CN115976076A - HBC-SpyTag003 fusion gene, expression vector thereof, monoclonal antibody and application thereof - Google Patents

Abstract

The invention discloses aHBC‑SpyTag003Fusion gene, expression vector thereof, monoclonal antibody and application thereof, aiming at solving the technical problems of complex and difficult purification of the SpyTag protein at present. The invention adopts gene engineering technology toSpyTag003 is displayed on the surface of HBV core antigen (HBc) at high density, expressed in escherichia coli and self-assembled to form nanoparticles, mice are immunized with the nanoparticles, and a high-affinity SpyTag003 monoclonal antibody is obtained by screening through a hybridoma method; covalently immobilizing the monoclonal antibody to an AminoLink ^TM The immunoaffinity chromatography column for purifying the SpyTag protein can be prepared on the coupling resin, has high purification efficiency and high protein purity, can effectively retain the assembly function of the purified protein, and provides a new way for the efficient purification of the SpyTag protein.

Description

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

Technical Field

The invention relates to the technical field of bioengineering, and particularly relates to an HBC-SpyTag003 fusion gene, an expression vector thereof, a monoclonal antibody and application thereof.

Background

The SpyTag/SpyCatcher is a star molecule for modular vaccine assembly, provides a simple protein covalent conjugation approach, has high robustness and universality, and is currently applied to various candidate vaccines and personalized tumor neoantigen therapeutic vaccines. This series ofThe system is generated by cleavage of the CnaB2 domain from the fibronectin binding protein Fbab of Streptococcus pyogenes, with spontaneous intramolecular isopeptide bonds between the two components, and allows rapid assembly at a certain temperature (at least 4-37 ℃), pH (5-8), buffer (no need for specific anions or cations), even when using 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 the SpyTag003/SpyCatcher003 the more preferable choice in a 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 useful after purification is complete and can also generate an undesirable immune response in vivo. When the purity of the eluted fractions is insufficient or the sample is not hung on the purification column at all due to insufficient exposure of the His tag, further purification methods are required. Furthermore, 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.

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 known to a person skilled in the art.

Disclosure of Invention

The inventor finds out through research that: because the molecular weight of the SpyTag is small, it is difficult to induce a sufficiently high antibody level, which is a bottleneck factor that restricts protein purification and separation based on the SpyTag tag protein; the nanoparticle display mode 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, so as 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 a SpyTag003 monoclonal antibody with high affinity can be obtained by screening through a hybridoma technology.

In view of the above, the high-affinity SpyTag003 monoclonal antibody is prepared by utilizing the multivalent display of the SpyTag003 label by the HBc nanoparticles in the application of the invention, and a brand-new and universal affinity immunochromatography purification method based on the monoclonal antibody is established for separation and purification of the 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 an aspect of the present disclosure, there is provided a method of manufacturing a semiconductor deviceHBC-SpyTag003The DNA sequence of the fusion gene is shown in SEQ ID NO. 1.

According to another aspect of the present disclosure, the aboveHBC-SpyTag003The fusion gene is cloned to a pET28a vector, and a pET28a-HBC-SpyTag003 prokaryotic expression vector is constructed.

In some embodiments of the disclosure, the prokaryotic expression vector is transformed into escherichia coli, and a pET28a-HBC-SpyTag003 prokaryotic expression strain is constructed; then carrying out induction expression on the obtained expression strain in the presence of IPTG; and (3) cracking the cells, purifying the cracked supernatant containing the HBC-SpyTag003, and finally obtaining purified HBC-SpyTag003 nano-particles.

In some embodiments of the disclosure, the escherichia coli strain transformed with the pET28a-HBC-SpyTag003 plasmid is pTf/BL 21 (DE 3), and an escherichia coli BL21 (DE 3) competent cell transformed with the chaperone pTf can help the foreign protein to be folded correctly and increase the proportion of soluble protein.

In some embodiments of the disclosure, the HBC-SpyTag003 nanoparticles are purified by precipitation with 5% volume saturated ammonium sulfate, dialysis to remove residual ammonium sulfate, and loading with Capto ^TM Core 700 packed chromatographic column, collection of flow-through is completed.

According to another aspect of the disclosure, a monoclonal antibody against SpyTag003 label is provided, which is prepared by using the nanoparticle as an immunogen and using a hybridoma method.

In some embodiments of the disclosure, the heavy chain variable region sequence of the anti-SpyTag 003 tag monoclonal antibody is a DNA sequence shown in SEQ ID No.2 or an amino acid sequence shown in SEQ ID No.3, or a sequence of an active fragment or conservative variant obtained by adding, deleting, replacing one or more amino acids based on 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, a mouse is immunized with the purified HBC-SpyTag003 nanoparticles described above, and mouse splenocytes are fused with mouse myeloma cells (Sp/20 cells) to obtain hybridoma cells. And screening by adopting an indirect ELISA method and a limiting dilution method to obtain the positive hybridoma cells. Inoculating the hybridoma cells into abdominal cavities of mice to collect ascites containing the monoclonal antibody, and evaluating the titer of the ascites containing the monoclonal antibody by an indirect ELISA method; and finally obtaining the SpyTag003 monoclonal antibody by purifying the ascites.

In some embodiments of the disclosure, the purified SpyTag003 monoclonal antibody described above is immobilized on an AminoLink ^TM Coupling the mixture on resin to obtain a universal immunoaffinity chromatography column (Spy) for the SpyTag protein&IAC). The resin is cross-linked bead agarose activated by aldehyde groups, has a rapid linear flow potential, and can be used for gravity flow and medium and low pressure applications. The fixing principle is as follows: the primary amine on the monoclonal antibody molecule reacts with the activated aldehyde group on the agarose carrier spontaneously to form Schiff base bond, and sodium cyanoborohydride (NaCNBH) is used as a mild reducing agent ₃ ) In the presence of this, it can be reduced to a stable secondary amine linkage, thus allowing simple and efficient covalent immobilization of the monoclonal antibody on an agarose support.

According to another aspect of the disclosure, the immunoaffinity chromatography column is used for efficient purification of N-terminal or C-terminal SpyTag tagged proteins from prokaryotic or eukaryotic expression systems.

In some embodiments of the disclosure, the immunoaffinity chromatography column is used to purify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein or Porcine Epidemic Diarrhea Virus (PEDV) core neutralizing epitope (COE) protein with SpyTag003 tag at the N-terminus or C-terminus.

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

1. by passingHBC-SpyTag003The fusion gene can display SpyTag003 on the surface of Hepatitis B Virus (HBV) core antigen (HBc) at high densityHBC-SpyTag003The fusion gene can be expressed in Escherichia coli and self-assembled to form HBC-SpyTag003 nanoparticles.

2. HBC-SpyTag003 nano-particles are used for immunizing mice, and a high-affinity SpyTag003 monoclonal antibody is screened by a hybridoma method.

3. The obtained SpyTag003 monoclonal antibody can be used for preparing a universal immunoaffinity chromatography column (Spy & IAC) to efficiently purify protein with SpyTag labels at the N-terminal or the C-terminal of a prokaryotic or eukaryotic expression system, and provides a universal new method for efficient purification of SpyTag protein.

Drawings

FIG. 1 is a schematic diagram of an HBc-SpyTag003 gene fusion construct, in accordance with one embodiment of the present disclosure.

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

FIG. 3 is a plot of the particle size of HBC and HBC-SpyTag003 nanoparticles measured by dynamic light scattering in one 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.

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 fusion construct of 4 SpyTag modular protein genes in 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 an embodiment of the present 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 a 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. 20-23 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. 24-27 are the validation of the assembly ability of the 4 SpyTag model proteins purified according to one embodiment of the 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: prokaryotic expression method and purification method of HBC-SpyTag003 nanoparticles

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

The SpyTag003 tag sequence (RGVPHIVMVDAYKRYK) was inserted between amino acids 80 and 81 of the Major Immunodominant Region (MIR) of the HBc protein (amino acids 1-149) flanked by one GGS linker 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. Expression 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 transformation product 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 to 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 5% was added to the supernatantThe volume of saturated ammonium sulphate solution was then left to stand at 4 ℃ for 6h. The pellet was resuspended in 20ml of 20mM Tris-HCl, 150mM NaCl solution at pH 8.0 by centrifugation at 12000 r/min for 20min at 4 ℃ and supernatant discarded. The 24 h was dialyzed against excess of the same buffer to remove residual 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 is: 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, and the result is shown in figure 3, and the DLS result shows that the obtained nano-particles have high purity and good 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 in both of the latter two immunizations, and the dose of each of the three immunizing proteins was 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. Determination of serum antibody titer in immunized 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 mul of blocking solution (5% skimmed milk powder + PBST) into each well, 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 with dilution buffer (PBST) was added to each well (initial dilution fold 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, and then the supernatant is discarded and 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 SO ₄ 50. Mu l of stop solution is used for stopping the reaction, and an enzyme-labeling instrument is used for determining an OD450 value.

(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) Taking out the spleen of the mouse in an ultra-clean workbench in a sterile operation manner, placing the spleen on a 200-mesh sterile filter screen, shearing the spleen by using small scissors, adding GNK washing liquid for washing, and filtering the spleen cells into a small sterile beaker;

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

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 10 ml in 1640 medium containing 10% FBS, thoroughly vortexed, and added at 100 μ l/well to 96 well cell culture plates previously plated with feeder cells (theoretically up to 1 hybridoma cell per well). Completing the subcloning of each hybridoma cell strain in the same way;

(4) Culturing for 7-10 days, and carrying out 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) The 3 strains of the finally screened positive monoclonal cell strains stably secreted are subjected to expanded culture according to the proportion of 1-2 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 supernatant of the ascites, and storing at-80 ℃ for later use.

(2) Determination of ascites titer of monoclonal antibody

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 the HBC-SpyTag003 nanoparticles with an ELISA coating solution (CBS), incubating overnight at 4 ℃ in a coating volume of 50 micrograms and 50 microliters per hole, discarding the coating solution, washing for 3 times with PBST, and drying residual liquid on a 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: 50 μ l each of mouse ascites diluted 2-fold by dilution buffer (PBST) was added to each well 50ul (initial dilution 1: 1000), 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, and then the supernatant is discarded and washed 6 times with 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 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 compiled and the maximum dilution was taken as the titer of this 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 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 absorbance profile at 280 nm and the orange line represents the change in the electrical conductivity 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 the 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 gene for SpyTag003 monoclonal antibody 6G3 VH and VL. 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 mapped as shown in fig. 8. As shown in the structural simulation of fig. 9, the CDRs are spatially close together 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: application of SpyTag003 monoclonal antibody in preparation of universal immunoaffinity column (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 producing about 50 mM) was added to the reaction slurry ₃ ) And mixed at 4 ℃ overnight with inversion. 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 have formed 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 purified and verified SpyTag model protein

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 the SARS-CoV-2N protein, the GGSGGS linker and the C-terminal SpyTag003 tag into 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 (pH 7.4 in PBS), sonicated, and centrifuged at 12000 rpm 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, the cell viability of 96 h decreased to 40-60% or less, at which time the expression of the protein in the cell supernatant was examined by 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 final eluted purified protein. 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 left sides of the two figures are the expression conditions of the protein in the cell supernatant detected by Western blot by using the SpyTag003 monoclonal antibody 6G3 prepared in the invention. The final purification of the protein was identified using SDS-PAGE on the right 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: spyTag protein purification method based on type immunoaffinity chromatography column (Spy & IAC)

The 4 SpyTag model protein supernatant samples were purified by a ̈ KTA pure chromatography system using a SpyTag protein-based 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 with pH 7.4), passing the filtered protein supernatant sample through the column at a flow rate of 0.5 ml/min;

(3) After the loading is complete, the column is equilibrated again with the binding buffer and unbound components are washed away. After the UV value plateaus (typically below 20 mAU), the bound protein is eluted with an elution buffer (0.1M glycine solution at 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 eluate 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 analysis

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 pg columns were used for the analysis of N-SpyTag003-COE and C-SpyTag003-COE proteins. As shown in fig. 12-15 and fig. 20-23, the purified 4 SpyTag pattern proteins were homogeneous and pure, with purity above 90%, as confirmed by the single main peak in the clear blot 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 4 purified SpyTag model proteins were bound to 2h at different molar ratios at 4 ℃ in SpyCatcher003-mi3 and aggregates were removed by centrifugation. SDS-PAGE was used to show the amount of covalent VLP-antigen conjugate formed at each coupling ratio. 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 molecules by 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) can 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 of 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 preferred embodiments and all such alterations and modifications as 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, the present invention is intended to include such modifications and variations, provided they come within the scope of the appended claims and their equivalents.

Claims (10)

1. A kind ofHBC-SpyTag003The DNA sequence of the fusion gene is shown in SEQ ID NO. 1.

2. A prokaryotic expression vector comprising the nucleic acid molecule of claim 1HBC-SpyTag003The fusion gene is cloned to pET28a vector to construct.

3. A nanoparticle is HBC-SpyTag003 protein particles obtained by transforming a prokaryotic expression strain formed by escherichia coli through the prokaryotic expression vector of claim 2 and carrying out IPTG induced expression, thallus lysis and purification.

4. The nanoparticle of claim 3, wherein the purifying step 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 (3) collecting flow-through liquid by using a chromatographic column filled with Core 700 to obtain purified nano particles.

5. An anti-SpyTag 003-tag monoclonal antibody prepared by a hybridoma method using the nanoparticle of claim 3 as an immunogen.

6. The monoclonal antibody against the SpyTag003 tag according to claim 5, wherein the heavy chain variable region sequence thereof is a DNA sequence shown in SEQ ID No.2 or an amino acid sequence shown in SEQ ID No.3, or a sequence of an active fragment or a conservative variant thereof obtained by addition, deletion, or substitution of one or more amino acids based on the SEQ ID No.3 sequence; and/or

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

7. The use of the anti-SpyTag 003 tag monoclonal antibody of claim 5 in the purification of a protein having a SpyTag003 tag.

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

9. The use of claim 7, wherein the anti-SpyTag 003-tag monoclonal antibody is conjugated to aldehyde-activated cross-linked bead sepharose on a solid support to form an immunoaffinity chromatography column, which is then used to purify SpyTag protein.

10. The use according to claim 8, wherein the aldehyde-activated agarose carrier is subjected to robust immobilization with the anti-SpyTag 003-tag monoclonal antibody under the action of a reducing agent, sodium cyanoborohydride.

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