Method for enhancing antibody detection sensitivity by covalent and non-covalent binding

Abstract

The invention discloses a method for enhancing antibody detection sensitivity by covalent and non-covalent binding, and relates to the technical field of medical detection. The preparation method comprises the following steps: firstly, adding a nano antibody heavy chain variable region (VHH) gene into a SpyTag tag through a pET22b vector plasmid, transforming the VHH gene into an escherichia coli BL21 competent cell to obtain a purified nano antibody protein, then transforming a pET28a-SpyCatch-C4BP-SnoopCatch-6xHis plasmid into the escherichia coli BL21 competent cell, culturing and dissociating, centrifugally collecting supernate, and purifying to obtain a soluble heptamer protein skeleton, finally, covalent binding is carried out through the reaction of the soluble heptamer fusion protein skeleton and the nano antibody protein, and the densely displayed soluble heptamer giant nano antibody molecule is formed. According to the invention, covalent and non-covalent methods are combined and then combined with SnoopTag-Biotin, so that a polymer macromolecular protein loaded with 14 nano antibodies and 7 Biotin molecules is formed, a nano antibody polymer is prepared, the display density and affinity of the antibodies are improved, the lowest detection limit is reduced, and the detection sensitivity is improved.

Description

Technical Field

[0001] The present invention relates to the field of medical detection technology, and in particular to a method for enhancing antibody detection sensitivity by utilizing covalent and non-covalent binding. Background Art

[0002] Accurate disease diagnosis is a key step in guiding clinical treatment. Traditional Y-shaped antibodies are used to identify specific proteins and have been widely used in scientific research, clinical diagnosis and treatment-related fields. However, the large size of traditional antibodies limits their sequence processing, expression and their application value as drugs in the body.

[0003] Compared with IgG antibodies, which have been successful in clinical development for decades, nanobodies are a young platform full of unknowns. Hamers and other scientists have found that there is a single-chain antibody (heavy chain antibodies, HCAb) in camelids that lacks light chains and the first constant region (CH1) and binds to antigens through the variable region on the heavy chain. It has a high sequence and structural similarity with the VH of human IgG3. Later, antibodies with similar structures were also found in cartilaginous fish such as sharks and rays. The variable domain of heavy chain antibody (VHH) of alpaca antibodies can bind to antigens. Because this VHH is 4nm long, 2.5nm in diameter, and has a molecular weight of 12-15kDa, it is the smallest known antigen-binding protein, only one-tenth of traditional antibodies, and is also called nanobody (Nb). Nanobodies have the advantages of large-scale and low-cost expression in a variety of systems, strong plasticity, good water solubility, high affinity and stability, strong specificity, small size, easy transformation, screening and purification, etc., and can recognize more hidden antigen epitopes that are difficult for traditional antibodies to recognize.

[0004] With the deepening of the application of nanoantibodies in the treatment of various diseases and detection, the application of nanoantibodies in imaging technology and disease diagnosis research requires higher accuracy and sensitivity. Due to the small molecular weight of nanoantibodies, VHH is more suitable for molecular modification to add tags, and is used as a bonding module to create antibody-related fusion proteins, which can achieve the purpose of precision treatment. For example, an antibody-enzyme conjugate has been invented, which has the ability to selectively activate the site of anticancer prodrugs, using antibodies as targeting partners. People are committed to continuous application innovation, so that in addition to monovalent nanoantibodies, bivalent, bispecific and VHH enriched by fusion protein scaffolds are studied to enhance their specificity and affinity for the target, which is more conducive to the drug to play its efficient role. Uchański et al. expressed nanoantibodies by fusing them into selected scaffold proteins to produce stable and effectively folded monomer chimeras and developed giant antibodies, which can promote the activity of antibodies and label specific tags. However, the solubility and stability of proteins produced by this method are greatly reduced, and the difficulty of protein purification and preservation becomes greater.

[0005] Nanobodies derived from camel heavy chain antibodies are one of the smallest antibody fragments designed for various applications. As a good bioengineering material, nanobodies can bind to a variety of reporter proteins. Although VHH fragments are highly stable and soluble, their disadvantages are that they dissociate quickly in the non-equilibrium environment of blood vessels and tissues, and their affinity does not meet the requirements due to their monovalent interaction with antigens.

[0006] Therefore, it is of great significance to improve the detection sensitivity of antibodies and reduce the dissociation rate of antigen-antibody. Therefore, we urgently need a production plan for dense display of nanoantibodies that is easy to operate, has good solubility, high stability, and modular splicing similar to building blocks. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a method for enhancing the sensitivity of antibody detection by utilizing a nano-antibody polymerization strategy, which specifically includes the following contents.

[0008] The present invention provides a method for enhancing antibody detection sensitivity by using covalent and non-covalent binding, comprising the following steps:

[0009] Step 1: The variable region (VHH) gene of the nanobody heavy chain is transformed into E. coli Top10 competent cells by adding the SpyTag tag through the pET22b-VHH1-VHH2-SpyTag-6xHis vector plasmid, and the dimeric nanobody protein VHH1-VHH2-SpyTag-6xHis is obtained by culture, lysis and purification;

[0010] Step 2: Heat transform the pET28a-SpyCatcher-C4BP-SnoopCatcher-6xHis plasmid into Escherichia coli BL21 competent cells, perform cell culture and cell dissociation, collect the supernatant by centrifugation, and purify it by affinity chromatography to obtain a soluble heptamer protein skeleton SpyCatcher-C4BP-SnoopCatcher-6xHis;

[0011] Step 3: The soluble heptameric fusion protein backbone SpyCatcher-C4BP-SnoopCatcher-6xHis is reacted and covalently bound with the dimeric nanobody protein VHH1-VHH2-SpyTag-6xHis to form a densely displayed soluble heptameric giant nanobody fusion protein molecule.

[0012] Preferably, in step 1, the VHH gene uses a signal peptide (PelB) as an N-terminal leader sequence and a 6xHis tag as a C-terminal metal affinity chromatography purification tag.

[0013] Preferably, in step 2, the specific operations of cell culture and cell dissociation are:

[0014] E. coli BL21 competent cells were cultured in 100 mL solution containing 10 μg / mL ampicillin at 250 r / min for 10 to 12 h at 25 to 37°C, and the cells were collected by centrifugation at 8000×g for 15 to 20 min at 4°C, and resuspended with 20 mL of equilibration buffer and 60 mg of lysozyme. The resuspended E. coli cells were disrupted by ultrasound in an ice-water bath.

[0015] Preferably, in step 3, the specific operation of the purification is:

[0016] The cell lysate containing VHH1-VHH2-SpyTag-6His was purified by Ni-NTA affinity chromatography, eluted with 300 mmol / L imidazole, and dialyzed against 10 mmol / L PBS buffer at a pH of 6.8 to 7.4.

[0017] Preferably, the method of enhancing antibody detection sensitivity by covalent and non-covalent binding comprises the following steps:

[0018] Step 1: The pET28a vector was digested with XhoI and purified with agarose gel. After homologous recombination with the flexible amino acid linker 3x (ggggs), it was combined with the gene sequence encoding the C4 bpα protein at 37-42°C for 90s. The combined product was electroporated into freshly prepared E. coli Top10 competent cells and cultured overnight on agar plates containing 100 μg / mL ampicillin at 37-42°C. The E. coli BL21 competent cells produced the dimeric nanobody protein VHH-SpyTag-6xHis by self-induction expression;

[0019] Step 2: Transform the recombinant vector pET28a-SpyCatcher-C4BP-SnoopCatcher-6xHis into Escherichia coli BL21 competent cells by thermal transformation at 37-42°C for 90-120s, culture in an induction solution at 25-37°C with self-induction shaking at 200-250r / min for 10-12h, collect the cells by centrifugation at 8000×g for 15-20min at 4°C, resuspend with 20mL of equilibration buffer and 60mg of lysozyme, destroy the resuspended Escherichia coli cells by ultrasound in an ice water bath to prevent degradation or denaturation of the target protein, and then collect the supernatant by centrifugation at 1000×g for 15-20min at 4°C, and filter through 0.22μm to obtain a soluble heptamer fusion protein;

[0020] Step 3: Purify the SpyCatcher-C4BP-SnoopCatcher-6xHis fusion protein using Ni-NTA affinity chromatography, elute with 300 mmol / L imidazole, and then dialyze with 10 mmol / L PBS buffer at a pH of 6.8-7.4 to obtain a purified soluble heptamer fusion protein.

[0021] Step 4: The soluble heptameric fusion protein skeleton SpyCatcher-C4BP-SnoopCatcher-6xHis is reacted and covalently bound with the dimeric nanobody protein VHH1-VHH2-SpyTag-6xHis and then combined with SnoopTag-Biotin to form a densely displayed soluble heptameric giant nanobody (14xVHH) and 7 Biotin molecules fusion protein molecule.

[0022] Preferably, in step 4, the soluble protein skeleton SpyCatcher-C4BP-SnoopCatcher-6xHis is reacted with the dimeric nanobody protein VHH1-VHH2-SpyTag-6His to form densely displayed soluble giant nanobody (14VHH) and 7 Biotin molecules through covalent and non-covalent binding.

[0023] Preferably, in step 2, the components and their concentrations in the equilibration buffer are: 2mmol / L KH2PO4, 8mmol / L Na2HPO4, 136mmol / L NaCl, 2.6mmol / L KCl, and 1mmol / L phenylmethylsulfonyl fluoride.

[0024] Preferably, in step 2, the concentration of ampicillin in the induction solution is 100 μg / mL, and the concentration of IPTG is 1 mM / L.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention transforms the VHH gene into Escherichia coli BL21 competent cells via a vector plasmid, performs cell culture and cell dissociation, and collects the supernatant by centrifugation to obtain a nanobody with a connection tag Spy-Tag.

[0027] (2) The present invention prepares a soluble heptamer backbone macromolecular protein, which can introduce a macromolecular nanobody display platform that can connect 14 VHH and 7 Biotin molecules by covalently linking dimers to reconstruct the heptamer and combining it with SnoopTag-Biotin, thereby promoting the modularization of nanoantibodies for more convenient large-scale application.

[0028] (3) The soluble heptamer fusion protein prepared by the present invention retains the antigen-specific recognition ability and improves the affinity of the monomeric VHH antibody. The C-terminus of the soluble heptamer fusion protein also provides more binding sites for analytical signal amplification.

[0029] (4) The multimeric protein loaded with nanoantibodies prepared by the present invention can theoretically show an additional 6 times sensitivity of a single antibody. The binding of the heptamer can improve the affinity and amplify the signal. This method can be applied to the determination of the new coronavirus and can serve as a powerful tool for rapid detection of antigens in samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The gene structure and plasmid map involved in Example 1 of the present invention;

[0031] Figure 2 This is a diagram showing the verification scheme of the magnetic bead sandwich method for the binding activity of nano antibodies in Example 3 of the present invention;

[0032] Figure 3 A prokaryotic synthesis diagram of a COVID-19 nanobody prepared using the soluble heptamer fusion protein prepared in Example 1 of the present invention;

[0033] Figure 4The molecular structure diagram and electron microscope diagram of the heptamer fusion protein prepared in the embodiment of the present invention;

[0034] Figure 5 This is a western blot immunoblot validation diagram in Example 4 of the present invention;

[0035] Figure 6 This is a diagram of the magnetic bead antigen-antibody sandwich detection in Example 3 of the present invention;

[0036] Figure 7 This is a graph showing the results of SPR detection of recombinant polymer affinity in Example 5 of the present invention;

[0037] Figure 8 This is the antigen sensitivity detected by the nanobody polymerized immune sandwich ELISA in Example 6 of the present invention. DETAILED DESCRIPTION

[0038] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Example 1

[0040] This embodiment provides a method for preparing a soluble nanobody protein

[0041] 1. The pET22b vector was digested with XhoI and purified with agarose gel. The gene sequence after homologous recombination with a flexible amino acid linker (GGGGS) was combined at 42°C for 90s. The combined nanobody protein VHH1-VHH2-SpyTag-6xHis was electroporated into freshly prepared E. coli Top10 competent cells and cultured overnight on an agar plate containing 100 μg / mL ampicillin at 37°C. The plasmid with correct sequencing connection was extracted, purified and frozen;

[0042] 2. Transform the recombinant vector pET22b-VHH1-VHH2-SpyTag-6xHis into E. coli BL21 competent cells by thermal transformation at 42°C for 90s, culture in the induction solution at 250r / min for 12h, collect the cells by centrifugation at 8000×g for 15min at 4°C, and resuspend them with 20mL of equilibrium buffer and 60mg of lysozyme. Destroy the resuspended E. coli cells by ultrasound in an ice water bath to prevent degradation or denaturation of the target protein, then collect the supernatant by centrifugation at 1000×g for 15min at 4°C, and filter through 0.22μm to obtain soluble nanobody protein;

[0043] The concentration of ampicillin in the induction solution was 100 μg / mL, and the concentration of IPTG was 1 mM / L.

[0044] 3. Purify the VHH-SpyTag-6His protein using Ni-NTA affinity chromatography, elute with 300 mmol / L imidazole, and then dialyze with 10 mmol / L PBS buffer at pH 7.4 to obtain purified VHH1-VHH2-SpyTag-6His nanobody protein.

[0045] Among them, the components and their concentrations in the equilibrium buffer are: 2mmol / L KH2PO4, 8mmol / LNa2HPO4, 136mmol / L NaCl, 2.6mmol / L KCl, 1mmol / L phenylmethylsulfonyl fluoride.

[0046] Example 2

[0047] This embodiment provides a method for preparing a soluble heptamer fusion protein

[0048] 1. The pET28a vector was digested with XhoI and purified by agarose gel. After homologous recombination with a flexible amino acid linker (GGGGS), it was combined with the gene sequence encoding the C4 bpα protein at 37°C for 90s. The combined product, the nanobody protein VHH-SpyTag-6His, was electroporated into freshly prepared E. coli Top10 competent cells and cultured overnight on an agar plate containing 100 μg / mL ampicillin at 37°C. The E. coli Top10 competent cells were amplified to produce the pET28a-SpyCatcher-C4BP-SnoopCatcher-6xHis plasmid, which was purified and frozen;

[0049] 2. Transform the recombinant vector pET28a-SpyCatcher-C4BP-SnoopCatcher-6xHis into E. coli BL21 competent cells by thermal transformation at 37°C for 90s, culture in the induction solution at 250r / min for 12h, collect the cells by centrifugation at 8000×g for 20min at 4°C, and resuspend them in 20mL of equilibrium buffer and 60mg of lysozyme. Destroy the resuspended E. coli cells by ultrasonication in an ice-water bath to prevent degradation or denaturation of the target protein, then collect the supernatant by centrifugation at 1000×g for 20min at 4°C, and filter through 0.22μm to obtain a soluble heptamer SpyCatcher-C4BP-SnoopCatcher-6xHis fusion protein lysate;

[0050] The concentration of ampicillin in the induction solution was 100 μg / mL, and the concentration of IPTG was 1 mM / L.

[0051] 3. Purify the SpyCatcher-C4BP-SnoopCatcher-6xHis fusion protein using Ni-NTA affinity chromatography, elute with 300 mmol / L imidazole, and then dialyze with 10 mmol / L PBS buffer at pH 7.4 to obtain the purified SpyCatcher-C4BP-SnoopCatcher-6xHis fusion protein.

[0052] Among them, the components and their concentrations in the equilibrium buffer are: 2mmol / L KH2PO4, 8mmol / L Na2HPO4, 136mmol / L NaCl, 2.6mmol / L KCl, 1mmol / L phenylmethylsulfonyl fluoride.

[0053] Example 3

[0054] This example provides a magnetic bead sandwich method to verify the binding activity of nanoantibodies

[0055] 1 mg of 2.8 μm diameter streptavidin magnetic beads (Biomag Biotech, Wuxi, 10 mg / ml) were washed three times with 1 ml PBS on a Themo magnetic collection stand, and the specificity and affinity index were evaluated by direct binding (sandwich method) of nanoantibodies immobilized on magnetic microspheres to capture SARS-CoV-2 NP protein. To prepare biotinylated labeled antibodies, a 10-fold excess of Spy-Tag-Biotin was added to each VHH-Spy-catcher at a concentration of 1 mg / mL for 30 minutes; ultrafiltration tubes were used to remove excess biotin, and 150 ng of the primary antibody VHH-SnoopC labeled with biotin was covalently linked to Snooptag-biotin for each test in triplicate. Streptavidin and biotin bind very quickly and are not easily affected by pH, temperature, organic solvents, and other denaturants after binding. It was then blocked with 1 mL 3% BSA at 37°C for 30 minutes. 200 ng of antigen was used for each test and grouped according to different antibody combination types. Fluorescent tag antibodies were constructed by combining VHH-Spy-tag->Spy-catcher-GFP protein components. Each step of the reaction was oscillated at room temperature for 30 minutes and then placed on a magnetic stand for magnetic separation. The supernatant was discarded and washed with PBS three times for antigen-antibody sandwich assay. Fluorescence microscopy was performed to verify virus binding at a total magnification of 400X, and digital images were captured using a Nikon confocal microscope.

[0056] Example 4

[0057] This example verifies the antigen capture effect of SpyCatcher-C4BP-SnoopCatcher-6xHis fusion protein

[0058] Western blot immunohybridization was used for verification. Five experimental groups and a blank control group were set up. The same concentration of COVID-19-NP nucleocapsid protein was added to each group. Monomers E2, C2 and B6 and two dimers C2-B6 and E2-E2 were added to each experimental group, respectively. The polymer and SnoopTag-Biotin were coupled with VHH-SnoopCatcher and SA-HRP, and ECL staining was performed. 5ug of antigen was loaded on a 15% SDS-PAGE gel (PAGE gel rapid preparation kit, Shanghai Yazyme Biotechnology Co., Ltd., PG114) and imaged in a gel imaging system.

[0059] Example 5

[0060] In this example, SPR was used to detect the affinity of the polymer.

[0061] The antigen recognition capture ability of the recombinant polymer was evaluated by testing the intermolecular affinity by surface plasmon resonance (SPR). We applied Biacore 8K (Cytiva, USA) according to 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / N-hydroxysulfosuccinimide (NHS) coupling chemistry, and the lanes of the universal layer dense chip were individually coated with COVID-19NP protein covalently linked to the chip. The binding kinetics of each antibody were tested at 25°C, and 6 antibodies with concentrations ranging from 300 to 0nM at 100μL / min were flowed through the antigen-coated chip, and then the dissociation was monitored for 600 seconds.

[0062] Test results: Example 5 detected the K of the soluble heptamer fusion protein D The value is 1.23nmol / L, the KD value of the monomer is 2.47nmol / L, K D It can characterize the strength of antibody affinity, which refers to the lowest antigen concentration that the antibody can recognize and bind to. D The smaller the value, the stronger the affinity of the antibody. It can be seen from this that the preparation of soluble heptamer fusion protein effectively improves the affinity of the monomeric VHH antibody.

[0063] Example 6

[0064] This example uses ELISA based on nanobodies to perform sample analysis

[0065] In order to apply the nanobody-based ELISA sandwich technology to sample analysis, the performance of the purified nanobody was tested by indirect competitive ELISA. First, the concentration of the coated nanobody and antigen was adjusted. 300 ng of VHH antibody polystyrene ELISA plate was coated with two dimeric nanobodies C2-B6 and E2-E2 in the polystyrene ELISA plate and non-specific sites were blocked with 2% bovine serum albumin (BSA), washed 3 times in 250 μl PBST, 5 minutes each time, and 300 ng NP protein was gently added to interact with the stationary phase antibody at room temperature for 1 hour. The plate was washed with PBST as above, incubated with VHH2-IMX-biotin for 30 minutes, then washed, and 100 μl of 1:2,000 streptavidin peroxidase stock solution SA-HRP in PBS was added to each well and gently mixed for 30 minutes at room temperature. Then the ECL color development kit was added for color development, and the binding was evaluated on the microplate reader. To improve the limit of detection (LOD) of N, the same assay format was repeated using heterologous and homologous bivalent constructs of the three best nanobodies, as well as C4bp as a heptameric backbone to enrich the luminescent signal and increase the antibody detection sensitivity. The signal / background ratio was calculated and used as the LOD determination because this ratio provides a signal that ensures that the difference is greater than the background and the data point is considered significant.

[0066] Sources of raw materials used in the examples: screening antibiotics, bacterial culture medium, isopropanol bD-1-thiogalactoside (IPTG) and all buffer salts were purchased from Shanghai Bio-Tech; nickel agarose gel high-performance resin and pre-packed columns, Superdex200 molecular sieves were purchased from Cytiva; anti-HIS tag antibody was purchased from Shanghai Bio-Tech (Cat. No. AF2873); prokaryotic expression vector pET22b was purchased from Guangzhou Ruiboxingke; all restriction enzymes and buffers used for cloning were purchased from Thermo Fisher, and all primers were ordered from Guangzhou Ruiboxingke.

[0067] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that all equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A method for enhancing antibody detection sensitivity by using covalent and non-covalent binding, characterized in that: The following steps are involved: Step 1: The nanobody heavy chain variable region (VHH) gene is transformed into Escherichia coli Top10 competent cells by adding the SpyTag tag through the pET22b-VHH1-VHH2-SpyTag-6His vector plasmid, and the dimeric nanobody protein VHH1-VHH2-SpyTag-6xHis is obtained after culture, lysis, affinity purification; Step 2: Heat transform the pET28a-SpyCatcher-C4BP-SnoopCatcher-6xHis plasmid into Escherichia coli BL21 competent cells, perform cell culture and cell dissociation, collect the supernatant by centrifugation, and purify it by affinity chromatography to obtain a soluble heptamer protein skeleton SpyCatcher-C4BP-SnoopCatcher-6xHis; Step 3: The soluble heptameric fusion protein skeleton SpyCatcher-C4BP-SnoopCatcher-6xHis is reacted and covalently bonded with the dimeric nanobody protein VHH1-VHH2-SpyTag-6xHis to form a densely displayed soluble heptameric giant fusion protein molecule containing 14 nanobodies (14xVHH) and 7 Biotin molecules.

2. A method for enhancing antibody detection sensitivity by using covalent and non-covalent binding according to claim 1, characterized in that: In step 1, the VHH gene uses a signal peptide (PelB) as an N-terminal leader sequence and a 6×His tag as a C-terminal metal affinity chromatography purification tag.

3. The method of claim 1, wherein the method comprises: In step 2, the specific operations of bacterial culture and dissociation are: E. coli BL21 competent cells were cultured in 100 mL solution containing 10 μg / mL ampicillin at 250 r / min for 10 to 12 h at 25 to 37°C, and the cells were collected by centrifugation at 8000×g for 15 to 20 min at 4°C, and resuspended with 20 mL of equilibration buffer and 60 mg of lysozyme. The resuspended E. coli cells were disrupted by ultrasound in an ice-water bath.

4. The method of claim 1, wherein: In step 3, the specific operation of the purification is: The cell lysate containing VHH1-VHH2-SpyTag-6xHis was purified by Ni-NTA affinity chromatography, eluted with 300 mmol / L imidazole, and dialyzed against 10 mmol / L PBS buffer with a pH of 6.8 to 7.

4.

5. The method of claim 1, wherein the method comprises: The specific steps include: Step 1: The pET28a vector was digested with XhoI and purified with agarose gel. After homologous recombination with the flexible amino acid linker 3x (ggggs), it was combined with the gene sequence encoding the C4 bpα protein at 37-42°C for 90s. The combined product was electroporated into freshly prepared E. coli Top10 competent cells and cultured overnight on agar plates containing 100 μg / mL ampicillin at 37-42°C. The E. coli BL21 competent cells produced the dimeric nanobody protein VHH1-VHH2-SpyTag-6xHis by self-induction expression; Step 2: Transform the recombinant vector pET28a-SpyCatcher-C4BP-SnoopCatcher-6xHis into Escherichia coli BL21 competent cells by thermal transformation at 37-42°C for 90-120s, culture in an induction solution at 25-37°C with self-induction shaking at 200-250r / min for 10-12h, collect the cells by centrifugation at 8000×g for 15-20min at 4°C, resuspend with 20mL of equilibration buffer and 60mg of lysozyme, destroy the resuspended Escherichia coli cells by ultrasound in an ice water bath to prevent degradation or denaturation of the target protein, and then collect the supernatant by centrifugation at 1000×g for 15-20min at 4°C, and filter through 0.22μm to obtain a soluble heptamer fusion protein lysate; Step 3: Purify the SpyCatcher-C4BP-SnoopCatcher-6xHis fusion protein using Ni-NTA affinity chromatography, elute with 300 mmol / L imidazole, and then dialyze with 10 mmol / L PBS buffer at a pH of 6.8-7.4 to obtain a purified soluble heptamer fusion protein. Step 4: Fully mix the soluble heptameric fusion protein skeleton SpyCatcher-C4BP-SnoopCatcher-6xHis and the dimeric nanobody protein VHH1-VHH2-SpyTag-6xHis, and react and covalently bind them under low temperature to form densely displayed soluble heptameric giant nanobody (14VHH) and 7 Biotin molecules fusion protein molecules.

6. The method of claim 5, wherein the method comprises: In step 2, the components and their concentrations in the equilibration buffer are: 2mmol / LKH2PO4, 8mmol / LNa2HPO4, 136mmol / L NaCl, 2.6mmol / L KCl, and 1mmol / L phenylmethylsulfonyl fluoride.

7. The method of claim 5, wherein the method comprises: In step 4, the soluble protein skeleton SpyCatcher-C4BP-SnoopCatcher-6xHis is reacted with the dimeric nanobody protein VHH1-VHH2-SpyTag-6xHis to form densely displayed soluble giant nanobody (14xVHH) molecules through covalent and non-covalent binding.

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