A universal immunoaffinity column for modular component spytag protein, its preparation method and purification method

Abstract

The application discloses a universal immunization affinity column for modular component SpyTag protein, a preparation method and a purification method, and aims to solve the technical problems of complex and difficult SpyTag protein purification. The application realizes high-density display of SpyTag003 on the surface of HBV core antigen (HBc) through genetic engineering technology, expresses and self-assembles the SpyTag003 into a nanoparticle in Escherichia coli, immunizes mice, and obtains a high-affinity SpyTag003 monoclonal antibody through a hybridoma method; the monoclonal antibody is covalently fixed on an AminoLink TM The coupling resin is used for preparing the immunization affinity column, the immunization affinity column can be used for efficiently purifying proteins with SpyTag labels at N-termini or C-termini from prokaryotic or eukaryotic expression systems, the column is simple to prepare, can be effectively regenerated, and has simple and conventional elution conditions, is low in cost, is high in purification efficiency, is high in protein purity, and can effectively reserve the assembly function of the purified protein, and provides a universal new method for efficient purification of SpyTag proteins.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioengineering, and particularly relates to a universal immunological affinity chromatography column for modular component SpyTag protein, a preparation method and a purification method. BACKGROUND

[0002] SpyTag / SpyCatcher is a star molecule for modular vaccine assembly, which provides a simple covalent conjugation approach for proteins, has high robustness and universality, and has been applied to various candidate vaccines and personalized tumor neoantigen therapeutic vaccines. This system is produced by splitting the CnaB2 domain from the fibronectin-binding protein FbaB of Streptococcus pyogenes, and the two components can spontaneously form an intramolecular isopeptide bond, and can be rapidly assembled under certain temperature (at least 4-37℃), pH value (5-8), buffer (no specific anion or cation) and even using non-ionic detergents. SpyTag003 / SpyCatcher003 is the latest version of this system, which solves the problem of low concentration of proteins in time resolution and labeling efficiency of SpyTag / SpyCatcher with medium reaction rate, and after modification, the reaction rate (5.5×10 5 M -1 s -1 ) is close to the diffusion limit, which is about 400 times faster than the original SpyTag / SpyCatcher reaction rate (1.4×10 3 M - 1 s -1 ). Even at low protein concentration (10nM), SpyTag003 / SpyCatcher003 reaction can be completed in 15min, and almost no original SpyTag / SpyCatcher reaction occurs. At present, the ultra-high efficiency of SpyTag003 / SpyCatcher003 makes it a better choice in the toolbox of synthetic biologists.

[0003] Based on the potential of SpyTag / SpyCatcher system in modular vaccine production, the purification of SpyTag protein, as one of the key components, is a real and urgent problem. At present, most of the SpyTag proteins are designed with additional tags, such as His tag, for purification. However, these tags usually have no use after the completion of purification, and also produce unnecessary immune response in vivo. When the purity of the eluted components is not enough, or the sample does not hang on the purification column at all due to the insufficient exposure of His tag, further purification methods are needed. In addition, in research, multiple candidate SpyTag antigens often need to be prepared, and when they need to be purified by different methods due to their differences, the production steps will become extremely complex. Therefore, if a more simple and universal purification method can be established for the original SpyTag tag, and the SpyTag tag can be directly used for purification in addition to its assembly function, the production efficiency of this modular component will be greatly improved.

[0004] Benefiting from the high specific affinity between antigens and antibodies, immunoaffinity chromatography (IAC) has developed into a powerful purification method. It is a special subclass of affinity chromatography, in which the stationary phase is composed of antibodies or antibody-related reagents. Using monoclonal antibodies against SpyTag as fixed ligands to specifically separate SpyTag tag proteins from complex samples is a promising and feasible purification method.

[0005] The information disclosed in this section is only for deepening the understanding of the background of the present disclosure, and should not be regarded as admitting or implying in any form that this information constitutes prior art known to those skilled in the art. SUMMARY

[0006] The inventors found through research that, due to the small molecular weight of SpyTag, it is difficult to induce high enough antibody levels, which is a bottleneck factor for purifying and separating proteins based on SpyTag tag proteins; and using the method of nanoparticle display can promote antigen drainage to lymph nodes, complement activation, antigen-presenting cell (APC) uptake and B cell receptor (BCR) crosslinking, thereby inducing a strong humoral response to the displayed antigen and producing higher levels of antibodies; further research found that by genetically engineering SpyTag003 to be highly displayed on the surface of hepatitis B virus (HBV) core antigen (HBc), it can be expressed in E. coli and self-assembled into nanoparticles; using it as an immunogen to immunize mice, high-affinity SpyTag003 monoclonal antibodies can be screened through hybridoma technology.

[0007] In view of this, in the present application, the SpyTag003 tag is displayed on the HBc nanoparticle by multivalent display, high-affinity SpyTag003 monoclonal antibodies are prepared, a new, universal, monoclonal antibody-based affinity immunochromatography method is established for the separation and purification of SpyTag proteins, and a plurality of different tag positions and different expression sources of the SpyTag model proteins are constructed as a purification verification model to characterize and confirm the purity and assembly capacity of the obtained proteins, thereby providing a new effective way for efficient separation and purification of SpyTag proteins.

[0008] According to one aspect of the present disclosure, an immunoaffinity chromatography column is provided, containing aldehyde-activated cross-linked agarose beads as a solid phase carrier and HBC-SpyTag003 monoclonal antibodies coupled thereto; the heavy chain variable region sequence of the anti-HBC-SpyTag003 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 based on the sequence of SEQ ID NO. 3; and / or

[0009] the light chain variable region DNA sequence is a DNA sequence as shown in SEQ ID NO. 4, or an amino acid sequence as shown in SEQ ID NO. 5, or a sequence of an active fragment or a conservative variant obtained by adding, deleting, or replacing one or more amino acids based on the sequence of SEQ ID NO. 5.

[0010] In some embodiments of the present disclosure, the HBC-SpyTag003 monoclonal antibody is prepared by the following method:

[0011] (1) a fusion gene with a DNA sequence as shown in SEQ ID NO. 1 is synthesized and cloned into a pET28a vector to construct a prokaryotic expression vector; HBC-SpyTag003

[0012] (2) the obtained prokaryotic expression vector is transformed into E. coli to form a prokaryotic expression strain, which is then induced by IPTG for expression, cell lysis, and purification steps to obtain a nanoparticle containing HBC-SpyTag003;

[0013] (3) the nanoparticle is used as an immunogen, and a HBC-SpyTag003 monoclonal antibody is prepared by a hybridoma method.

[0014] In some embodiments of the present disclosure, in the step (2), the purification method comprises:

[0015] ① the cell lysate is precipitated with saturated ammonium sulfate solution, and residual ammonium sulfate is removed by dialysis;​

[0016] ②Then load the Capto TM Core 700 packed column, collect the flow-through to obtain the purified nanoparticles.

[0017] According to another aspect of the present disclosure, a method for preparing an immunoaffinity chromatography column is provided, comprising the following steps:

[0018] (1) Preparation of monoclonal antibody

[0019] ①Synthesize the DNA sequence shown in SEQ ID NO. 1 HBC-SpyTag003 The fusion gene is cloned into the pET28a vector to construct a prokaryotic expression vector;

[0020] ②The obtained prokaryotic expression vector is transformed into E. coli to form a prokaryotic expression strain, and then induced by IPTG, lysed, and purified to obtain nanoparticles containing HBC-SpyTag003;

[0021] ③Using the nanoparticles as an immunogen, a HBC-SpyTag003 monoclonal antibody is prepared by the hybridoma method;

[0022] (2) Immobilization of monoclonal antibody

[0023] In the chromatography column, under the action of the reducing agent sodium cyanoborohydride, the aldehyde-activated agarose carrier is stably fixed with the HBC-SpyTag003 monoclonal antibody;

[0024] (3) Stop remaining active sites: add a quenching buffer and a cyanoborohydride solution to the chromatography column under ventilation conditions, shake and mix for 25-35 min, and then discharge the liquid in the chromatography column;

[0025] (4) Wash the column: wash the chromatography column with 1M Nacl solution, and finally wash the resin with a PBS solution containing 0.05% sodium azide at pH 7.2, and store the chromatography column therein.

[0026] In some embodiments of the present disclosure, in the step (2), AminoLink TM The coupling resin is packed in an empty chromatography column, the packing buffer is naturally flowed out, the chromatography column is equilibrated with a PBS solution at pH 7.2, the purified HBC-SpyTag003 monoclonal antibody is added to the chromatography column, a cyanoborohydride solution is added to the reaction slurry under ventilation conditions, and the mixture is inverted at 4°C overnight, and then the resin is washed with a coupling buffer.

[0027] In some embodiments of the present disclosure, in the step (3), the resin is first washed with 1 M Tris-Hcl solution with pH 7.4, and then the quenching buffer and cyanoborohydride solution are added to the chromatography column under ventilation.

[0028] According to another aspect of the present disclosure, a SpyTag protein purification method is provided, which is based on the above-mentioned immunoaffinity chromatography column and comprises the following steps:

[0029] (1) Equilibrate the immunoaffinity chromatography column to room temperature;

[0030] (2) Equilibrate the chromatography column with the binding buffer, and pass the filtered supernatant sample of the SpyTag protein to be purified through the chromatography column at a flow rate of 0.5 ml / min;

[0031] (3) After the sample is completed, equilibrate the chromatography column with the binding buffer again, and wash away the unbound components; when the UV value tends to be stable, elute the bound protein with the elution buffer;

[0032] (4) When the conductance value starts to decrease rapidly, collect the eluate; and add neutralization buffer to the collected eluate to adjust the pH value to neutral;

[0033] (5) Collect the eluted components together, and exchange them into storage buffer through Sephadex G-25.

[0034] In some embodiments of the present disclosure, the SpyTag protein is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein or a porcine epidemic diarrhea virus (PEDV) core neutralization epitope (COE) protein with a SpyTag003 tag at the N-terminus or C-terminus.

[0035] According to another aspect of the present disclosure, a regeneration method of an immunoaffinity chromatography column is provided, which comprises the following steps:

[0036] (1) After the protein elution is completed, immediately wash the chromatography column with PBS solution with pH 7.4 to remove residual proteins and reactivate the resin;

[0037] (2) Then, equilibrate the chromatography column with the binding buffer containing 0.05% sodium azide, and store the chromatography column therein.

[0038] The one or more technical solutions provided in the embodiments of the present disclosure have at least the following technical effects or advantages:

[0039] 1. The first universal immunoaffinity chromatography column (Spy&IAC) based on SpyTag003 monoclonal antibody, which can efficiently purify N-terminal or C-terminal SpyTag-labeled proteins derived from prokaryotic or eukaryotic expression systems.

[0040] 2. The preparation method of the immunoaffinity chromatography column is simple, can be effectively regenerated, and has simple and conventional elution conditions, which is conducive to reducing costs; the purification efficiency is high, the harvested protein has high purity, and the assembly function of the purified protein can be effectively retained, thereby providing a universal new method for efficient purification of SpyTag proteins. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 A mode diagram of an HBc-SpyTag003 gene fusion construct according to an embodiment of the present disclosure.

[0042] Figure 2 An SDS-PAGE diagram of HBC-SpyTag003 nanoparticles after purification according to an embodiment of the present disclosure, wherein each band is as follows: M is a protein marker, and lane 1 is the purified HBC-SpyTag003 nanoparticles.

[0043] Figure 3 A particle size diagram of HBC and HBC-SpyTag003 nanoparticles determined by dynamic light scattering according to an embodiment of the present disclosure.

[0044] Figure 4 A serum titer diagram of mice collected at 28 and 42 days after immunization with HBC-SpyTag003 nanoparticles according to an embodiment of the present disclosure.

[0045] Figure 5 An ascites titer diagram containing different monoclonal antibodies according to an embodiment of the present disclosure.

[0046] Figure 6 An elution curve of 6G3 ascites purification according to an 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 in conductance value during the elution process.

[0047] Figure 7 A PCR diagram of the amplification of the VH and VL genes of the SpyTag003 monoclonal antibody 6G3 according to an embodiment of the present disclosure.

[0048] Figure 8 A diagram of the complementary determining regions (CDRs) and framework regions (FRs) of the light chain and heavy chain variable regions of the SpyTag003 monoclonal antibody 6G3 according to an embodiment of the present disclosure.

[0049] Figure 9Figure 1 is a structural modeling diagram of the VH and VL regions of SpyTag003 monoclonal antibody 6G3 in an embodiment of the present disclosure.

[0050] Figure 10 Figure 2 is a chemical schematic diagram of the preparation of a universal immunoaffinity column (Spy&IAC) for SpyTag proteins in an embodiment of the present disclosure.

[0051] Figure 11 Figure 3 is a schematic diagram of four SpyTag model protein gene fusion constructs in an embodiment of the present disclosure.

[0052] Figure 12 , Figure 13 Figure 4 is a SDS-PAGE analysis of the expression and purification of N-SpyTag003-N and C-SpyTag003-N, respectively, in an embodiment of the present disclosure; M is a protein marker, lane 1 is whole cell lysate, and lane 2 is the final eluted purified protein.

[0053] Figure 14 , Figure 15 Figure 5 is a SDS-PAGE analysis of the expression and purification of N-SpyTag003-COE and C-SpyTag003-COE, respectively, in an embodiment of the present disclosure; and in the same figure, the left panel is a Western blot analysis of the expression of proteins in cell supernatant using SpyTag003 monoclonal antibody 6G3 prepared in the Examples, and the right panel is a SDS-PAGE analysis of the final purification of the proteins; M is a protein marker, lane 1 is cell supernatant, and lane 2 is the final eluted purified protein.

[0054] Figures 16-19 Figures 6A and 6B are elution curves of four SpyTag model proteins purified by Spy&IAC columns, respectively, in an embodiment of the present disclosure; wherein the blue line represents the distribution of absorbance at 280 nm, and the orange line represents the change in conductance value during the elution process.

[0055] Figures 20 to 23 Figures 7A and 7B are SEC spectra of four SpyTag model proteins purified by Spy&IAC columns, respectively, in an embodiment of the present disclosure; wherein the blue line represents the distribution of absorbance at 280 nm.

[0056] Figures 24 to 27 Figures 8A, 8B, 8C and 8D are assembly capability verification of four SpyTag model proteins purified, respectively, in an embodiment of the present disclosure; the purified four 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

[0057] The experimental methods described in the following examples are all conventional methods unless otherwise specified; those skilled in the art should understand that the reagents, enzymes, carriers, etc. used in the following examples are all commercially available analytical grade reagents or enzymes, carriers unless otherwise specified. The materials, methods and examples are for illustration only and are not limiting.

[0058] In order to better understand the technical solutions of the present application, the above technical solutions will be described in detail below in combination with the drawings of the specification and specific embodiments.

[0059] Example 1: Obtaining HBC-SpyTag003 Nanoparticles

[0060] 1. Construction of pET28a-HBC-SpyTag003 Prokaryotic Expression Vector

[0061] The SpyTag003 tag sequence (RGVPHIVMVDAYKRYK) was inserted between the 80th and 81st amino acids of the major immunodominant region (MIR) of the HBc protein (1st-149th amino acids), with a GGS linker on both sides of the sequence to increase the flexibility of the protein chain. The entire sequence was optimized for E. coli preference codons, synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd., and inserted into the pET28a vector using Ncol and Xhol restriction sites. As shown in BamHI and HindIII The synthesized gene (as shown in SEQ ID NO. 1) was inserted into the pET28a vector using Ncol and Xhol restriction sites. As shown in Figure 1 is the mode diagram of the HBc-SpyTag003 gene fusion construct.

[0062] 2. Preparation of HBC-SpyTag003 Nanoparticles

[0063] To perform soluble expression, the constructed pET28a-HBC-SpyTag003 plasmid was transformed into pTf16 / BL21(DE3) E. coli expression competent cells, the transformation product was spread on LB agar plates containing 50 pg / mL kanamycin and 100 pg / mL ampicillin, and incubated at 37 °C overnight. A single colony was picked into 5 ml LB medium containing 50 pg / mL kanamycin and 100 pg / mL ampicillin, and incubated at 37 °C with 200 r / min shaking for 16 h. Then the pre-culture was diluted 1 : 100 into 500 ml LB medium, 50 pg / mL kanamycin, 100 pg / mL ampicillin and 2 mg / ml L-arabinose were added, and incubated at 37 °C with 200 r / min shaking. When the culture reached OD600 around 0.6, IPTG was added to a final concentration of 0.2 mM, and the induction was continued at 16 °C with 200 r / min shaking for 18 h. The bacterial cells were collected by centrifugation, and the centrifuged bacterial cells were resuspended in a lysis buffer (50 ml of 20 mM Tris-Hcl, 150 mM NaCl solution with pH 8.0) at a volume ratio of 10: 1 (original bacterial liquid volume: lysis buffer), and a portion of the resuspended liquid was used for SDS-PAGE identification.

[0064] 3. Purification and identification of HBC-SpyTag003 nanoparticles

[0065] (1) Purification of HBC-SpyTag003 nanoparticles

[0066] The bacterial cell suspension obtained in the previous step was subjected to ultrasonic disruption treatment, and centrifuged at 12000 r / min for 20 min at 4 °C to collect the supernatant. The collected supernatant was filtered using a 0.22 pm filter, and then 5% saturated ammonium sulfate solution was added to the supernatant, which was then placed at 4 °C for 6 h. Centrifugation was performed at 12000 r / min for 20 min at 4 °C, and the supernatant was discarded, and the precipitate was resuspended in 20 ml of 20 mM Tris-Hcl, 150 mM NaCl solution with pH 8.0. Dialsed with an excess of the same buffer for 24 h to remove residual ammonium sulfate. The dialysed HBC-SpyTag003 was centrifuged at 6000 r / min for 20 min at 4 °C, and the supernatant was filtered using a 0.22 pm filter to further remove any insoluble material. Finally, the supernatant was passed through a column packed with Capto Core 700 filler at a flow rate of 0.75 ml / min, and the flow-through was collected. The purified HBC-SpyTag003 was identified using SDS-PAGE gel electrophoresis. TM Core 700 filler, and the flow-through was collected. The purified HBC-SpyTag003 was identified using SDS-PAGE gel electrophoresis. Figure 2is SDS-PAGE figure after purification, each band is respectively: M is protein marker, lane 1 is purified HBC-SpyTag003 nanoparticle.

[0067] (2) Dynamic light scattering (DLS) detection

[0068] The purified HBC-SpyTag003 nanoparticles obtained were detected by dynamic light scattering (DLS) technology, and the results are shown in Figure 3 According to the DLS results, the obtained nanoparticles have high purity and good structural uniformity.

[0069] Example Two: Preparation of SpyTag003 monoclonal antibody

[0070] 1. Mouse immunization: The purified HBC-SpyTag003 nanoparticles obtained in Example One were emulsified with Freund's complete adjuvant at a volume ratio of 1:1. Five 6-8 week old female BALB / c mice were immunized by subcutaneous injection at multiple sites on the back at an interval of 14 days. The last two immunizations used Freund's incomplete adjuvant, and the protein dose of each immunization was 10 μg per mouse, and the total dose was 200 μl per mouse. Mouse tail blood was collected on the 28th and 42nd days after the first immunization, and the mouse with the highest serum antibody titer was selected for hyperimmunization by indirect ELISA. Hyperimmunization was performed by intraperitoneal injection, and the immunization was pure HBC-SpyTag003 nanoparticles without adjuvant, and the dose was 50 μg per mouse.

[0071] 2. Determination of serum antibody titer of immunized mice: The serum titer of mice collected on the 28th and 42nd days after the first immunization was detected by indirect ELISA, and the steps were as follows:

[0072] (1) Coating: Dilute the purified HBC-SpyTag003 nanoparticles to 1 μg / mL with ELISA coating solution (CBS), add 50 μl of coating solution to each well, incubate at 4°C overnight, discard the coating solution, wash the plate with PBST 3 times, and shake off the residual liquid on the plate;

[0073] (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;

[0074] (3) Primary antibody: Add 50ul of the serum to be tested diluted with dilution buffer (PBST) at a dilution ratio of 2:1 to each well (the initial dilution ratio is 1:100), incubate at 37°C for 1 h, discard the supernatant, and wash with PBST 6 times;

[0075] (4) Second antibody: 50 μl of HRP labeled goat anti-mouse IgG diluted 1:5000 with dilution buffer was added to each well, and after 45 min of incubation at 37°C, the supernatant was discarded and the wells were washed 6 times with PBST;

[0076] (5) Color development: 50 μl of DAB color developing solution was added to each well, and after 20 min of action at room temperature in the dark, 2 M H2SO4 50 μl was added to stop the reaction, and the OD450 value was determined using an enzyme marker.

[0077] (6) The data were analyzed and arranged using Excel, and the serum titers of each mouse at each time period were calculated.

[0078] The results are shown in Figure 4 Table 1, and the ELISA results showed that the serum ELISA titers of 3 BALB / c mice were relatively high, reaching 1:51 200. No. 3 mouse was randomly selected for cell fusion to prepare monoclonal antibodies.

[0079] 3. Cell fusion: According to the results determined in the previous step, No. 3 BALB / c mouse was taken for hyperimmunization, and was executed by cervical dislocation after 3 days (blood was taken from the eye socket before execution to preserve the positive control serum). The body surface was disinfected with 75% alcohol, and the specific steps of cell fusion were as follows:

[0080] (1) 2-5 x 10 7 cells of well-grown sp2 / 0 tumor cells were collected in a centrifuge tube;

[0081] (2) The mouse spleen was taken out under sterile conditions in a clean bench, placed on a 200-mesh sterile screen, and the spleen was cut into small pieces with small scissors. The spleen cells were washed with GNK washing solution to make them single-filtered into a sterile small beaker;

[0082] (3) The spleen cell suspension was transferred to the centrifuge tube, and GNK washing solution was added to 40 ml. Together with the tumor cells, it was centrifuged at 1200 r / min for 10 min;

[0083] (4) The supernatant was discarded, and the cell mass was bounced. GNK washing solution 10 ml was added, and the spleen cell suspension was transferred to the tumor cell tube. GNK washing solution was added to 40 ml, and it was centrifuged at 1200 r / min for 10 min, and the supernatant was discarded;

[0084] (5) The cell mass was gently dispersed, 1 ml of fusion agent 50% PEG1500 was added dropwise, and attention was paid to add it within one minute. It was static for 90 s. Then 15 ml of GNK washing solution was slowly added to terminate the fusion (1 ml was added in the first half minute, 3 ml was added in the second half minute, and then the remaining 11 ml was gradually added), and it was stably incubated at 37°C for 5 min. GNK washing solution was added to 40 ml, and it was centrifuged at 1000 r / min for 10 min;

[0085] (6) After centrifugation, discard the supernatant, gently disperse the cell mass, add HAT selection medium, and gently suspend the cells. Be careful not to blow the fused cells too hard, which may damage them;

[0086] (7) Disperse the suspended cells into a 96-well cell culture plate, and add 250 μl of cell suspension to each well;

[0087] (8) After 3-4 days of culture, small cell masses can be observed under a microscope, and the supernatant of the hybridoma cells can be detected after 9-12 days.

[0088] 4. Screening and identification of hybridoma cells

[0089] On the 11th day after cell fusion, the supernatant of the hybridoma cells was detected by indirect ELISA. HBC-SpyTag003 nanoparticles and HBC nanoparticles were used as coating antigens, and the positive hybridoma cell line that could specifically recognize the SpyTag003 label and not react with the HBC nanoparticle scaffold was screened by combining "positive screening" and "reverse screening". The steps of indirect ELISA are as follows:

[0090] (1) Coating: HBC-SpyTag003 nanoparticles and HBC nanoparticles were used as coating antigens, diluted with ELISA coating solution (CBS), and incubated at 4°C overnight at a coating dose of 50 μg per well and a coating volume of 50 μl. Discard the coating solution, wash the plate with PBST 3 times, and spin to remove residual liquid;

[0091] (2) Blocking: Add 200 μl of blocking solution (5% skim milk powder + PBST) to each well and incubate at 37°C for 2 hours. Wash the plate in the same way;

[0092] (3) Primary antibody: Add 50 ul of hybridoma cell supernatant to each well, and set the mouse serum before fusion as a positive control and the mouse serum before immunization as a negative control. Incubate at 37°C for 30 minutes, discard the supernatant, and wash with PBST 6 times;

[0093] (4) Secondary antibody: Add 50 μl of HRP-labeled goat anti-mouse IgG diluted 1:5000 with dilution buffer to each well. Incubate at 37°C for 30 minutes, discard the supernatant, and wash with PBST 6 times;

[0094] (5) Color development: Add 50 μl of DAB color developing solution to each well, and develop at room temperature for 20 minutes in the dark.

[0095] According to the color results, select the positive hybridoma cell strains with good reactivity and transfer them into 24-well cell culture plates. Add HT selection culture solution for expansion culture. After 2-3 days of culture, observe the growth state of the hybridoma cells, and identify the hybridoma cell strains again by the aforementioned indirect ELISA method. Transfer the reaction wells with positive identification and cell density of more than 80% to 6-well cell culture plates. At this time, the conventional 1640 culture medium containing 10% FBS can be used. Note that re-screening should be performed after each transfer to ensure the stability of the positive hybridoma cells.

[0096] 5. Subcloning of hybridoma cells

[0097] Select the positive hybridoma cells in the 6-well cell culture plates finally identified in step 4 above with cell density of more than 50% and good growth state for subcloning. The specific operation steps are as follows:

[0098] (1) One day before subcloning, prepare feeder cells to cover multiple 96-well cell culture plates with 100 μl / well for standby;

[0099] (2) Gently blow up and mix the cells in each well to be subcloned, and then take 200 μl and add to the first vertical column of a 96-well cell culture plate, and perform serial dilution with a gradient of 2 times from left to right. Then observe each well under a medium power lens. When the number of cells in the field of view is 10-15 (equivalent to the total number of cells in the whole well being less than 100), it is determined that the dilution is appropriate;

[0100] (3) Add the cells in the appropriate dilution well to 10 ml of 1640 culture medium containing 10% FBS, and mix thoroughly by blowing. Add 100 μl / well to the 96-well cell culture plate previously covered with feeder cells (theoretically, a maximum of 1 hybridoma cell per well). In the same way, complete the subcloning of each hybridoma cell strain;

[0101] (4) Culture for 7-10 days, and when the cell mass grows to 1 / 5 of the bottom of the well, perform ELISA detection, as described above;

[0102] (5) For positive wells, secondary subcloning can be performed until a stable secreting monoclonal hybridoma cell strain is obtained. In this experiment, 3 monoclonal cell strains against SpyTag003 tag were obtained, designated as 6G3, 5D2 and 2C10;

[0103] (6) Expand the culture of the 3 stable secreting positive monoclonal cell strains finally screened, and freeze them at a cell number of 1-2 x 10 6 / pipe.

[0104] 6. Preparation and purification of anti-SpyTag003 tag monoclonal antibody ascites

[0105] (1) Preparation of monoclonal antibody ascites

[0106] Select female BALB / c mice of older age, intraperitoneally inject 500 μl of sterilized paraffin. 7-10 days later, centrifuge the 3 strains of SpyTag003 monoclonal cells expanded in step 5 above, collect the cells and resuspend with PBS, and intraperitoneally inject about 1x10 7 cells per mouse. Observe the state of the mice, and collect the ascites about 10 days later, centrifuge at 4000 r / min for 20 min to remove oil and cell precipitate, and collect the ascites supernatant for storage at -80°C for standby use.

[0107] (2) Determination of monoclonal antibody ascites titer

[0108] Determine the mouse ascites titer by indirect ELISA method. The steps are as follows:

[0109] ① Coating: use HBC-SpyTag003 nanoparticles as coating antigen, dilute with ELISA coating solution (CBS), and incubate at 4°C overnight at a coating dose of 50 μg per well and a coating volume of 50 μl, discard the coating solution, wash the plate 3 times with PBST, and shake off the residual liquid on the plate;

[0110] ② Blocking: add 200 μl of blocking solution (5% skim milk powder + PBST) per well at 37°C for 2 h, and wash the plate in the same way;

[0111] ③ Primary antibody: add 50 ul of mouse ascites diluted with dilution buffer (PBST) at a dilution ratio of 2 times per well, 50 μl each (initial dilution ratio is 1:1000), incubate at 37°C for 1 h, discard the supernatant, and wash 6 times with PBST;

[0112] ④ Secondary antibody: add 50 μl of HRP-labeled goat anti-mouse IgG diluted with dilution buffer at a dilution ratio of 1:5000 per well, incubate at 37°C for 45 min, discard the supernatant, and wash 6 times with PBST;

[0113] ⑤ Color development: add 50 μl of DAB color developing solution to each well, develop at room temperature for 20 min in the dark, add 2 M H2SO4 50 μl of termination solution to terminate the reaction, and measure the OD450 value with an enzyme marker.

[0114] ⑥ Organize and analyze the data, and take the maximum dilution ratio as the titer of the ascites.

[0115] The results are shown in Figure 5 Table 1, and the ELISA detection results show that the ascites titer of 6G3 is the highest, which is 1:512 000.

[0116] (3) Purification of ascites

[0117] The 6G3 ascites was purified by HiTrap Protein G pre-packed column through ÄKTA pure chromatography system, and the purified SpyTag003 monoclonal antibody was obtained. As shown in Figure 6 is 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 of conductance value during the elution process.

[0118] Example Three: Sequencing and bioinformatics analysis of variable region of SpyTag003 monoclonal antibody

[0119] Total RNA of SpyTag003 6G3 hybridoma cells was extracted and reverse transcribed into cDNA. Then, the universal primers for the heavy chain variable region (VH) and the light chain variable region (VL) of mouse IgG were used for PCR to amplify the variable region genes 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 extension at 72℃ for 10 min. The PCR product was gel purified and sequenced by GenScript Biotech (Shanghai) Co., Ltd. The monoclonal antibody sequences were subjected to homology search and analysis 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 monoclonal antibody was simulated using SWISS-MODEL (https: / / swissmodel.expasy.org), and analyzed by PyMOL.

[0120] Figure 7 is the PCR map of the amplified VH and VL genes of SpyTag003 monoclonal antibody 6G3. The sequences of the heavy chain variable region and the light chain variable region of SpyTag003 monoclonal antibody 6G3 are shown in SEQ ID NO. 2, SEQ ID NO. 4, respectively, and the deduced amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO. 3, SEQ ID NO. 5, respectively. The VH and VL gene sequences were aligned in the antibody gene database, and the results showed that both VH and VL contained 3 complementarity determining regions (CDRs) and 4 framework regions (FRs), as shown in Figure 8 is the positioning of CDRs and FRs in the antibody variable region. As shown in Figure 9 ​Structural modeling shows that CDRs are in close spatial proximity in the VH and VL monomer structures and are looped around each other in the overall variable region structure, together forming the antigen binding site (antigen binding pocket) of SpyTag003.

[0121] Example Four: Preparation of a general-purpose immunoaffinity column for SpyTag proteins (Spy&IAC)

[0122] Using the SpyTag003 monoclonal antibody obtained in Example Two, a general-purpose immunoaffinity column (Spy&IAC) for SpyTag proteins was prepared, Figure 10 is a chemical schematic of the preparation. The specific preparation method is as follows:

[0123] 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 times the column volume) of the coupling buffer (PBS solution with pH 7.2), and the contents were drained. Then, 5 mL of the purified HBC-SpyTag003 monoclonal antibody (dissolved in the coupling buffer) was added to the column, and 0.1 mL of the sample was saved for subsequent determination of the coupling efficiency. In a fume hood, 100 μL of cyanoborohydride solution was added to the reaction slurry (resulting in ~50 mM NaCNBH3), and the mixture was inverted overnight at 4°C. The next day, the top cap was carefully removed, and the bottom cap was also removed, as some pressure might have been generated during the reaction in the fume hood. The effluent from the column was collected, and the coupling efficiency was determined by comparing the protein concentration of the unbound fraction with that of the starting sample. Subsequently, the resin was washed with 10 ml of the coupling buffer;

[0124] 2. Blocking remaining active sites: The resin was washed with 10 mL of the quenching buffer (1 M Tris-Hcl solution with pH 7.4). In a fume hood, 5 mL of the quenching buffer and 100 μL of the cyanoborohydride solution (resulting in ~50 mM NaCNBH3 when mixed with the resin) were added to the column, and the mixture was gently mixed by end-to-end shaking for 30 min. Then, the top and bottom caps were carefully removed, and the contents of the column were drained;

[0125] 3. Washing the column: The column was washed with at least 25 mL (5 resin bed volumes) of the washing solution (1 M Nacl solution), and the final wash was monitored for the presence of protein, which, if present, was continued to be washed until no protein flowed out. Finally, the resin was washed with 15 mL of PBS solution with pH 7.2 containing 0.05% sodium azide, and the column was stored in this solution.

[0126] Example Five: Construction of a SpyTag model protein for purification verification

[0127] 1. Construction of four SpyTag model protein expression vectors

[0128] Figure 11 is the schematic diagram of four SpyTag model protein gene fusion constructs. All sequences are optimized for E. coli preference codons and synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd.:

[0129] (1) pET28a-N-SpyTag003-N is generated by inserting N-terminal SpyTag003 tag, GGSGGS linker and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein in pET28a plasmid;

[0130] (2) pET28a-C-SpyTag003-N is generated by inserting SARS-CoV-2 N protein, GGSGGS linker and C-terminal SpyTag003 tag in pET28a plasmid;

[0131] (3) pcDNA3.1-N-SpyTag003-COE is generated by inserting N-terminal SpyTag003 tag, GGSGGS linker and core neutralizing epitope (COE) protein of porcine epidemic diarrhea virus (PEDV) in pcDNA3.1 plasmid;

[0132] (4) pcDNA3.1-C-SpyTag003-COE is generated by inserting PEDV COE protein, GGSGGS linker and C-terminal SpyTag003 tag in pcDNA3.1 plasmid.

[0133] 2. Expression of four SpyTag model proteins

[0134] The expression method of two model proteins for prokaryotic expression is similar to the method described in step 2 of Example 1. Briefly, N-SpyTag003-N and C-SpyTag003-N were expressed in E. coli BL21(DE3) competent cells. After picking up single colonies, 10 mL overnight culture was diluted into 1 L LB with 50 µg / mL kanamycin. When the OD600 reached about 0.6 at 37℃, 200 r / min, IPTG was added to a final concentration of 0.2 mM, and the induction was continued at 16℃, 200 r / min for 12 h. The bacterial culture was harvested and resuspended in binding buffer (PBS solution at pH 7.4), and after ultrasonic disruption treatment, centrifuged at 12 000 r / min for 20 min at 4℃. The supernatant was collected and the expression was identified by SDS-PAGE.

[0135] N-SpyTag003-COE and C-SpyTag003-COE were expressed in Expi293F cells. According to the manufacturer's recommendations, Expi293F cells were cultured for at least 5 passages at 37°C in an orbital shaker incubator at 125 r / min with 5% (v / v) CO2 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 flask. At 96 h post-transfection, cell viability dropped to below 40-60%, at which time the expression of proteins in cell supernatant was detected by western blot using the SpyTag003 monoclonal antibody 6G3 prepared in Example Two.

[0136] Figure 12 and Figure 13 are the analysis of the expression and purification of N-SpyTag003-N and C-SpyTag003-N, respectively, using SDS-PAGE. M is a protein marker, lane 1 is whole cell lysate, and lane 2 is the final eluted purified protein. Figure 14 and Figure 15 are the identification of the expression and purification of N-SpyTag003-COE and C-SpyTag003-COE, respectively. In both figures, the left side is the detection of the expression of proteins in cell supernatant by western blot using the SpyTag003 monoclonal antibody 6G3 prepared in the present application. The right side of both figures is the final purification of proteins using SDS-PAGE. M is a protein marker, lane 1 is cell supernatant, and lane 2 is the final eluted purified protein.

[0137] Example Six: Purification of SpyTag Proteins

[0138] The supernatant samples of the four SpyTag mode proteins prepared in Example Four were purified by the ÄKTA pure chromatography system using the universal immunoaffinity chromatography column (Spy&IAC).

[0139] The specific method is as follows:

[0140] (1) Before use, the prepared immunoaffinity chromatography column (Spy&IAC) is equilibrated to room temperature. Do not let the resin bed dry during the whole process. Pay attention to degassing all buffers to avoid introducing air bubbles into the chromatography column. The protein supernatant sample is filtered using a 0.22 μm filter for standby;

[0141] (2) First, equilibrate the chromatography column with binding buffer (PBS solution with pH 7.4), and pass the filtered protein supernatant sample through the chromatography column at a flow rate of 0.5 ml / min;

[0142] (3) After loading, equilibrate the column again with binding buffer to wash away unbound components. When the UV value tends to be stable (usually below 20 mAU), elute the bound proteins with elution buffer (0.1 M glycine solution, pH 2.7);

[0143] (4) Carefully observe the change of system conductivity. When the conductivity value begins to decrease rapidly, pay attention to collect the eluate. Adjust the pH value to neutral by adding 100 μL neutralization buffer (1 M Tris-Hcl solution, pH 9.0) to every 1 mL collected eluate;

[0144] (5) Monitor the elution by 280 nm absorbance throughout. Pool the collected eluate fractions together and exchange into appropriate storage buffer, such as PBS solution, pH 7.4, by Sephadex G-25.

[0145] As Figures 16-19 is the elution profile of four SpyTag pattern proteins purified by Spy&IAC column. The blue line represents the distribution of absorbance at 280 nm, and the orange line represents the change of conductivity value during elution.

[0146] Example Seven: Purification Analysis of Four SpyTag003 Pattern Proteins

[0147] 1. Protein Purity Analysis

[0148] SDS-PAGE and size exclusion chromatography (SEC) were used to analyze the purity of four SpyTag003 pattern proteins purified by Spy&IAC column, a general-purpose immunoaffinity chromatography column for SpyTag proteins. Superdex TM 200Increase 10 / 300 GL chromatography column was used for analysis of N-SpyTag003-N and C-SpyTag003-N proteins, HiLoad TM 16 / 600 Superdex TM 200 pg chromatography column was used for analysis of N-SpyTag003-COE and C-SpyTag003-COE proteins. As shown in FIGS. Figures 12-15 and Figures 20-23 The four purified SpyTag pattern proteins were uniform and pure, with a purity of more than 90%, as confirmed by clear bands in SDS-PAGE and single main peaks in SEC chromatograms.

[0149] 2. Protein Assembly Ability Verification

[0150] To assess the functionality of the SpyTag model proteins purified using the universal immunoaffinity chromatography column for SpyTag proteins (Spy&IAC), i.e. the assembly ability with its "partner" SpyCatcher. The purified four 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. As shown in FIG. 6, SDS-PAGE confirmed efficient covalent reaction with all SpyTag model proteins efficiently displayed on SpyCatcher003-mi3. When incubated at equimolar concentrations, although the changes in N-SpyTag003-COE and C-SpyTag003-COE were less obvious, most of the reactions produced minimal residual unbound antigen, consistent with previous literature reports. That is, the SpyTag proteins purified using the universal immunoaffinity chromatography column for SpyTag proteins (Spy&IAC) exhibited satisfactory assembly ability. Figures 24-27

[0151] Example Eight: Regeneration and storage method of the universal immunoaffinity chromatography column (Spy&IAC)

[0152] The chromatography column should be regenerated immediately after each elution to prevent damage to the immobilized monoclonal antibody molecules from the low pH elution buffer. After the protein elution is complete, immediately wash the chromatography column with a large volume of binding buffer (PBS solution at pH 7.4) to remove any residual protein and re-activate the resin. Subsequently, equilibrate the chromatography column with binding buffer containing 0.05% sodium azide and store the chromatography column therein. A spring valve (or syringe) can be added on top of the chromatography column, which can apply pressure to the resin bed to avoid its resuspension or leakage, thereby preventing the chromatography column from drying.

[0153] Store the chromatography column vertically at 4°C and attempt to use it multiple times over a longer time span to verify its service life. The expression and purification of the aforementioned four SpyTag model proteins were actually carried out successively within 4 months. The experimental results of each experiment showed that the universal immunoaffinity chromatography column for SpyTag proteins (Spy&IAC) exhibited good regeneration and storage stability.

[0154] While there have been shown and described and pointed out fundamental novel features as applied to preferred embodiments, it will be understood that various omissions and substitutions and changes of the form of the methods described herein can be made by those skilled in the art, without departing from the spirit of the disclosure. Accordingly, the appended claims are intended to embrace all such alterations, permutations, and modifications as fall within the true spirit and scope of the disclosure. In the claims, means-plus-function clauses are used where the function of a claim element is described in a means- plus-function claim element. Means-plus-function

[0155] ​Obviously, many modifications and variations of the present application are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims and their equivalents, the application can be practiced otherwise than as specifically described.

Claims

1. An immunoaffinity chromatography column, characterized in that, The aldehyde-activated cross-linked beaded agarose contains a solid phase carrier and the HBC-SpyTag003 monoclonal antibody coupled thereto; the heavy chain variable region sequence of the anti-HBC-SpyTag003 monoclonal antibody is the amino acid sequence as shown in SEQ ID NO. 3; and the light chain variable region sequence is the amino acid sequence as shown in SEQ ID NO.

5.

2. A method of SpyTag protein purification, characterized by, Based on the immunoaffinity chromatography column of claim 1, comprising the following steps: (1) Equilibrate the immunoaffinity chromatography column to room temperature; (2) Equilibrate the chromatography column with binding buffer, and pass the filtered supernatant sample of the SpyTag protein to be purified through the chromatography column at a flow rate of 0.5 ml / min; (3) After the sample is completed, equilibrate the chromatography column again with binding buffer to wash away the unbound components; when the UV value tends to be stable, elute the bound protein with elution buffer; (4) When the conductance value begins to decrease rapidly, collect the eluate; adjust the pH value of the collected eluate to neutral by adding neutralization buffer; (5) Pool the collected elution components together and exchange them into storage buffer through Sephadex G-25.

3. The SpyTag protein purification method of claim 2, wherein, The SpyTag protein is a severe acute respiratory syndrome coronavirus 2 nucleocapsid protein or a porcine epidemic diarrhea virus core neutralizing epitope protein with a SpyTag003 tag at the N-terminus or C-terminus.

4. The method for regenerating the immunoaffinity column according to claim 1, characterized in that, Comprising the following steps: (1) After the protein elution is completed, immediately wash the chromatography column with a PBS solution with a pH of 7.4 to remove residual proteins and reactivate the resin; (2) Then equilibrate the chromatography column with binding buffer containing 0.05% sodium azide, and store the chromatography column therein.

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