CN111423486A - Renaturation method of new type coronavirus recombinant protein inclusion body - Google Patents

Abstract

The invention discloses a renaturation method of a novel coronavirus recombinant protein inclusion body. The renaturation method of the novel coronavirus recombinant protein inclusion body provided by the invention has the advantages that the renaturation is carried out by carrying out gradient elution on the denatured inclusion body dissolved product through a nickel chelating column and then using renaturation liquid containing a denaturant with the concentration gradient reduction of the denaturant at normal temperature, so that the flowing renaturation is carried out, the cost is reduced, the time required by the renaturation of the novel coronavirus recombinant protein inclusion body is obviously shortened, the renaturation of the novel coronavirus recombinant protein inclusion body at a low cost in a short time and in a large scale is favorably carried out, the recovery rate of the protein is high, the purity is high, the stability is strong, and the recovery method is favorable for obtaining a large amount of high-quality novel coronavirus protein for novel coronavirus detection and antibody preparation at a. The renaturation method of new type coronavirus RBD recombinant protein inclusion body is applicable to renaturation of new type coronavirus RBD recombinant protein inclusion body or new type coronavirus N recombinant protein inclusion body.

Description

Renaturation method of new type coronavirus recombinant protein inclusion body

Technical Field

The invention belongs to the technical field of biology, and relates to a renaturation method of a novel coronavirus recombinant protein inclusion body.

Background

The novel coronavirus (SARS-CoV-2 or COVID-19) is a new strain of coronavirus discovered in human body for the first time in 2019, is β coronavirus as the SARS coronavirus, has homology of 80% compared with the SARS coronavirus, is different from common cold or influenza, mainly causes lower respiratory tract infection, causes symptoms such as fever, cough, weakness, dyspnea and the like, and poses serious threat to public health.

At present, the effective detection means aiming at the novel coronavirus is PCR detection of virus nucleic acid and IgM/IgG detection of serum virus antibody. The existing virus nucleic acid detection kit for carrying out PCR detection on virus nucleic acid has high cost and long detection period. And the time can be obviously shortened by detecting the serum virus antibody IgM/IgG, and the result can be obtained in tens of minutes generally. Research shows that the S protein and the N protein used for detecting the virus antibody have better detection effect. Therefore, how to obtain a large amount of S protein and N protein in a short time at a low cost is significant. The Escherichia coli expression system is low in cost and short in period, and is undoubtedly a better choice.

The results of the present research show that the N recombinant protein obtained by the E.coli expression system is partially soluble, but still contains a large amount of inclusion bodies, and the inclusion bodies need to be further renatured to obtain a large amount of soluble N recombinant protein. The RBD protein, an important component of the S protein, is responsible for binding of the virus to ACE2, which infects host cells, and initiates cell infection. RBD is used as an important binding epitope for neutralizing antibody therapy, is used for blocking virus binding with cells, and is one of means for effectively preventing and treating novel coronavirus. A large amount of RBD protein is needed for detection and immunization in the antibody preparation and detection processes, and the quality requirements of the prepared antibody and the protein required for detection, such as purity, are high. The prior research shows that the RBD protein can be expressed and purified by a mammalian cell expression system and an insect cell baculovirus expression system, but the two methods have high cost and long time and need about 2 weeks. And the RBD protein is expressed by an inclusion body, and can be applied to novel coronavirus detection and antibody preparation only by further renaturation.

The conventional dilution renaturation method and dialysis renaturation method have the problems of long time consumption, low recovery rate and unsuitability for large-scale operation. How to renaturate the novel coronavirus recombinant protein inclusion body in a short time at a low cost on a large scale and improve the recovery rate and the quality of the novel coronavirus recombinant protein to obtain a large amount of high-quality novel coronavirus protein is of great significance to infection detection and treatment research of the novel coronavirus.

Disclosure of Invention

The first purpose of the invention is to provide a novel method for renaturation of coronavirus recombinant protein inclusion body. The renaturation method of the novel coronavirus recombinant protein inclusion body solves the problems of large amount of protein precipitation and low recovery rate brought by the conventional dialysis renaturation method at present through direct hanging column flowing renaturation, obviously shortens the time required by the renaturation of the recombinant protein inclusion body and reduces the cost.

According to one aspect of the present invention, there is provided a novel coronavirus recombinant protein inclusion body renaturation method, comprising the steps of:

(1) denaturing and dissolving the novel coronavirus recombinant protein inclusion body by using a denaturing solution containing a denaturant, centrifuging and taking supernatant to obtain an inclusion body dissolving product;

(2) after the inclusion body dissolved product passes through a nickel chelating column after balance, gradient elution is carried out at normal temperature by using renaturation liquid containing a denaturant and having the concentration of the denaturant reduced in a gradient manner, and an eluent is discarded;

(3) and (3) washing the nickel chelating column by using a washing buffer solution, then adding an elution buffer solution for elution, and collecting the eluent to obtain the nickel chelating column.

In some embodiments, the novel coronavirus RBD recombinant protein may be a novel coronavirus RBD recombinant protein or a novel coronavirus N recombinant protein. The renaturation method of the novel coronavirus RBD recombinant protein inclusion body provided by the invention is used for renaturating the novel coronavirus RBD recombinant protein inclusion body or the novel coronavirus N recombinant protein inclusion body which is produced by the expression of a prokaryotic expression system, the RBD recombinant protein and the N recombinant protein obtained after renaturation have high recovery rate and stable protein state, and the protein purity is higher than 95 percent and the stability is strong as proved by SDS-PAGE electrophoresis.

In some embodiments, the denaturant may be selected from one of urea, guanidine hydrochloride, and sarcosyl (Sarkosyl).

In some embodiments, the denaturing liquid may comprise: 10-100mM Tris, 100-.

In some embodiments, the denaturing liquid may comprise: 50mM Tris, 150mM NaCl and 8M urea, pH 8.0.

In some embodiments, the nickel chelating column is fully equilibrated with an equilibration buffer before passing the inclusion body lysate through the nickel chelating column, wherein the equilibration buffer may comprise: 50-200mM NaH₂PO₄5-20mM Tris and 6-10M urea, and the pH is 7.0-8.5.

In some embodiments, the equilibration buffer may comprise: 100mM NaH₂PO₄10mM Tris and 8M urea, pH 8.0.

In some embodiments, the naturaceutical composition further comprises: 300-800mM NaCl, 10-30mM Tris and 10-30% glycerol according to the volume percentage of the renaturation solution, wherein the pH value of the renaturation solution is 7.0-8.0.

In some embodiments, the naturaceutical composition further comprises: 500mM NaCl, 20mM Tris and 20% glycerol by volume percent of the renaturation solution, wherein the pH of the renaturation solution is 7.4.

In some embodiments, the column-passing time of the renaturation solution of each denaturant concentration gradient can be 0-20 min.

In some embodiments, the wash buffer may comprise: 10-100mM Tris, 50-200mM NaCl and 5-50mM imidazole, pH 7.0-8.5.

In some embodiments, the wash buffer may comprise: 50mM Tris, 150mM NaCl and 20mM imidazole, pH 8.0.

In some embodiments, the elution buffer may comprise: 10-100mM NaH₂PO₄50-500mM NaCl, 10-200mM imidazole, pH 7.5-8.5.

In some embodiments, the elution buffer may comprise: 50mM NaH₂PO₄300mM NaCl, 100mM imidazole, pH 8.0

In some embodiments, the method for renaturation of inclusion bodies of recombinant proteins of novel coronavirus may further comprise the following steps: the collected eluate was dialyzed using a dialysis buffer. Therefore, the method can remove small molecular substances in the eluent and has the function of buffer solution replacement, can achieve the aim of further purification, and is favorable for long-term storage of the protein.

In some embodiments, the dialysis buffer can include: 20-100mM Tris and 50-200mM NaCl, pH 7.0-8.5.

In some embodiments, the dialysis buffer can include: 50mM Tris and 150mM NaCl, pH 8.0.

The second purpose of the invention is to provide a preparation method of a novel coronavirus RBD recombinant protein. The preparation method of the novel coronavirus RBD recombinant protein provided by the invention comprises the steps of constructing a novel coronavirus RBD recombinant protein prokaryotic expression system, expressing and generating the RBD recombinant protein by utilizing the prokaryotic expression system, and renaturing and purifying the RBD recombinant protein inclusion body by combining the renaturation method of the recombinant protein inclusion body provided by the invention, wherein the required cost is only about 20-30% of that of a mammalian cell expression system and an insect cell baculovirus expression system, the cycle only needs about 3 days, and the prepared protein has high purity and strong stability. The preparation method of the novel coronavirus RBD recombinant protein provided by the invention has high yield, is suitable for large-scale operation, and can obtain a large amount of novel coronavirus RBD protein in a short time at low cost.

According to another aspect of the present invention, there is provided a method for preparing a novel coronavirus RBD recombinant protein, comprising the steps of:

1) constructing recombinant escherichia coli expressing a novel coronavirus RBD recombinant protein;

2) inoculating and culturing the recombinant escherichia coli, carrying out IPTG induced expression, and centrifugally collecting thalli sediment;

3) carrying out ultrasonic crushing on the thallus precipitate, then centrifuging and removing supernatant to obtain a novel coronavirus RBD recombinant protein inclusion body;

4) the novel coronavirus RBD recombinant protein inclusion body is renatured by using the renaturation method of the novel coronavirus RBD recombinant protein inclusion body provided by the invention, so that the novel coronavirus RBD recombinant protein is obtained.

In some embodiments, step 1) comprises:

amplifying a coding gene of the novel coronavirus RBD recombinant protein by using an upstream primer shown as SEQ ID NO. 3 and a downstream primer shown as SEQ ID NO. 4;

constructing a recombinant expression vector for expressing a novel coronavirus RBD recombinant protein;

and (3) transforming the recombinant expression vector into escherichia coli to obtain the recombinant escherichia coli for expressing the novel coronavirus RBD recombinant protein.

Drawings

FIG. 1 is a schematic diagram of pET-24a-RBD expression vector;

FIG. 2 is a schematic representation of the pET-24a-N expression vector;

FIG. 3 shows the SDS-PAGE result of the RBD recombinant protein of the novel coronavirus;

FIG. 4 shows SDS-PAGE electrophoresis results of N recombinant proteins of the novel coronavirus, wherein lane A is an electrophoresis lane of the N recombinant proteins obtained after elution, collection, and concentration by dialysis after renaturation by the renaturation method of the present invention, lane B is an electrophoresis lane of the N recombinant proteins obtained after renaturation by conventional renaturation by dialysis and concentration by dialysis, and lane C is an electrophoresis lane of the soluble N recombinant proteins obtained after purification, collection, and concentration by dialysis by a Ni column.

Detailed Description

The present invention will be described in further detail with reference to the following examples and the accompanying drawings. Unless otherwise specified, the reagents used in the examples are all conventional products commercially available, and the technical means used are conventional means well known to those skilled in the art, and the specific conditions are not specified in the examples, and are performed according to conventional conditions or conditions recommended by the manufacturer.

Example 1 expression of novel coronavirus RBD recombinant protein and inclusion body renaturation

Expression of novel coronavirus RBD recombinant protein

(1) Introducing a BamH I restriction enzyme site at the N-terminal of the novel coronavirus RBD protein, and introducing an Xho I restriction enzyme site at the C-terminal to obtain the novel coronavirus RBD recombinant protein. Designing a synthetic primer according to the gene sequence of the coding novel coronavirus RBD recombinant protein for PCR amplification, and purifying PCR amplification products to obtain the PCR amplification products of the coding gene of the novel coronavirus RBD recombinant protein. Wherein, the whole gene sequence of the novel coronavirus RBD protein is shown as SEQ ID NO. 1, and the upstream primer of PCR amplification is as follows: acacggatccAGAGTCCAACCAACAGAATC (SEQ ID NO:3), and the downstream primer is: agagctcgagGAAATTGACACATTTGTTTTTAACC (SEQ ID NO: 4).

(2) Respectively carrying out enzyme digestion treatment on a PCR amplification product of a coding gene of the novel coronavirus RBD recombinant protein and a pET-24a vector, then connecting the PCR amplification product and the pET-24a vector, inserting the novel coronavirus RBD protein gene between BamH I and Xho I enzyme digestion sites of the pET-24a expression vector to obtain the pET-24a-RBD expression vector, then converting the pET-24a-RBD expression vector into DH5a competent cells, selecting a single clone bacterial colony which is correct through sequencing verification, culturing, and then extracting a plasmid. A schematic diagram of the pET-24a-RBD expression vector is shown in FIG. 1.

(3) The plasmid is transformed into B L21 (DE3) escherichia coli, a monoclonal expression colony is obtained by plating culture, the monoclonal expression colony is picked up and cultured in L B culture medium containing 3m L kanamycin (Kanr) at 37 ℃ and 250rpm overnight, the cultured bacterial liquid is transferred to 200m L L B culture medium containing Kanr according to the ratio of 1: 100 on the next day, and the culture is carried out for about 3 hours until the OD is 0.6-0.8, so that the target protein expression can be induced.

(4) 1m L bacterial liquid is taken and sample preparation is taken as an uninduced control group, IPTG (Isopropyl- β -D-Thiogalactoside ) with the final concentration of 1mM is added into the other bacterial liquid, the culture is continued for 16 to 18 hours at 26 ℃ and 250rpm, and then the bacterial sedimentation is collected at 4 ℃ and 4500rpm after centrifugation for 15 min.

(5) Carrying out ultrasonic disruption on the escherichia coli thallus precipitate, wherein an ultrasonic buffer solution is as follows: 50mM Tris, 500mM NaCl, pH 8.0. The ultrasonic conditions are as follows: 4 ℃, 40% of power and 15min of ultrasonic time. And (3) placing the bacterial liquid after the ultrasonic crushing in a high-speed refrigerated centrifuge, and centrifuging for 10min at 4 ℃ and 12000 rpm. The supernatant was discarded and the inclusion bodies of the novel coronavirus RBD recombinant protein remained in the pellet.

Second, renaturation of new type coronavirus RBD recombinant protein inclusion body

(1) Adding a denaturing solution into a novel coronavirus RBD recombinant protein inclusion body, and performing denaturation and dissolution at the temperature of 4 ℃ for 1 hour to denature and dissolve the inclusion body, wherein the denaturing solution contains: 50mM Tris, 150mM NaCl and 8M urea, pH 8.0. And (3) placing the denatured solution in a high-speed refrigerated centrifuge, centrifuging at 4 ℃ and 12000rpm for 10min, removing undissolved precipitate, and reserving a supernatant part after denaturation and dissolution to obtain an inclusion body lysate.

(2) After passing an equilibration buffer solution through a nickel chelating column (Ni Focure 6FF, column volume 2m L) to fully equilibrate the nickel chelating column, adding an inclusion body lysate, passing the inclusion body lysate through the column, reasonably controlling the flow-through speed and monitoring the A280 value of the effluent liquid in real time to verify whether the protein has been bound to the nickel chelating column, wherein the equilibration buffer solution contains 100mM NaH₂PO₄10mM Tris and 8M urea, pH 8.0.

(3) After the inclusion body lysate passes through the column, renaturation liquid containing a denaturant and with the concentration of the denaturant being reduced in a gradient manner is used for gradient elution at normal temperature, so that the protein is recovered to a normal folding structure from a completely extended denaturation state. Wherein, the renaturation liquid contains: 500mM NaCl, 20mM Tris, 1-6M urea and 20% glycerol (by volume of the renaturation solution) and a pH value of 7.4. The concentration change of the denaturant urea is 6M-5M-4M-3M-2M-1M, the gradient is reduced, and the column passing time of the renaturation solution of each denaturant concentration gradient is 0-20 min. Specific gradient renaturation procedures are shown in table 1.

TABLE 1 gradient renaturation procedure

Time of day	Concentration of urea in renaturation liquid	Volume of renaturation liquid
			0-20min	6M	4mL
20-40min	5M	4mL
			40-60min	4M	4mL
60-80min	3M	4mL
			80-100min	2M	4mL
100-120min	1M	4mL

(4) After all renaturation liquid passes through the column, adding washing buffer solution into the nickel chelating column to remove part of impure protein and precipitate impuritiesAnd finally, adding an elution buffer solution, and eluting and collecting the eluent. Wherein the wash buffer comprises: 50mM Tris, 150mM NaCl, 20mM imidazole (imidazole), pH 8.0; elution buffer containing 50mM NaH₂PO₄300mM NaCl, 100mM imidazole, pH 8.0.

(5) And (3) dialyzing the collected eluent by using a 5kb filter membrane at the temperature of 4 ℃ for liquid exchange to obtain the novel coronavirus RBD recombinant protein. The dialysis buffer contained: 50mM Tris, 150mM NaCl, pH 8.0.

And (3) carrying out UV detection and quantification on the obtained novel coronavirus RBD recombinant protein, and carrying out SDS-PAGE electrophoresis quality control on the protein quality, wherein the electrophoresis result is shown in figure 3.

The novel coronavirus RBD recombinant protein finally obtained by expressing by using a prokaryotic expression system and combining the renaturation method provided by the invention has the purity of more than 95 percent, strong stability and high recovery rate, and 0.4mg of protein can be obtained per ml of bacteria by the renaturation method provided by the invention.

Example 2 expression of novel coronavirus N recombinant protein and inclusion body renaturation

The method comprises the following steps:

(1) introducing Sac I restriction enzyme cutting site at the N end of the novel coronavirus N protein, and introducing 6 × His and Not I restriction enzyme cutting site at the C end to obtain the novel coronavirus N recombinant protein. Designing a synthetic primer according to the gene sequence of the N recombinant protein of the novel coronavirus for PCR amplification, and purifying a PCR amplification product to obtain a PCR amplification product of the coding gene of the N recombinant protein of the novel coronavirus, wherein the whole gene sequence of the N protein of the novel coronavirus is shown as SEQ ID NO:2, and the upstream primer of the PCR amplification is as follows: tgtcgagctcATGTCTGATAATGGACCC (SEQ ID NO:5), the downstream primer is: gagtgcggccgcTTAGTGATGGTGGTGATGGTGGGCCTGAGTTGAGTCAGC (SEQ ID NO: 6).

(2) Respectively carrying out enzyme digestion on a PCR amplification product of a coding gene of the novel coronavirus N recombinant protein and a pET-24a vector, then connecting the PCR amplification product and the pET-24a vector, inserting the novel coronavirus N protein gene into the pET-24a expression vector to obtain the pET-24a-N expression vector, then converting the pET-24a-N expression vector into DH5a competent cells, selecting a single clone colony which is verified to be correct through sequencing for culture, and then extracting a plasmid. A schematic diagram of the pET-24a-N expression vector is shown in FIG. 2.

(3) Transforming B L (DE3) escherichia coli by the plasmid, performing plate-laying culture to obtain a monoclonal expression colony, picking the monoclonal colony in a L B culture medium containing Kanr at 3m L, culturing at 37 ℃ and 250rpm overnight, transferring the cultured bacterial liquid into a L B culture medium containing Kanr at a ratio of 1: 100 on the next day, and culturing for about 3 hours until OD is 0.6-0.8, so that the target protein can be induced to express.

(4) Collecting bacterial solution of 1m L, preparing sample as non-induced control group, adding IPTG with final concentration of 1mM, culturing at 26 deg.C and 250rpm for 16-18 hr, centrifuging at 4 deg.C and 4500rpm, and collecting thallus precipitate after 15 min.

(5) Carrying out ultrasonic disruption on the escherichia coli thallus precipitate, wherein an ultrasonic buffer solution is as follows: 50mM Tris, 500mM NaCl, pH 8.0. The ultrasonic conditions are as follows: 4 ℃, 40% of power and 15min of ultrasonic time. And (3) placing the ultrasonically crushed bacterial liquid in a high-speed freezing centrifuge, centrifuging at 4 ℃ and 12000rpm for 10min, respectively collecting supernatant and precipitate, and keeping the novel coronavirus N recombinant protein inclusion body in the precipitate. Purifying the supernatant according to a conventional nickel chelating column purification process to obtain the soluble N recombinant protein. Precipitation the inclusion bodies of the novel coronavirus N recombinant protein were renatured as described in example 1 for inclusion bodies of the novel coronavirus RBD recombinant protein.

Taking the novel coronavirus N recombinant protein obtained after dialysis and liquid exchange for UV detection and quantification, performing SDS-PAGE electrophoresis quality control on the protein quality, and renaturing to obtain the novel coronavirus N recombinant protein, wherein the purity of the novel coronavirus N recombinant protein is more than 95%, the stability is high, the recovery rate is high, and about 0.6mg of protein can be obtained per milliliter of bacteria.

In order to confirm the quality of renaturation protein, the N recombinant protein (FIG. 4 lane A) obtained after renaturation and dialysis concentration according to the renaturation method of the present example, the N recombinant protein (FIG. 4 lane B) obtained after renaturation and dialysis concentration according to the conventional dialysis renaturation method, and the soluble N recombinant protein (FIG. 4 lane C) collected by Ni column purification and dialysis concentration were subjected to electrophoresis at the same time. From the results of fig. 4, it can be seen that: (1) the N recombinant protein obtained by the renaturation method on the column has high purity and the molecular weight is the same as that of the soluble N recombinant protein; (2) the yield of the N recombinant protein obtained by the renaturation method is obviously higher than that of the N recombinant protein and soluble N recombinant protein obtained by conventional dialysis renaturation; (3) the lane C contains multiple bands, which indicates that the purity of the N recombinant protein obtained by the method of the invention is obviously superior to that of the soluble N recombinant protein which is roughly purified by a Ni column.

What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention.

Sequence listing

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Claims (10)

1. The renaturation method of new-type coronavirus recombinant protein inclusion body includes the following steps:

(1) denaturing and dissolving the novel coronavirus recombinant protein inclusion body by using a denaturing solution containing a denaturant, centrifuging and taking supernatant to obtain an inclusion body dissolving product;

(2) after the inclusion body dissolved product passes through a nickel chelating column after balance, gradient elution is carried out at normal temperature by using renaturation liquid containing a denaturant and having the concentration of the denaturant reduced in a gradient manner, and an eluent is discarded;

(3) and (3) washing the nickel chelating column by using a washing buffer solution, then adding an elution buffer solution for elution, and collecting the eluent to obtain the nickel chelating column.

2. Renaturation process according to claim 1, characterized in that said novel coronavirus recombinant protein is a novel coronavirus RBD recombinant protein or a novel coronavirus N recombinant protein.

3. The renaturation method according to claim 2, characterized in that said denaturant is selected from one of urea, guanidine hydrochloride and sodium lauryl sarcosinate.

4. Renaturation method according to claim 3, characterized in that said denaturing liquids comprise: 10-100mM Tris, 100-; preferably, the denaturing solution comprises: 50mM Tris, 150mM NaCl and 8M urea, pH 8.0.

5. The renaturation method according to claim 4, characterized in that said renaturation liquid further comprises: 300-800mM NaCl, 10-30mM Tris and 10-30% glycerol according to the volume percentage of the renaturation liquid, wherein the pH value of the renaturation liquid is 7.0-8.0; preferably, the renaturation liquid further comprises: 500mM NaCl, 20mM Tris and 20% glycerol by volume percent of the renaturation solution, wherein the pH of the renaturation solution is 7.4.

6. Renaturation method according to claim 5, characterized in that said washing buffer comprises: 10-100mM Tris, 50-200mM NaCl and 5-50mM imidazole, pH 7.0-8.5; preferably, the wash buffer comprises: 50mM Tris, 150mM NaCl and 20mM imidazole, pH 8.0.

7. Renaturation method according to claim 6, characterized in that said elution buffer comprises: 10-100mM NaH₂PO₄50-500mM NaCl, 10-200mM imidazole, pH 7.5-8.5; preferably, the elution buffer comprises: 50mM NaH₂PO₄300mM NaCl, 100mM imidazole, pH 8.0.

8. Renaturation method according to any one of claims 1 to 7, characterized in that it further comprises the following steps: dialyzing the collected eluate against a dialysis buffer, wherein the dialysis buffer comprises: 20-100mM Tris and 50-200mM NaCl, pH 7.0-8.5; preferably, the dialysis buffer comprises: 50mM Tris and 150mM NaCl, pH 8.0.

9. The preparation method of the novel coronavirus RBD recombinant protein comprises the following steps:

1) constructing recombinant escherichia coli expressing a novel coronavirus RBD recombinant protein;

2) inoculating and culturing the recombinant escherichia coli, carrying out IPTG induced expression, and centrifugally collecting thalli sediment;

3) carrying out ultrasonic crushing on the thallus precipitate, then centrifuging and removing supernatant to obtain a novel coronavirus RBD recombinant protein inclusion body;

4) renaturation of novel inclusion bodies of recombinant proteins of coronavirus RBD using the method for renaturation of inclusion bodies of recombinant proteins of coronavirus RBD according to any one of claims 1 to 8, thereby obtaining novel recombinant proteins of coronavirus RBD.

10. The method for preparing according to claim 9, wherein the step 1) includes:

amplifying a coding gene of the novel coronavirus RBD recombinant protein by using an upstream primer shown as SEQ ID NO. 3 and a downstream primer shown as SEQ ID NO. 4;

constructing a recombinant expression vector for expressing a novel coronavirus RBD recombinant protein;

and (3) transforming the recombinant expression vector into escherichia coli to obtain the recombinant escherichia coli for expressing the novel coronavirus RBD recombinant protein.

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