Expression and purification method of coronavirus protein

Abstract

The invention belongs to the technical field of biology, and particularly relates to an expression and purification method of coronavirus protein, through designed recombinant plasmids, optimized host strain selection, an accurate induction expression strategy and an efficient multi-stage purification technology, the method not only improves the total yield of the protein, but also ensures the biological activity of the protein, and the method is suitable for industrial production. According to the method, the coronavirus protein is more suitable for serving as a vaccine candidate component or a research tool, so that the method shows wide application potential in the field of biomedicine, the problems that the coronavirus protein is poor in solubility and difficult to purify in a traditional expression system are solved, powerful support is provided for related research of the coronavirus, and the method is suitable for popularization and application. The method aims at remarkably improving the soluble expression level of protein in escherichia coli and simplifying the purification process at the same time so as to obtain high-purity and high-activity coronavirus protein.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for expressing and purifying coronavirus proteins. Background Art

[0002] In the biomedical field, especially in vaccine development and disease research, high-quality coronavirus proteins are crucial resources. However, coronavirus proteins are often difficult to express in high yields and maintain good solubility in conventional expression systems. Although Escherichia coli, a commonly used protein expression host, has the advantages of low cost and fast growth, coronavirus proteins often form inclusion bodies when expressed in vivo, resulting in complex protein recovery and impaired activity.

[0003] The main problem encountered when traditional expression methods are used to produce coronavirus proteins in E. coli is the low protein expression, especially in soluble form, which limits its efficacy and practicality in subsequent applications. In addition, the formation of inclusion bodies increases the difficulty of downstream purification and renaturation, consuming time and resources. Summary of the invention

[0004] The purpose of the present invention is to provide a method for expressing and purifying a coronavirus protein, thereby improving the soluble expression level of the protein in Escherichia coli and simplifying the purification process to obtain a high-purity and high-activity coronavirus protein, so as to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention adopts the following technical scheme: a method for expressing and purifying coronavirus protein, comprising the following steps:

[0006] Constructing a recombinant plasmid, wherein the recombinant plasmid comprises a coronavirus protein gene and a promoter sequence, wherein the promoter sequence can be recognized by Escherichia coli to promote the transcription of the target protein;

[0007] Transforming the recombinant plasmid into a host Escherichia coli cell, wherein the host Escherichia coli has a regulatory system that matches the promoter;

[0008] Cultivating the E. coli strain to start the expression of coronavirus protein, and when the bacteria grow to the logarithmic phase, adding an inducer to enhance the expression level of the coronavirus protein;

[0009] The E. coli is lysed by ultrasonic disruption or osmotic pressure shock treatment to release the coronavirus protein expressed in the cell;

[0010] Affinity chromatography is used to perform preliminary purification based on the tag on the coronavirus protein. The preliminary purified coronavirus protein is further purified by size exclusion chromatography or ion exchange chromatography, and the purified coronavirus protein is buffer replaced and concentrated;

[0011] Test and evaluate the purity and activity of coronavirus proteins to ensure they meet standards for biomedical research or vaccine preparation.

[0012] Preferably, the construction of the recombinant plasmid comprises:

[0013] Select and synthesize coronavirus protein coding sequences, ensure that they are compatible with E. coli codon preference, and design and clone promoter sequences that are temperature-sensitive and allow for enhanced transcription of coronavirus protein genes at specific temperatures;

[0014] Connect the coronavirus protein coding sequence and the temperature-sensitive promoter to form a fusion gene, which is then inserted into the vector backbone;

[0015] Verify the constructed recombinant plasmid by sequencing to confirm that the coronavirus protein coding sequence and promoter sequence are correctly integrated into the vector.

[0016] Preferably, the step of transforming the recombinant plasmid into a host Escherichia coli cell comprises:

[0017] Prepare competent E. coli cells, which have been pre-cooled to increase their membrane permeability so that they can absorb foreign DNA;

[0018] Mix the prepared competent cells with the recombinant plasmid and incubate briefly on ice to allow the plasmid DNA to fully contact the cells;

[0019] Through heat shock treatment, the E. coli cells in the mixture are prompted to absorb the recombinant plasmid and complete the transformation process;

[0020] The treated cells were cultured in a recovery medium to restore cell viability and screen out E. coli clones that successfully carried the recombinant plasmid.

[0021] Preferably, the culturing of the Escherichia coli strain comprises:

[0022] The selected E. coli single clone that successfully carries the recombinant plasmid is inoculated into a selective medium containing antibiotics to inhibit the growth of untransformed strains;

[0023] The bacterial solution was cultured using a fed-batch culture strategy, and the growth status of the bacteria during the culture process was monitored. When the OD600 value reached a predetermined range, the bacteria entered the logarithmic growth phase and were ready to induce the expression of coronavirus proteins;

[0024] Adding an inducer to the culture medium activates the promoter linked to the coronavirus protein gene, thereby initiating efficient expression of the coronavirus protein.

[0025] Preferably, the adding of an inducer to enhance the expression level of coronavirus protein comprises:

[0026] Determine the bacterial density and calculate the amount of inducer required based on the results;

[0027] The calculated induction dose was added to the bacterial culture and the culture conditions were adjusted;

[0028] Bacterial cultures are sampled and analyzed regularly to monitor the expression of coronavirus proteins until the expected level is reached.

[0029] Preferably, the release of intracellularly expressed coronavirus proteins comprises:

[0030] Collect the E. coli cells that have completed protein expression, remove the culture medium residue by centrifugation, and retain the cell precipitate;

[0031] Resuspend the bacterial pellet in a mild buffer to prepare for pre-lysis to protect the protein structure;

[0032] Ultrasonic disruption was used to treat bacterial suspensions to selectively destroy cell walls and membrane structures and release intracellular proteins.

[0033] The cell debris and protein mixture produced after the lysis reaction are separated and centrifuged or filtered to obtain the supernatant, which contains the target coronavirus protein.

[0034] Preferably, the preliminary purification comprises:

[0035] Prepare an affinity chromatography column loaded with a specific ligand that can specifically bind to the tag on the coronavirus protein in the cleavage product;

[0036] The supernatant containing coronavirus protein is slowly passed through the affinity chromatography column to allow the protein tag to bind to the ligand with high affinity;

[0037] The column was washed with a buffer solution to remove non-specifically adsorbed impurities. The target coronavirus protein bound to the column was gradually eluted by changing the composition of the mobile phase, and the eluate was collected for the next step of purification.

[0038] Preferably, the further purification by size exclusion chromatography or ion exchange chromatography comprises:

[0039] Select a size exclusion chromatography or ion exchange chromatography column that is appropriate for the target coronavirus protein, optimized based on its molecular size or charge characteristics;

[0040] Load the initially purified coronavirus protein sample onto the selected chromatography column to start the purification process;

[0041] Control the flow rate and elution conditions so that the loaded protein sample undergoes separation in the chromatographic column, and different components elute in order of molecular size or charge distribution;

[0042] The eluted components were collected, the purity and homogeneity of the coronavirus protein were tested by analysis, and the peak segments with higher purity were selected and merged.

[0043] Preferably, the buffer replacement and concentration of the purified coronavirus protein comprises:

[0044] Prepare buffer systems to replace residual salts or other solvent components during purification;

[0045] The purified coronavirus protein solution is fully exchanged with the buffer system using dialysis technology;

[0046] After buffer exchange, the protein concentration was assessed and the protein solution was concentrated by ultrafiltration.

[0047] Preferably, the detection and evaluation of the purity and activity of coronavirus proteins comprises:

[0048] The concentrated coronavirus proteins were analyzed by SDS-PAGE to assess their purity;

[0049] Utilize ELISA to quantitatively detect the antigenicity of the analyzed coronavirus protein and confirm its biological activity;

[0050] Conduct stability tests to monitor changes in the activity of tested coronavirus proteins under different storage conditions to ensure they remain stable over a specified period of time;

[0051] Based on the test results, a comprehensive assessment is made as to whether the coronavirus protein meets the purity and activity standards required for biomedical research or vaccine preparation.

[0052] Technical effects and advantages of the present invention: Compared with the prior art, the method for expressing and purifying coronavirus protein proposed in the present invention has the following advantages:

[0053] The present invention uses designed recombinant plasmids, optimized host strain selection, precise induced expression strategy and efficient multi-stage purification technology. This method not only improves the overall protein yield, but also ensures the biological activity of the protein, making it more suitable as a vaccine candidate component or research tool, thereby showing broad application potential in the biomedical field. This method overcomes the problems of poor solubility and difficulty in purification of coronavirus proteins in traditional expression systems, provides strong support for coronavirus-related research, and aims to significantly improve the soluble expression level of proteins in Escherichia coli, while simplifying the purification process to obtain high-purity and high-activity coronavirus proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The present invention is a flowchart of a method for expressing and purifying coronavirus protein. DETAILED DESCRIPTION

[0055] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0056] The present invention provides a method for expressing and purifying coronavirus protein, such as Figure 1 As shown, the following steps are included:

[0057] Constructing a recombinant plasmid, wherein the recombinant plasmid comprises a coronavirus protein gene and a promoter sequence, wherein the promoter sequence can be recognized by Escherichia coli to promote the transcription of the target protein;

[0058] Construction of the recombinant plasmid further includes: selecting and synthesizing a coronavirus protein coding sequence to ensure that it is compatible with the codon preference of Escherichia coli, designing and cloning a promoter sequence, wherein the promoter is temperature-sensitive and allows enhanced transcription of the coronavirus protein gene at a specific temperature; connecting the coronavirus protein coding sequence and the temperature-sensitive promoter to form a fusion gene, which is then inserted into the vector backbone; verifying the constructed recombinant plasmid by sequencing to confirm that the coronavirus protein coding sequence and the promoter sequence are correctly integrated into the vector.

[0059] The specific steps are as follows:

[0060] Step 1: Select and synthesize coronavirus protein coding sequences

[0061] Gene sequence acquisition: First, obtain the gene sequence of the target coronavirus protein from the NCBI database or other reliable resources.

[0062] Codon optimization: Bioinformatics software is used to optimize the codons of the original gene sequence to ensure that it matches the codon usage preference of E. coli to improve translation efficiency and protein expression.

[0063] Step 2: Design and clone a temperature-sensitive promoter

[0064] Promoter design: Choose a temperature-sensitive promoter, such as the λ phage promoter, which barely initiates transcription at lower temperatures but significantly increases transcription activity at higher temperatures (such as 42°C).

[0065] PCR amplification: Using a plasmid template containing a promoter sequence, design a pair of primers and amplify the desired promoter fragment through PCR reaction.

[0066] Restriction enzyme digestion: Treat the PCR product with the corresponding restriction enzyme to prepare for ligation with the vector.

[0067] Step 3: Construction of recombinant plasmid

[0068] Vector preparation: Select a suitable expression vector, such as the pET series, and perform the same restriction endonuclease digestion to remove the original promoter region.

[0069] Ligation: Ligate the promoter fragment digested in step 2 with the vector treated in step 3 to form a fusion gene. Ligation reactions are usually performed at room temperature or lower using T4 DNA ligase.

[0070] Transformation: The ligation product was transformed into E. coli DH5α competent cells to amplify the recombinant plasmid.

[0071] Step 4: Verify the recombinant plasmid

[0072] Colony PCR: Select positive clones and preliminarily screen strains containing recombinant plasmids through colony PCR.

[0073] Plasmid extraction: Extract plasmid DNA from the screened strains for subsequent sequencing verification.

[0074] Sequencing verification: The extracted plasmid DNA is sent to a sequencing service provider for bidirectional sequencing of the coronavirus protein coding sequence and promoter sequence to confirm the correctness and integrity of the sequence.

[0075] Sequence alignment analysis: Align the sequencing results with the original designed sequence to ensure that no mutations or errors have been introduced.

[0076] Through the above steps, a recombinant plasmid containing the coronavirus protein coding sequence and the temperature-sensitive promoter can be constructed, laying a solid foundation for subsequent protein expression and purification.

[0077] Transforming the recombinant plasmid into a host Escherichia coli cell, wherein the host Escherichia coli has a regulatory system that matches the promoter;

[0078] Transforming the recombinant plasmid into the host Escherichia coli cells further includes: preparing competent Escherichia coli cells, the cells have been pre-cooled to increase their membrane permeability so that they absorb exogenous DNA; mixing the prepared competent cells with the recombinant plasmid, and briefly incubating on ice to allow the plasmid DNA to fully contact the cells; through heat shock treatment, the Escherichia coli cells in the mixture are prompted to absorb the recombinant plasmid to complete the transformation process; and culturing the treated cells in a recovery medium to restore cell viability and screen out Escherichia coli clones that successfully carry the recombinant plasmid.

[0079] The specific steps are as follows:

[0080] Step 1: Prepare competent E. coli cells

[0081] Cell culture: Pick a single E. coli colony from a plate containing appropriate antibiotics, inoculate it into LB liquid medium, and culture it in a shaking incubator at 37°C overnight.

[0082] Dilution inoculation: The next day, take an appropriate amount of overnight culture and inoculate it into fresh LB liquid medium at a ratio of 1:100, and continue to culture at 37°C until OD 600 About 0.5-0.6.

[0083] Precooling: Place the culture on ice for about 10 minutes, then add 100 mL of precooled CaCl per 500 mL of culture. 2 Solution (0.1 M) was gently shaken to mix and ensure even coverage of the cells.

[0084] Prepare competent cells: centrifuge the cells at 4000 rpm for 5 minutes at 4°C, discard the supernatant, and wash with pre-cooled CaCl 2 The cell pellet was resuspended in 1 mL CaCl 2 in solution.

[0085] Step 2: Mix plasmid DNA with competent cells

[0086] Plasmid DNA: Take a certain amount of recombinant plasmid DNA (usually 50ng-1μg) and add it to a small centrifuge tube containing competent cells.

[0087] Incubation: Place on ice for 30 minutes to allow the plasmid DNA to fully contact the cells.

[0088] Step 3: Heat shock treatment

[0089] Heat shock: Rapidly transfer the mixture containing plasmid DNA and competent cells to a 42°C water bath, heat shock for 90 seconds, and immediately return to ice to cool for 2 minutes.

[0090] Step 4: Resuscitation and screening

[0091] Recovery: Add 1 mL of LB liquid medium to the mixture and incubate on a shaker at 37°C for 1 hour to restore cell viability.

[0092] Plate coating: Take an appropriate amount of the revived cell suspension and spread it on an LB solid plate containing the corresponding antibiotics, ensuring that the concentration of the antibiotics is sufficient to inhibit the growth of untransformed cells but will not affect the cells carrying the recombinant plasmid.

[0093] Culture: Place the plate upside down in a 37°C constant temperature incubator overnight and observe whether single colonies are formed.

[0094] Screening: The next day, pick single colonies from the plate and verify whether these colonies successfully carry the recombinant plasmid by colony PCR or small amount of plasmid extraction and sequencing.

[0095] Through the above steps, the recombinant plasmid can be effectively introduced into E. coli cells to prepare for subsequent protein expression experiments.

[0096] Cultivating the E. coli strain to start the expression of coronavirus protein, and when the bacteria grow to the logarithmic phase, adding an inducer to enhance the expression level of the coronavirus protein;

[0097] The culturing of the Escherichia coli strain further includes: inoculating the screened Escherichia coli monoclone that successfully carries the recombinant plasmid into a selective culture medium containing antibiotics to inhibit the growth of untransformed strains; culturing the bacterial solution using a fed-batch culture strategy, monitoring the growth state of the bacteria during the culture process, and when the OD600 value reaches a predetermined range, that is, the bacteria enter a logarithmic growth phase, preparing to induce the expression of coronavirus protein; adding an inducer to the culture medium to activate a promoter connected to the coronavirus protein gene, thereby initiating efficient expression of the coronavirus protein.

[0098] Adding an inducer to enhance the expression level of coronavirus protein further includes: measuring the bacterial density, and calculating the amount of inducer required based on the measurement results; adding the calculated inducer dose to the bacterial culture and adjusting the culture conditions; regularly sampling and analyzing the bacterial culture to monitor the expression level of the coronavirus protein until the expected level is reached.

[0099] The details are as follows:

[0100] Step 1: Inoculation and cultivation

[0101] Inoculation: Pick a single colony from a selective LB plate containing antibiotics, inoculate it into 5 mL of LB liquid medium containing the same antibiotics, and culture it overnight at 37°C and 220 rpm with shaking to prepare seed solution.

[0102] Expand the culture: The next day, take 1 mL of the overnight culture and inoculate it into 500 mL of LB liquid culture medium containing the corresponding antibiotics at a ratio of 1:100. Use a fed-batch culture strategy, that is, gradually replenish nutrients as the bacteria grow to avoid growth stagnation caused by nutrient depletion.

[0103] Step 2: Monitor growth status

[0104] Timed sampling: Take 1 mL of culture medium every hour and measure OD using a spectrophotometer 600value to monitor bacterial growth.

[0105] Logarithmic period judgment: when OD 600 When the value reaches 0.6-0.8, the bacteria enter the logarithmic growth phase, during which the cell metabolism is active, and it is an ideal time to add inducers.

[0106] Step 3: Induce protein expression

[0107] Inducer preparation: Depending on the type of promoter used, select a suitable inducer, such as IPTG (isopropylthiogalactoside) for PLAC promoter, or other inducers such as lactose, arabinose, etc., and adjust the concentration as needed.

[0108] Adding inducer: Calculate and add an appropriate amount of inducer to the culture medium, usually the concentration of IPTG is 1mM. At the same time, adjust the culture temperature as needed, for example, some proteins are better expressed at lower temperatures (such as 16°C or 30°C).

[0109] Expression monitoring: After induction, continue to culture the bacteria and take samples at regular intervals to detect the expression level of coronavirus protein using methods such as SDS-PAGE or Western blot until the expected expression level is reached.

[0110] Step 4: Adjust culture conditions

[0111] pH adjustment: As the culture progresses, the pH of the culture medium may change and should be adjusted in time to maintain a suitable growth environment.

[0112] Oxygen saturation monitoring: Especially in large-scale fermentation processes, it is necessary to ensure that there is sufficient dissolved oxygen in the culture medium, which can be achieved by adjusting the stirring speed and ventilation volume.

[0113] Through the above steps, the expression of coronavirus protein can be effectively initiated and enhanced in Escherichia coli, laying the foundation for subsequent protein purification and application.

[0114] The E. coli is lysed by ultrasonic disruption or osmotic pressure shock treatment to release the coronavirus protein expressed in the cell;

[0115] The method of releasing the intracellularly expressed coronavirus protein further includes: collecting Escherichia coli cells that have completed protein expression, removing culture medium residues through centrifugation, and retaining the cell precipitate; resuspending the cell precipitate with a mild buffer to prepare for pretreatment before lysis to protect the protein structure; applying ultrasonic disruption to the cell suspension to selectively destroy the cell wall and membrane structure to release the intracellular protein; separating the cell fragments and protein mixture produced after the lysis reaction, and obtaining the supernatant by centrifugation or filtration, which contains the target coronavirus protein.

[0116] The details are as follows:

[0117] Step 1: Bacteria collection and pretreatment

[0118] Bacterial collection: After the coronavirus protein expression is completed, the E. coli culture is centrifuged at 4°C, 4000 rpm for 15 minutes, the bacterial precipitate is collected, and the supernatant (i.e., the culture medium residue) is removed.

[0119] Resuspend the cells: Use an appropriate amount of PBS (phosphate buffered saline) or a buffer containing protease inhibitors to gently resuspend the cell pellet to ensure that the cells are evenly dispersed and prepare for lysis.

[0120] Step 2: Ultrasonication

[0121] Set the ultrasonic instrument parameters: adjust the working parameters of the ultrasonic disruptor according to the volume of the bacteria. Generally, the power is set at 20% to 50%, and the pulse mode is 2 seconds on / 2 seconds off to reduce damage to the protein structure.

[0122] Lysis treatment: Place the resuspended bacteria in an ice bath and use an ultrasonic disruptor. The ultrasonic time should not exceed 30 seconds each time, with intermittent cooling until the cells are completely lysed. Multiple cycles are usually required.

[0123] Step 3: Lysate Clarification

[0124] Centrifugation clarification: The lysate after ultrasonic disruption was centrifuged at 12,000 rpm for 30 min at 4°C to separate unbroken cell debris and other insoluble particles.

[0125] Collect the supernatant: Carefully aspirate the supernatant and transfer it to a new centrifuge tube. This is the crude extract containing soluble coronavirus protein.

[0126] Step 4: Protein detection and storage

[0127] Protein detection: Use the BCA protein quantification kit to determine the concentration of protein in the supernatant, and preliminarily identify the presence and integrity of coronavirus proteins by methods such as SDS-PAGE or Western blot.

[0128] Protein preservation: Aliquot the supernatant containing coronavirus protein, add an appropriate amount of glycerol, and store it at -80°C for a long time pending the subsequent purification steps.

[0129] Through the above steps, the intracellularly expressed coronavirus protein can be effectively released from Escherichia coli, preparing for subsequent purification and application.

[0130] Affinity chromatography is used to perform preliminary purification based on the tag on the coronavirus protein. The preliminary purified coronavirus protein is further purified by size exclusion chromatography or ion exchange chromatography, and the purified coronavirus protein is buffer replaced and concentrated;

[0131] The preliminary purification further includes: preparing an affinity chromatography column loaded with a specific ligand, which can specifically bind to the tag on the coronavirus protein in the cleavage product; slowly passing the supernatant containing the coronavirus protein through the affinity chromatography column to allow the protein tag to bind to the ligand with high affinity; washing the chromatography column with a buffer solution to remove non-specifically adsorbed impurities, gradually eluting the target coronavirus protein bound to the chromatography column by changing the composition of the mobile phase, and collecting the eluate for the next step of purification.

[0132] Further purification by size exclusion chromatography or ion exchange chromatography further includes: selecting a size exclusion chromatography or ion exchange chromatography column suitable for the target coronavirus protein, and optimizing it according to its molecular size or charge characteristics; loading the preliminary purified coronavirus protein sample onto the selected chromatographic column to start the purification process; controlling the flow rate and elution conditions so that the loaded protein sample undergoes separation in the chromatographic column, and different components are eluted in order of molecular size or charge distribution; collecting the eluted components, analyzing and detecting the purity and homogeneity of the coronavirus protein, and selecting peak segments with higher purity for merging.

[0133] Buffer replacement and concentration of purified coronavirus protein further includes: preparing a buffer system to replace residual salts or other solvent components during the purification process; using dialysis technology to fully exchange the purified coronavirus protein solution with the buffer system; after buffer replacement, evaluating the protein concentration, and concentrating the protein solution by ultrafiltration.

[0134] The details are as follows:

[0135] Step 1: Initial purification by affinity chromatography

[0136] Affinity chromatography column preparation: Select an affinity chromatography column with a His-tag or GST-tag ligand, and select the corresponding ligand according to the type of protein tag. Pre-equilibrate the column with an equilibrium buffer (such as PBS containing 20 mM imidazole).

[0137] Loading: Slowly flow the supernatant containing coronavirus protein through the affinity chromatography column at an appropriate flow rate to ensure that the tagged protein is fully bound to the ligand on the column.

[0138] Washing: Use a washing buffer (such as PBS containing 50 mM imidazole) to flow through the chromatography column to remove non-specifically bound proteins and impurities.

[0139] Elution: Gradually increase the imidazole concentration of the elution buffer (e.g., PBS containing 250 mM imidazole) to elute the specifically bound coronavirus protein. Collect the eluate for subsequent further purification.

[0140] Step 2: Further purification by size exclusion chromatography or ion exchange chromatography

[0141] Select a chromatographic column: Based on the molecular size or charge properties of the coronavirus protein, select a size exclusion column with an appropriate pore size or an ion exchange column with an appropriate charge.

[0142] Loading: Load the initially purified coronavirus protein solution onto the chromatographic column.

[0143] Elution: Use gradient elution to control the flow rate and composition of the eluent so that protein components of different sizes or charges are eluted in sequence.

[0144] Collection and detection: Collect the elution peaks, detect the purity and homogeneity by SDS-PAGE or HPLC, and select the peak segments with higher purity for merging.

[0145] Step 3: Buffer exchange and concentration

[0146] Buffer preparation: Prepare appropriate buffer according to subsequent application requirements to remove residual salts or other solvents during the purification process.

[0147] Dialysis exchange: The further purified coronavirus protein solution is placed in a dialysis bag and placed in a buffer, and the external buffer is replaced through a dialysis process until the salt concentration of the internal solution drops to the desired level.

[0148] Ultrafiltration concentration: Use an ultrafiltration membrane (select a membrane with an appropriate cutoff value based on the molecular weight of the protein) to concentrate the dialyzed protein solution until the ideal protein concentration is reached.

[0149] Through the above steps, coronavirus proteins can be efficiently purified from complex lysates while ensuring their activity and stability, providing high-quality protein materials for subsequent research or applications.

[0150] Test and evaluate the purity and activity of coronavirus proteins to ensure they meet standards for biomedical research or vaccine preparation.

[0151] Testing and evaluating the purity and activity of coronavirus proteins further include: using SDS-PAGE to analyze the concentrated coronavirus proteins to evaluate their purity; using enzyme-linked immunosorbent assay to quantitatively detect the antigenicity of the analyzed coronavirus proteins and confirm their biological activity; conducting stability tests to monitor changes in the activity of the tested coronavirus proteins under different storage conditions to ensure that they remain stable within a specified time; and based on the test results, comprehensively evaluating whether the coronavirus proteins meet the purity and activity standards required for biomedical research or vaccine preparation.

[0152] The details are as follows:

[0153] Step 1: Purity analysis by SDS-PAGE

[0154] Sample preparation: Mix the purified coronavirus protein with protein loading buffer (containing a reducing agent such as β-mercaptoethanol) and boil for 5 minutes to denature the protein so that SDS can fully bind.

[0155] Electrophoresis: Use 10% or higher concentration polyacrylamide gel for SDS-PAGE electrophoresis and select the appropriate gel concentration according to the molecular weight of the protein.

[0156] Staining and analysis: After electrophoresis, use Coomassie Brilliant Blue staining to evaluate the clarity and purity of the protein bands on the gel. A single, distinct band indicates high protein purity.

[0157] Step 2: Enzyme-linked immunosorbent assay (ELISA) to detect antigenicity

[0158] Coating: Coat the purified coronavirus protein onto a microplate, usually at a concentration of 1-10 μg / mL, overnight at 4°C.

[0159] Blocking: Use blocking solution (such as PBS containing BSA) to block nonspecific binding sites to prevent nonspecific adsorption of antibodies.

[0160] Antibody testing: Add specific antibodies against coronavirus proteins, usually mouse or rabbit polyclonal or monoclonal antibodies against coronavirus proteins.

[0161] Color development: Add enzyme-labeled secondary antibodies, then add substrates for color development, read the absorbance value with an enzyme reader, and quantitatively analyze the protein antigenicity.

[0162] Step 3: Stability test

[0163] Sample packaging: The purified coronavirus protein is divided into several small portions and stored under different conditions (such as -80°C, -20°C, 4°C).

[0164] Regular testing: Take out samples at set time points (such as 1 week, 1 month, 3 months, etc.), repeat the tests in steps 1 and 2, and compare the purity and activity changes of the protein under different storage conditions.

[0165] Step 4: Comprehensive Assessment

[0166] Data collation: The results of SDS-PAGE, ELISA and stability tests were summarized to evaluate the purity, antigenicity and stability of the protein.

[0167] Standard control: Compare the test results with known standards or data reported in the literature to determine whether the coronavirus protein meets the requirements for biomedical research or vaccine preparation.

[0168] Conclusion and suggestions: If the protein purity and activity meet the standards, it can be used for subsequent experiments or vaccine preparation. If it does not meet the standards, the purification process needs to be reviewed, the problem needs to be found, and the conditions need to be optimized before testing.

[0169] In summary, the present invention not only improves the overall yield of the protein, but also ensures the biological activity of the protein through the designed recombinant plasmid, optimized host strain selection, precise induced expression strategy and efficient multi-stage purification technology, making it more suitable as a vaccine candidate component or research tool, thus showing a wide range of application potential in the biomedical field. This method overcomes the problems of poor solubility and difficult purification of coronavirus proteins in traditional expression systems, provides strong support for coronavirus-related research, and aims to significantly improve the soluble expression level of the protein in Escherichia coli, while simplifying the purification process to obtain high-purity and high-activity coronavirus proteins.

[0170] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for expressing and purifying coronavirus protein, It is characterized in that The following steps are involved: Constructing a recombinant plasmid, wherein the recombinant plasmid comprises a coronavirus protein gene and a promoter sequence, wherein the promoter sequence can be recognized by Escherichia coli to promote the transcription of the target protein; Transforming the recombinant plasmid into a host Escherichia coli cell, wherein the host Escherichia coli has a regulatory system that matches the promoter; Cultivating the E. coli strain to initiate the expression of coronavirus protein, and when the bacteria grow to the logarithmic phase, adding an inducer to enhance the expression level of the coronavirus protein; The E. coli is lysed by ultrasonic disruption or osmotic pressure shock treatment to release the coronavirus protein expressed in the cell; Affinity chromatography is used to perform preliminary purification based on the tag on the coronavirus protein. The preliminary purified coronavirus protein is further purified by size exclusion chromatography or ion exchange chromatography, and the purified coronavirus protein is buffer replaced and concentrated; Test and evaluate the purity and activity of coronavirus proteins to ensure they meet standards for biomedical research or vaccine preparation.

2. A method for expressing and purifying coronavirus protein according to claim 1, Features: The construction of the recombinant plasmid comprises: Select and synthesize coronavirus protein coding sequences, ensure that they are compatible with E. coli codon preference, and design and clone promoter sequences that are temperature-sensitive and allow for enhanced transcription of coronavirus protein genes at specific temperatures; Connect the coronavirus protein coding sequence and the temperature-sensitive promoter to form a fusion gene, which is then inserted into the vector backbone; Verify the constructed recombinant plasmid by sequencing to confirm that the coronavirus protein coding sequence and promoter sequence are correctly integrated into the vector.

3. A method for expressing and purifying coronavirus protein according to claim 2, Features: The method of transforming the recombinant plasmid into a host Escherichia coli cell comprises: Prepare competent E. coli cells, which have been pre-cooled to increase their membrane permeability so that they can absorb foreign DNA; Mix the prepared competent cells with the recombinant plasmid and incubate briefly on ice to allow the plasmid DNA to fully contact the cells; Through heat shock treatment, the E. coli cells in the mixture are prompted to absorb the recombinant plasmid and complete the transformation process; The treated cells were cultured in a recovery medium to restore cell viability and screen out E. coli clones that successfully carried the recombinant plasmid.

4. A method for expressing and purifying coronavirus protein according to claim 3, Features: The cultured Escherichia coli strain comprises: The selected E. coli single clone that successfully carries the recombinant plasmid is inoculated into a selective medium containing antibiotics to inhibit the growth of untransformed strains; The bacterial solution was cultured using a fed-batch culture strategy, and the growth status of the bacteria during the culture process was monitored. When the OD600 value reached a predetermined range, the bacteria entered the logarithmic growth phase and were ready to induce the expression of coronavirus proteins; Adding an inducer to the culture medium activates the promoter linked to the coronavirus protein gene, thereby initiating efficient expression of the coronavirus protein.

5. A method for expressing and purifying coronavirus protein according to claim 4, Features: The inducing agent is added to enhance the expression level of coronavirus protein, including: Determine the bacterial density and calculate the amount of inducer required based on the results; The calculated induction dose was added to the bacterial culture and the culture conditions were adjusted; Bacterial cultures are sampled and analyzed regularly to monitor the expression of coronavirus proteins until the expected level is reached.

6. A method for expressing and purifying coronavirus protein according to claim 5, Features: The release of intracellularly expressed coronavirus proteins comprises: Collect the E. coli cells that have completed protein expression, remove the culture medium residue by centrifugation, and retain the cell precipitate; Resuspend the bacterial pellet in a mild buffer to prepare for pre-lysis to protect the protein structure; Ultrasonic disruption was used to treat bacterial suspensions to selectively destroy cell walls and membrane structures and release intracellular proteins. The cell debris and protein mixture produced after the lysis reaction are separated and centrifuged or filtered to obtain the supernatant, which contains the target coronavirus protein.

7. A method for expressing and purifying coronavirus protein according to claim 6, Features: The preliminary purification comprises: Prepare an affinity chromatography column loaded with a specific ligand that can specifically bind to the tag on the coronavirus protein in the cleavage product; The supernatant containing coronavirus protein is slowly passed through the affinity chromatography column to allow the protein tag to bind to the ligand with high affinity; The column was washed with a buffer solution to remove non-specifically adsorbed impurities. The target coronavirus protein bound to the column was gradually eluted by changing the composition of the mobile phase, and the eluate was collected for the next step of purification.

8. A method for expressing and purifying coronavirus protein according to claim 7, Features: The further purification by size exclusion chromatography or ion exchange chromatography comprises: Select a size exclusion chromatography or ion exchange chromatography column that is appropriate for the target coronavirus protein, optimized based on its molecular size or charge characteristics; Load the initially purified coronavirus protein sample onto the selected chromatography column to start the purification process; Control the flow rate and elution conditions so that the loaded protein sample undergoes separation in the chromatographic column, and different components elute in order of molecular size or charge distribution; The eluted components were collected, the purity and homogeneity of the coronavirus protein were tested by analysis, and the peak segments with higher purity were selected and merged.

9. A method for expressing and purifying coronavirus protein according to claim 8, Features: The method of performing buffer replacement and concentration on the purified coronavirus protein comprises: Prepare buffer systems to replace residual salts or other solvent components during purification; The purified coronavirus protein solution is fully exchanged with the buffer system using dialysis technology; After buffer exchange, the protein concentration was assessed and the protein solution was concentrated by ultrafiltration.

10. A method for expressing and purifying coronavirus protein according to claim 9, Features: The detection and evaluation of the purity and activity of coronavirus proteins include: The concentrated coronavirus proteins were analyzed by SDS-PAGE to assess their purity; Utilize ELISA to quantitatively detect the antigenicity of the analyzed coronavirus protein and confirm its biological activity; Conduct stability tests to monitor changes in the activity of tested coronavirus proteins under different storage conditions to ensure they remain stable over a specified period of time; Based on the test results, a comprehensive assessment is made as to whether the coronavirus protein meets the purity and activity standards required for biomedical research or vaccine preparation.

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