素修饰的磁性微珠表面，快速的诱发自组装功能融合蛋白快速生长在其末端。
(2)p24蛋白纳米线-磁性微珠复合物的制备：如附图1所示，将亲和素修饰的磁性微珠与过量制备好的生物素化的种子在37℃孵育，通过磁场分离洗涤，加入功能化的融合蛋白Sup35-p24单体，在室温下孵育，使蛋白纳米线在磁性微珠表面进行生长，通过磁场分离洗涤，即获得Sup35-p24/Sup35-BAP基于蛋白纳 米线的3D探针-磁性微珠复合物，复合物表面高密度固定了抗原分子p24。
实施例2基于p24蛋白纳米线-磁性微珠复合物的高灵敏3D免疫检测
本发明一具体实施方式中，将p24蛋白纳米线-磁性微珠应用于高灵敏3D免疫检测，对p24抗体进行快速、高灵敏检测。
抗体检测原理参照附图2，由于蛋白纳米线具有着较大的比表面积，展示其上的亲和分子能够更充分的与溶液中目标分子相结合，从而提高目标分子的捕获效率；配合磁性微珠可以快速分离的性质，可以实现对样品中目标分子的快速、高灵敏检测。
免疫检测的具体步骤如下：
(1)将100μL待测样品与100μL酶标二抗(2μg/mL)进行混合；
(2)将实施例1制备好的p24蛋白纳米线-磁性微珠复合物33.3μg与上述待测样品-酶标二抗在37℃下孵育5-15min；
(3)磁场分离1-2min，用100μL PBS洗涤6次；
(4)加入200μL TMB显色液，显色10min；
(5)加入50μL 2M硫酸终止反应；
(6)在450nm波长下测定吸光度。
本发明还采用常规方法(p24-磁性微珠以及商业ELISA试剂)用于p24抗体检测，将本发明p24蛋白纳米线-磁性微珠复合物替换为p24抗原-磁性微珠复合物和商业ELISA试剂，其余步骤和参数与本发明相通同。
检测结果如附图3所示，由附图3可知，采用本发明所述的基于蛋白纳米线的3D探针-磁性微珠的检测方法的检测灵敏度相对于常规的ELISA方法提高了100倍以上，而整个检测的时间大大减少至半个小时以内。当配合化学发光检测体系，或者新型的信号放大系统，检测灵敏度将能提高至更高的水平。
本领域技术人员知晓，本发明p24蛋白纳米线-磁性微珠复合物，可根据实际需求，将功能配体替换为其它捕获抗原，可实现对不同目标分子(抗体)的捕获和检测。例如功能配体可为感染性病原体的抗原(比如HIV、HBV、HCV、Ebola、EV71、各种病毒和细菌乃至寄生虫等)，可以检测血液中的抗体，从而得知人体是否感染或感染过这类型的疾病。
下面结合具体实施例3-4，以Sup35-SPG/Sup35-BAP基于蛋白纳米线的3D 探针-磁性微珠复合物(以下简称SPG蛋白纳米线-磁性微珠复合物)为例，对本发明基于蛋白纳米线的3D探针-磁性微珠复合物的制备及其在免疫分析中的应用作进一步阐述。
实施例3 SPG蛋白纳米线-磁性微珠复合物的制备
A.功能化融合蛋白的制备
(1)功能化蛋白Sup35-SPG的克隆：通过分子克隆将酵母朊蛋白Sup35自组装结构域(第1-61位氨基酸)与功能配体蛋白G(Protein G，简称SPG)通过柔性连接多肽基因融合连接形成融合蛋白Sup35-SPG。
(2)功能化蛋白Sup35-BAP的克隆：在线性蛋白Sup35的基础上在其C末端融合BAP，形成可生物素化融合蛋白Sup35-BAP，该融合蛋白中的BAP标签可以在大肠杆菌生物素连接酶(Biotin-protein ligase(EC 6.3.4.15)，BirA)的作用下被生物素化。
(3)功能蛋白Sup35-SPG表达、纯化：融合蛋白Sup35-SPG的基因被克隆入表达载体pET28(该蛋白可在多种表达载体和表达宿主中良好表达，这里仅描述在大肠杆菌中的表达、纯化)，将构建的功能蛋白表达载体转化到大肠杆菌BL21表达株中，卡那霉素、链霉素双抗平板挑取阳性克隆。将阳性克隆二次活化到卡那霉素、链霉素双抗LB培养基，37℃、200rpm振荡培养至对数生长期(OD值约为0.5)。向培养物中加入工作终浓度为1mM的IPTG，25℃、120rpm振荡培养诱导蛋白表达8小时。8000rpm离心收集菌体5分钟，超声破碎菌体，10000×g离心30分钟去除细胞碎片，取上清Ni亲和层析纯化目标蛋白，即获得纯化的功能性融合蛋白Sup35-SPG。
(4)功能蛋白Sup35-BAP表达、纯化：融合蛋白Sup35-BAP的基因被克隆入表达载体pET28中，得到pET28-Sup35-BAP。同时克隆大肠杆菌生物素蛋白连接酶BirA到共表达载pCDFDuet中，得到pCDFDuet-BirA。将两个载体共转化入大肠杆菌表达菌株BL21，卡那霉素、链霉素双抗平板挑取阳性克隆。将挑取的阳性克隆E.coli BL21(Sup35-BAP/pCDFDuet-BirA)二次活化到卡那霉素、链霉素双抗LB培养基，37℃、200rpm振荡培养至对数生长期(OD值约为0.5)。向培养物中加入工作终浓度为1mM的IPTG和工作中浓度为50μM的生物素，25℃、120rpm振荡培养诱导蛋白表达8小时。8000rpm离心收集菌体5分钟， 超声破碎菌体，10000×g离心30分钟去除细胞碎片，取上清Ni亲和层析纯化目标蛋白，即获得纯化的生物素化的融合蛋白Sup35-BAP。
本领域技术人员知晓，本发明上述具体实施方式中蛋白的表达与纯化中具体参数(例如浓度、时间、温度等)数值并不用于限制本发明，本领域技术人员可以依据实际需求作调整。
在一个具体的实施方式中，克隆通过链式酶聚合反应(PCR)完成的。将BirA克隆入pCDFDuet-1具体包括：获取BirA基因的核苷酸序列，设计引物，在上、下游引物中分别加入限制性内切酶NcoI和SalI的酶切位点，通过PCR扩增BirA基因；将PCR产物BirA和表达载体pCDFDuet-1进行双酶切反应，收集酶切产物；将收集到的酶切产物BirA和pCDFDuet-1载体按物质的量比以6：1进行连接反应，得到BirA-pCDFDuet-1。
在一个具体的实施方式中，表达载体可为质粒载体，包括但不限于pET-28、pET-32、pET-15或pET-11质粒载体等；表达宿主还可为枯草芽孢杆菌、巨大芽孢杆菌、棒状杆菌、酿酒酵母、毕赤酵母或哺乳动物细胞等能够进行蛋白表达的宿主。
B.SPG蛋白纳米线-磁性微珠复合物的制备
(1)生物素化种子的制备：将生物素化的融合蛋白Sup35-BAP单体置于4℃孵育一周后，通过超声所产生的剪切力将长的纳米线打断成为纳米线片段制备种子，该种子通过生物素结合在亲和素修饰的磁性微珠表面，快速的诱发自组装功能融合蛋白快速生长在其末端。
(2)SPG蛋白纳米线-磁性微珠复合物的制备：如附图4所示，将亲和素修饰的磁性微珠与过量制备好的生物素化的种子在37℃孵育，通过磁场分离洗涤，加入功能化的融合蛋白Sup35-SPG单体，在室温下孵育，使蛋白纳米线在磁性微珠表面进行生长，通过磁场分离洗涤，即获得Sup35-SPG/Sup35-BAP基于蛋白纳米线的3D探针-磁性微珠复合物，复合物表面高密度固定了用于特异性固定抗体的SPG。
实施例4基于SPG蛋白纳米线-磁性微珠复合物的高灵敏3D免疫检测
本发明一具体实施方式中，将SPG蛋白纳米线-磁性微珠应用于高灵敏3D夹心免疫检测，对目标抗原(p24)进行快速、高灵敏检测。
抗体检测原理参照附图4，由于蛋白纳米线具有着较大的比表面积，展示其上的亲和分子SPG能够更充分的与溶液中固定抗体相结合，从而提高目标分子p24的捕获效率；配合磁性微珠可以快速分离的性质，可以实现对样品中目标分子的快速、高灵敏检测。
夹心免疫检测的具体步骤如下：
(1)将100μL待测样品与等体积的酶标一抗进行混合；
(2)将实施例3制备好的SPG蛋白纳米线-磁性微珠复合物33.3μg与上述步骤(1)所得的待测样品-酶标抗体混合，在37℃下孵育5-15min；
(3)磁场分离1-2min，用100μL PBS洗涤6次；
(4)加入200μL TMB显色液，显色10min；
(5)加入50μL 2M硫酸终止反应；
(6)在450nm波长下测定吸光度。
检测结果如附图5所示，由附图5可知，采用本发明复合物对于目标分子p24的检测浓度可低至0.01ng/mL。
本领域技术人员知晓，功能配体SPG也可采用其它对抗体具有特异性结合能力的蛋白如SPA、SPL等，采用夹心免疫的方法，当结合不同的捕获抗体时，可对与捕获抗体发生特异性结合的相应目标分子(例如巨球蛋白、降钙素、癌症相关抗原等)进行检测。

Claims (11)

  1. 一种基于蛋白纳米线的3D探针-磁性微珠复合物，包括磁性微珠和蛋白纳米线，所述蛋白纳米线通过成线蛋白自组装形成，且所述蛋白纳米线表面包括至少一种功能配体和至少一种连接配体，所述蛋白纳米线通过连接配体连接在磁性微珠表面。
  2. 如权利要求1所述的基于蛋白纳米线的3D探针-磁性微珠复合物，其特征在于：所述成线蛋白为酵母朊蛋白Sup35、淀粉样蛋白Ure2、丝素蛋白；优选的，所述成线蛋白为酵母朊蛋白Sup35。
  3. 如权利要求1所述的基于蛋白纳米线的3D探针-磁性微珠复合物，其特征在于：所述功能配体和连接配体可相同或不同，并独立的选自以下组中的一种或多种：抗原、具有特异性结合能力的功能化抗体、SPA、SPG、SPL、BJP、β2m、CT、hCG、ACTG、PHT、荧光蛋白、生物素和亲和素。
  4. 如权利要求3所述的基于蛋白纳米线的3D探针-磁性微珠复合物，其特征在于：所述连接配体为生物素；所述功能配体选自抗原、SPG、SPA或SPL，优选自以下组中的一种或多种：HIV-p24、HIV-gp41、HIV-gp120、HIV-gp160、HIV-nef、HA1、HAV、HBV、HCV、HDV、HEV、HBsAg、HBcAg、Ebola、EV71、SV40、HTLV-Ⅰ、CBV、EB、SARS、CEA、AFP、PSA、POA、PSCA、PSMA、CA125、CA19-9、CA15-3、CA50、CA242、SPA、SPG和SPL。
  5. 如权利要求1-4任一项所述的基于蛋白纳米线的3D探针-磁性微珠复合物，其特征在于：所述蛋白纳米线通过生物素-亲和素相互作用、化学共价交联或特异性DNA-蛋白相互作用的方式连接在磁性微珠表面；优选的，所述蛋白纳米线通过生物素-亲和素相互作用的方式连接在磁性微珠表面。
  6. 一种如权利要求1-5任一项所述的基于蛋白纳米线的3D探针-磁性微珠复合物的制备方法，包括以下步骤：
    (1)通过分子克隆将成线蛋白的自组装结构域与至少一种功能配体融合后形成融合蛋白基因LP-L，经表达纯化后得到融合蛋白LP-L；
    (2)通过分子克隆将成线蛋白的自组装结构域与至少一种连接配体融合后形成融合蛋白基因LP-CL，经表达纯化后得到融合蛋白LP-CL；或者，
    通过分子克隆将成线蛋白的自组装结构域与至少一种功能配体融合后，再与至少一种连接配体融合，形成融合蛋白基因LP-CL，经表达纯化后得到融合蛋白LP-CL；
    (3)将步骤(2)得到的融合蛋白LP-CL破碎作为种子，并将所述种子连接在磁性微珠表面，得到种子-磁性微珠复合物；
    (4)将步骤(3)得到的种子-磁性微珠复合物表面进行种子诱导自组装，将步骤(1)得到的融合蛋白LP-L组装成蛋白纳米线，得到所述基于蛋白纳米线的3D探针-磁性微珠复合物。
  7. 如权利要求6所述的制备方法，其特征在于：所述连接配体为生物素，所述步骤(2)具体包括如下步骤：
    通过分子克隆将成线蛋白的自组装结构域与生物素接受多肽融合后形成融合蛋白基因LP-BAP；或者，通过分子克隆将成线蛋白的自组装结构域与至少一种功能配体融合后，再与生物接受多肽融合，形成融合蛋白基因LP-BAP；
    将融合蛋白基因LP-BAP克隆入表达载体，将生物素蛋白连接酶克隆入共表达载体中；
    将所述表达载体、共表达载体转入表达宿主中培养，活化至对数生长期后加入诱导表白蛋白和生物素；
    经破碎、纯化后制得生物素化的融合蛋白LP-BAP。
  8. 如权利要求7所述的制备方法，其特征在于：所述步骤(3)具体包括以下步骤：
    将生物素化的融合蛋白LP-BAP置于4℃下孵育一周后，形成纳米线，通过超声将纳米线破碎为纳米线片段，制备生物素化的LP-BAP种子；将亲和素修饰的磁性微珠与过量生物素化的种子LP-BAP在37℃下孵育，通过磁场分离洗涤，得到种子-磁性微珠复合物。
  9. 如权利要求6-8任一项所述的制备方法，其特征在于：所述步骤(4)具体包括以下步骤：
    将步骤(3)得到的种子-磁性微珠复合物表面进行种子诱导自组装，加入含功能配体的融合蛋白LP-L，在室温下孵育，使蛋白纳米线在磁性微珠表面进行生长，通过磁场分离洗涤，得到所述基于蛋白纳米线的3D探针-磁性微珠复合 物。
  10. 一种用于免疫分析的产品，其特征在于：所述产品包括如权利要求1-5任一项所述的基于蛋白纳米线的3D探针-磁性微珠复合物或如权利要求6-9任一项所述制备方法制得的基于蛋白纳米线的3D探针-磁性微珠复合物。
  11. 一种如权利要求1-5任一项所述的基于蛋白纳米线的3D探针-磁性微珠复合物或如权利要求6-9任一项所述制备方法制得的基于蛋白纳米线的3D探针-磁性微珠复合物在免疫分析中的应用。
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