A SARS-CoV-2 coronavirus glycosylated RBD protein and its preparation and application

Abstract

The present invention discloses a glycosylated RBD protein of SARS-CoV-2 coronavirus. This glycosylated RBD protein, abbreviated as Tn-RBD, is composed of the RBD domain of the coronavirus S protein shown in the amino acid sequence as SEQ ID NO.1, with GalNAc residues conjugated at positions T323, T345, and S514. It is obtained by using conventional methods to obtain a non-glycosylated RBD protein, and then using human N-acetylgalactosaminyltransferase GalNAc-T to synthesize Tn-RBD in vitro and purifying it. The SARS-CoV-2 coronavirus RBD glycoprotein provided by the present invention helps to form vaccines or antiviral drugs for the prevention and treatment of SARS-CoV-2 coronavirus, providing a new approach for preventing the infection of novel coronavirus and having broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a glycosylated RBD protein of SARS-CoV-2 coronavirus, a preparation method thereof, and an application thereof. Background Art

[0002] As a key factor for coronavirus 2 (SARS-CoV-2) to enter host cells, the Spike (S) protein is a highly glycosylated protein composed of S1 and S2 subunits, which plays a role in host cell recognition (S1) and viral host cell membrane fusion (S2). The receptor binding domain (RBD, residues 319-541) on the S1 subunit directly contacts the human ACE2 (hACE2) receptor and plays an important role in the process of virus infection. Therefore, it is the most attractive antigen for inducing human immune responses and is also the main target for the development of therapeutic drugs, neutralizing antibodies, and vaccines. In RBD, two N-glycosylation sites (N331, N343) and multiple O-glycosylation sites have been identified, which play a key role in the binding to hACE2.

[0003] Recombinant RBD proteins have been used in candidate vaccines or therapeutic drugs, and multiple related products have been marketed or are in different clinical stages. Experiments have confirmed that different glycosylation modifications can affect the stability of recombinant proteins and therapeutic effects. Therefore, finding an RBD protein with better glycosylation modification as a candidate vaccine or therapeutic glycoprotein drug for SARS-CoV-2 has important theoretical significance and application value and has become a research hotspot at present. Retrieval shows that there are no relevant reports at home and abroad on the Tn (GalNAc-) modified RBD protein, its preparation method of in vitro glycosylation, and its application as a candidate vaccine or antiviral drug for novel coronavirus. Summary of the Invention

[0004] The purpose of the present invention is to provide a glycosylated RBD protein of SARS-CoV-2 coronavirus, a preparation method thereof, and an application thereof. The glycosylated RBD protein of SARS-CoV-2 coronavirus has potential important effects in preventing or treating novel coronavirus infection.

[0005] The glycosylated RBD protein of SARS-CoV-2 coronavirus described in the present invention is characterized in that: the glycosylated RBD protein, abbreviated as Tn-RBD, is composed of the RBD domain of the coronavirus S protein shown in the amino acid sequence of SEQ ID NO.1 modified and bound with GalNAc residues at positions T323, T345, and S514, wherein the Tn glycosylation modification of the Thr323 glycosylation site is 100%, the Tn glycosylation modification of the Thr345 glycosylation site is 7.95%, and the Tn glycosylation modification of the Ser514 glycosylation site is 2.56%.

[0006] The preparation method of the above-mentioned SARS-CoV-2 coronavirus glycosylated RBD protein comprises the following steps:

[0007] (1) The RBD domain protein with the amino acid sequence shown in SEQ ID NO.1 is prepared by using a conventional method;

[0008] Specifically, it includes cloning the nucleotide sequence encoding the RBD domain into an Escherichia coli expression vector, and then transforming the vector into an Escherichia coli expression host bacterium for expression and purification; when expressing and purifying, a purification tag is added at the end, and expression and purification can be carried out in a soluble or inclusion body form.

[0009] (2) Using the RBD domain protein as a substrate, Tn-RBD is synthesized by an in vitro glycosylation modification method using human N-acetylgalactosaminyltransferase GalNAc-T; the synthesis steps are as follows:

[0010] 1) According to the final concentration, in a reaction system of 10-300 mM Tris buffer containing 0.8-1.0 mg / mL of RBD domain protein, 3-10 mM of UDP-GalNAc, 10 mM of MnCl2, and pH 7.0-8.0, 2-10 μg / mL of human N-acetylgalactosaminyltransferase GalNAc-T is added;

[0011] 2) Then, under the water bath condition at 30-37 °C, after reacting for 4 h, UDP-GalNAc with a final concentration of 3-10 mM is added, and the reaction continues for 4-40 h;

[0012] 3) After the reaction is completed, the GalNAc-modified glycosylated RBD protein Tn-RBD is purified by gel chromatography.

[0013] In the above preparation method, the preferred embodiment is: in the synthesis step of step (2), 1) according to the final concentration, in a reaction system of 25 mM Tris buffer containing 1.0 mg / mL of RBD domain protein, 5 mM of UDP-GalNAc, 10 mM of MnCl2, and pH 7.5, 5 μg / mL of human N-acetylgalactosaminyltransferase GalNAc-T is added; 2) then, under the water bath condition at 37 °C, after reacting for 4 h, UDP-GalNAc with a final concentration of 5 mM is added, and the reaction continues for 4 h; 3) after the reaction is completed, Tn-RBD is purified by using pea lectin VVA / VVL gel chromatography.

[0014] The application of the SARS-CoV-2 coronavirus glycosylated RBD protein of the present invention in the preparation of anti-SARS-CoV-2 coronavirus drugs.

[0015] Use of the SARS-CoV-2 coronavirus glycosylated RBD protein in the preparation of an anti-SARS-CoV-2 coronavirus subunit vaccine using aluminum hydroxide adjuvant.

[0016] The SARS-CoV-2 coronavirus glycosylated RBD protein provided by the present invention is a Tn(GalNAc-) modified RBD protein (Tn-RBD) prepared by an in vitro glycosylation method. It has the characteristics of a simple preparation process route, convenient purification, high recovery rate, and high purity. The purified protein sample can be used as the vaccine stock solution and prepared into a vaccine product together with an adjuvant, and the antibodies produced by its immunization significantly enhance the inhibitory ability of RBD to bind to hACE2. Retrieval shows that there are no relevant reports on the application of Tn(GalNAc-) modified RBD protein as a candidate vaccine for COVID-19 at home and abroad. The present invention provides a simple method for the synthesis of glycoprotein vaccines, which has important theoretical significance and application value. Brief Description of the Drawings

[0017] Figure 1 It is an SDS-PAGE electrophoresis analysis diagram of the purified RBD protein domain.

[0018] Figure 2 It is a Western blot analysis diagram of the glycosylation level of the RBD protein domain catalyzed by GalNAc-T.

[0019] A) Western Blot analysis diagram using anti-His antibody; B) Lectin Blot analysis diagram using VVA-Biotin; where lane 1 is the purified non-glycosylated RBD protein domain; lane 2 is the Tn-RBD protein domain catalyzed by GalNAc-T.

[0020] Figure 3 It is a MALDI-TOF analysis diagram of the glycosylation level of the RBD protein domain catalyzed by GalNAc-T.

[0021] A) is the purified non-glycosylated RBD protein domain; B) is the Tn-RBD protein domain catalyzed by GalNAc-T.

[0022] Wherein: [M+H] of RBD + = 26248.4017, [M+H] of Tn-RBD + 26718.1779, and the difference between the two is ~470 Da.

[0023] Figure 4 It is a schematic diagram of the Tn(GalNAc-) modified RBD protein (Tn-RBD) prepared by an in vitro glycosylation method.

[0024] Figure 5 Analysis of glycosylation sites and occupancy of the Tn-RBD protein domain by high-resolution liquid chromatography-mass spectrometry.

[0025] Figure 6 Graph showing the detection of the titer of IgG antibodies against Tn-RBD in sera at different times after immunizing mice with Tn-RBD as an antigen.

[0026] Figure 7 Graph showing the analysis of the content of different antibody subtypes in the Tn-RBD immune antiserum.

[0027] Figure 8 Graph showing the analysis of the ability of the Tn-RBD immune antiserum to inhibit the binding of human ACE2 receptor protein to the RBD protein.

[0028] Among them: The inhibitory ability of the Tn-RBD antiserum is stronger than that of the RBD antiserum and the VVA lectin. Detailed implementation mode

[0029] The content of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The following examples are only the preferred embodiments of the present invention. It should be noted that the following description is only for explaining the present invention and does not limit the present invention in any form. Any simple modification, equivalent change and modification made to the embodiments based on the technical essence of the present invention all fall within the scope of the technical solution of the present invention.

[0030] In the following examples, the materials, reagents, plasmids, strains, etc. used are all obtained from commercial sources unless otherwise specified.

[0031] The test methods without specific conditions indicated in the following examples are basically carried out according to the conditions described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press), or according to the conditions recommended by the relevant reagent or kit manufacturers.

[0032] Example 1. Preparation of the RBD domain of the Spike protein of SARS-CoV-2 coronavirus

[0033] 1. Construction of a recombinant plasmid containing the RBD domain of the Spike protein

[0034] According to the gene sequence of SARS-CoV-2 coronavirus Spike protein coding gene (Gene ID: 43740568) published by NCBI, the coding gene of the RBD domain (Arg319–Phe541, such as SEQ ID NO.1) of the novel coronavirus Spike protein was synthesized by full gene and recombined into the NdeI and XhoI multiple cloning sites of the pET21a vector (purchased from Novagen) to construct the pET21a-RBD expression vector.

[0035] 2. Expression and purification of RBD protein

[0036] 1) Construction of recombinant expression strains

[0037] The pET21a-RBD plasmid was transformed into competent cells of Escherichia coli BL21 (E. coli BL21(DE3)pLysS), and the recombinant Escherichia coli strain containing pET21a-RBD was obtained by screening with ampicillin (final concentration: 100 μg / mL).

[0038] 2) Fermentation of recombinant strains

[0039] Single colonies of the constructed recombinant strains were separately picked into 25 mL test tubes containing 5 mL of LB medium, added with ampicillin to a final concentration of 100 μg / mL, and cultured at 37 °C and 200 r / min for 12 h.

[0040] The overnight cultured bacterial solution was inoculated into a 100 mL Erlenmeyer flask containing 50 mL of LB medium at an inoculation amount of 1% (v / v), added with ampicillin to a final concentration of 100 μg / mL, and cultured at 37 °C and 200 r / min.

[0041] When the OD 600 of the bacterial solution reached 0.6 - 0.8, isopropyl β-D-1-thiogalactopyranoside (IPTG) with a final concentration of 0.8 mM was added to the recombinant strain containing pET21a-RBD, and the culture was continued at 37 °C and 200 r / min for 4 h for induction expression.

[0042] 3) Extraction of recombinant protein

[0043] The overnight cultured bacterial solution was centrifuged at 4 °C and 13000 r / min for 5 min, and all the supernatant was discarded to collect the bacterial cells.

[0044] The bacteria were disrupted and dissolved using a high-pressure homogenizer, centrifuged at 4 °C and 13000 r / min for 10 min, and the precipitate was collected. The target protein was present in the inclusion bodies.

[0045] The inclusion bodies were washed three times with 50 mM Tris, pH 7.5, 2 M urea, centrifuged at 13,000 r / min for 30 min at 4°C, and the precipitate was collected.

[0046] The inclusion bodies were dissolved with 50 mM Tris, pH 9.0, 8 M urea, 10 mM β-mercaptoethanol.

[0047] The denatured recombinant inclusion body protein was purified using a nickel metal ion chromatography column (Ni-NTA).

[0048] a) Column packing: 0.5 mL of Ni-NTA was poured into a 2.5×10 cm column, and the column was equilibrated at 4°C with 10 column volumes of binding buffer (20 mM Tris-HCl buffer, pH 8.0; 0.3 M sodium chloride; 10 mM imidazole; 8 M urea).

[0049] b) Sample loading: The denatured inclusion body protein was loaded, and the chromatography column was washed with 10 column volumes of washing buffer (20 mM Tris-HCl buffer, pH 8.0; 0.3 M sodium chloride; 20 mM imidazole; 8 M urea).

[0050] c) Elution: The target protein was eluted with 6 column volumes of elution buffer (20 mM Tris-HCl buffer, pH 8.0; 250 mM imidazole; 0.3 M sodium chloride, 8 M urea), and the eluate containing the RBD protein domain was collected.

[0051] d) Inclusion body renaturation: The eluate containing the target protein was rapidly diluted in renaturation buffer (50 mM Tris, pH 9.0, 0.4 M arginine, 5 mM GSH, 0.5 mM GSSG) to renature the RBD protein.

[0052] e) Desalting: The separately collected protein eluates were ultrafiltered at 4°C and 4000 r / min, and desalted with PBS buffer to obtain a solution of the RBD protein domain.

[0053] 4) Determination of the molecular weight and concentration of the RBD protein domain

[0054] The obtained RBD protein domain was detected by 12% SDS-PAGE electrophoresis.

[0055] The analysis and detection results are shown in Figure 1 。

[0056] The detection results showed that an RBD protein domain with a purity > 95% was obtained, and the protein concentration was 1 mg / ml.

[0057] Example 2: Catalytic synthesis of Tn-RBD glycoprotein using GalNAc glycosyltransferase (GalNAc-T)

[0058] 1) In a reaction system containing 25 mM Tris buffer with a final concentration of 1.0 mg / mL recombinant RBD domain protein, 5 mM UDP-GalNAc, 10 mM MnCl2, and pH 7.5, add GalNAc glycosyltransferase (GalNAc-T) at a final concentration of 5 μg / mL.

[0059] 2) Incubate at 37 °C in a water bath for 4 h.

[0060] 3) Add 5 mM UDP-GalNAc and continue the reaction at 37 °C in a water bath for 4 h.

[0061] 4) Separate and purify the supernatant obtained in step 3) using a VVA-Agarose gel (Vector Laboratories, AL-1233-2) column. The purification process is as follows:

[0062] a) Column packing: Pour 0.2 mL of VVA-Agarose into a 2.5 × 10 cm column and equilibrate the column with 20 column volumes of binding buffer (25 mM Tris pH 7.5, 150 mM NaCl, 1 M Urea, 1 mM CaCl2 / MgCl2 / MnCl2 / ZnCl2) at 4 °C.

[0063] b) Sample loading: Dilute the reaction solution with binding buffer and load the sample, then wash the chromatography column with 20 column volumes of binding buffer.

[0064] c) Elution: Elute the target protein with 4 column volumes of elution buffer (25 mM Tris pH 7.5, 150 mM NaCl, 1 M Urea, 1 mM CaCl2 / MgCl2 / MnCl2 / ZnCl2, 0.2 M GalNAc) and collect the eluate containing the RBD protein domain.

[0065] d) Desalting: Ultrafilter the protein eluate collected above at 4000 r / min at 4 °C and perform desalting treatment with PBS buffer to obtain a solution of the RBD protein domain of Tn-RBD.

[0066] Detect the purified protein using Western Blot (anti-His antibody) and Lectin Blot (VVA-Biotin).

[0067] Detect the purified protein using MALDI TOF mass spectrometry.

[0068] The analysis and detection results are shown in Figure 2 , Figure 3 , Figure 4 .

[0069] Figure 2 The results showed that the molecular weight of the glycosylated product after the GalNAc-T reaction increased and could be recognized by VVA lectin.

[0070] Figure 3 The results showed that the molecular weight of the glycosylated product after the GalNAc-T reaction increased by about 470 Da, which was equivalent to 2 - 3 GalNAc residues.

[0071] Figure 4 Schematic diagram of the Tn(GalNAc-) - modified RBD protein (Tn - RBD) prepared by the in vitro glycosylation method.

[0072] Example 3. Determination of the glycosylation site of Tn - RBD

[0073] The purified Tn - RBD protein was hydrolyzed with Trypsin protease and then detected by mass spectrometry to determine the glycosylation site. The specific steps are as follows:

[0074] 1) Take 10 μg of Tn - RBD protein, add DTT with a final concentration of 10 mM, and treat at 60 °C for 45 min;

[0075] 2) Add iodoacetamide with a final concentration of 20 mM, and treat in the dark at room temperature for 30 min;

[0076] 3) Add DTT with a final concentration of 10 mM, and treat in the dark at room temperature for 30 min;

[0077] 4) Add 1 μg of Trypsin protease;

[0078] 5) Place it in a water bath at 37 °C and react for 12 h;

[0079] 6) Add TFA with a final concentration of 0.1% to terminate the reaction;

[0080] 7) Desalt the sample using a C18 chromatography column;

[0081] 8) Analyze the glycosylation site using high - resolution mass spectrometry;

[0082] The analysis and detection results are shown in Figure 5 .

[0083] The results showed that three sites on Tn-RBD were glycosylated. Among them, nearly 100% of the Thr323 site had Tn glycosylation modification. At the same time, 7.94% of the Thr345 and 2.56% of the Ser514 sites had Tn glycosylation modification.

[0084] Example 4. Preparation of SARS-CoV-2 coronavirus Tn-RBD protein subunit vaccine

[0085] Use the Tn-RBD protein prepared in Example 2 to prepare a SARS-CoV-2 coronavirus subunit vaccine.

[0086] Dilute the purified recombinant Tn-RBD protein to 2 times the target antigen concentration, mix and adsorb it with 1.2 mg / mL aluminum hydroxide adjuvant at a ratio of 1:1 (w / w), and stir vigorously on a magnetic stirrer for 40 - 120 min to obtain the semi-finished subunit vaccine, in which the residual protein content in the supernatant is less than 10% of the total protein content.

[0087] Example 5. Experiment on immunizing mice with Tn-RBD subunit vaccine

[0088] 1) Take 8 - 10-week-old BALB / c experimental mice and inject 100 μL of the prepared subunit vaccine subcutaneously into the mice.

[0089] Injection;

[0090] 2) Use the immune solution to boost the immunity on the 14th, 28th, and 42nd days after the primary immunization respectively.

[0091] 3) Take mouse blood samples on the 0th, 21st, 35th, 49th, and 63rd days after the primary immunization respectively.

[0092] 4) After coagulation, centrifuge the blood sample at 1200 × g for 20 min, and store the supernatant, that is, the serum sample, at -20°C.

[0093] 5) Use the ELISA method to detect the antibody titer in the mouse serum. The specific procedure is as follows:

[0094] a) Add various antigens (100 ng / well, 100 μL) diluted with carbonate buffer (15 mM Na2CO3 and 35 mM NaHCO3) to a 96-well plate and incubate overnight at 4°C.

[0095] b) After antigen coating, wash the plate three times with PBST (200 μL / well), and block it at room temperature with 1% BSA / PBS (100 μL / well) for 1 h. Then wash the plate with PBST and incubate it with serially diluted serum in 0.1% BSA / PBS (100 μL / well) at 37°C for 2 h.

[0096] c) After washing the plate three times, add the HRP-labeled secondary antibody at an appropriate dilution. Subsequently, incubate the 96-well plate at 37 °C for 1 h, and then wash it three times with PBST. Add the TMB working solution (100 μL / well), incubate for 15 min in the dark, and then add 0.5 M sulfuric acid (50 μL / well) to stop the reaction.

[0097] d) Measure the absorbance at 450 nm using a BioTek CytationTM5. All samples are in triplicate.

[0098] The analysis and test results are shown in Figure 6 , Figure 7 .

[0099] Figure 6 The results showed that the antibody titer in the serum reached a peak 49 days after vaccination and remained at a high level until 63 days.

[0100] Figure 7 The results showed that the main antibodies in the serum after vaccination were IgM and IgG2b.

[0101] Example 6. SARS-CoV-2 surrogate virus neutralization assay of mouse serum

[0102] 1) Dilute the mouse serum at different dilution multiples;

[0103] 2) Seed the HEK293 cells expressing the RBD domain in a 96-well plate;

[0104] 3) Mix hACE2 (His-tag) with the serum diluted at different multiples;

[0105] 4) Add the mixture to a 96-well plate pre-coated with the RBD expressed by HEK293 cells and incubate at room temperature for 1 h. Then add the HRP-conjugated anti-His-tag antibody and incubate at room temperature for another 1 h. Subsequently, wash the plate three times.

[0106] Add the TMB working solution (100 μL / well), incubate in the dark for 15 min, and then add 0.5 M sulfuric acid (50 μL / well)

[0107] to stop the reaction.

[0108] 5) Measure the absorbance at 450 nm using a BioTek CytationTM5. All samples are in triplicate.

[0109] The analysis and test results are shown in Figure 8 .

[0110] The results showed that the sera after immunization with the Tn-RBD vaccine had a higher inhibitory effect than the sera after immunization with RBD prepared by the same method as in Example 4.

Claims

1. A SARS-CoV-2 coronavirus glycosylated RBD protein, characterized in that: The glycosylated RBD protein, abbreviated as Tn-RBD, is composed of the RBD domain of the coronavirus S protein shown in the amino acid sequence SEQ ID NO.1, with GalNAc residues conjugated at its T323, T345, and S514 positions. Among them, the Tn glycosylation modification at the Thr323 glycosylation site is 100%, the Tn glycosylation modification at the Thr345 glycosylation site is 7.95%, and the Tn glycosylation modification at the Ser514 glycosylation site is 2.56%.

2. The preparation method of the SARS-CoV-2 coronavirus glycosylated RBD protein according to claim 1, the steps are: (1) Prepare the RBD domain protein with the amino acid sequence shown in SEQ ID NO.1 by using a conventional method; (2) Using the RBD domain protein as a substrate, synthesize Tn-RBD by an in vitro glycosylation modification method using human N-acetylgalactosaminyltransferase GalNAc-T; wherein, The synthesis steps are as follows: 1) In a reaction system of 10 - 300 mM Tris buffer containing 0.8 - 1.0 mg / mL of the RBD domain protein, 3 - 10 mM of UDP-GalNAc, 10 mM of MnCl2, and pH 7.0 - 8.0, add 2 - 10 μg / mL of human N-acetylgalactosaminyltransferase GalNAc-T according to the final concentration; 2) Then, under the water bath condition of 30 - 37 °C, after reacting for 4 h, add UDP-GalNAc with a final concentration of 3 - 10 mM, and continue to react for 4 - 40 h; 3) After the reaction is completed, use gel chromatography to purify to obtain the GalNAc-modified glycosylated RBD protein Tn-RBD.

3. According to the method described in claim 2, characterized in that: The synthesis steps described in step (2) are as follows: 1) In a reaction system of 25 mM Tris buffer containing 1.0 mg / mL of the RBD domain protein, 5 mM of UDP-GalNAc, 10 mM of MnCl2, and pH 7.5, add 5 μg / mL of human N-acetylgalactosaminyltransferase GalNAc-T; 2) Then, under the water bath condition of 37 °C, after reacting for 4 h, add UDP-GalNAc with a final concentration of 5 mM, and continue to react for 4 h; 3) After the reaction is completed, use pea lectin VVA / VVL gel chromatography to purify to obtain Tn-RBD.

4. The application of the SARS-CoV-2 coronavirus glycosylated RBD protein according to claim 1 in the preparation of a vaccine for preventing SARS-CoV-2 coronavirus infection.

5. The application of the SARS-CoV-2 coronavirus glycosylated RBD protein according to claim 1 in the preparation of an anti-SARS-CoV-2 coronavirus subunit vaccine using aluminum hydroxide adjuvant.

Images