HBC-SpyTag003 fusion gene, its expression vector, monoclonal antibody and its application

Abstract

The present invention discloses a HBC‑SpyTag003 The fusion gene, its expression vector, monoclonal antibody and its application are intended to solve the current technical problems of complex and difficult purification of SpyTag protein. The present invention uses genetic engineering technology to display SpyTag003 at high density on the surface of HBV core antigen (HBc), express it in Escherichia coli and self-assemble to form nanoparticles, immunize mice with it, and screen for high-affinity SpyTag003 monoclonal antibodies by hybridoma method; the monoclonal antibody is covalently fixed on AminoLink TM The coupled resin can be used to prepare an immunoaffinity chromatography column for purifying SpyTag protein, which has high purification efficiency and high protein purity, and can effectively retain the assembly function of the purified protein, providing a new approach for the efficient purification of SpyTag protein.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to an HBC-SpyTag003 fusion gene, an expression vector thereof, a monoclonal antibody and applications thereof. Background Art

[0002] SpyTag / SpyCatcher is a star molecule for modular vaccine assembly. It provides a simple protein covalent conjugation pathway with high robustness and versatility. It has been applied to a variety of candidate vaccines and personalized tumor neoantigen therapeutic vaccines. This system is produced by cleaving the CnaB2 domain from the fibronectin binding protein FbaB of Streptococcus pyogenes. The two components can spontaneously form intramolecular isopeptide bonds, and rapid assembly can be achieved at a certain temperature (at least 4 to 37°C), pH value (5 to 8), buffer (no specific anions or cations required), and even when using non-ionic detergents. SpyTag003 / SpyCatcher003 is the latest version of this system. It solves the problem of SpyTag / SpyCatcher's moderate reaction rate limiting the time resolution and labeling efficiency of low-concentration proteins. After modification, the reaction rate (5.5×10 5 M -1 s -1 ) is close to the diffusion limit, which is much faster than the original SpyTag / SpyCatcher reaction rate (1.4×10 3 M - 1 s -1 ) is about 400 times faster; even at low protein concentrations (10nM), SpyTag003 / SpyCatcher003 reactions can be completed in more than 90% within 15min, while there is almost no reaction of the original SpyTag / SpyCatcher. Currently, the ultra-efficient reactivity of SpyTag003 / SpyCatcher003 makes it a better choice in the toolkit of synthetic biologists.

[0003] Based on the potential of the SpyTag / SpyCatcher system in modular vaccine production, the purification of SpyTag protein, one of its key components, is a realistic and urgent problem to be solved. At present, most SpyTag proteins are designed with additional tags, such as His tags, for purification. However, these tags usually have no use after purification and will produce unhelpful immune responses in the body. When the purity of the eluted components is not enough, or the sample does not hang on the purification column at all due to insufficient exposure of the His tag, other purification methods are required. In addition, it is often necessary to prepare a variety of candidate SpyTag antigens in research. When they need to be purified by different methods due to their own differences, the production steps will become extremely complicated. Therefore, if a simpler and more universal purification method can be established for the original SpyTag tag, so that the SpyTag tag can be directly used for purification in addition to the assembly function, the production efficiency of this modular component will be greatly improved.

[0004] Using monoclonal antibodies against SpyTag as immobilized ligands to specifically separate SpyTag-tagged proteins from complex samples is a promising and feasible purification method.

[0005] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0006] Through research, the inventors found that due to the small molecular weight of SpyTag, it is difficult to induce a sufficiently high level of antibodies, which is a bottleneck factor restricting the purification and separation of proteins based on SpyTag tag proteins; the use of nanoparticle display can promote antigen drainage to lymph nodes, complement activation, antigen presenting cell (APC) uptake and B cell receptor (BCR) cross-linking, and then induce a strong humoral response to the displayed antigen and produce higher levels of antibodies; further research found that SpyTag003 was displayed at a high density on the surface of hepatitis B virus (HBV) core antigen (HBc) through genetic engineering technology, expressed in Escherichia coli and self-assembled to form nanoparticles; it was used as an immunogen to immunize mice, and high-affinity SpyTag003 monoclonal antibodies were screened through hybridoma technology.

[0007] In view of this, in the present invention application, HBc nanoparticles are used to multivalently display the SpyTag003 tag to prepare high-affinity SpyTag003 monoclonal antibodies, and a new, universal, monoclonal antibody-based affinity immunochromatography purification method is established for the separation and purification of SpyTag proteins; and the constructed SpyTag model proteins with various tag positions and different expression sources are used as purification verification models, and the purity and assembly ability of the obtained proteins are characterized and confirmed, thereby providing a new and effective way for the efficient separation and purification of SpyTag proteins.

[0008] According to one aspect of the present disclosure, there is provided a HBC-SpyTag003 The fusion gene, whose DNA sequence is shown as SEQ ID NO.1.

[0009] According to another aspect of the present disclosure, the above HBC-SpyTag003 The fusion gene was cloned into pET28a vector and the prokaryotic expression vector pET28a-HBC-SpyTag003 was constructed.

[0010] In some embodiments of the present disclosure, the above-mentioned prokaryotic expression vector is transformed into Escherichia coli to construct the pET28a-HBC-SpyTag003 prokaryotic expression strain; the obtained expression strain is then induced for expression in the presence of IPTG; the cells are lysed, and then the lysis supernatant containing the above-mentioned HBC-SpyTag003 is purified to finally obtain purified HBC-SpyTag003 nanoparticles.

[0011] In some embodiments of the present disclosure, the E. coli strain transformed with the pET28a-HBC-SpyTag003 plasmid is pTf16 / BL21(DE3), a competent cell of E. coli BL21(DE3) transformed with the molecular chaperone pTf16, which can help exogenous proteins to fold correctly and increase the proportion of soluble proteins.

[0012] In some embodiments of the present disclosure, the HBC-SpyTag003 nanoparticles are purified by first precipitating with 5% volume saturated ammonium sulfate, dialyzing to remove residual ammonium sulfate, and then filling with Capto TM Core 700 column, collecting the flow-through.

[0013] According to another aspect of the present disclosure, an anti-SpyTag003 monoclonal antibody is provided, which is a monoclonal antibody prepared by a hybridoma method using the nanoparticles as an immunogen.

[0014] In some embodiments of the present disclosure, the heavy chain variable region sequence of the anti-SpyTag003 tag monoclonal antibody is a DNA sequence as shown in SEQ ID NO.2 or an amino acid sequence as shown in SEQ ID NO.3, or a sequence of an active fragment or a conservative variant obtained by adding, deleting or replacing one or more amino acids on the basis of the SEQ ID NO.3 sequence; and / or

[0015] The light chain variable region DNA sequence is the DNA sequence shown in SEQ ID NO.4, or the amino acid sequence shown in SEQ ID NO.5, or the sequence of an active fragment or conservative variant obtained by adding, deleting or replacing one or more amino acids on the basis of the SEQ ID NO.5 sequence.

[0016] In some embodiments of the present disclosure, mice were immunized with the purified HBC-SpyTag003 nanoparticles, and mouse spleen cells were fused with mouse myeloma cells (Sp / 20 cells) to obtain hybridoma cells. Positive hybridoma cells were screened by indirect ELISA and limiting dilution methods. The hybridoma cells were inoculated into the peritoneal cavity of mice to collect ascites containing monoclonal antibodies, and the titer of ascites containing monoclonal antibodies was evaluated by indirect ELISA; finally, SpyTag003 monoclonal antibodies were obtained by purifying the ascites.

[0017] In some embodiments of the present disclosure, the purified SpyTag003 monoclonal antibody is immobilized on AminoLink TM The coupling resin is used to obtain a universal immunoaffinity chromatography column (Spy&IAC) for SpyTag protein. The resin is an aldehyde-activated cross-linked beaded agarose with a fast linear flow potential and can be used for gravity flow and medium and low pressure applications. The fixing principle is that the primary amine on the monoclonal antibody molecule spontaneously reacts with the activated aldehyde group on the agarose carrier to form a Schiff base bond, which can be reduced to a stable secondary amine bond in the presence of a mild reducing agent, sodium cyanoborohydride (NaCNBH3), thereby simply and effectively covalently fixing the monoclonal antibody on the agarose carrier.

[0018] According to another aspect of the present disclosure, the immunoaffinity chromatography column is used to efficiently purify proteins with SpyTag tags at the N-terminus or C-terminus from prokaryotic or eukaryotic expression systems.

[0019] In some embodiments of the present disclosure, the immunoaffinity chromatography column is used to purify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein or porcine epidemic diarrhea virus (PEDV) core neutralizing epitope (COE) protein with a SpyTag003 tag at the N-terminus or C-terminus.

[0020] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0021] 1. By HBC-SpyTag003 The fusion gene can display SpyTag003 at a high density on the surface of hepatitis B virus (HBV) core antigen (HBc). HBC-SpyTag003 The fusion gene can be expressed in Escherichia coli and self-assembled to form HBC-SpyTag003 nanoparticles.

[0022] 2. Mice were immunized with HBC-SpyTag003 nanoparticles, and high-affinity SpyTag003 monoclonal antibodies were screened through the hybridoma method.

[0023] 3. The obtained SpyTag003 monoclonal antibody can be used to prepare a universal immunoaffinity chromatography column (Spy&IAC) for efficient purification of proteins with SpyTag tags at the N-terminus or C-terminus from prokaryotic or eukaryotic expression systems, providing a universal new method for efficient purification of SpyTag proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the HBc-SpyTag003 gene fusion construct according to one embodiment of the present disclosure.

[0025] Figure 2 This is an SDS-PAGE diagram of the purified HBC-SpyTag003 nanoparticles according to an embodiment of the present disclosure, wherein the bands are: M is a protein marker, and lane 1 is the purified HBC-SpyTag003 nanoparticles.

[0026] Figure 3 This is a graph showing the particle size of HBC and HBC-SpyTag003 nanoparticles measured by dynamic light scattering in one embodiment of the present disclosure.

[0027] Figure 4 This is a graph of mouse serum titers collected on days 28 and 42 after the first immunization with HBC-SpyTag003 nanoparticles in an embodiment of the present disclosure.

[0028] Figure 5 Graph showing the titer of ascites containing different monoclonal antibodies in one embodiment of the present disclosure.

[0029] Figure 6 : This is the elution curve of 6G3 ascites purification in one embodiment of the present disclosure; wherein, the blue line represents the distribution diagram of absorbance at 280 nm, and the orange line represents the change of conductivity value during the elution process.

[0030] Figure 7PCR diagram of SpyTag003 monoclonal antibody 6G3 VH and VL gene amplification in one embodiment of the present disclosure.

[0031] Figure 8 In one embodiment of the present disclosure, the complementarity determining regions (CDRs) and framework regions (FRs) of the light chain and heavy chain variable regions of the SpyTag003 monoclonal antibody 6G3 were located respectively.

[0032] Fig. 9 This is a structural modeling diagram of the VH and VL regions of the SpyTag003 monoclonal antibody 6G3 in one embodiment of the present disclosure.

[0033] Fig.10 The chemical principle diagram of preparing a universal immunoaffinity column (Spy&IAC) for SpyTag protein in one embodiment of the present disclosure.

[0034] Fig.11 This is a schematic diagram of four SpyTag model protein gene fusion constructs in one embodiment of the present disclosure.

[0035] Fig.12 , Fig.13 In one embodiment of the present disclosure, SDS-PAGE was used to analyze the expression and purification of N-SpyTag003-N and C-SpyTag003-N respectively; M is a protein marker, lane 1 is a whole cell lysate, and lane 2 is the final eluted purified protein.

[0036] Fig.14 , Fig.15 The expression and purification of N-SpyTag003-COE and C-SpyTag003-COE were identified in an embodiment of the present disclosure; and in the same figure, the left figure shows the expression of the protein in the cell supernatant detected by Western blot using the SpyTag003 monoclonal antibody 6G3 prepared in the embodiment, and the right figure shows the final purification of the protein identified by SDS-PAGE; M is a protein marker, lane 1 is the cell supernatant, and lane 2 is the final eluted purified protein.

[0037] Figure 16-19 They are respectively the elution curves of the four SpyTag model proteins in one embodiment of the present disclosure after being purified by Spy&IAC columns; wherein the blue line represents the distribution diagram of absorbance at 280 nm, and the orange line represents the change of conductivity value during the elution process.

[0038] Figure 20-23 They are SEC spectra of four SpyTag model proteins purified by Spy&IAC column in one embodiment of the present disclosure; the blue line represents the distribution diagram of absorbance at 280 nm.

[0039] Figure 24-27 The assembly capabilities of the four SpyTag model proteins purified in one embodiment of the present disclosure were verified respectively; the four purified SpyTag model proteins were coupled with SpyCatcher003-mi3 at different molar ratios at 4°C, and the final assembly was verified by SDS-PAGE. DETAILED DESCRIPTION

[0040] The experimental methods described in the following examples are conventional methods unless otherwise specified. It should be understood by those skilled in the art that the reagents, enzymes, carriers, etc. used in the following examples are analytically pure reagents, enzymes, and carriers commercially available from reagent companies unless otherwise specified. Materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

[0041] In order to better understand the technical solution of the present application, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0042] Example 1: Prokaryotic expression and purification method of HBC-SpyTag003 nanoparticles

[0043] 1. Construction of pET28a-HBC-SpyTag003 prokaryotic expression vector

[0044] The SpyTag003 tag sequence (RGVPHIVMVDAYKRYK) was inserted between amino acids 80 and 81 of the major immunodominant region (MIR) of the HBc protein (amino acids 1-149), with a GGS linker on each side of the sequence to increase the flexibility of the protein chain. The entire sequence was codon-optimized for E. coli preference and synthesized by Sangon Biotech (Shanghai) Co., Ltd. B H and HindIII The synthesized gene (as shown in SEQ ID NO.1) was inserted into the pET28a vector. Figure 1 This is a schematic diagram of the HBc-SpyTag003 gene fusion construct.

[0045] 2. Expression of HBC-SpyTag003 Nanoparticles

[0046] For soluble expression, the pET28a-HBC-SpyTag003 plasmid constructed above was transformed into pTf16 / BL21(DE3) E. coli expression competent cells, and the transformation products were spread on LB agar plates containing 50μg / mL kanamycin and 100μg / mL ampicillin and incubated overnight at 37℃. Single clones were picked into 5ml LB medium containing 50μg / mL kanamycin and 100μg / mL ampicillin, and incubated at 37℃ with shaking at 200 r / min for 16 h. Then the preculture was diluted 1:100 into 500ml LB medium, 50μg / mL kanamycin, 100μg / mL ampicillin and 2mg / ml L-arabinose were added, and the culture was shaken at 37℃ and 200r / min. When the OD600 reaches about 0.6, add IPTG with a final concentration of 0.2mM, and continue to induce at 16℃, 200r / min for 18h. Collect the cells by centrifugation, add lysis buffer (50ml 20mM Tris-Hcl, 150mM NaCl solution with pH 8.0) to the cells after centrifugation at a volume ratio of 10:1 (original bacterial liquid volume: lysis buffer) to resuspend the cells, and take a part of the resuspended liquid for SDS-PAGE identification.

[0047] 3. Purification and characterization of HBC-SpyTag003 nanoparticles

[0048] (1) Purification of HBC-SpyTag003 nanoparticles

[0049] The heavy suspension of the bacteria obtained in the previous step was subjected to ultrasonic disruption, centrifuged at 12000 r / min for 20 minutes at 4°C, and the supernatant was collected. The collected supernatant was filtered through a 0.22μm filter, and then a 5% volume of saturated ammonium sulfate solution was added to the supernatant and allowed to stand at 4°C for 6 hours. Centrifuged at 12000 r / min for 20 minutes at 4°C, the supernatant was discarded, and the precipitate was resuspended in 20 ml of 20mM Tris-Hcl, 150mM NaCl solution with a pH of 8.0. Dialyze with an excess of the same buffer for 24 hours to remove residual ammonium sulfate. Centrifuge the dialyzed HBC-SpyTag003 at 6000r / min for 20 minutes at 4°C, and filter the supernatant with a 0.22μm filter to further remove any insoluble matter. Finally, the supernatant was passed through a Capto TM The column was filled with Core 700 and the flow-through was collected. The purified HBC-SpyTag003 was identified by SDS-PAGE gel electrophoresis. Figure 2It is the SDS-PAGE image after purification, and the bands are: M is the protein marker, and lane 1 is the purified HBC-SpyTag003 nanoparticles.

[0050] (2) Dynamic light scattering (DLS) detection

[0051] The purified HBC-SpyTag003 nanoparticles were detected by dynamic light scattering (DLS) technology. Figure 3 As shown, according to the DLS results, the obtained nanoparticles have high purity and good structural uniformity.

[0052] Example 2: Preparation of SpyTag003 monoclonal antibody

[0053] 1. Mouse immunization: The purified HBC-SpyTag003 nanoparticles obtained in Example 1 were emulsified with Freund's complete adjuvant in a volume ratio of 1:1. Five female BALB / c mice aged 6 to 8 weeks were immunized three times at intervals of 14 days by multiple subcutaneous injections on the back. Freund's incomplete adjuvant was used for the last two immunizations, and the protein dose for the three immunizations was 10 μg / mouse, and the total dose was 200 μl / mouse. Blood was collected from the tails of mice on the 28th and 42nd days after the first immunization, and mice with the highest serum antibody titers were selected by indirect ELISA for super immunization. Super immunization was performed by intraperitoneal injection of pure HBC-SpyTag003 nanoparticles without adjuvant at a dose of 50 μg / mouse.

[0054] 2. Determination of serum antibody titer in immunized mice

[0055] The titer of mouse sera collected on days 28 and 42 after the first immunization was detected by indirect ELISA method, the steps are as follows:

[0056] (1) Coating: Dilute the purified HBC-SpyTag003 nanoparticles to 1 µg / mL with ELISA coating buffer (CBS), add 50 µl of coating buffer to each well, incubate overnight at 4°C, discard the coating buffer, wash three times with PBST, and spin dry the residual liquid on the plate;

[0057] (2) Blocking: Add 200 µl of blocking solution (5% skim milk powder + PBST) to each well and block at 37°C for 2 h. Wash the plate in the same way.

[0058] (3) Primary antibody: Add 50 μl of the serum to be tested diluted 2-fold with dilution buffer (PBST) to each well (initial dilution factor is 1:100), incubate at 37°C for 1 h, discard the supernatant, and wash 6 times with PBST;

[0059] (4) Secondary antibody: 50 μl of HRP-labeled goat anti-mouse IgG diluted 1:5000 in dilution buffer was added to each well. After incubation at 37°C for 45 min, the supernatant was discarded and the cells were washed 6 times with PBST.

[0060] (5) Color development: Add 50 µl of DAB color development solution to each well and incubate at room temperature in the dark for 20 min. Then add 50 µl of 2 M H2SO4 stop solution to terminate the reaction and measure the OD450 value using a microplate reader.

[0061] (6) Use Excel to organize and analyze the data and calculate the serum titer of each mouse at each time period.

[0062] The results are as follows Figure 4 As shown, the ELISA results showed that the serum ELISA titers of 3 BALB / c mice were all high, reaching 1:51 200. Mouse No. 3 was randomly selected from them for cell fusion to prepare monoclonal antibodies.

[0063] 3. Cell Fusion

[0064] According to the results of the previous step, BALB / c mice No. 3 were super-immunized and killed by cervical stenosis 3 days later (orbital bleeding was performed before killing to retain positive control serum), and the body surface was disinfected with 75% alcohol. The specific steps of cell fusion are as follows:

[0065] (1) Collect 2 to 5 × 10 well-growing sp2 / 0 tumor cells 7 in a centrifuge tube;

[0066] (2) Remove the mouse spleen under aseptic operation in a clean bench, place it on a 200-mesh sterile filter, chop the spleen with small scissors, add GNK wash solution to rinse, and filter the spleen cells individually into a sterile beaker;

[0067] (3) Transfer the spleen cell suspension into a centrifuge tube, add GNK wash solution to 40 ml, and centrifuge it together with the tumor cells at 1200 rpm for 10 min.

[0068] (4) Discard the supernatant, flick off the cell pellet, add 10 ml of GNK wash solution to each tube, transfer the spleen cell suspension into the tumor cell tube, add GNK wash solution to 40 ml, centrifuge at 1200 r / min for 10 min, and discard the supernatant;

[0069] (5) Gently break up the cell clusters and add 1 ml of fusion agent 50% PEG1500. Make sure to add it within one minute and let it stand for 90 seconds. Then slowly add 15 ml of GNK wash solution to terminate the fusion (add 1 ml in the first half minute, 3 ml in the second half minute, and then gradually increase the speed of adding the remaining 11 ml). Stable in a 37°C water bath for 5 minutes, add GNK wash solution to 40 ml, and centrifuge at 1000 r / min for 10 minutes.

[0070] (6) After centrifugation, discard the supernatant, gently break up the cell clumps, add HAT selection medium and gently suspend the cells. Remember not to blow vigorously to avoid damaging the fused cells.

[0071] (7) Disperse the suspended cells into a 96-well cell culture plate and add 250 μl of cell suspension to each well;

[0072] (8) Small cell clusters can be observed under a microscope after 3 to 4 days of culture, and the hybridoma cell supernatant can be detected after 9 to 12 days.

[0073] 4. Screening and identification of hybridoma cells

[0074] On the 11th day after cell fusion, the hybridoma cell supernatant was detected by indirect ELISA. HBC-SpyTag003 nanoparticles and HBC nanoparticles were used as coating antigens, and positive hybridoma cell lines that could specifically recognize the SpyTag003 tag and did not react with the HBC nanoparticle scaffold were screened by combining "positive screening" and "reverse screening". The steps of indirect ELISA are as follows:

[0075] (1) Coating: HBC-SpyTag003 nanoparticles and HBC nanoparticles were used as coating antigens, respectively. After diluting with ELISA coating solution (CBS), the plates were incubated at 50 µg per well and 50 µl per well overnight at 4°C. The coating solution was discarded, the plates were washed three times with PBST, and the residual liquid on the plates was dried.

[0076] (2) Blocking: Add 200 µl of blocking solution (5% skim milk powder + PBST) to each well and block at 37°C for 2 h. Wash the plate in the same way.

[0077] (3) Primary antibody: Add 50 ul of hybridoma cell supernatant to each well, set pre-fusion mouse serum as positive control and pre-immune mouse serum as negative control, incubate at 37°C for 30 min, discard the supernatant, and wash with PBST 6 times;

[0078] (4) Secondary antibody: 50 μl of HRP-labeled goat anti-mouse IgG diluted 1:5000 in dilution buffer was added to each well, incubated at 37°C for 30 min, the supernatant was discarded, and the cells were washed 6 times with PBST;

[0079] (5) Color development: Add 50 µl of DAB color development solution to each well and incubate at room temperature in the dark for 20 min.

[0080] According to the color development results, select the positive hybridoma cell lines with good reactivity and transfer them to 24-well cell culture plates, add HT selection culture medium for expansion culture. After 2-3 days of culture, observe the growth status of hybridoma cells, and identify the hybridoma cell lines again by the aforementioned indirect ELISA method. Transfer the reaction wells with positive identification and cell density reaching more than 80% to 6-well cell culture plates, and then replace them with conventional 1640 culture medium containing 10% FBS. Note that rescreening should be performed after each transfer to ensure the stability of positive hybridoma cells.

[0081] 5. Subcloning of hybridoma cells by limiting dilution

[0082] Select the positive hybridoma cells in the 6-well cell culture plate finally identified in step 4 above, with a cell density of more than 50% and a good growth state for subcloning. The specific operation steps are as follows:

[0083] (1) One day before subcloning, prepare feeder layer cells and spread them on multiple 96-well cell culture plates at 100 μl / well for later use;

[0084] (2) Gently blow up the cells in each well to be subcloned and mix them evenly. Pipette 200µl of each well and add them to the first vertical column of a 96-well cell culture plate, and dilute them continuously from left to right in a 2-fold gradient. Then observe each well under a medium power microscope. When the number of cells in the field of view is 10 to 15 (equivalent to a total number of cells in the entire well of about 100), the well is determined to be at an appropriate dilution multiple.

[0085] (3) Add the cells in the appropriate dilution wells to 10 ml of 1640 medium containing 10% FBS, pipette thoroughly to mix, and add 100 μl / well to a 96-well cell culture plate pre-covered with feeder cells (theoretically, a maximum of 1 hybridoma cell can be added to each well). Subclone each hybridoma cell line in the same way;

[0086] (4) After culturing for 7 to 10 days, when the cell clusters have grown to 1 / 5 of the bottom of the well, ELISA testing can be performed using the method described above;

[0087] (5) Secondary subcloning can be performed on the positive wells until a stable secreting monoclonal hybridoma cell line is obtained. In this experiment, three monoclonal cell lines resistant to SpyTag003 were obtained, named 6G3, 5D2 and 2C10;

[0088] (6) The three stable secretion positive monoclonal cell lines finally screened were expanded and cultured at a rate of 1 to 2 × 10 6 Cryopreservation was performed at a cell number of 1:1.

[0089] 6. Preparation and purification of anti-SpyTag003 monoclonal antibody ascites

[0090] (1) Preparation of monoclonal antibody ascites

[0091] Select female BALB / c mice of older age and inject 500 μl of sterilized paraffin intraperitoneally. After 7 to 10 days, centrifuge the three SpyTag003 monoclonal cells expanded in step 5 above, collect the cells and resuspend them in PBS. 7 The mice were observed and the ascites was collected about 10 days later. The ascites was centrifuged at 4000 r / min for 20 min to remove fat and cell precipitation. The ascites supernatant was collected and stored at -80°C for later use.

[0092] (2) Determination of monoclonal antibody ascites titer

[0093] The titer of mouse ascites was determined by indirect ELISA method. The steps are as follows:

[0094] ① Coating: Use HBC-SpyTag003 nanoparticles as coating antigens, dilute with ELISA coating solution (CBS), incubate overnight at 4°C with a coating dose of 50 µg per well and a coating volume of 50 µl, discard the coating solution, wash with PBST three times, and spin dry the residual liquid on the plate;

[0095] ② Blocking: Add 200 µl of blocking solution (5% skim milk powder + PBST) to each well and block at 37°C for 2 h, and wash the plate in the same way;

[0096] ③ Primary antibody: Add 50 ul of mouse ascites diluted 2-fold with dilution buffer (PBST) to each well (initial dilution factor is 1:1000), incubate at 37°C for 1 h, discard the supernatant, and wash 6 times with PBST;

[0097] ④ Secondary antibody: Add 50 μl of HRP-labeled goat anti-mouse IgG diluted 1:5000 in dilution buffer to each well, incubate at 37°C for 45 min, discard the supernatant, and wash 6 times with PBST;

[0098] ⑤ Color development: Add 50 µl DAB color development solution to each well, incubate at room temperature in the dark for 20 min, then add 50 µl 2 M H2SO4 stop solution to terminate the reaction, and measure the OD450 value with a microplate reader.

[0099] ⑥ Arrange and analyze the data and use the maximum dilution multiple as the titer of the ascites.

[0100] The results are as follows Figure 5 As shown, the ELISA test results showed that 6G3 ascites had the highest titer, which was 1:512 000.

[0101] (3) Purification of ascites

[0102] The 6G3 ascites was purified using HiTrap Protein G prepacked columns with the ÄKTA pure chromatography system to obtain the purified SpyTag003 monoclonal antibody. Figure 6 The figure shows the elution curve of 6G3 ascites purification. The blue line represents the distribution of absorbance at 280 nm, and the orange line represents the change in conductivity during the elution process.

[0103] Example 3: Sequencing and bioinformatics analysis of the variable region of SpyTag003 monoclonal antibody

[0104] Total RNA of SpyTag003 6G3 hybridoma cells was extracted and reverse transcribed into cDNA. Then, PCR was performed using universal primers for the heavy chain variable region (VH) and light chain variable region (VL) of mouse IgG to amplify the variable region gene of SpyTag003 monoclonal antibody. The amplification cycle was: 98℃ for 5 min; 35 cycles of 98℃ for 10 s, 60℃ for 15 s, 72℃ for 1 min, and further stretching at 72℃ for 10 min. The PCR product was gel purified and sequenced by Sangon Biotech (Shanghai) Co., Ltd. The monoclonal antibody sequence was searched and analyzed for homology by IMGT / V-QUEST (https: / / www.imgt.org / IMGT_vquest / vquest) and IGBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / igblast.cgi). The tertiary structure of SpyTag003 mAb was simulated using SWISS-MODEL (https: / / swissmodel.expasy.org) and analyzed by PyMOL.

[0105] like Figure 7: This is a PCR diagram of the amplification of the VH and VL genes of the SpyTag003 monoclonal antibody 6G3. The heavy chain variable region and light chain variable region gene sequences of the SpyTag003 monoclonal antibody 6G3 were determined to be SEQ ID NO.2 and SEQ ID NO.4, respectively, and the amino acid sequences of the heavy chain variable region and light chain variable region deduced therefrom were shown in SEQ ID NO.3 and SEQ ID NO.5, respectively. The VH and VL gene sequences were compared in the antibody gene database, and the results showed that both VH and VL contained three complementary determining regions (CDRs) and four framework regions (FRs), as shown in FIG. Figure 8 The CDR and FR of the antibody variable region are positioned as shown. Fig. 9 Structural simulation showed that the CDRs were close in space in the VH and VL monomer structures and surrounded each other in the entire variable region structure, together forming the antigen binding site (antigen binding pocket) of SpyTag003.

[0106] Example 4: Application of SpyTag003 monoclonal antibody in the preparation of universal immunoaffinity column (Spy&IAC)

[0107] The SpyTag003 monoclonal antibody obtained in Example 2 was used to prepare a universal immunoaffinity chromatography column (Spy&IAC) based on SpyTag protein. Fig.10 This is the chemical principle diagram of the preparation. The specific preparation method is as follows:

[0108] (1) Monoclonal antibody immobilization: 5 ml AminoLink TM The coupling resin was packed in an empty column and the packing buffer was allowed to flow out naturally. The column was equilibrated by adding 15 ml (3 column volumes) of coupling buffer (PBS solution with a pH of 7.2) and the contents were allowed to drain. Next, 5 mL of purified HBC-SpyTag003 monoclonal antibody (dissolved in coupling buffer) was added to the column, and 0.1 mL of the sample was saved for later determination of coupling efficiency. In a fume hood, 100 μL of cyanoborohydride solution (producing approximately 50 mM NaCNBH3) was added to the reaction slurry and mixed by inversion overnight at 4°C. The next day, the top cover was carefully removed in the fume hood, as some air pressure may have formed during the reaction, and then the bottom cover was removed. The effluent from the column was collected and the coupling efficiency was determined by comparing the protein concentration of the non-bound fraction with that of the starting sample. The resin was then washed with 10 ml of coupling buffer;

[0109] (2) Block remaining active sites: Wash the resin with 10 mL of quenching buffer (1 M Tris-HCl solution at pH 7.4). In a fume hood, add 5 mL of quenching buffer and 100 μL of cyanoborohydride solution (resulting in ~50 mM NaCNBH3 when mixed with the resin) to the column and gently mix by shaking end-to-end for 30 min. Then carefully remove the top and bottom caps and allow the column contents to drain;

[0110] (3) Washing the column: Wash the column with at least 25 mL (5 resin bed volumes) of washing solution (1M NaCl solution). Pay attention to monitor whether there is protein in the final washing. If there is, continue washing until no protein flows out. Finally, wash the resin with 15 mL of PBS solution with pH 7.2 containing 0.05% sodium azide and store the column in it.

[0111] Example 5: Purification and verification of the construction of SpyTag model protein

[0112] 1. Construction of four SpyTag model protein expression vectors

[0113] Fig.11 This is a schematic diagram of the fusion constructs of 4 SpyTag model proteins. All sequences have been optimized for E. coli preference codons and synthesized by Sangon Biotech (Shanghai) Co., Ltd.:

[0114] (1) pET28a-N-SpyTag003-N was generated by inserting the N-terminal SpyTag003 tag, GGSGGS linker, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein into the pET28a plasmid;

[0115] (2) pET28a-C-SpyTag003-N was generated by inserting SARS-CoV-2 N protein, GGSGGS linker, and C-terminal SpyTag003 tag into the pET28a plasmid;

[0116] (3) pcDNA3.1-N-SpyTag003-COE was generated by inserting the N-terminal SpyTag003 tag, GGSGGS linker, and the core neutralizing epitope (COE) protein of porcine epidemic diarrhea virus (PEDV) into the pcDNA3.1 plasmid;

[0117] (4) pcDNA3.1-C-SpyTag003-COE was generated by inserting PEDV COE protein, GGSGGS linker, and C-terminal SpyTag003 tag into the pcDNA3.1 plasmid.

[0118] 2. Expression of four SpyTag model proteins

[0119] The expression methods of the two model proteins for prokaryotic expression are similar to the method described in step 2 of Example 1. In brief, N-SpyTag003-N and C-SpyTag003-N were expressed in E. coli BL21 (DE3) competent cells. After picking a single colony, 10 mL of overnight culture was diluted to 1 L LB and 50 µg / mL kanamycin was added. Cultured at 37°C and 200 r / min until OD600 reached about 0.6, IPTG was added at a final concentration of 0.2 mM, and induction was continued at 16°C and 200 r / min for 12 h. The bacterial culture was harvested and resuspended in binding buffer (PBS solution with a pH of 7.4), and after ultrasonic disruption, centrifuged at 12,000 r / min for 20 min at 4°C. The supernatant was collected and the expression was identified using SDS-PAGE.

[0120] N-SpyTag003-COE and C-SpyTag003-COE were expressed in Expi293F cells. According to the manufacturer's recommendations, Expi293F cells were cultured in an orbital shaker incubator at 37°C, 5% (v / v) CO2, and 125 r / min for at least 5 generations before transient transfection. Recombinant DNA was transiently transfected using PEI. All proteins were expressed in a total culture volume of 1 L in a ventilated cell shaker. 96 h after transfection, the cell viability dropped to below 40-60%, and the SpyTag003 monoclonal antibody 6G3 prepared in Example 2 was used to detect the expression of proteins in the cell supernatant by western blot.

[0121] Fig.12 and Fig.13 The expression and purification of N-SpyTag003-N and C-SpyTag003-N were analyzed by SDS-PAGE. M is a protein marker, lane 1 is the whole cell lysate, and lane 2 is the final eluted purified protein. Fig.14 and Fig.15 The expression and purification of N-SpyTag003-COE and C-SpyTag003-COE are identified respectively. Among them, the left side of both figures are the expression of proteins in cell supernatants detected by Western blot using the SpyTag003 monoclonal antibody 6G3 prepared in the present invention. The right side of both figures are the final purification of proteins identified by SDS-PAGE. M is a protein marker, lane 1 is the cell supernatant, and lane 2 is the final eluted purified protein.

[0122] Example 6: SpyTag protein purification method based on type 2 immunoaffinity chromatography column (Spy&IAC)

[0123] The universal immunoaffinity chromatography column (Spy&IAC) based on SpyTag protein prepared in Example 4 was used to purify the supernatant samples of four SpyTag model proteins using the ÄKTA pure chromatography system. The specific method is as follows:

[0124] (1) Before use, equilibrate the prepared immunoaffinity chromatography column (Spy&IAC) to room temperature. Do not let the resin bed dry out during the entire process. Be careful to degas all buffers to avoid introducing bubbles into the chromatographic column. Filter the protein supernatant sample using a 0.22 μm filter and set aside;

[0125] (2) First, the chromatographic column was equilibrated with binding buffer (PBS solution with a pH of 7.4), and the filtered protein supernatant sample was passed through the chromatographic column at a flow rate of 0.5 ml / min;

[0126] (3) After loading, re-equilibrate the column with binding buffer to wash away unbound components. When the UV value stabilizes (generally below 20 mAU), use elution buffer (0.1 M glycine solution at pH 2.7) to elute the bound protein.

[0127] (4) Pay attention to the changes in the conductivity of the system. When the conductivity value begins to decrease rapidly, you should collect the eluate. Adjust the pH value to neutral by adding 100 μL of neutralization buffer (1 M Tris-HCl solution with a pH of 9.0) to each 1 mL of the collected eluate;

[0128] (5) The elution was monitored by absorbance at 280 nm. The collected elution fractions were pooled and exchanged through Sephadex G-25 into an appropriate storage buffer, such as PBS solution with a pH of 7.4.

[0129] like Figure 16-19 The following are the elution curves of four SpyTag model proteins purified by Spy&IAC columns. The blue line represents the distribution of absorbance at 280 nm, and the orange line represents the change in conductivity during the elution process.

[0130] Example 7: Purification analysis of four SpyTag003 model proteins

[0131] 1. Protein Purity Analysis

[0132] The purity of four SpyTag003 model proteins purified by a universal immunoaffinity chromatography column (Spy&IAC) for SpyTag proteins was analyzed by SDS-PAGE and size exclusion chromatography (SEC). TM 200Increase 10 / 300GL column for analysis of N-SpyTag003-N and C-SpyTag003-N proteins, HiLoad TM 16 / 600 Superdex TM The 200 pg column was used for the analysis of N-SpyTag003-COE and C-SpyTag003-COE proteins. Figure 12-15 and Figure 20-23 As shown, the purified four SpyTag model proteins were uniform and pure, with a purity of more than 90%, which was confirmed by clear blots in SDS-PAGE and a single main peak in the SEC chromatogram.

[0133] 2. Verification of protein assembly capability

[0134] In order to evaluate the function of SpyTag model proteins purified using a universal immunoaffinity chromatography column (Spy&IAC) for SpyTag proteins, namely, their ability to assemble with their "partner" SpyCatcher. The four purified SpyTag model proteins were combined with SpyCatcher003-mi3 at different molar ratios at 4°C for 2 h, and the aggregates were removed by centrifugation. SDS-PAGE was used to show the amount of covalent VLP-antigen conjugates formed at each coupling ratio. Figure 24-27 As shown, SDS-PAGE confirmed the effective covalent reaction, and all SpyTag model proteins were efficiently displayed on SpyCatcher003-mi3. When incubated at equimolar concentrations, although the changes in N-SpyTag003-COE and C-SpyTag003-COE were not obvious, most reactions produced minimal residual non-bound antigen, which was consistent with previous literature reports. In other words, the SpyTag protein purified using a universal immunoaffinity chromatography column (Spy&IAC) for SpyTag proteins showed satisfactory assembly ability.

[0135] Example 8: Regeneration and storage method of universal immunoaffinity chromatography column (Spy&IAC)

[0136] The column should be regenerated immediately after each elution to prevent the low pH elution buffer from damaging the immobilized monoclonal antibody molecules. When the protein elution is completed, the column is immediately washed with a large amount of binding buffer (PBS solution with a pH of 7.4) to remove any residual protein and reactivate the resin. The column is then equilibrated with binding buffer containing 0.05% sodium azide and stored in it. A spring valve (or syringe) can be added to the top of the column, which can apply pressure to the resin bed to prevent it from resuspending or leaking, thereby preventing the column from drying out.

[0137] The columns were stored vertically at 4°C and used multiple times over a long period of time to verify their service life. The expression and purification of the four SpyTag model proteins were actually carried out over a period of 4 months. The results of various experiments showed that the universal immunoaffinity chromatography column (Spy&IAC) for SpyTag proteins exhibited good regeneration and storage stability.

[0138] Although some preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0139] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of this application and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A HBC-SpyTag003 The fusion gene, whose DNA sequence is shown in SEQ ID NO.

1.

2. A prokaryotic expression vector, as claimed in claim 1 HBC-SpyTag003 The fusion gene was cloned into the pET28a vector.

3. A nanoparticle, which is a prokaryotic expression strain formed by transforming the prokaryotic expression vector of claim 2 into Escherichia coli, and is obtained by IPTG-induced expression, bacterial cell lysis, and purification steps to obtain HBC-SpyTag003 protein particles.

4. The nanoparticle according to claim 3, characterized in that The purification step comprises: (1) Precipitate the bacterial lysate with a saturated ammonium sulfate solution and remove the residual ammonium sulfate by dialysis; (2) Then fill Capto TM The purified nanoparticles were obtained by collecting the flow-through liquid from the column filled with Core 700.

5. An anti-SpyTag003 monoclonal antibody, which is prepared by a hybridoma method using the nanoparticles according to claim 3 as an immunogen.

6. The anti-SpyTag003 monoclonal antibody according to claim 5, characterized in that: The heavy chain variable region sequence is the DNA sequence shown in SEQ ID NO.2 or the amino acid sequence shown in SEQ ID NO.3; and / or the light chain variable region DNA sequence is the DNA sequence shown in SEQ ID NO.4 or the amino acid sequence shown in SEQ ID NO.

5.

7. Use of the anti-SpyTag003 monoclonal antibody according to claim 5 in purifying proteins with SpyTag003 tags.

8. The use according to claim 7, characterized in that: The protein is a severe acute respiratory syndrome coronavirus 2 nucleocapsid (N) protein or a porcine epidemic diarrhea virus core neutralizing epitope protein with a SpyTag003 tag at the N-terminus or C-terminus.

9. The use according to claim 7, characterized in that: The anti-SpyTag003 tag monoclonal antibody is coupled to the corresponding solid phase carrier aldehyde-activated cross-linked beaded agarose to prepare an immunoaffinity chromatography column, and then the SpyTag protein is purified by using the immunoaffinity chromatography column.

10. The use according to claim 8, characterized in that: Under the action of reducing agent sodium cyanoborohydride, the aldehyde-activated agarose carrier and the anti-SpyTag003 monoclonal antibody were robustly fixed.

Images