CN115970338A

CN115970338A - Universal immunoaffinity chromatography column for modular component SpyTag protein, and preparation method and purification method thereof

Info

Publication number: CN115970338A
Application number: CN202210827939.XA
Authority: CN
Inventors: 张改平; 丁培阳; 王爱萍; 刘红亮; 陈玉梅; 周景明; 朱习芳; 梁超; 祁艳华
Original assignee: Longfor Modern Immunology Laboratory; Zhengzhou University
Current assignee: Longfor Modern Immunology Laboratory; Zhengzhou University
Priority date: 2022-07-13
Filing date: 2022-07-13
Publication date: 2023-04-18

Abstract

The invention discloses a universal immunoaffinity chromatography column for modular component SpyTag protein, a preparation method and a purification method thereof, and aims to solve the technical problems of complexity and difficulty in purification of the SpyTag protein at presentThe problem of operation. SpyTag003 is displayed on the surface of an HBV core antigen (HBc) at high density by a genetic engineering technology, expressed in escherichia coli and self-assembled to form nano particles, mice are immunized by the nano particles, and a high-affinity SpyTag003 monoclonal antibody is obtained by screening by a hybridoma method; covalently immobilizing the monoclonal antibody to an AminoLink ^TM Preparing an immunoaffinity chromatography column on the coupling resin, wherein the immunoaffinity chromatography column can be used for efficiently purifying protein with an N-terminal or a C-terminal SpyTag label from a prokaryotic or eukaryotic expression system; the chromatographic column is simple and convenient to prepare, can be effectively regenerated, and has simple and conventional elution conditions and low cost; the purification efficiency is high, the protein purity is high, the assembly function of the purified protein can be effectively reserved, and a universal new method is provided for efficient purification of the SpyTag protein.

Description

Universal immunoaffinity chromatography column for modular component SpyTag protein, and preparation method and purification method thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to a universal immunoaffinity chromatography column for modular component SpyTag protein, a preparation method and a purification method thereof.

Background

The SpyTag/SpyCatcher is a star molecule used for modular vaccine assembly, and provides a simple protein covalent conjugateSynthetic approaches, with high robustness and versatility, are currently used in a variety of candidate vaccines and personalized tumor neoantigen therapeutic vaccines. This system is generated by cleavage of the CnaB2 domain from the fibronectin binding protein Fbab of Streptococcus pyogenes, with spontaneous intramolecular isopeptide bond formation between the two components, and rapid assembly at a certain temperature (at least 4-37 ℃), pH (5-8), buffer (no need for specific anions or cations), even with non-ionic detergents. The SpyTag003/SpyCatcher003 is the latest version of the system, solves the problems that the intermediate reaction rate of the SpyTag/SpyCatcher limits the time resolution and the marking efficiency of low-concentration protein, and the reaction rate after modification (5.5 multiplied by 10) ⁵ M ^-1 s ^-1 ) Near the diffusion limit, compared to the original SpyTag/SpyCatcher reaction rate (1.4X 10) ³ M ^- ¹ s ^-1 ) About 400 times faster; even at low protein concentrations (10 nM), the SpyTag003/SpyCatcher003 reaction completed 90% or more within 15min, when there was almost no reaction of the original SpyTag/SpyCatcher. At present, the ultra-efficient reactivity of SpyTag003/SpyCatcher003 makes it a more preferred choice in the synthetic biologist toolkit.

Based on the potential of the SpyTag/SpyCatcher system in modular vaccine production, purification of SpyTag protein, one of its key components, is a realistic and urgent problem to be solved. Most SpyTag proteins are currently designed with an additional tag, such as a His tag, for purification. However, these tags are generally not used after purification is complete and can also generate an undesirable immune response in vivo. When the purity of the eluted fractions was insufficient, or the sample was not hung on the purification column at all due to insufficient exposure of the His tag, further purification methods were required. In addition, it is often necessary to prepare multiple candidate SpyTag antigens during the study, and the production steps become exceptionally complicated when they need to be purified by different methods due to their own differences. Therefore, if a simpler and more convenient and universal purification method can be established for the original SpyTag tag, the SpyTag tag can be directly used for purification besides the assembly function, and the production efficiency of the module component can be greatly improved.

Thanks to the highly specific affinity between antigen and antibody, immunoaffinity chromatography (IAC) has evolved as a powerful purification method. It is a special subclass of affinity chromatography, wherein the stationary phase consists of antibodies or antibody-related reagents. The use of monoclonal antibodies against SpyTag as immobilized ligands to specifically isolate SpyTag tag proteins from complex samples is a highly desirable and feasible purification method.

The information disclosed in this background section is only for enhancement of understanding of the background of the disclosure and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is already known to a person skilled in the art.

Disclosure of Invention

The inventor discovers through research that: because the molecular weight of the SpyTag is small, the induction of a high enough antibody level is difficult, and the SpyTag is a bottleneck factor for restricting the purification and separation of protein based on the SpyTag tag protein; the mode of utilizing 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 strong humoral response to the displayed antigen to generate higher-level antibodies; further research shows that SpyTag003 is displayed on the surface of a Hepatitis B Virus (HBV) core antigen (HBc) in high density by a genetic engineering technology, and is expressed and self-assembled in escherichia coli to form nanoparticles; the monoclonal antibody can be used as an immunogen to immunize a mouse, and the high-affinity SpyTag003 monoclonal antibody can be screened by a hybridoma technology.

In view of the above, the application of the invention utilizes HBc nanoparticles to display the SpyTag003 label in a multivalent manner to prepare the high-affinity SpyTag003 monoclonal antibody, and establishes a brand-new and universal affinity immunochromatography method based on the monoclonal antibody for separation and purification of SpyTag protein; and the constructed SpyTag model proteins with different label positions and different expression sources are used as purification verification models, and the purity and the assembly capacity of the obtained protein are characterized and confirmed, so that a new effective way is provided for efficient separation and purification of the SpyTag protein.

According to one aspect of the present disclosure, there is provided an immunoaffinity chromatography column comprising a solid support, aldehyde-activated cross-linked bead sepharose and coupled thereto a HBC-SpyTag003 monoclonal antibody; the heavy chain variable region sequence of the anti-HBC-SpyTag 003 monoclonal antibody is a DNA sequence shown as SEQ ID NO.2 or an amino acid sequence shown as SEQ ID NO.3, or a sequence of an active fragment or a conservative variant obtained by adding, deleting and replacing one or more amino acids on the basis of the SEQ ID NO.3 sequence; and/or

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

In some embodiments of the disclosure, the HBC-SpyTag003 monoclonal antibody is made by the following method:

(1) The synthetic DNA sequence is shown as SEQ ID NO.1HBC-SpyTag003Fusing genes, and cloning the genes to a pET28a vector to construct a prokaryotic expression vector;

(2) Transforming the prokaryotic expression vector into escherichia coli to form a prokaryotic expression strain, and then carrying out IPTG induced expression, thallus cracking and purification to obtain nano-particles containing HBC-SpyTag 003;

(3) The nano particles are used as immunogen to prepare HBC-SpyTag003 monoclonal antibody by a hybridoma method.

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

(1) precipitating the thallus lysate with saturated ammonium sulfate solution, and removing residual ammonium sulfate through dialysis;

(2) then filling with Capto ^TM And (4) a chromatographic column filled with Core 700, and collecting the flow-through liquid to obtain the purified nano particles.

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

(1) Preparation of monoclonal antibodies

(2) transforming the prokaryotic expression vector into escherichia coli to form a prokaryotic expression strain, and performing IPTG induced expression, thallus cracking and purification to obtain nano-particles containing HBC-SpyTag 003;

(3) preparing the HBC-SpyTag003 monoclonal antibody by using the nano particles as immunogen through a hybridoma method;

(2) Monoclonal antibody immobilization

In a chromatographic column, under the action of a reducing agent sodium cyanoborohydride, stably fixing the agarose carrier activated by aldehyde groups and the HBC-SpyTag003 monoclonal antibody;

(3) Blocking the remaining active sites: adding a quenching buffer solution and a cyanoborohydride solution into the chromatographic column under a ventilation condition, shaking and mixing for 25-35 min, and then discharging a content liquid of the chromatographic column;

(4) Washing the column: the column was washed with 1M NaCl solution and finally the resin was washed with 0.05% sodium azide in PBS at pH 7.2 and the column was stored in it.

In some embodiments of the present disclosure, in the step (2), an AminoLink is taken ^TM The coupling resin is filled in a hollow chromatographic column, so that the filling buffer solution naturally flows out, a PBS solution with the pH value of 7.2 is added to balance the chromatographic column, the purified HBC-SpyTag003 monoclonal antibody is added into the chromatographic column, a cyanoborohydride solution is added into the reaction slurry under the ventilation condition, the mixture is inverted overnight at the temperature of 4 ℃, and then the resin is washed by the coupling buffer solution.

In some embodiments of the present disclosure, in step (3), the resin is washed with 1M Tris-Hcl solution at pH 7.4, and then the quench buffer and cyanoborohydride solution are added to the column under aeration.

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

(1) Equilibrating the immunoaffinity chromatography column to room temperature;

(2) Equilibrating the chromatographic column with a binding buffer solution, and passing the filtered SpyTag protein supernatant sample to be purified through the chromatographic column at a flow rate of 0.5 ml/min;

(3) After the loading is finished, the chromatographic column is equilibrated again by the binding buffer solution, and the unbound components are washed away; eluting the binding protein by using an elution buffer solution after the UV value tends to be stable;

(4) Collecting the eluate when the conductivity begins to decrease rapidly; adding a neutralization buffer solution into the collected eluent to adjust the pH value to be neutral;

(5) The collected fractions were pooled together and exchanged into storage buffer by Sephadex G-25.

In some embodiments of the 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 neutralizing epitope (COE) protein with a SpyTag003 tag at the N-terminus or C-terminus.

According to another aspect of the present disclosure, there is provided a method for regenerating an immunoaffinity chromatography column, comprising the steps of:

(1) Immediately after completion of protein elution, the column was washed with PBS solution at pH 7.4 to remove residual protein and reactivate the resin;

(2) The column was then equilibrated with binding buffer containing 0.05% sodium azide and stored therein.

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

1. the universal immunoaffinity chromatography column (Spy & IAC) is prepared based on the SpyTag003 monoclonal antibody for the first time, and can efficiently purify protein with SpyTag labels at the N-terminal or the C-terminal of a prokaryotic or eukaryotic expression system.

2. The immunoaffinity chromatography column has simple and convenient preparation method, can be effectively regenerated, has simple and conventional elution conditions, and is beneficial to reducing the cost; the purification efficiency is high, the purity of the harvested protein is high, the assembly function of the purified protein can be effectively reserved, and a universal new method is provided for efficient purification of the SpyTag protein.

Drawings

FIG. 1 is a schematic diagram of an HBc-SpyTag003 gene fusion construct according to one embodiment of the present disclosure.

Fig. 2 is a SDS-PAGE map of purified HBC-SpyTag003 nanoparticles of an embodiment of the disclosure, wherein each band: m is a protein marker, lane 1 is purified HBC-SpyTag003 nanoparticles.

FIG. 3 is a graph of the particle size of HBC and HBC-SpyTag003 nanoparticles measured by dynamic light scattering in an embodiment of the present disclosure.

FIG. 4 is a graph of serum titers from mice collected at 28 and 42 days after first immunization with HBC-SpyTag003 nanoparticles in one embodiment of the disclosure.

FIG. 5 is a graph of ascites titers containing different monoclonal antibodies in one example of the disclosure.

FIG. 6 is an elution profile for purification of 6G3 ascites in one embodiment of the disclosure; where the blue line represents the absorbance profile at 280 nm and the orange line represents the change in conductivity during elution.

FIG. 7 shows PCR amplification of the 6G3 VH and VL genes of the SpyTag003 monoclonal antibody in one embodiment of the disclosure.

FIG. 8 shows an example of the positioning of the Complementarity Determining Regions (CDRs) and Framework Regions (FRs) of the light chain and heavy chain variable regions of SpyTag003 monoclonal antibody 6G3, respectively, in accordance with an embodiment of the present disclosure.

FIG. 9 is a schematic block diagram of the VH and VL regions of SpyTag003 monoclonal antibody 6G3 in one embodiment of the present disclosure.

FIG. 10 is a chemical schematic diagram of a generalized immunoaffinity column (Spy & IAC) for preparation of a spyTag protein according to one embodiment of the present disclosure.

FIG. 11 is a schematic diagram of a 4 SpyTag model protein gene fusion constructs according to one embodiment of the present disclosure.

FIGS. 12 and 13 show SDS-PAGE analysis of expression and purification of N-SpyTag003-N and C-SpyTag003-N, respectively, in one embodiment of the disclosure; m is the protein marker, lane 1 is the whole cell lysate and lane 2 is the final eluted purified protein.

FIGS. 14 and 15 show the expression and purification of N-SpyTag003-COE and C-SpyTag003-COE, respectively, in one embodiment of the present disclosure; in the same figure, the left figure shows the expression condition of protein in cell supernatant detected by Western blot by using the SpyTag003 monoclonal antibody 6G3 prepared in the example, and the right figure shows the final purification condition of protein identified by SDS-PAGE; m is the protein marker, lane 1 is the cell supernatant and lane 2 is the final eluted purified protein.

FIGS. 16-19 are elution profiles of 4 SpyTag model proteins purified by Spy & IAC column according to one embodiment of the present disclosure; where the blue line represents the absorbance profile at 280 nm and the orange line represents the change in conductivity during elution.

FIGS. 20 to 23 are SEC profiles of 4 SpyTag model proteins purified by a Spy & IAC column according to an embodiment of the present disclosure, respectively; wherein the blue line represents the profile of absorbance at 280 nm.

FIGS. 24 to 27 are the verification of the assembly ability of 4 SpyTag model proteins purified according to an embodiment of the present disclosure; the 4 purified SpyTag model proteins were coupled to SpyCatcher003-mi3 at different molar ratios at 4 ℃ and final assembly was verified by SDS-PAGE.

Detailed Description

The experimental methods described in the following examples are all conventional methods unless otherwise specified; it will be understood by those skilled in the art that the reagents, enzymes, carriers, etc. used in the examples below are, unless otherwise specified, analytical grade reagents or enzymes, carriers, etc. commercially available from reagent companies. The materials, methods, and examples are illustrative only and not intended to be limiting.

For better understanding of the technical solutions of the present application, the technical solutions will be described in detail below with reference to the drawings and specific embodiments.

The first embodiment is as follows: acquisition of HBC-SpyTag003 nanoparticles

1. Construction of prokaryotic expression vector pET28a-HBC-SpyTag003

The SpyTag003 tag sequence (RGVPHIVMVDAYKRYK) is inserted into the main immunodominant region of HBc protein (amino acids 1-149)Between amino acids 80 and 81 of the domain (MIR), a GGS linker was placed on each side of the sequence to increase flexibility of the protein chain. The whole sequence is optimized by the preferred codon of Escherichia coli, synthesized and utilized by the company of biological engineering (Shanghai)BamHIAndHindIIIthe enzyme cleavage site inserts the synthesized gene (shown as SEQ ID NO. 1) into the pET28a vector. FIG. 1 is a schematic diagram of the HBc-SpyTag003 gene fusion construct.

2. Preparation of HBC-SpyTag003 nanoparticles

For soluble expression, the pET28a-HBC-SpyTag003 plasmid constructed above was transformed into pTf/BL 21 (DE 3) E.coli expression competent cells, and the transformant was spread on LB agar plates containing 50. Mu.g/mL kanamycin and 100. Mu.g/mL ampicillin, and incubated overnight at 37 ℃. Single colony colonies were picked into 5mL LB medium containing 50. Mu.g/mL kanamycin and 100. Mu.g/mL ampicillin, and incubated at 37 ℃ with shaking at 200r/min for 16 h. The preculture was then diluted to 500 mL of LB medium at 1. When the culture reached an OD600 of about 0.6, IPTG was added at a final concentration of 0.2mM and induction was continued at 200r/min at 16 ℃ for 18h. And (3) centrifugally collecting thalli, and mixing the thalli after centrifugation according to the ratio of 10:1 (amount of original cells: lysis buffer) was added to lysis buffer (50 ml of 20mM Tris-HCl having pH 8.0, 150mM NaCl solution) to resuspend the cells, and a portion of the resuspension was identified by SDS-PAGE.

3. Purification and identification of HBC-SpyTag003 nanoparticles

(1) Purification of HBC-SpyTag003 nanoparticles

And (3) carrying out ultrasonic disruption treatment on the thallus heavy suspension obtained in the previous step, centrifuging at 12000 r/min for 20min at 4 ℃, and collecting a supernatant. The collected supernatant was filtered using a 0.22 μm filter, and then a 5% by volume saturated ammonium sulfate solution was added to the supernatant and allowed to stand at 4 ℃ for 6 hours. Centrifugation was carried out at 12000 r/min at 4 ℃ for 20min, the supernatant discarded, and the pellet resuspended in 20ml of 20mM Tris-HCl, 150mM NaCl solution, pH 8.0. Dialyzing 24 h against excess of the same buffer to removeResidual ammonium sulfate. The dialyzed HBC-SpyTag003 was centrifuged at 6000r/min for 20min at 4 deg.C, and the supernatant was filtered through a 0.22 μm filter to further remove any insoluble material. Finally, the supernatant was passed through the fill Capto at a flow rate of 0.75 ml/min ^TM Core 700 packed column, collecting the flow-through. The purified HBC-SpyTag003 was identified by SDS-PAGE gel electrophoresis. FIG. 2 is a diagram of SDS-PAGE after purification, wherein each band: m is a protein marker, lane 1 is purified HBC-SpyTag003 nanoparticles.

(2) Dynamic Light Scattering (DLS) detection

The HBC-SpyTag003 nano-particles obtained by purification are detected by a Dynamic Light Scattering (DLS) technology, the result is shown in figure 3, and the DLS result shows that the obtained nano-particles have high purity and better structural uniformity.

Example two: preparation of SpyTag003 monoclonal antibody

1. Mouse immunization: purified HBC-SpyTag003 nanoparticles obtained in example one were mixed with freund's complete adjuvant in a ratio of 1:1, was emulsified. 5 female BALB/c mice, 6-8 weeks old, were immunized 3 times at 14-day intervals by dorsal subcutaneous multi-site injection. Freund's incomplete adjuvant was used for both of the latter two immunizations, and the three immunoprotein doses were 10. Mu.g/mouse, and the total dose was 200. Mu.l/mouse. And (3) respectively carrying out tail blood collection on 28 th and 42 th days after the first immunization, and selecting the mouse with the highest serum antibody titer by indirect ELISA for superstrong immunization. The super-strong immunity adopts an intraperitoneal injection mode, pure HBC-SpyTag003 nano-particles without adjuvant are immunized, and the dosage is 50 mu g per particle.

2. And (3) measuring the serum antibody titer of the immune mice: the indirect ELISA method is used for detecting the titer of the mouse serum collected at 28 th and 42 th days after the first immunization, and the steps are as follows:

(1) Coating: diluting purified HBC-SpyTag003 nano particles to 1 microgram/mL by using ELISA coating solution (CBS), adding 50 microliter of coating solution into each hole, incubating overnight at 4 ℃, discarding the coating solution, washing for 3 times by using PBST, and throwing off residual liquid on the plate;

(2) And (3) sealing: adding 200 μ l of blocking liquid (5% skimmed milk powder + PBST) into each hole, blocking at 37 ℃ for 2h, and washing the plate by the same method;

(3) A first antibody: 50ul of each 50 μ l of serum to be tested diluted 2-fold by dilution buffer (PBST) is added to each well (initial dilution factor is 1;

(4) Secondary antibody: each well was added with dilution buffer at 1:50 mul of each HRP-labeled goat anti-mouse IgG diluted by 5000 is incubated at 37 ℃ for 45 min, the supernatant is discarded, and the mixture is washed 6 times by PBST;

(5) Color development: adding 50 mul DAB color development liquid into each well, and adding 2M H after keeping out of the sun at room temperature for 20min ₂

SO

₄ 50. And (5) stopping the reaction by using mul stop solution, and determining an OD450 value by using an enzyme-labeling instrument.

(6) The analytical data were collated by Excel and the serum titers of the individual mice were calculated at each time period.

The results are shown in FIG. 4, and the ELISA results show that the serum ELISA titers of 3 BALB/c mice are all higher and can reach 1:51 200, randomly selecting No.3 mice for cell fusion to prepare monoclonal antibodies.

3. Cell fusion: according to the results of the above determination, BALB/c mouse No.3 is taken for super-strong immunization, the neck is killed after 3 days (orbital bleeding before sacrifice to retain positive control serum), the body surface is sterilized by 75% alcohol, and the cell fusion is specifically carried out as follows:

(1) Collecting well-grown sp2/0 tumor cells at 2-5 × 10 ⁷ Putting the mixture into a centrifuge tube;

(2) Aseptically taking out mouse spleen in an ultraclean workbench, placing on 200-mesh sterile filter screen, cutting spleen with small scissors, adding GNK lotion for washing, and filtering spleen cells into a small aseptic beaker;

(3) Transferring the splenocyte suspension into a centrifuge tube, adding GNK lotion to 40ml, and centrifuging 1200 r/min for 10min together with the tumor cells;

(4) Discarding the supernatant, flicking the cell mass, adding GNK lotion 10 ml respectively, transferring the spleen cell suspension into tumor cell tube, adding GNK lotion to 40ml, centrifuging at 1200 r/min for 10min, and discarding the supernatant;

(5) The cell pellet was gently broken up and 1 ml fusogen 50% PEG1500 was added dropwise, taking care that the addition was complete within one minute and allowed to stand for 90s. Then slowly dripping 15 ml GNK lotion to terminate fusion (dripping 1 ml in the first half minute, dripping 3 ml in the second half minute, then gradually and quickly dripping the rest 11 ml), stabilizing in a 37 ℃ water bath for 5min, adding GNK lotion to 40ml, and centrifuging at 1000 r/min for 10 min;

(6) Centrifuging, removing supernatant, gently scattering cell clusters, adding HAT selection culture solution to gently suspend cells, cutting and beating the cells without force so as to prevent fused cells from being damaged;

(7) Dispersing the suspension cells into a 96-well cell culture plate, and adding 250 mu l of cell suspension to each well;

(8) The culture can be carried out for 3-4 days, small cell masses can be observed under a microscope, and hybridoma cell supernatants are detected for about 9-12 days.

4. Screening and identification of hybridoma cells

On day 11 after cell fusion, hybridoma cell supernatants were assayed by indirect ELISA. HBC-SpyTag003 nano-particles and HBC nano-particles are respectively used as coating antigens, and positive hybridoma cell strains which can specifically recognize SpyTag003 labels and do not react with HBC nano-particle scaffolds are screened out in a mode of combining 'positive screening' with 'reverse screening'. The indirect ELISA procedure was as follows:

(1) Coating: respectively using HBC-SpyTag003 nanoparticles and HBC nanoparticles as coating antigens, diluting with an ELISA coating solution (CBS), incubating overnight at 4 ℃ in a coating dose of 50 mug per hole and a coating volume of 50 mul, discarding the coating solution, washing for 3 times by PBST, and throwing off residual liquid on a plate;

(2) And (3) sealing: adding 200 mul of sealing liquid (5% skimmed milk powder + PBST) into each hole, sealing for 2h at 37 ℃, and washing the plate by the same method;

(3) A first antibody: adding 50ul hybridoma cell supernatant into each well, setting the serum of the mouse before fusion as a positive control and the serum of the mouse before immunization as a negative control, incubating for 30 min at 37 ℃, removing the supernatant, and washing for 6 times by PBST;

(4) Secondary antibody: each well was added with dilution buffer at 1: 5000. 50 mul of each diluted HRP-labeled goat anti-mouse IgG is incubated at 37 ℃ for 30 min, the supernatant is discarded, and the mixture is washed 6 times by PBST;

(5) Color development: and adding 50 mul of DAB color development solution into each well, and keeping the mixture at room temperature in a dark place for 20 min.

And (4) selecting positive hybridoma cell strains with good reactivity according to the color development result, transferring the positive hybridoma cell strains into a 24-hole cell culture plate, and adding an HT selection culture solution for amplification culture. After culturing for 2 to 3 days, the growth state of the hybridoma cells was observed, and the hybridoma cell lines were identified again by the indirect ELISA method described above. Reaction wells that were positive for identification and had a cell density of 80% or more were transferred to 6-well cell culture plates, at which time the conventional 1640 medium containing 10% FBS was used instead. Note that rescreening was performed after each rotation to ensure stability of positive hybridoma cells.

5. Subcloning of hybridoma cells

And (4) selecting the positive hybridoma cells which have the cell density of more than 50% and have a good growth state in the 6-well cell culture plate finally identified in the step 4 for subcloning. The specific operation steps are as follows:

(1) One day before subcloning, feeder layer cells were prepared and used in 96-well cell culture plates of 100. Mu.l/Kong Puman;

(2) And slightly blowing and uniformly mixing the cells in each hole to be subcloned, respectively sucking 200 mu l of the cells to be added to the first vertical column of a 96-hole cell culture plate, and continuously diluting the cells from left to right by 2-time gradient. Observing each hole under a medium-magnification microscope, and determining the hole as an appropriate dilution factor when the number of cells in the visual field range is 10-15 (which is equivalent to the total number of cells in the whole hole being within about 100);

(3) Cells in wells of appropriate dilution factor were added to 1640 medium containing 10% FBS in 10 ml, thoroughly pipetted well and mixed well, and added to 96 well cell culture plates previously plated with feeder cells at 100. Mu.l/well (theoretically, a maximum of 1 hybridoma cell per well). Completing the subcloning of each hybridoma cell strain in the same way;

(4) Culturing for 7-10 days, and performing ELISA detection when the cell mass grows to 1/5 of the bottom of the hole, wherein the method is as described above;

(5) Secondary subcloning can be performed for positive wells until stably secreted monoclonal hybridoma cell lines are obtained. In the experiment, 3 monoclonal cell strains resisting the SpyTag003 label are obtained in the experiment and are respectively named as 6G3,5D2 and 2C10;

(6) Will eventuallyThe 3 strains of positive monoclonal cell strains which are stably secreted are obtained by screening and are cultured in an expanding way according to the proportion of 1 to 2 multiplied by 10 ⁶ Cell number/tube frozen.

6. Preparation and purification of anti-SpyTag 003-tag monoclonal antibody ascites

(1) Preparation of monoclonal antibody ascites

Female BALB/c mice of older weeks were selected and injected intraperitoneally with 500. Mu.l of sterile paraffin. After 7 to 10 days, 3 SpyTag003 monoclonal cells which were expanded in the above step 5 were centrifuged, the cells were collected and resuspended in PBS at about 1X 10 per mouse ⁷ The number of individual cells was injected intraperitoneally. Observing the state of the mouse, collecting ascites after about 10 days, centrifuging for 20min at 4000 r/min to remove grease and cell precipitate, collecting the ascites supernatant, and storing at-80 ℃ for later use.

(2) Monoclonal antibody ascites titer determination

The ascites titer of mice was determined by an indirect ELISA method. The method comprises the following steps:

(1) coating: using HBC-SpyTag003 nanoparticles as coating antigens, diluting with an ELISA coating solution (CBS), incubating overnight at 4 ℃ in a coating volume of 50 μ g per well and 50 μ l, discarding the coating solution, washing for 3 times with PBST, and drying residual liquid on a plate;

(3) a first antibody: 50 μ l each of mouse ascites diluted 2-fold with dilution buffer (PBST) was added to each well of 50ul (initial dilution multiple 1: 1000), and after incubation at 37 ℃ for 1 h, the supernatant was discarded and washed 6 times with PBST;

(4) secondary antibody: each well was added with dilution buffer at 1: 5000. 50 mul of each diluted HRP-labeled goat anti-mouse IgG is incubated at 37 ℃ for 45 min, the supernatant is discarded, and the mixture is washed 6 times by PBST;

(5) color development: adding 50 mul DAB color development liquid into each hole, and adding 2M H after the DAB color development liquid is kept in the dark at room temperature for 20min ₂ And stopping the reaction by using SO4 mu l of stop solution, and measuring an OD450 value by using an enzyme-labeling instrument.

(6) The analytical data were collated and the maximum dilution was taken as the titer of the ascites.

The results are shown in fig. 5, and the ELISA test results showed that the 6G3 ascites titer was the highest and was 1:512 000.

(3) Purification of ascites

The purified SpyTag003 monoclonal antibody was obtained by purifying 6G3 ascites using a HiTrap Protein G prepacked column using an a ̈ KTA pure chromatography system. The elution profile for the purification of 6G3 ascites is shown in FIG. 6. Where the blue line represents the profile of absorbance at 280 nm and the orange line represents the change in conductivity value during elution.

Example three: spyTag003 monoclonal antibody variable region sequencing and bioinformatics analysis

Total RNA of SpyTag003 6G3 hybridoma cells is extracted and is reversely 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 the SpyTag003 monoclonal antibody. The amplification cycle was: 5min at 98 ℃;35 cycles, 98 ℃ 10 s,60 ℃ 15s,72 ℃ 1min,72 ℃ further 10min of stretching. The PCR product was gel purified and sequenced by Biotechnology engineering (Shanghai) Inc. Homology searches and analyses were performed on mAb sequences by IMGT/V-QUEST (https:// www.imgt.org/IMGT _ vquest/vquest) and IGBLAST (https:// www.ncbi.nlm.nih.gov/IGBLAST. The tertiary structure of SpyTag003 mab was simulated using SWISS-MODEL (https:// swissmodule.expasy.org) and analyzed by PyMOL.

FIG. 7 shows PCR amplification of the 6G3 VH and VL genes of SpyTag003 monoclonal antibody. The gene sequences of the heavy chain variable region and the light chain variable region of the SpyTag003 monoclonal antibody 6G3 are respectively shown in SEQ ID NO.2 and SEQ ID NO.4, and the amino acid sequences of the heavy chain variable region and the light chain variable region deduced from the gene sequences are respectively shown in SEQ ID NO.3 and SEQ ID NO. 5. Comparison of the VH and VL gene sequences in the antibody gene database revealed that both VH and VL comprise 3 Complementarity Determining Regions (CDRs) and 4 Framework Regions (FRs), and that the CDRs and FRs of the antibody variable regions were located as shown in FIG. 8. As shown in the structural simulation of fig. 9, the CDRs are closely spaced in the VH and VL monomeric structures and surround each other throughout the variable domain structure, together constituting the antigen binding site (antigen binding pocket) of SpyTag 003.

Example four: preparation of general immunoaffinity column for SpyTag protein (Spy & IAC)

The SpyTag003 monoclonal antibody obtained in example two was used to prepare a SpyTag protein-based universal immunoaffinity chromatography column (Spy & IAC), and fig. 10 is a chemical schematic diagram of the preparation. The preparation method comprises the following steps:

1. monoclonal antibody immobilization: 5ml of AminoLink was added ^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 pH 7.2) and the contents were drained. Then, 5mL of purified HBC-SpyTag003 monoclonal antibody (dissolved in coupling buffer) was added to the column, and a sample of 0.1 mL was stored for subsequent use in determining coupling efficiency. In a fume hood, 100. Mu.L of cyanoborohydride solution (NaCNBH yielding about 50 mM) was added to the reaction slurry ₃ ) And mixed by inversion overnight at 4 ℃. The next day, the top cover was carefully removed in the fume hood and the bottom cover was removed again, as some air pressure may build up during the reaction. The effluent from the column was collected and the coupling efficiency was determined by comparing the protein concentration of the unbound fraction to the starting sample. The resin was then washed with 10 ml coupling buffer;

2. blocking the remaining active sites: the resin was washed with 10 mL quench buffer (1M Tris-HCl solution at pH 7.4). In a fume hood, 5mL quench buffer and 100 μ L cyanoborohydride solution (50 mM NaCNBH when mixed with resin as a result) were added to the column ₃ ) And gently mixed by shaking end-to-end for 30 min. Then, the top cover and the bottom cover are carefully disassembled to discharge the content liquid in the chromatographic column;

3. washing the column: the column was washed with at least 25 mL (5 resin bed volumes) of wash solution (1M Nacl solution) taking care to monitor the final wash for the presence of protein and if present, washing continued until no protein flowed out. Finally, the resin was washed with 15 mL containing 0.05% sodium azide in PBS solution at pH 7.2 and the column was stored therein.

Example five: construction of SpyTag model protein for purification validation

1. Construction of four SpyTag model protein expression vectors

FIG. 11 is a schematic diagram of a gene fusion construct of 4 SpyTag model proteins. All sequences were optimized by E.coli preferred codons and synthesized by Biotechnology engineering (Shanghai) GmbH:

(1) pET28a-N-SpyTag003-N is generated by inserting into pET28a plasmid an N-terminal SpyTag003 tag, GGSGGS linker and Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein;

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

(3) pcDNA3.1-N-SpyTag003-COE was generated by inserting into pcDNA3.1 plasmid an N-terminal SpyTag003 tag, a GGSGGS linker and a core neutralizing epitope (COE) protein of Porcine Epidemic Diarrhea Virus (PEDV);

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

2. Expression of four SpyTag model proteins

The expression method for the two model proteins for prokaryotic expression is similar to the method described in step 2 of example one. Briefly, N-SpyTag003-N and C-SpyTag003-N were expressed in E.coli BL21 (DE 3) competence. After picking to obtain a single colony, 10 mL overnight cultures were diluted to 1L LB and 50 μ g/mL kanamycin was added. Culturing at 37 deg.C and 200r/min until OD600 reaches about 0.6, adding IPTG with final concentration of 0.2mM, and inducing at 16 deg.C and 200r/min for 12 hr. Bacterial cultures were harvested and resuspended in binding buffer (PBS solution pH 7.4), sonicated, and centrifuged at 12000 r/min for 20min at 4 ℃. Supernatants were collected and expression was characterized using SDS-PAGE.

N-SpyTag003-COE and C-SpyTag003-COE were expressed in Expi293F cells. Expi293F cells were cultured for at least 5 passages at 37 ℃ in an orbital shaker incubator containing 5% (v/v) CO2 at a rotation rate of 125 r/min according to the manufacturer's recommendations prior to transient transfection. PEI was used for transient transfection of recombinant DNA. All proteins were expressed in a total culture volume of 1L in ventilated cell shake flasks. After transfection, 96 h, the cell viability was reduced to below 40-60%, and the expression of protein in the cell supernatant was examined using western blot using SpyTag003 mab 6G3 prepared in example two.

FIGS. 12 and 13 show SDS-PAGE analysis of the expression and purification of N-SpyTag003-N and C-SpyTag003-N, respectively. M is the protein marker, lane 1 is the whole cell lysate and lane 2 is the purified protein that is finally eluted. FIGS. 14 and 15 show the identification of the expression and purification of N-SpyTag003-COE and C-SpyTag003-COE, respectively. In the two figures, the expression of the protein in the cell supernatant was detected by Western blot using SpyTag003 monoclonal antibody 6G3 prepared in the present invention. The final purification of the protein was identified using SDS-PAGE on the right hand side of both figures. M is the protein marker, lane 1 is the cell supernatant and lane 2 is the final eluted purified protein.

Example six: for purification of SpyTag protein

The 4 SpyTag model protein supernatant samples were purified by a ̈ KTA pure chromatography system using the universal immunoaffinity chromatography column (Spy & IAC) prepared in example four.

The specific method comprises the following steps:

(1) The prepared immunoaffinity chromatography column (Spy & IAC) was equilibrated to room temperature before use. The resin bed was not allowed to dry throughout the process. Care was taken to degas all buffers to avoid introducing air bubbles into the column. Filtering the protein supernatant sample by using a 0.22 mu m filter for later use;

(2) First equilibrating the column with binding buffer (PBS solution at pH 7.4) and passing the filtered protein supernatant sample through the column at a flow rate of 0.5 ml/min;

(3) After the loading was complete, the column was again equilibrated with binding buffer and the unbound fraction was washed away. After the UV value plateaued (typically to below 20 mAU), the bound protein was eluted with elution buffer (0.1M glycine pH 2.7);

(4) The change in conductivity of the system is observed by noting that the eluate is collected as the conductivity begins to decrease rapidly. The pH was adjusted to neutral by adding 100. Mu.L of a neutralization buffer (1M Tris-HCl solution at pH 9.0) to each eluate collected at 1 mL;

(5) Elution was monitored throughout by absorbance of 280 nm. The collected fractions are pooled together and exchanged by Sephadex G-25 into an appropriate storage buffer, such as PBS at pH 7.4.

FIGS. 16-19 show the elution profiles of 4 SpyTag model proteins purified by Spy & IAC column. Where the blue line represents the profile of absorbance at 280 nm and the orange line represents the change in conductivity value during elution.

Example seven: purification analysis of 4 SpyTag003 model proteins

1. Protein purity assay

4 of the fractions passed through a universal immunoaffinity chromatography column for SpyTag protein (Spy) by SDS-PAGE and Size Exclusion Chromatography (SEC)&IAC) purified SpyTag003 model protein. Wherein Superdex ^TM 200 Increate 10/300GL chromatography column for analysis of N-SpyTag003-N and C-SpyTag003-N proteins, hiLoad ^TM 16/600 Superdex ^TM 200 The pg column was used for the analysis of N-SpyTag003-COE and C-SpyTag003-COE proteins. As shown in FIGS. 12-15 and FIGS. 20-23, the 4 purified SpyTag model proteins were homogeneous and pure, with purity above 90%, as confirmed by the single main peak in the clear blots and SEC chromatograms in SDS-PAGE.

2. Protein assembly capacity validation

To evaluate the function of SpyTag model protein purified using a universal immunoaffinity chromatography column for SpyTag protein (Spy & IAC), i.e., the ability to assemble with its "partner" SpyCatcher. The purified 4 SpyTag model proteins were conjugated to SpyCatcher003-mi3 at different molar ratios at 4 ℃ for 2h and aggregates were removed by centrifugation. The amount of covalent VLP-antigen conjugate formed at each coupling ratio is shown using SDS-PAGE. As shown in FIGS. 24-27, SDS-PAGE demonstrated efficient covalent reactions, and all SpyTag pattern proteins were efficiently displayed on SpyCatcher003-mi 3. Although the changes in N-SpyTag003-COE and C-SpyTag003-COE were less pronounced when incubated at equimolar concentrations, most reactions produced the least residual unbound antigen, consistent with previous literature reports. That is, the SpyTag protein purified using a general-purpose immunoaffinity chromatography column (Spy & IAC) for SpyTag protein exhibited satisfactory assembling ability.

Example eight: regeneration and storage method of universal immunoaffinity chromatography column (Spy & IAC)

The column should be regenerated immediately after each elution to prevent damage to the immobilized monoclonal antibody molecule from the low pH elution buffer. When protein elution was complete, the column was immediately washed with a large amount of binding buffer (PBS solution at pH 7.4) to remove any residual protein and reactivate the resin. The column was then equilibrated with binding buffer containing 0.05% sodium azide and stored therein. A spring valve (or syringe) may be added at the top of the column to apply pressure to the resin bed to prevent resuspension or leakage, thereby preventing drying of the column.

The columns were stored upright at 4 ℃ and attempted to be used multiple times over a longer time span to verify their service life. The expression and purification of the aforementioned 4 SpyTag pattern proteins was actually performed sequentially within 4 months. The experimental results show that the universal immunoaffinity chromatography column (Spy & IAC) for SpyTag protein shows good regeneration and storage stability.

While certain preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including the preferred embodiment and all changes and modifications that fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present application and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. An immunoaffinity chromatography column, which is characterized by comprising cross-linked bead agarose activated by aldehyde group of a solid phase carrier and HBC-SpyTag003 monoclonal antibody coupled with the cross-linked bead agarose; the heavy chain variable region sequence of the anti-HBC-SpyTag 003 monoclonal antibody is a DNA sequence shown as SEQ ID NO.2 or an amino acid sequence shown as SEQ ID NO.3, or an active fragment or conservative variant sequence is obtained by adding, deleting and replacing one or more amino acids on the basis of the SEQ ID NO.3 sequence; and/or

The DNA sequence of the light chain variable region 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 a conservative variant obtained by adding, deleting and replacing one or more amino acids on the basis of the SEQ ID NO.5 sequence.

2. The immunoaffinity chromatography column according to claim 1, wherein said HBC-SpyTag003 monoclonal antibody is produced by:

3. The immunoaffinity chromatography column according to claim 2, wherein in step (2), the purification method comprises:

4. A method for preparing an immunoaffinity chromatography column of claim 1, comprising the steps of:

(1) Preparation of monoclonal antibodies

(2) Monoclonal antibody immobilization

5. The method of claim 4, wherein in step (2), the AminoLink is taken ^TM The coupling resin is filled in a hollow chromatographic column, so that the filling buffer solution naturally flows out, a PBS solution with the pH value of 7.2 is added to balance the chromatographic column, the purified HBC-SpyTag003 monoclonal antibody is added into the chromatographic column, a cyanoborohydride solution is added into the reaction slurry under the ventilation condition, the mixture is inverted overnight at the temperature of 4 ℃, and then the resin is washed by the coupling buffer solution.

6. The method of claim 4, wherein in step (3), the resin is washed with 1M Tris-HCl solution at pH 7.4, and then the quenching buffer and the cyanoborohydride solution are added to the column under aeration.

7. A method for purifying SpyTag protein, which is carried out based on the immunoaffinity chromatography column of claim 1, and comprises the following steps:

(1) Equilibrating the immunoaffinity chromatography column to room temperature;

(4) Collecting the eluate when the conductance begins to decrease rapidly; adding a neutralization buffer solution into the collected eluent to adjust the pH value to be neutral;

(5) The collected eluate fractions were pooled together and exchanged into storage buffer by Sephadex G-25.

8. The method for purifying a SpyTag protein according to claim 7, wherein the SpyTag protein is 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.

9. The method for regenerating immunoaffinity chromatography column of claim 1, comprising the steps of:

(1) Immediately after the protein elution was completed, the column was washed with PBS solution at pH 7.4 to remove residual protein and reactivate the resin;