Preparation method of recombinant protein of novel coronavirus

Abstract

The present invention relates to the field of vaccine fermentation technology, and particularly to a method for preparing a recombinant protein of novel coronavirus. In the present invention, the DNA sequence of the ELABELA signal peptide and the novel coronavirus antigen spike-RBD protein gene are introduced into Pichia pastoris, induced to culture, and purified to obtain a recombinant strain of Pichia pastoris and a recombinant protein of novel coronavirus. The present invention has the advantages of low culture cost, high protein yield, good batch-to-batch stability, short culture period, and few impurities for subsequent treatment; at the same time, in the early stage of the present invention, the gene sequence inserted into Pichia pastoris and during the culture, the overall quality of Pichia pastoris can be improved, which is beneficial to the quality of the final product.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine fermentation, and particularly relates to a method for preparing a recombinant protein of novel coronavirus. Background Art

[0002] Coronaviruses are known to cause colds and more severe diseases such as Middle East Respiratory Syndrome and Severe Acute Respiratory Syndrome. The novel coronavirus is a new strain of coronavirus that has never been found in humans before. Common symptoms after human infection with coronavirus include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. The treatment of coronavirus disease 2019 mainly focuses on symptomatic treatment and is supplemented by vaccine defense. The main vaccines currently available on the market still have defects such as low protection rate and inability to cope with virus mutant strains. The overall technical routes for the development of novel coronavirus vaccines can be generally divided into three categories: The first category is the classical technical route; the second major category is virus vector vaccines mainly based on viral genetic material; the third major category is viral protein antigens expressed by means of genetic engineering, including subunit vaccines and virus-like particle vaccines. Currently, the main expression vectors selected for recombinant subunit vaccines and VLPs vaccines are Vero cells, which have disadvantages such as relatively high culture costs, easy contamination by microorganisms, many types of impurity proteins and difficult separation, and low content of target proteins. Summary of the Invention

[0003] In view of this, the present invention provides a method for preparing a recombinant protein of novel coronavirus, which has the advantages of low culture cost, high protein yield, good batch-to-batch stability, short culture period, and few impurity proteins, facilitating subsequent processing.

[0004] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides a method for preparing a recombinant protein of novel coronavirus, comprising the following steps:

[0006] Step (1): Introduce the coding genes of the ELABELA signal peptide and the novel coronavirus S-RBD protein into Pichia pastoris to obtain a recombinant Pichia pastoris strain;

[0007] Step (2): Perform monoclonal picking, culturing, and cryopreserving on the recombinant Pichia pastoris strain obtained in step (1) to obtain Pichia pastoris engineering bacteria seeds;

[0008] Step (3): Take the Pichia pastoris engineering bacteria seeds obtained in step (2), inoculate them into a culture medium for activation until the OD of the culture medium 600 reaches 4.0 - 7.0 to obtain an activation solution; culture the activation solution until the OD of the activation solution 600 reaches 6.0 - 9.0 to obtain fermentation broth I;

[0009] Step (4): Mix the fermentation broth I described in step (3) with the basal salt medium BSM, ferment until the wet cell weight reaches 120 - 160 g / L, mix with an inducer, and induce to obtain fermentation broth II;

[0010] Step (5): Centrifuge the fermentation broth II described in step (4), collect the supernatant to obtain the recombinant protein of the novel coronavirus.

[0011] In some specific embodiments of the present invention, the coding genes of the ELABELA signal peptide and the novel coronavirus S-RBD protein in the above preparation method have the nucleotide sequence shown in SEQ ID NO.1; and / or

[0012] The ELABELA signal peptide and the novel coronavirus S-RBD protein have the amino acid sequence shown in SEQ ID NO.2.

[0013] In some specific embodiments of the present invention, the inoculation in step (3) of the above preparation method includes taking 200 μL of the Pichia pastoris engineered bacteria seeds and inoculating them into 20 mL of the medium; and / or

[0014] The medium includes YPD medium; and / or

[0015] The rotation speed of the activation is 160 - 180 r / min; and / or

[0016] The temperature of the activation is 30 - 32 °C; and / or

[0017] The time of the activation is 18 - 30 h.

[0018] In some specific embodiments of the present invention, the cultivation in step (3) of the above preparation method includes the step of taking 2 - 5 mL of the activation solution and inoculating it into 200 - 500 mL of YPD medium; and / or

[0019] The rotation speed of the cultivation is 180 r / min; and / or

[0020] The temperature of the cultivation is 30 °C; and / or

[0021] The time of the cultivation is 18 - 30 h.

[0022] In some specific embodiments of the present invention, the preparation method of 5 L of the basic salt medium BSM in step (4) of the above preparation method includes: mixing 2.0 - 3.0 g of K2SO4, 8.0 - 12.0 g of MgSO4, 0.5 - 1.0 g of CaSO4, 20.0 - 25.0 g of ammonium dihydrogen phosphate, 1 - 2 mL of H3PO4, 2 - 3 g of KOH, 200 - 250 g of glycerol, 20 - 30 mL of PTM1, and 0.5 - 1.0 mL of antifoaming agent, adding deionized water to make the volume up to 5 L, and adjusting the pH to 4.5 - 5.0 with phosphoric acid; and / or

[0023] The H3PO4 includes 85% concentrated phosphoric acid; and / or

[0024] The antifoaming agent includes Antifoam produced by SIGMA; and / or

[0025] The preparation method of the PTM1 includes: weighing 6.0 g of copper sulfate, 0.09 g of potassium iodide, 3.0 g of manganese sulfate, 0.2 g of sodium molybdate dihydrate, 0.02 g of boric acid, 0.5 g of cobalt chloride, 20 g of zinc chloride, 65 g of ferrous sulfate, and 5.0 mL of sulfuric acid, adding water to make the volume up to 1000 mL, and filtering and sterilizing with a 0.22 μm filter membrane; and / or

[0026] The copper sulfate includes copper(II) sulfate pentahydrate; and / or

[0027] The manganese sulfate includes manganese sulfate monohydrate; and / or

[0028] The cobalt chloride includes cobalt(II) chloride hexahydrate; and / or

[0029] The ferrous sulfate includes ferrous sulfate heptahydrate.

[0030] In some specific embodiments of the present invention, the volume ratio of the fermentation broth I to the basic salt medium BSM in step (4) of the above preparation method is 1:15 - 1:7.5; or

[0031] In step (4), the volume of the basic salt medium BSM is 5 L, and the volume of the fermentation broth I is 333 mL - 667 mL.

[0032] In some specific embodiments of the present invention, the fermentation temperature in step (4) of the above preparation method is 29.0 - 30.0 °C; and / or

[0033] The initial pH of the fermentation is 5.0 - 5.3; and / or

[0034] The starting rotation speed of the fermentation is 200 rpm; and / or

[0035] The dissolved oxygen of the fermentation is 10% - 50%; and / or

[0036] The fermentation time is 16 to 20 hours.

[0037] In some specific embodiments of the present invention, the induction in step (4) of the above preparation method includes: feeding an inducer at a feeding rate of 2.0 to 4.0 g / L / h; and / or

[0038] The dissolved oxygen during the induction is 10% to 50%; and / or

[0039] The inducer includes methanol or a mixture of methanol and glycerol; and / or

[0040] The induction time is 48 to 72 hours.

[0041] In some specific embodiments of the present invention, the rotation speed of the centrifugation in step (5) of the above preparation method is 5000 rpm; and / or

[0042] The recombinant protein of the novel coronavirus includes the S-RBD protein of the novel coronavirus.

[0043] The present invention also provides an antigen protein, the coding gene of which has the nucleotide sequence shown in SEQ ID NO.1.

[0044] The present invention also provides the use of the recombinant protein of the novel coronavirus or the above antigen protein prepared by the above preparation method in the preparation of a novel coronavirus vaccine.

[0045] The method of the present invention has the following effects:

[0046] The present invention has the advantages of low culture cost, high protein yield, good batch-to-batch stability, short culture period, and few impurity proteins, which is convenient for subsequent processing; at the same time, in the early stage of inserting the gene sequence into Pichia pastoris and culturing, the overall quality of Pichia pastoris can be improved, which is beneficial to the quality of the final product. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0048] Figure 1 Electrophoresis diagram of Example 1 is shown;

[0049] Figure 2 Electrophoresis diagram of Example 2 is shown;

[0050] Figure 3 Electrophoresis diagram of Example 3 is shown;

[0051] Figure 4 Electrophoresis diagram of the comparative example is shown. Detailed implementation mode

[0052] The present invention discloses a method for preparing a recombinant protein of novel coronavirus. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The method and application of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0053] The present invention provides the following technical solution: A fermentation method for preparing a novel coronavirus vaccine using Pichia pastoris, comprising the following steps:

[0054] Step 1: Pretreatment of Pichia pastoris. Add the coding gene of the novel coronavirus S-RBD and the DNA sequence of the ELABELA signal peptide into Pichia pastoris to obtain a Pichia pastoris genetic engineering strain capable of secreting and expressing the recombinant novel coronavirus S-RBD protein;

[0055] Step 2: Isolation and purification of the Pichia pastoris genetic engineering strain. Streak and isolate the genetic engineering bacteria obtained in Step 1 on a solid plate, pick a single colony for culture and freeze preservation to obtain the Pichia pastoris engineering bacteria seeds;

[0056] Step 3: Secondary amplification culture of Pichia pastoris seeds. Take out the Pichia pastoris engineering bacteria seeds, after melting, pipette 200 μL and inoculate it into 20 mL of YPD medium, culture at 180 r / min and 30 °C for 24 h, OD 600 At 4.1, and there is no contamination of miscellaneous bacteria under microscopic examination. Pipette 5 mL of the qualified activation solution into 500 mL of YPD medium, culture at 180 rpm and 30 °C for 24 h, OD 600 At 5.4, and there is no contamination of miscellaneous bacteria under microscopic examination to obtain the culture solution;

[0057] Step 4: Add 5 L of the basic salt medium BSM to the fermenter, perform sterilization at 121 °C, after sterilization and cooling, add 500 mL of the culture solution obtained in Step 3, and culture at pH 5.0, an initial rotation speed of 200 rpm, and a fermentation temperature of 29.5 °C for 16 h, and the wet weight reaches 132.5 g / L;

[0058] Step 5: Add a pure methanol solution to the fermentation broth in Step 4 at a rate of 2 g / L / h. Continue to culture for 48 h to obtain a fermentation broth containing the recombinant novel coronavirus S-RBD protein;

[0059] Step 6: Harvest the fermentation supernatant. Centrifuge the fermentation broth with a refrigerated centrifuge and collect the fermentation supernatant, and weigh to obtain 4.1 L of the fermentation supernatant;

[0060] Step 7: The fermented supernatant harvested in Step 6 can be directly used for purifying the recombinant novel coronavirus S-RBD protein, or can be stored short-term at 2-8°C or long-term at -20°C or below.

[0061] As a preferred technical solution of the present invention, the inducer in Step 5 is any one of methanol, a mixture of methanol and glycerol.

[0062] As a preferred technical solution of the present invention, the ratio of the culture medium in Step 4 to the BSM fermentation broth is 1:15 to 1:7.5.

[0063] As a preferred technical solution of the present invention, the BSM medium in Step 4 is prepared with 2.0 - 3.0 g of K2SO4, 8.0 - 12 g of MgSO4, 0.5 - 1.0 g of CaSO4, 1 - 2 mL of H3PO4, 2 - 3 g of KOH, 200 g of glycerol, 20 - 30 mL of PTM1, 0.5 mL of antifoaming agent, and deionized water is added to 5 L, and the sterilization condition is 121°C for 30 minutes, and after sterilization, it is cooled to 32°C; inoculated at a ratio of 1:15, the fermentation temperature is 32.0 ± 0.5°C, the initial pH is 5.30 ± 0.05, and the starting rotation speed is 200 rpm for cultivation.

[0064] As a preferred technical solution of the present invention, the signal peptide in Step 1 is purified by cation exchange chromatography at a pH between 10.0 and 13.0.

[0065] As a preferred technical solution of the present invention, in Step 3, the recombinant Pichia pastoris and the virus mutant strain genes are mixed and self-assembled into an anti-gene mutation encoding.

[0066] As a preferred technical solution of the present invention, in Step 7, the labeling information of the fermentation broth includes indicating the batch number, date, and weight.

[0067] The sequence information involved in the present invention is as follows:

[0068] The coding gene sequences (codon-optimized) of the ELABELA signal peptide and the novel coronavirus S-RBD protein:

[0069]

[0070]

[0071] The amino acid sequences of the ELABELA signal peptide and the novel coronavirus S-RBD protein:

[0072]

[0073] Unless otherwise specified, the raw materials, reagents, consumables and instruments used in the present invention are all ordinary commercially available products and can be purchased from the market.

[0074] The present invention will be further described below in conjunction with embodiments:

[0075] Embodiment 1

[0076] The present invention provides a technical solution: a fermentation method for preparing an anti-SARS-CoV-2 vaccine using Pichia pastoris, comprising the following steps:

[0077] Step 1: Pretreatment of Pichia pastoris. Add the SARS-CoV-2 S-RBD gene and the DNA sequence of the signal peptide into Pichia pastoris to obtain a genetically engineered strain of Pichia pastoris capable of secreting and expressing recombinant SARS-CoV-2 S-RBD protein.

[0078] Step 2: Isolation and purification of the genetically engineered strain of Pichia pastoris. Streak and isolate the genetically engineered bacteria obtained in Step 1 on a solid plate, pick a single colony for culture and cryopreservation to obtain the seed of the Pichia pastoris engineering bacteria.

[0079] Step 3: Secondary amplification culture of the Pichia pastoris seeds. Take out the seeds of the Pichia pastoris engineering bacteria, after melting, pipette 200 μL and inoculate it into 20 mL of YPD medium, culture at 180 r / min and 30 °C for 24 h, OD 600 At 4.1, and there is no contamination of miscellaneous bacteria under microscopic examination. Pipette 2 mL of the qualified activation solution into 200 mL of YPD medium, culture at 180 rpm and 30 °C for 24 h, OD 600 At 5.4, and there is no contamination of miscellaneous bacteria under microscopic examination to obtain the culture solution.

[0080] Step 4: Add 5 L of the basic salt medium BSM into the fermenter, sterilize at 121 °C, after sterilization and cooling, add 500 mL of the culture solution obtained in Step 3, culture at pH 5.0, an initial rotation speed of 200 rpm and a fermentation temperature of 29.5 °C for 16 h, and the wet weight reaches 132.5 g / L.

[0081] Step 5: Add pure methanol solution to the fermentation broth obtained in Step 4 at a rate of 2 g / L / h. Continue to culture for 48 h to obtain a fermentation broth containing recombinant SARS-CoV-2 S-RBD protein.

[0082] Step 6: Harvest the fermentation supernatant. Centrifuge the fermentation broth with a refrigerated centrifuge and collect the fermentation supernatant, weigh to obtain 4.1 L of the fermentation supernatant.

[0083] Step 7: The fermentation supernatant harvested in Step 6 can be directly used for purifying recombinant SARS-CoV-2 S-RBD protein, or can be stored short-term at 2-8 °C or long-term at -20 °C or below.

[0084] In this embodiment, the inducer in step five is pure methanol, or a mixture of methanol and glycerol can also be selected as the inducer.

[0085] In this embodiment, the culture medium BSM in step four is composed of 3.0 g of K2SO4, 9.1 g of MgSO4, 0.6 g of CaSO4, 1 mL of H3PO4, 25.0 g of ammonium dihydrogen phosphate, 2 g of KOH, 200 g of glycerol, adding 30 mL of PTM1 (6.0 g of CuSO4·5H2O, 0.008 g of KI, 3.0 g of MnSO4, 0.2 g of NaMoO4, 0.02 g of H3BO3, 20.0 g of ZnSO4, 0.5 g of CoCl2, 65.0 g of FeSO4·7H2O, 0.2 g of biotin, 5.0 mL of H2SO4, deionized water is added to 1 L), 0.5 mL of antifoaming agent, and deionized water is added to 5 L, and the sterilization conditions are 121 °C for 30 minutes, and after sterilization, it is cooled to 32 °C;

[0086] In this embodiment, the culture is inoculated at a ratio of 1:10 in step four, the fermentation temperature is set at 29.5 °C, the initial pH is 5.0, the initial rotation speed is 200 rpm for cultivation, the ventilation volume is 0.5 L / min, the DO control is set at 30%, the initial growth stage is about 16 hours, the dissolved oxygen value is maintained not less than 20%, when the carbon source is consumed, the dissolved oxygen value rises rapidly, and the wet cell weight reaches about 132.5 g / L.

[0087] In this embodiment, the flow rate of the inducer in step five is 2 mL / L / h (that is, 0.2% is added per hour).

[0088] Example 2

[0089] The differences from Example 1 are as follows:

[0090] Step 3: Secondary amplification culture of Pichia pastoris seeds. Take out the Pichia pastoris engineering bacteria seeds, after melting, suck 200 μL and inoculate it into 20 mL of YPD medium, cultivate at 180 r / min and 30 °C for 30 h, OD 600 At 4.8, suck 5 mL of the above liquid and inoculate it into 500 mL of YPD medium, cultivate at 180 rpm and 30 °C for 30 h, OD 600 At 6.0, microscopically check for no contamination by miscellaneous bacteria;

[0091] Step 4: Sterilize 5 L of the basic salt medium BSM at 121 °C and cool it, add 350 mL of the fermentation broth, and cultivate at pH 5.3, an initial rotation speed of 200 rpm, and a fermentation temperature of 30.5 °C for 18 h, and the wet weight reaches 128.5 g / L;

[0092] Step 5: The inducer is a methanol-glycerol mixture. Add a methanol-glycerol solution (methanol:glycerol = 4:1) to the fermentation broth from Step 4 at a rate of 3 g / L / h. Continue culturing for 48 h to obtain a fermentation broth containing the recombinant novel coronavirus S-RBD protein.

[0093] Example 3

[0094] It is different from those in Example 1 and Example 2 in that:

[0095] Step 4: The culture medium BSM is made of 2.5 g of K2SO4, 12.0 g of MgSO4, 1.0 g of CaSO4, 20.0 g of ammonium dihydrogen phosphate, 250 g of glycerol, add 20 mL of PTM1, 0.5 mL of antifoaming agent, and add deionized water to 5 L, and the sterilization conditions are 121 °C for 30 minutes;

[0096] Step 5: The inducer is a methanol-glycerol mixture (methanol:glycerol = 1:1). Add it to the fermentation broth from Step 4 at a rate of 4 g / L / h. Continue culturing for 60 h to obtain a fermentation broth containing the recombinant novel coronavirus S-RBD protein.

[0097] Comparative Example

[0098] It is different from that in Example 1 in that:

[0099] Step 5: The inducer is methanol. Add it to the fermentation broth from Step 4 at a rate of 8 g / L / h. Continue culturing for 60 h to obtain a fermentation broth containing the recombinant novel coronavirus S-RBD protein.

[0100] Results Figure 4 As shown, different from Examples 1 to 3 (the results are as Figures 1 to 3 shown), the expression level of the target protein reaches the highest at 36 h of fermentation, and there is an obvious tendency for the target protein to degrade after 36 h of induction.

[0101] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a recombinant protein of novel coronavirus, characterized in that, It includes the following steps: Step (1): Introduce the coding genes of the ELABELA signal peptide and the novel coronavirus S-RBD protein into Pichia pastoris to obtain a recombinant Pichia pastoris strain; Step (2): Pick monoclonal colonies from the recombinant Pichia pastoris strain in step (1), culture and cryopreserve them to obtain the seeds of the Pichia pastoris engineering bacteria; Step (3): Take the Pichia pastoris engineering bacteria seeds described in step (2), inoculate them into the culture medium for activation until the OD of the culture medium 600 reaches 4.0 - 7.0 to obtain the activation solution; Cultivate the activation solution until the OD of the activation solution 600 reaches 5.0 - 8.0 to obtain Fermentation Broth I; Step (4): Mix the fermentation broth I in step (3) with the basal salt medium BSM, ferment until the wet cell weight reaches 120-160 g / L, mix with an inducer, and induce to obtain fermentation broth II; Step (5): Centrifuge the fermentation broth II in step (4), collect the supernatant to obtain the novel coronavirus recombinant protein; The nucleotide sequences of the coding genes of the ELABELA signal peptide and the novel coronavirus S-RBD protein are as shown in SEQ ID NO.1; and / or The amino acid sequences of the ELABELA signal peptide and the novel coronavirus S-RBD protein are as shown in SEQ ID NO.2; The preparation method of 5 L of the basal salt medium BSM in step (4) includes: Mix 2.0-3.0 g of K2SO4, 8.0-12.0 g of MgSO4, 0.5-1.0 g of CaSO4, 20.0-25.0 g of ammonium dihydrogen phosphate, 1-2 mL of H3PO4, 2-3 g of KOH, 200-250 g of glycerol, 20-30 mL of PTM1 and 0.5-1.0 mL of antifoaming agent, add deionized water to make up the volume to 5 L, and adjust the pH to 4.5-5.0 with phosphoric acid; and / or The H3PO4 includes 85% concentrated phosphoric acid; and / or The antifoaming agent includes Antifoam; and / or The preparation method of the PTM1 includes: Weigh 6.0 g of copper sulfate, 0.09 g of potassium iodide, 3.0 g of manganese sulfate monohydrate, 0.2 g of sodium molybdate dihydrate, 0.02 g of boric acid, 0.5 g of cobalt chloride hexahydrate, 20 g of zinc chloride, 65 g of ferrous sulfate heptahydrate and 5.0 mL of concentrated sulfuric acid, add water to make up the volume to 1000 mL, and filter and sterilize with a 0.22 μm filter membrane; and / or The copper sulfate includes copper (II) sulfate pentahydrate; and / or The manganese sulfate includes manganese sulfate monohydrate; and / or The cobalt chloride includes cobalt chloride hexahydrate; and / or The ferrous sulfate includes ferrous sulfate heptahydrate; The induction in step (4) includes: Feeding the inducer at a feeding rate of 2.0-4.0 g / L / h; The dissolved oxygen of the induction is 10%-50%; The inducer includes methanol or a mixture of methanol and glycerol; The induction time is 48-72 hours.

2. The preparation method according to claim 1, characterized in that, The inoculation in step (3) includes taking 200 μL of the seeds of the Pichia pastoris engineering bacteria and inoculating them into 20 mL of the medium; and / or The medium includes YPD medium; and / or The rotation speed of the activation is 160-180 r / min; and / or The activation temperature is 30-32 °C; and / or The activation time is 18-30 h.

3. The preparation method according to claim 2, characterized in that, The cultivation described in step (3) includes the step of adding 2 - 5 mL of the activation solution into 200 - 500 mL of YPD medium; and / or The rotation speed of the cultivation is 180 r / min; and / or The temperature of the cultivation is 30 °C; and / or The time of the cultivation is 18 - 30 h.

4. The preparation method according to any one of claims 1 to 3, characterized in that, In step (4), the volume ratio of the fermentation broth I to the basal salt medium BSM is 1:15 - 1:7.5; or In step (4), the volume of the basal salt medium BSM is 5 L, and the volume of the fermentation broth I is 333 mL - 667 mL.

5. The preparation method according to claim 4, characterized in that, In step (4), the temperature of the fermentation is 29.0 - 30.0 °C; and / or The initial pH of the fermentation is 5.0 - 5.3; and / or The starting rotation speed of the fermentation is 200 rpm; and / or The dissolved oxygen of the fermentation is 10% - 50%; and / or The time of the fermentation is 16 - 20 hours.

6. The preparation method according to claim 5, characterized in that, In step (5), the rotation speed of the centrifugation is 5000 rpm; and / or The recombinant protein of the novel coronavirus includes the S-RBD protein of the novel coronavirus.

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