CN118515783B - Novel coronavirus N protein and S1 antigen-encoding mRNA molecule combined bionic virus structural vaccine and preparation method thereof - Google Patents
Novel coronavirus N protein and S1 antigen-encoding mRNA molecule combined bionic virus structural vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a novel coronavirus N protein and S1 antigen-encoding mRNA molecule combined bionic virus structural vaccine and a preparation method thereof, belonging to the technical field of biological medicine. In a buffer solution system, N protein of a prokaryotic expression purified novel coronavirus nucleocapsid structure is combined with mRNA molecules for encoding S1 antigen to form a biosynthesis body with a chromatin-like bead-like structure. The lipid particle structure formed by the biosynthesis body is wrapped by the LNP lipid carrier, and the eukaryotic expressed S1 protein is further loaded on the surface of the lipid particle structure wrapping the biosynthesis body to form the bionic virus structure vaccine, and the vaccine can induce a host to generate a strong specific antibody against the S antigen and can also generate a specific antibody against the N antigen after immunization of animals. The bionic virus structure vaccine can induce organisms to generate strong specific T cell responses of the S antigen and the N antigen, and strengthen the protection effect of the vaccine immune response in virus attack.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and in particular relates to a novel coronavirus N protein and S1 antigen encoding mRNA molecule combined bionic virus structural vaccine and a preparation method thereof.
Background
In the research and development and application of various SARS-CoV-2 virus vaccine, mRNA vaccine is prepared in artificial synthesis mode, and can express endogenous virus antigen in cell, so that it can activate natural immune system to start the antiviral immune reaction of body, and its immune effect is obviously superior to that of other inactivated vaccine and polypeptide vaccine. Previous studies have shown that this vaccine has the advantage of delivering replicative or non-autonomously replicative mRNA molecules, which may rely on intracellular translation expression systems, into the cytoplasm through a nanolipid delivery system (lipid nanoparticles DELIVERING SYSTEM), expressing antigen proteins, and in the process, first activating the innate immune response system by binding to the pattern recognition receptors (pattern recognition receptor, PRR) of the innate immune system within the cell through different forms of exogenously introduced mRNA molecules and expressed antigen proteins, thereby recruiting antigen presenting cells including dendritic cells, macrophages within the tissue through immune signaling molecules released by the cells, phagocytosing antigens expressed by mRNA molecules in these cells, and thereby presenting the treated antigens to the T cell system, activating the acquired immune response. Based on the fact that this complete process of activating innate/adaptive immunity by the mRNA molecule expression process produces a good immune response, it is shown that mRNA vaccines have the potential to mimic the viral infection process and elicit an immune response. However, it is notable that since the lipid delivery system used in mRNA vaccines is designed to achieve mRNA delivery by fusing with different cell membranes in a non-specific manner, it has the characteristics of lower entry efficiency and non-specific entry into a variety of cells compared to the entry of viruses into cells by binding their membrane surface proteins to cell surface specific receptors.
Based on this, by analyzing the structure of SARS-CoV-2 virus, the characteristic of virus surface spike protein as neutralizing antigen and the characteristic of binding with DC-SIGN receptor on the surface of dendritic cell and macrophage besides ACE2 receptor are fully utilized, so that the virus-like structure VLS (virus-like-structure vaccine) vaccine is constructed, which is formed by coating mRNA molecule by lipid carrier and carrying S1 protein on the surface, and patent is granted (publication No. CN 116139108A). However, based on the in-depth analysis of the structure of SARS-CoV-2 virus and the related analysis of the antigen profile and immunoprotection response of the virus during infection of the host, one trend is that in the analysis of the immune response against the Covid-19 population, in addition to the important recognition of the S antigen by the immune system and the generation of corresponding neutralizing antibodies to block infection of the virus, the nucleocapsid structural protein N antigen of the virus was also found to be an important antigenic molecule capable of eliciting specific T cell responses of the immune system. This response is often associated with the host formation of a killer T cell response that controls viral proliferation. None of the antigens used in the existing mRNA vaccines contemplate the use of an N antigen component.
Disclosure of Invention
Based on the immunological analysis conclusion related to SARS-CoV-2 infection, the present invention further designs a technology for simulating SARS-CoV-2 virus complete structure on the premise of obtaining virus-like structure VLS vaccine patent (CN 116139108A), firstly, in the buffer solution system designed by the present invention, the prokaryotic expression virus nucleocapsid main structural component N protein is combined with the encoding S1 antigen mRNA molecule to form the biosynthesis body of the bead-like structure of the chromatin. After the complete combination of N protein and mRNA molecule is confirmed by ChIP experiment, the protein is wrapped by LNP lipid carrier to form lipid particle structure, and S1 protein expressed in eukaryotic is loaded on the surface of the lipid particle structure to form the bionic virus structure vaccine. Through vaccine feature identification and comprehensive immunological analysis, it is proved that the obtained bionic virus structure vaccine can induce the host to produce stronger specific antibody against S antigen and can produce specific antibody against N antigen. More importantly, the vaccine can induce the organism to generate strong specific T cell reaction of resisting S antigen and N antigen, thereby strengthening and improving the protective effect of vaccine immune reaction in virus attack.
Based on this, a first object of the present invention is to provide a buffer system that allows the binding of the novel coronavirus N protein to the mRNA molecule encoding the S1 antigen to form a bead-like, viroid nucleocapsid-like structure of the biosynthesized body. A second object of the present invention is to provide a lipid particle structure coated with a novel coronavirus N protein and a biosynthesized body bound to an mRNA molecule encoding an S1 antigen, and a method for preparing the same; the third object of the invention is to provide a novel coronavirus N protein and a biomimetic virus structural vaccine combined with an encoding S1 antigen mRNA molecule and a preparation method thereof.
The buffer system (the "buffer system" used in the following description of the invention is the buffer of the present component when no special description is made): dissolving NaAc, naCl, mgCl 2, DTT and Fucose in water as solvent to obtain buffer solution with pH of 6.0-6.4 and NaAc and 20mM concentration; naCl,10mM; mgCl 2, 10mM; DTT,1mM; fucose,0.001% w/v.
The preparation method of the novel coronavirus N protein and the biosynthesis body combined with the mRNA molecule of the coded S1 antigen uses the buffer solution system, and comprises the following steps:
(1) Dissolving the mRNA of the novel coronavirus encoding S1 antigen in TE Buffer with the pH value of between 6.0 and 6.4, and configuring the concentration to be 1000ug/ml;
(2) Dissolving the prokaryotic expression new coronavirus N protein in TE Buffer with pH=6.8-7.2, and configuring the concentration to be 200 ug/ml;
(3) (3) adding the mRNA of the step (1) and the N protein of the step (2) into the buffer solution system in a concentration ratio of 5:1, and reacting for 28-32min at the temperature of 35-39 ℃ to obtain a novel biosynthesis body combined by the coronavirus N protein and the mRNA molecule of the coding S1 antigen.
A method for preparing a lipid particle structure encapsulating a novel coronavirus N protein and a biosynthesized body encoding an S1 antigen mRNA molecule binding, comprising the steps of:
(1) Lipids DHA-1, DOTAP, DOPC and mPEG-DTA-1-2K were combined at 56:10:27:1.6 molar ratio mixing in absolute ethanol; simultaneously diluting the biosynthesized of claim 4 with a buffer containing NaAc 20mM,NaCL 10mM, mgCl 2 mM, DTT 1mM and trehalose at 0.001% (w/v);
(2) Preparing a lipid particle structure encapsulating the biosynthesized body using a microfluidic device NanoAssemblr R IgniteTM Model;
(3) Removing ethanol from the liposome particle structure wrapped with the biosynthesized body prepared in the step (2) to obtain the liposome particle structure wrapped with the biosynthesized body, wherein the nitrogen-phosphorus molar ratio is 15-18:1.
The novel coronavirus N protein and the S1 antigen mRNA molecule-encoding combined bionic virus structural vaccine comprises novel coronavirus N protein and S1 antigen mRNA molecule-encoding combined biosynthesized body.
The novel coronavirus N protein and the S1 antigen-encoding mRNA molecule combined bionic virus structure vaccine comprises a lipid particle structure which is wrapped with a biosynthesis body and has a nitrogen-phosphorus molar ratio of 15-18:1.
The preparation method of the novel coronavirus N protein and S1 antigen-encoding mRNA molecule combined bionic virus structural vaccine is realized by loading the S1 protein in the lipid particle structure according to claim 6, in particular,
The lipid particle structure was modified to encode S1 antigen mRNA and S1 protein at 4:1, adding S1 protein, rotating and mixing at room temperature, standing at 4deg.C for 16 hr, and diluting with buffer solution containing NaAc 20 mM, naCL 10 mM, trehalose 0.001% w/v and glycerol 2.5%.
The invention has the beneficial effects that:
The buffer solution system designed by the invention can combine N protein of prokaryotic expression new coronavirus nucleocapsid structure with the mRNA molecule of coding S1 antigen to form a chromatin-like bead-like structure. After the complete combination of N protein and mRNA molecule is verified by ChIP experiment, the protein is wrapped by LNP lipid carrier to form lipid particle structure, and eukaryotic expressed S1 protein is loaded on the surface of the lipid particle structure to obtain the bionic virus structure vaccine. Through structural feature identification and comprehensive immunological analysis, the bionic virus structural vaccine is proved to be capable of inducing a host to generate a strong specific antibody against an S antigen and generating a specific antibody against an N antigen. More importantly, the bionic virus structure vaccine can induce organisms to generate strong specific T cell responses of the S antigen and the N antigen, so that the protection effect of the vaccine immune response in virus attack experiments is enhanced and improved.
Drawings
FIG. 1 is an electron microscopic view of the biosynthesized body of example 1 of the present invention;
FIG. 2 is a comparison of copies of mRNA levels in a biosynthesized body obtained in example 1 of the present invention;
FIG. 3 is a graph showing the comparison of lipid particle structure with LNP electron microscopy for mRNA encapsulation in accordance with example 2 of the present invention;
FIG. 4 shows the S1 mRNA copy number in supernatants from IP qPCR assays at various time points of Lipofectin-coated biosynthesized and lipid particle structure transfection of 293T cells according to example 2 of the present invention;
FIG. 5 shows the IP WB assay eluate and N protein in the permeate at various time points when the Lipofectin-coated biosynthesized and lipid particle structures of example 2 of the present invention were transfected into 293T cells;
FIG. 6 is an electron microscope image of a biomimetic viral structural vaccine and VLS according to example 3 of the present invention;
FIG. 7 shows the qPCR detection result of the biomimetic viral structural vaccine of example 3 of the present invention
FIG. 8 shows the detection results of the biomimetic viral structural vaccine WB according to example 3 of the present invention;
FIG. 9 shows the lipid particle structure and neutralizing antibody titers of the biomimetic viral structure vaccine of example 3 of the present invention after immunization of Balb/C mice;
FIG. 10 shows the results of cellular immunity after immunization of animals with the biomimetic viral construct vaccine and VLS of example 3 of the present invention;
FIG. 11 shows the viral loads of different tissues after challenge of the biomimetic viral structured vaccine of example 3 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, the technical solutions of the present invention will be described in detail below, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, other embodiments that may be obtained by those skilled in the art without making any inventive effort are within the scope of the present invention.
In order to more clearly illustrate the present invention, the following examples are provided.
Example 1
A preparation method of a prokaryotic expression novel coronavirus N protein and an S1 antigen-encoding mRNA molecule combined biosynthesis body comprises the following steps:
(1) The novel coronavirus encoding S1 antigen mRNA was dissolved in TE Buffer at ph=6.2, and the mRNA was configured at a concentration of 1000ug/ml.
(2) The prokaryotic expressed novel coronavirus N protein was dissolved in TE Buffer at ph=7.0, with a configured concentration of N-protein of 200ug/ml.
(3) MRNA: the amount of N protein was added to the following buffer system in a concentration ratio of 5:1 to carry out the reaction:
buffer system used (solvent is sterile and asepsis water):
NaAc 20mM (pH=6.2)
NaCl 10mM
MgCl2 10mM
DTT 1mM
Fucose 0.001%w/v
standing at 37deg.C for 30 min, and storing at 4deg.C for use.
The novel coronavirus N protein and the mRNA molecule for encoding the S1 antigen are combined to obtain a biosynthesized body (hereinafter referred to as biosynthesized body), and the biosynthesized body is in a bead-like and virus-like nucleocapsid structure (shown in figure 1) through an electron microscope picture.
The biosynthesis bodies which are combined with mRNA are collected by precipitation by using a modified ChIP-q-RT-PCR method and a specific antibody of SARA-CoV-2N protein, and the collected biosynthesis bodies are quantitatively detected by using a q-RT-PCR method by using a specific primer. The method comprises the following steps:
(1) mu.L of anti-SARS-CoV-2N protein specific antibody was added to 200. Mu.L of the biosynthesized sample and incubated at room temperature for 2h.
(2) 30 Mu L of beaver IgG magnetic beads are added, fully mixed and placed on a rotator for 20min (15-20 rpm/min), then placed in a magnetic bead separator for standing for 2min, at the moment, the magnetic beads are adsorbed on the tube wall, and the supernatant sample is slowly sucked out (penetrating fluid).
(3) The magnetic beads were thoroughly mixed (eluent) in 200. Mu.L of elution buffer added to the kit.
(4) Adding 100 μl of each of the sample, penetrating fluid and eluent into 300 μl of Trizol extract, mixing thoroughly, centrifuging at 12000rpm for 10min at 4deg.C, collecting supernatant, placing into new EP tube, adding 250 μl of isopropanol, mixing uniformly, standing at room temperature for 15min, centrifuging at 12000rpm at 4deg.C for 15min, discarding supernatant, and observing trace white precipitate; washing with 500. Mu.L of an absolute ethanol solution, centrifuging at 12000rpm for 10min at 4 ℃, discarding the supernatant ethanol solution, air-drying at room temperature, and dissolving RNA with 100. Mu.L of sterile, enzyme-free water.
(5) Q-RT-PCR detection (Kit: takara One STEP PRIMESCRIPT ™ RT-PCR Kit, code No. RR 064A) was performed on the dissolved RNA, the mRNA molecule encoding the S1 antigen was used as mRNA standard, 10-fold gradient dilution was performed from 1 ng/. Mu.L to 0.1 ng/. Mu.L to 0.00000001 ng/. Mu.L, 2. Mu.L of penetrating solution was taken, the eluent was used to quantitatively detect the extracted RNA, and the primers required for the detection were:
F:TGGATCTGGAGGGAAAGCAGGGCAACT；
R: CCGATTGGCAGATCCACCAGAGGTTC；
Probe primer: 5'-6 FAM-ATGGCTACTTCAAGATCTATAGCAAGC-TAMRA-3';
The reaction procedure is shown in the following table.
TABLE 1 q-RT-PCR reaction System
TABLE 2 q-RT-PCR reaction procedure
The results showed that the copy number of mRNA in the biosynthesized bodies collected by antibody precipitation against SARS-CoV-2N protein was about 80% of the positive control group (FIG. 2) compared to the positive control group (positive control of mRNA extracted from biosynthesized bodies). This demonstrates that the buffer system designed in the present invention can effectively bind N protein and mRNA molecules to form a viroid nucleocapsid beaded structure.
Example 2
A method of preparing a lipid particle structure encapsulating the biosynthesized body of example 1:
(1) Lipids DHA-1, DOTAP, DOPC and mPEG-DTA-1-2K were added at 56:10:27:1.6 molar ratio mixing in absolute ethanol; the N protein-encapsulated mRNA molecule-like viral nucleocapsid biosynthesized from example 1 was simultaneously diluted with buffer containing NaAc 20mM,NaCL 10mM, mgCl 2 mM, DTT 1mM and trehalose at 0.001% (w/v).
(2) The preparation of the lipid microsphere system was accomplished with microfluidic equipment (NanoAssemblr R IgniteTM Model) with the following parameters: FRR:1:3, a step of; TFR is 20ml/min; wastevol 0.65+0.05 ml/3:9, a liposome-entrapped lipid particle system was obtained.
(3) The liposome-encapsulated lipid particle structure prepared in step (2) was diluted with 30 volumes of A1 buffer. The A1 buffer contained NaAc 20mM,NaCL 10mM,0.001% (w/v) trehalose and 2.5% glycerol. After dilution, the mixture was concentrated by ultrafiltration with a 100 Kda ultrafiltration membrane to obtain a lipid particle structure (hereinafter referred to as "lipid particle structure") having a nitrogen-phosphorus molar ratio of 15 to 18:1 and encapsulating the mRNA-binding biosynthesized body.
The electron microscope and physical and chemical characteristic analysis of the prepared lipid particle structure show that: the lipid particle structure was not significantly different in shape from the conventional mRNA/LNP, and was spherical (FIG. 3). Is characterized by a particle size of 100-150nm, a particle aggregation index (PDI) of less than 0.2 and a surface potential of 10-20mV (Table 3)
TABLE 3 physical and chemical Properties detection of lipid particle Structure
Biological characterization of the structure of the resulting lipid particles:
To confirm the biological function of our designed biosynthesized body, which is a virus-like nucleocapsid structure formed by the combination of the prokaryotic expressed novel coronavirus N protein and the mRNA molecule encoding the S1 antigen. Using the lipid delivery system and the transfection reagent lipofectin 2000 as control delivery systems published in patent CN115998714a (a lipid nanoparticle, delivery system and method for preparing the delivery system), 293 cells were transfected after encapsulation of the bound biosynthesized, cells were transfected simultaneously with the lipid particle structure obtained in this example, and cells were collected at time points of 20 minutes, 40 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes after transfection and disrupted with a sonicator, supernatant was collected, and mRNA molecules encapsulated by N protein were collected using the modified immunoprecipitation method, as follows:
(1) Paving 293T cells in a 6-hole plate 24h in advance, transfecting the biosynthesis body by Lipofectin 2000 in the 293T cells (Lipofectin 2000 transfection system: 5 mu g of the biosynthesis body of example 1 is added into 500 mu L of Opti-MEM buffer solution and slowly mixed, 15 mu L of Lipofectin 2000 transfection reagent is added into 500 mu L of Opti-MEM buffer solution and slowly mixed, then the two are mixed for 10min at room temperature to form 1mL transfection system, the complete culture medium in the 293T cells is completely sucked out, and 1mL transfection system is added); meanwhile, 5. Mu.g of the lipid particle structure of example 2 was taken and added to 293T cells.
(2) After 20min,40min,60min,90min,120min and 180min, respectively, taking cells, discarding cell supernatant, adding 500 μl of PBS solution, performing ultrasonic disruption for 10min (power 30%, ultrasonic disruption for 10 s), centrifuging at 12000rpm for 10min at 4deg.C, and collecting supernatant.
(3) 200 Mu L of the supernatant in the step (2) is taken, 2 mu L of an anti-SARS-CoV-2N protein specific antibody is added, and the mixture is incubated for 2 hours at room temperature.
(4) 30 Mu L of beaver IgG magnetic beads are added, fully mixed and placed on a rotator for 20min (15-20 rpm/min), then placed in a magnetic bead separator for standing for 2min, at the moment, the magnetic beads are adsorbed on the tube wall, and the supernatant sample is slowly sucked out (penetrating fluid).
(5) The magnetic beads were thoroughly mixed (eluent) in 200. Mu.L of elution buffer added to the kit.
Quantitative q-RT-PCR is carried out to detect the structural change characteristics of the lipid particle structure after entering cells, and the structural change characteristics are as follows:
① Adding 100 mu L of each of the supernatant, the penetrating fluid and the eluent after sample crushing into 300 mu lTrizol of extracting solution, fully and uniformly mixing, centrifuging at 12000rpm and 4 ℃ for 10min, placing the supernatant into a new EP tube, adding 250 mu L of isopropanol, uniformly mixing, standing at room temperature for 15min, centrifuging at 12000rpm and 4 ℃ for 15min, and discarding the supernatant, wherein trace white precipitation can be observed; washing with 500. Mu.L of an absolute ethanol solution, centrifuging at 12000rpm for 10min at 4 ℃, discarding the supernatant ethanol solution, air-drying at room temperature, and dissolving RNA with 100. Mu.L of sterile, enzyme-free water.
② The dissolved RNA was subjected to q-RT-PCR detection (Kit: takara One STEP PRIMESCRIPT ™ RT-PCR Kit, code No. RR 064A), wherein mRNA molecules encoding S1 antigen were used as mRNA standard substances and subjected to gradient dilution, 10-fold gradient dilution was performed from 1 ng/. Mu.L to 0.1 ng/. Mu.L to 0.00000001 ng/. Mu.L, 2. Mu.L of penetrating solution was taken, and the RNA extracted from the eluent was quantitatively detected, with the primers required for the detection:
F: TGGATCTGGAGGGAAAGCAGGGCAACT；
R: CCGATTGGCAGATCCACCAGAGGTTC；
Probe primer: 5'-6 FAM-ATGGCTACTTCAAGATCTATAGCAAGC-TAMRA-3';
The reaction procedure is shown in the following table.
TABLE 4 q-RT-PCR reaction System
TABLE 5 q-RT-PCR reaction procedure
Experiments show that after 20 minutes of the conventional transfection reagent, the N protein component and mRNA molecules are completely decomposed, while the lipid particle structure is slower than that of the conventional transfection reagent, and the dissociation of the N protein component and mRNA occurs after 40 minutes (FIG. 4). Although the mechanism is not yet clear, this phenomenon suggests: after the lipid particle structure of the invention enters cells, mRNA and N proteins can respectively play the functions of the lipid particle structure.
Immunoblotting detection:
(1) Samples were collected at different time points by 40. Mu.L, and the above-mentioned penetration solution and eluent after immunoprecipitation treatment were added to 10. Mu.L of loading buffer (SDS-PAGE 5X reducing loading buffer) and boiled for 10min.
(2) The 10. Mu.L treated sample was added to a PAGE gel well and run according to 120V 15min,180V 30min.
(3) After transfer through SDS-PAGE gel, PBS containing 5% skimmed milk powder was blocked for 1h at room temperature, and incubated overnight with N protein-specific antibodies (diluted in 1:1000PBS buffer); then using PBST solution to clean for 5 times at room temperature for 5min each time; adding secondary antibody (1:5000 PBS buffer solution for dilution use), and incubating for 1h at room temperature; then using PBST solution room temperature cleaning 5 times, each time 5 minutes, finally through ECL exposure color development. The results showed that the presence of N protein was undetectable after 40 minutes in the IP eluate of the sample coated with the conventional transfection reagent, and the lipid particle structure was similar to that of the conventional transfection reagent (fig. 5).
Example 3
A preparation method of a novel coronavirus N protein and S1 antigen-encoding mRNA molecule combined bionic virus structural vaccine comprises the following steps:
(1) The lipid particle structure obtained in example 2 was expressed as 4 in terms of the amount of mRNA encoding S1 antigen and the amount of S1 protein: 1 (corresponding to 20 mug of S1 mRNA and 5 mug of S1 protein in each dose of bionic virus structure vaccine) and mixing for 30min at room temperature by rotating (15 rpm/min), and then placing at 4 ℃ for 16 hours.
(2) The overnight treated sample was diluted with a buffer (NaAc 20 mM, naCL 10mM, trehalose 0.001% (w/v) and 2.5% glycerol) and the concentration of S1 mRNA was determined to be 20. Mu.g per dose, thereby obtaining a novel coronavirus N protein and a biomimetic virus structural vaccine encoding the combination of S1 antigen mRNA molecules, (hereinafter referred to as S1 protein-loaded biomimetic virus structural vaccine).
The morphological and physicochemical characteristics of the S1 protein loaded biomimetic virus structural vaccine were examined, and the particles were also spherical particles in terms of their electron microscopic shape (FIG. 6). The physical and chemical properties result parameters are similar to those of the VLS disclosed in patent CN116139108 a.
Physical and chemical property detection of bionic virus structural vaccine and VLS vaccine
The structural form is detected by using a modified immunoprecipitation method, and the structural form is specifically as follows:
(1) 2 mu L of anti-SARS-CoV-2 S1 protein specific antibody is added into 200 mu L of bionic virus structure vaccine sample, and incubated for 2h at room temperature.
(2) 30 Mu L of beaver IgG magnetic beads are added, fully mixed and placed on a rotator for 20min (15-20 rpm/min), then placed in a magnetic bead separator for standing for 2min, at the moment, the magnetic beads are adsorbed on the tube wall, and the supernatant sample is slowly sucked out (penetrating fluid).
(3) The magnetic beads were thoroughly mixed (eluent) in 200. Mu.L of elution buffer added to the kit.
(4) Adding 100 μl of each of the sample, penetrating fluid and eluent into 300 μ LTrizol extractive solution, mixing thoroughly, centrifuging at 12000rpm for 10min, collecting supernatant, placing into new EP tube, adding 250 μl of isopropanol, mixing uniformly, standing at room temperature for 15min, centrifuging at 12000rpm for 15min, discarding supernatant, and observing trace white precipitate; washing with 500. Mu.L of an absolute ethanol solution, centrifuging at 12000rpm for 10min at 4 ℃, discarding the supernatant ethanol solution, air-drying at room temperature, and dissolving RNA with 100. Mu.L of sterile, enzyme-free water.
(5) Q-RT-PCR detection (Kit: takara One STEP PRIMESCRIPT ™ RT-PCR Kit, code No. RR 064A) was performed on the dissolved RNA, the mRNA molecule encoding the S1 antigen was used as mRNA standard, the mRNA standard was subjected to gradient dilution, 10-fold gradient dilution was performed from 1 ng/. Mu.L to 0.1 ng/. Mu.L to 0.00000001 ng/. Mu.L, 2. Mu.L of penetrating solution was taken, and the RNA extracted from the eluent was quantitatively detected, with the primers required for the detection:
F: TGGATCTGGAGGGAAAGCAGGGCAACT；
R: CCGATTGGCAGATCCACCAGAGGTTC；
Probe primer: 5'-6 FAM-ATGGCTACTTCAAGATCTATAGCAAGC-TAMRA-3';
The reaction system and the reaction procedure are shown in the following table.
Q-RT-PCR reaction system
Q-RT-PCR reaction procedure
(6) Adding 10 μL of loading buffer (SDS-PAGE 5X reductive loading buffer) into 40 μL of sample, penetrating fluid and eluent, boiling for 10min;
(7) Adding the 10 mu L treated sample into a PAGE gel hole, and running gel according to 120V 15min,180V 30min;
(8) After passing through SDS-PAGE gel, blocking for 1h at room temperature by using PBS solution containing 5% skimmed milk powder, and respectively adding anti-SARS-CoV-2N protein specific antibody and S1 protein specific antibody (1:1000 PBS buffer for dilution) and incubating overnight; then using PBST solution to clean for 5 times at room temperature for 5min each time; adding secondary antibody (1:5000 PBS buffer solution for dilution use), and incubating for 1h at room temperature; then using PBST solution room temperature cleaning 5 times, each time 5 minutes, finally through ECL exposure color development.
QPCR and WB detection results prove that the structural form of the bionic virus structural vaccine is a bionic virus particle structure internally containing mRNA combined with N protein and externally loaded with S1 protein (figures 7 and 8).
And (3) analyzing the immunological effect of the obtained bionic virus structural vaccine:
Our earlier work (patent CN116139108 a) showed that only mRNA molecules were encapsulated and that the anti-SARS-CoV-2 VLS vaccine, externally loaded with S1 protein, could provide a strong immune response to antibodies in animal immunity, including endogenous and exogenous antigen signal stimuli, especially in the production of high titers of antibodies against S antigen.
According to the immunization mode of 0 day and 21 days, the Balb/c mice are immunized by intramuscular injection with a lipid particle structure (obtained in example 2), a bionic virus structure vaccine (obtained in example 3) and a VLS vaccine (patent CN 116139108A), tail vein blood collection is carried out on the 28 th day after the immunization is enhanced, serum is separated, and detection of a binding antibody and a neutralizing antibody is carried out; spleen lymphocytes were isolated simultaneously for ELISPOT detection of the cellular immune response of TL-4 and IFN-gamma.
According to the immunization modes of 0 day and 21 days, the K18-ACE2 transgenic mice are immunized by a bionic virus structure vaccine through intramuscular injection, the virus (WT) is tapped 45 days after the immunity is enhanced, and tissues such as heart, liver, spleen, lung, kidney, brain, spinal cord, lymph node, nasal cavity and the like are sampled 3 days and 5 days after the virus is tapped to carry out disease load detection.
Detection of bound antibodies: ELISA kits for Omicron (B.1.1.529/BA.4 & BA.5), WT, alpha (B.1.1.7), beta (B.1.351), gamma (P.1) and Delta (B.1.617.2) strains were tested in parallel for sera 28 days after the immunization of the lipid particle structure and the bionic virus structure vaccine immunization groups;
Neutralizing antibodies: and (3) detecting neutralizing antibodies of serum WT strain viruses 28 days after the immune groups of the lipid particle structure and the bionic virus structure vaccine are subjected to booster immunization by using a Vero cell system.
Immunological analysis showed that the biomimetic viral structural vaccine of the present invention can induce a strong humoral immune response, which is manifested by binding antibodies against different types of SARS-CoV-2 antigens up to 10 7-108, and neutralizing antibodies of 10 2-103 (fig. 9).
ELISPOT detection: taking spleen from mice 28 days after the boost immunization of the bionic virus structure vaccine and the VLS vaccine group, grinding, and utilizing lymphocyte separation liquid (4 ml of mouse lymphocyte separation liquid is added into a 15ml centrifuge tube, and cell liquid of the ground spleen is slowly added into the 15ml centrifuge tube, taking care that lower lymphocyte separation liquid is not to be flushed up, and then placing the mice in a horizontal centrifuge for centrifugation at 800rpm for 30min at room temperature, wherein the rotating speed is adjusted to be 3-3); the medium layer lymphocyte liquid is sucked out, washed once by 1640 serum-free medium, the supernatant is discarded, and then the prepared lymphocyte is resuspended by serum-free medium. The pre-plated plates were then washed 3 times with PBS, the amount of cells added during IL-4 detection was 2x10ζ5/100. Mu.L per well, IFN- γ detection was added 1x10ζ5/100. Mu.L and 3. Mu.g/100. Mu.L of S1-RBD peptide (BA.4 & BA.5, B.1.529, BA.2.12.1, BA.2.75.2, BQ.1.1, XBB.1) and N protein (WT, BA.4, B.1.1.529) of different strains, respectively, were stimulated, incubated in a 37℃incubator for 36-40h, plates were removed for cold water cell disruption, IFN- γ and IL-4 antibodies (PBS buffer 1:1000 dilution), incubated 1.5h at 37℃for 4-5 times with PBS, then the corresponding secondary antibodies (PBS buffer 1:1000 dilution) were added for 1h at 37℃and the PBS wash plates were incubated 4-5 times, finally color development was performed with addition of color development solution.
The results show that the biomimetic virus structural vaccine obtained by the invention can induce strong specific T cell responses respectively aiming at the S antigen and the N antigen (figure 10).
According to the immunization mode of 0 day and 21 days, the K18-ACE2 transgenic mice are immunized by intramuscular injection with the bionic virus structure vaccine, the virus is counteracted (WT) by nasal cavity mode 45 days after the immunity is enhanced, and tissues such as heart, liver, spleen, lung, kidney, brain, spinal cord, lymph node, nasal cavity and the like are taken for 3 days and 5 days after the virus is counteracted to carry out disease load detection. The results show that the bionic virus structure vaccine obtained by the invention has better protection effect on virus attack, can obviously inhibit the load of the virus in each organ of an attack animal (figure 11), and avoids damage to organ tissues.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (7)
1. A buffer solution system is characterized in that the buffer solution system is obtained by taking water as a solvent and dissolving NaAc, naCl, mgCl 2, DTT and Fucose, the pH value of the buffer solution system is 6.0-6.4,
NaAc20mM；NaCl10mM； MgCl210mM； DTT1mM； Fucose0.001%w/v；
The buffer system can combine the N protein of the novel coronavirus with the mRNA molecule encoding the S1 antigen to form a bead-like biosynthesis body with a virus-like nucleocapsid structure.
2. A method for preparing a novel coronavirus N protein and S1 antigen-encoding mRNA molecule-bound biosynthesized body using the buffer system of claim 1, characterized in that: the method comprises the following steps:
(1) Dissolving mRNA of the novel coronavirus encoding S1 antigen in TE Buffer with pH=6.0-6.4, and configuring the concentration to be 1000ug/ml;
(2) Dissolving the prokaryotic expression new coronavirus N protein in TE Buffer with pH=6.8-7.2, and configuring the concentration to be 200 ug/ml;
(3) Adding the mRNA of the step (1) and the N protein of the step (2) into the buffer solution system of claim 1 in a concentration ratio of 5:1, and reacting at 35-39 ℃ for 28-32min to obtain a novel biosynthesis body formed by combining the coronavirus N protein and the mRNA molecule encoding the S1 antigen.
3. A novel coronavirus N protein obtained by the method of claim 2 and a biosynthesized body which encodes an S1 antigen mRNA molecule.
4. A gene delivery system comprising the biosynthesized body of claim 3 and a nanolipid particle; the nano lipid particles consist of DHA-1, DOTAP, DOPC and mPEG-DTA-1-2K.
5. A method of preparing a lipid particle structure encapsulating the biosynthesized body of claim 3, comprising the steps of:
(1) Lipids DHA-1, DOTAP, DOPC and mPEG-DTA-1-2K were combined at 56:10:27:1.6 molar ratio mixing in absolute ethanol; simultaneously diluting the biosynthesized body of claim 3 with a buffer containing NaAc 20mM,NaCL 10mM, mgCl 2 mM, DTT 1mM and trehalose at 0.001%;
(2) Preparing a lipid particle structure encapsulating the biosynthesized body using a microfluidic device NanoAssemblr R IgniteTM Model;
(3) Removing ethanol from the lipid particle structure of the wrapped biosynthesized body prepared in the step (2) to obtain a nitrogen-phosphorus molar ratio of 15-18:1, a liposome particle structure in which a biosynthesized body is encapsulated.
6. A lipid particle structure encapsulating a biosynthesized body obtained by the production method according to claim 5.
7. A method for preparing a novel coronavirus N protein and S1 antigen-encoding mRNA molecule combined biomimetic virus structural vaccine, which is characterized in that the vaccine is obtained by loading S1 protein in the lipid particle structure as claimed in claim 6,
The lipid particle structure was modified according to the mRNA encoding S1 antigen and S1 protein at 4:1, adding S1 protein, rotating and mixing at room temperature, standing at 4deg.C for 16 hr, and diluting with buffer solution containing NaAc 20 mM, naCL 10 mM, trehalose 0.001% w/v and glycerol 2.5%.
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| CN105073136A (en) * | 2013-03-15 | 2015-11-18 | 葛兰素史克知识产权第二有限公司 | Low concentration antibody formulations |
| CN116139108A (en) * | 2023-04-23 | 2023-05-23 | 威瑞生物科技(昆明)有限责任公司 | Lipid delivery system and viroid structure vaccine formed by same |
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| US20230024494A1 (en) * | 2021-06-08 | 2023-01-26 | Nirmidas Biotech, Inc. | New saliva-based lateral-flow antibody test platform for assessing infections and vaccination efficacy |
| CN116808190A (en) * | 2023-06-28 | 2023-09-29 | 苏州祥纳生物医药科技有限公司 | Preparation of SARS-Cov-2 mRNA universal vaccine |
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| CN105073136A (en) * | 2013-03-15 | 2015-11-18 | 葛兰素史克知识产权第二有限公司 | Low concentration antibody formulations |
| CN116139108A (en) * | 2023-04-23 | 2023-05-23 | 威瑞生物科技(昆明)有限责任公司 | Lipid delivery system and viroid structure vaccine formed by same |
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