A rbd-fc fusion protein based on a delta strain of sars-cov-2 and applications thereof

Abstract

The application discloses a RBD-Fc fusion protein based on a delta strain of a novel coronavirus and application thereof; and a subunit vaccine prepared from the RBD-Fc fusion protein based on the delta strain of the novel coronavirus can induce mice to produce high-titer specific IgG antibodies and neutralizing antibodies, the high-titer antibody level can be sustained for 7 months, and the subunit vaccine can also induce the mice to produce specific cellular immune responses.

Description

Technical Field

[0001] This invention provides an RBD-Fc fusion protein of the SARS-CoV-2 delta strain, its preparation method, and its application, belonging to the field of biotechnology. Background Technology

[0002] Like SARS and MERS viruses, SARS-CoV-2 belongs to the β-coronavirus genus. It can be transmitted via aerosols and, being an RNA virus, is highly prone to mutation. The Delta mutant strain (B.1.617.2) originating from India has three important mutations in its S protein: L452R, T478K, and P681R. These mutations may increase the affinity between the SARS-CoV-2 S protein and its receptor, enhancing its infectivity. Clinically, the Delta mutant strain has shown stronger transmissibility and a higher risk of immune evasion than other SARS-CoV-2 strains. The Delta strain was the dominant circulating strain globally in the second half of 2021. Although the dominant global strain has now shifted from Delta to Omicron, and the pathogenicity of the Delta strain is significantly higher than that of the Omicron strain, given the highly mutable nature of SARS-CoV-2, it is essential to strengthen research and reserves on the Delta strain to prepare for future variants.

[0003] The control and eradication of human infectious diseases ultimately depend on the large-scale application of vaccines. Global research institutions and vaccine companies are fully committed to the research and development of COVID-19 vaccines. Currently, there are four types of vaccines available globally: inactivated vaccines, mRNA vaccines, recombinant protein vaccines, and adenovirus vector vaccines. Recombinant protein vaccines are constructed by recombinantly expressing the target antigen gene onto an expression vector, inducing the expression of the antigen protein in vitro, and finally purifying it into a protein vaccine. Recombinant protein vaccines have stable antigens, are easy to transport, and are suitable for regions with poor storage and transportation conditions, such as Africa. Furthermore, recombinant protein vaccines have fewer adverse reactions, lower biosafety risks, high technological maturity, and are easy to mass-produce, meeting the vaccination needs of large populations; the hepatitis B vaccine is a good example. Currently, there are not many marketed recombinant protein vaccines for COVID-19. Among recombinant protein vaccines, there is only one fusion protein vaccine, which is limited in variety and lacks specificity against the delta variant of the SARS-CoV-2 virus. Research and development of other recombinant protein vaccines for COVID-19 should be intensified to provide more means and technological reserves to cope with the ever-changing SARS-CoV-2 virus. Summary of the Invention

[0004] The purpose of this invention is to provide an RBD-Fc fusion protein based on a delta variant of SARS-CoV-2, its preparation method, and its application. The RBD-Fc fusion protein based on a delta variant of SARS-CoV-2 provided by this invention has the ability to induce mice to produce high levels of neutralizing antibodies and can be used to prepare a recombinant protein vaccine for SARS-CoV-2.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides an RBD-Fc fusion protein based on a delta variant of the novel coronavirus, the amino acid sequence of which is shown in SEQ ID No. 1.

[0007] Secondly, the present invention also provides a gene encoding the above-mentioned RBD-Fc fusion protein based on the delta variant of the novel coronavirus.

[0008] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID No. 2.

[0009] Thirdly, the present invention provides an expression plasmid containing the above-mentioned encoding gene.

[0010] Furthermore, the expression plasmid is expressed using pLVX-puro as a vector.

[0011] Furthermore, the expression plasmid is constructed by inserting the coding gene between the Xhol restriction site and the Xbal restriction site of the vector pLVX-puro.

[0012] Fourthly, the present invention provides an expression cell for the above-mentioned RBD-Fc fusion protein based on the delta variant of the novel coronavirus.

[0013] Furthermore, the host cell for the expressed cells is HEK293T cells.

[0014] Fifthly, the present invention specifically provides a method for preparing the above-mentioned RBD-Fc fusion protein based on the delta variant of the novel coronavirus:

[0015] The encoding gene of the RBD-Fc fusion protein based on the delta variant of SARS-CoV-2 was inserted between the Xhol and Xbal restriction enzyme sites of the pLVX-puro vector to obtain the expression plasmid of the RBD-Fc fusion protein. The expression plasmid of the RBD-Fc fusion protein was transfected into HEK293T cells for expression. The resulting cell culture supernatant was purified and freeze-dried to obtain the RBD-Fc fusion protein based on the lambda variant of SARS-CoV-2.

[0016] Further, the purification process involves sequentially subjecting the cell culture supernatant to Protein A column purification and Superdex molecular sieve purification. The eluent for Protein A column purification is a 30% (v / v) aqueous solution of glacial acetic acid, and the eluent for Superdex molecular sieve purification is PBS buffer.

[0017] Sixthly, the present invention also provides the application of the above-mentioned RBD-Fc fusion protein based on the delta variant of the novel coronavirus in the preparation of a novel coronavirus subunit vaccine.

[0018] Specifically, the novel coronavirus subunit vaccine is prepared by mixing the RBD-Fc fusion protein with aluminum hydroxide adjuvant to obtain the novel coronavirus subunit vaccine.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: the subunit vaccine can induce mice to produce high titers of specific IgG antibodies and neutralizing antibodies, and the high titer antibody level can last for 7 months. At the same time, it can also induce mice to produce specific cellular immune responses. Attached Figure Description

[0020] 1) Figure 1 This is a schematic diagram of the RBD-Fc fusion protein based on the delta variant of the novel coronavirus.

[0021] 2) Figure 2 Figure A shows the chromatogram of the purified RBD-Fc fusion protein and its purity analysis. Figure B shows the SDS-PAGE chromatogram of the purified RBD-Fc fusion protein. Figure C shows the Western blotting of the purified RBD-Fc fusion protein.

[0022] 3) Figure 3 Affinity assay of RBD-Fc fusion protein in Vero E6 cells.

[0023] 4) Figure 4 A time-series diagram of serum IgG antibody titers induced in mice immunized with the RBD-Fc fusion protein vaccine.

[0024] 5) Figure 5 A time-series diagram of serum neutralizing antibody titers induced in mice immunized with the RBD-Fc fusion protein vaccine.

[0025] 6) Figure 6 Cellular immunization of mice immunized with RBD-Fc fusion protein vaccine: Figure A shows the number of spleen cells that produce IFN-γ after antigen stimulation; Figure B shows the number of spleen cells that produce IL-4 after antigen stimulation. Detailed Implementation

[0026] Example 1

[0027] 1. Preparation of RBD-Fc fusion protein based on SARS-CoV-2 delta variant

[0028] The RBD at residues 310-531 of the S protein of the SARS-CoV-2 delta variant was fused with a modified Fc fragment of human IgG1. (See schematic diagram below.) Figure 1RBD and hFc were spliced ​​together via a flexible linker (Gly-Gly-Gly-Gly-Ser, gene sequence SEQ ID No. 2) to increase the flexibility between fragments without affecting their function (this linker sequence has been validated in multiple studies). Through extension PCR, an Xhol restriction site (CTCGAG), a kozak sequence (ACCACC), and the HEK293 cell signal peptide (amino acid sequence: MDWTWRILFLVAAATGAHS, gene sequence SEQ ID No. 2) were introduced upstream. A stop codon (TGA) and an Xbal restriction site (TCTAGA) were introduced downstream. Finally, the double-digested PCR product was ligated into the pLVX-puro eukaryotic expression vector, and the ligation product was transformed into DH5α *E. coli*. Single clones with correct sequencing were selected and maintained. The recombinant plasmid was transiently transfected into human embryonic kidney cells (HEK293 cells) for expression.

[0029] The culture supernatant was harvested and purified using a Protein A column with a 30% aqueous solution of glacial acetic acid as the eluent. It was then further purified using a Superdex molecular sieve to obtain the protein, with a 1*PBS buffer as the eluent. The purified chromatogram is shown below. Figure 2 A. SDS-PAGE analysis of the purified product is shown in [the original text]. Figure 2 B. The band approximately 55 kDa represents the RBD-Fc fusion protein. The purified product was analyzed using Western blotting. Figure 2 C) A band of approximately 55 kDa was found to contain an Fc fragment.

[0030]

[0031] PCR procedure:

[0032] denaturation at 98℃ for 5 seconds

[0033] Annealing at 65℃ for 15 seconds

[0034] Extend at 72℃ for 1 minute

[0035] (30-35 cycles)

[0036] Template: The nucleotide sequence of RBD (Delta) synthesized by a biotechnology company was inserted between the Xhol and Xbal sites of the nucleotide sequence shown in SEQ ID NO:2 (i.e., the 310 to 531 amino acids of RBD).

[0037] Primers: (Primers are used to add the desired fragment.)

[0038] Upstream primer:

[0039] CGCTACCGGACTCAGATCTCGAGGCCACCATGAAGACCATCATCGCCCTGAGC

[0040] Downstream primer: CCCTGTCTCCGGGTAAATGATCTAGATAATTCTA

[0041] Transformation:

[0042] 1. Remove the ligation product and pre-cool it on ice. At the same time, remove DH5α competent cells and place them on ice.

[0043] 2. In a clean bench, take 3-7 μl of the ligation product and attach it to competent cells, then place it on ice for 30 min.

[0044] 3. Heat shock: Place at 42℃ for 90 seconds, then place on ice for 5 minutes.

[0045] 4. Add 1 ml of antibiotic-free LB medium to competent cells and place on a shaker at 37°C for 1 hour.

[0046] 5. Centrifuge the competent cells at 3000g for 6 minutes.

[0047] 6. Remove most of the culture medium, blow the remaining approximately 100 μl of culture medium to mix the precipitate, and spread it evenly onto the prepared LB solid medium (containing 50-100 ug / ml ampicillin) using a spreader.

[0048] Incubate overnight at 7.37℃ for 12 hours.

[0049] Monoclonal selection:

[0050] 1. A single colony grew on the solid culture dish the day after transformation.

[0051] 2. Select 5 single colonies and place them in an ep tube (1 ml of ampicillin-resistant LB medium).

[0052] 3. Send to the company for sequencing.

[0053] 4. Compare sequencing results, select single clones with correct sequencing results for amplification culture, and extract plasmids.

[0054] Transfection:

[0055] 1. Add the plasmid to be transfected to DMEM culture medium (antibiotic-free), and add polyethyleneimine (PEI) to another EP tube and vortex mix.

[0056] 2. Pour the liquid from the PEI tube into the plasmid, vortex to mix, and let stand for 15 minutes.

[0057] 3. Prepare HEK-293T cells in advance (approximately 4 large dishes) with a cell density of 70%. Remove the 293T cells, aspirate the supernatant, and add an appropriate amount of DMEM culture medium.

[0058] 4. Add the liquid from step 2 evenly dropwise into the petri dish.

[0059] 5. Place in the incubator for 4 hours.

[0060] 6. Fluid replacement: Replenish with culture medium containing serum and antibiotics to bring the final serum concentration to 4%.

[0061] 7. Incubate for approximately 72 hours to express the protein (the protein will be in the cell supernatant).

[0062] 2. Flow cytometry analysis of the activity of the RBD-hFc fusion protein

[0063] Vero E6 cells (ACE2 receptor positive) were cultured, and 1×10⁶ cells were collected. 6 Vero E6 cells (ACE2 receptor positive) were incubated with RBD-hFc recombinant protein vaccine (10 μg / ml) at 4°C for 30 min. After washing three times with PBS, 200 μl of FITC-labeled goat anti-mouse secondary antibody (1:1000) was added to the incubation tubes. After incubation at 4°C for 30 min, the cells were washed three times with PBS and then detected by flow cytometry.

[0064] The affinity of the RBD-hFc fusion protein for Vero E6 cells (ACE2 receptor positive) detected by flow cytometry was mainly expressed by the mean fluorescence intensity of FITC after secondary antibody labeling. Figure 3 As shown, the affinity of the RBD recombinant protein for 293T cells was significantly higher than that for the negative control group, therefore the purified RBD-hFc recombinant protein still maintained its native conformation.

[0065] 3. Purification of RBD-Fc fusion protein based on the Lambda variant of SARS-CoV-2

[0066] The culture supernatant of human embryonic kidney cells (HEK293 cells) was harvested, purified first using a Protein A column, eluted with 30% glacial acetic acid aqueous solution, and then further purified to obtain protein using a Superdex molecular sieve. The eluent was 1*PBS buffer. The chromatogram of the purified protein is shown in the figure. Figure 2 A. SDS-PAGE analysis of the purified product is shown in [the original text]. Figure 2 B. The band approximately 55 kDa represents the RBD-Fc fusion protein. The purified product was analyzed using Western blotting. Figure 2 C) A band of approximately 55 kDa was found to contain an Fc fragment.

[0067] 4. Immunization of mice with RBD-hFc recombinant protein vaccine

[0068] Eight-week-old female BALB / c mice were randomly divided into five groups of five mice each. After lyophilization and quantification of the purified protein, high-dose (8 μg) and low-dose (2 μg) RBD-hFc fusion protein were mixed with aluminum hydroxide adjuvant (InvivoGen, catalog number: vac-alu-250) at a final concentration of 0.4–0.5 mg / mL and administered intramuscularly to the mice at a dose of 100 μL per mouse. The immunization schedule included vaccination on day 0, a booster on day 7, and blood collection via the orbital sinus at weeks 2, 4, 6, 8, 12, 20, and 28 post-immunization. Spleens were collected after euthanasia at week 28 for cellular immunoassay. PBS containing 0.4–0.5 mg / mL aluminum hydroxide served as a control.

[0069] 5. Time-series detection of serum IgG-specific antibody titers in immunized mice

[0070] The titer of IgG-specific antibodies in mouse serum at different post-immunization periods was detected using the following technical protocol:

[0071] 1) Add 50 ng of RBD protein to each well of the ELISA plate and coat it overnight at 4°C.

[0072] 2) Add 200 μL of 0.5% Tween-20 PBS to each well, wash the plate 5 times, and pat dry.

[0073] 3) Add 10% FBS and 3% BSA to the PBS solution as blocking and diluent, add 200 μL of blocking solution to each well, and incubate at 37°C for 1 hour for blocking.

[0074] 4) Serially dilute the serum samples, 100 μl of diluent per well, and incubate at 37°C for 1 hour.

[0075] 5) Wash the plate 5 times and pat it dry.

[0076] 6) Add 100 μl of rabbit anti-mouse IgG-HRP conjugate antibody diluted 1:20000 to each well and incubate at 37°C for 1 hour.

[0077] 7) Add 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate to each well for color development, and develop at 37°C for 12 min.

[0078] 8) Add 50 μl of 2M dilute sulfuric acid to each well to stop the color development.

[0079] 9) Measure the absorbance and OD at 450 nm using an ELISA reader. 450 A value greater than 2.1 times the background value is considered positive.

[0080] 6. Timing detection of neutralizing antibodies in mouse serum after immunization

[0081] 1) Inactivate the serum of immunized mice at 56°C for 0.5 hours.

[0082] 2) Mix serum that has been serially diluted 4-fold with the same volume of the original SARS-CoV-2 strain (100 TCID50). 50 Mix the culture medium and incubate at 37°C for 1 hour.

[0083] 3) Transfer the virus-serum mixture to pre-filled Vero E6 cells in a 96-well plate.

[0084] 4) After incubating at 37°C for another hour, discard the mixture.

[0085] 5) Spread cells in the virus culture medium with 0.6% agarose gel.

[0086] 6) Two days later, add a second agarose layer containing 0.1% neutral red.

[0087] 7) One day later, calculate the number of plaques. The neutralizing titer is calculated as the reciprocal of the serum dilution that results in a 50% reduction in plaques (PRNT). 50 ).

[0088] 7. Post-immunization mouse cellular immunity detection

[0089] Twelve weeks after booster immunization, all mice in the eight groups were euthanized. Spleens were collected and separated into individual spleen cell suspensions by syringe pressing. Spleen cells were then cultured in ELISPOT plates coated with either IFN-γ or IL-4 antibodies at a density of 1 × 10⁶ cells per well. 6 Cells were cultured at appropriate densities and stimulated with or without SARS-CoV-2 RBD-hFc (2 μg / well). After incubation at 37°C and 5% CO2 for 16 h, the number of spleen cells producing IFN-γ and IL-4 was measured using a mouse enzyme-linked immunospot (ELISBOT) kit according to the manufacturer's instructions. The spotted cells (SFCs) were imaged using an imaging system, and the relevant data were statistically analyzed.

[0090] SEQ ID No.1

[0091] RBD Delta -hFc amino acid sequence:

[0092] GIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVAD

[0093] YSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKI

[0094] ADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDIST

[0095] EIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPAT

[0096] VCGPKKSTN

[0097] EFGGGGSPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVV

[0098] VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDW

[0099] LNGKEYKCAVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT

[0100] CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0101] SEQ ID No.2

[0102] RBD Delta -hFc nucleotide sequence:

[0103] GGCATCTATCAGACATCCAATTTCAGGGTGCAGCCAACCGAGTCTATCGTG

[0104] CGCTTTCCTAATATCACAAACCTGTGCCCATTTGGCGAGGTGTTCAACGCA

[0105] ACCCGCTTCGCCAGCGTGTACGCCTGGAATAGGAAGCGGATCAGCAACTG

[0106] CGTGGCCGACTATAGCGTGCTGTACAACTCCGCCTCTTTCAGCACCTTTAA

[0107] GTGCTATGGCGTGTCCCCCACAAAGCTGAATGACCTGTGCTTTACCAACGT

[0108] CTACGCCGATTCTTTCGTGATCAGGGGCGACGAGGTGCGCCAGATCGCCCC

[0109] CGGCCAGACAGGCAAGATCGCAGACTACAATTATAAGCTGCCAGACGATTT

[0110] CACCGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATTCCAAAGTGG

[0111] GCGGCAACTACAATTATCGGTACCGGCTGTTTAGAAAGAGCAATCTGAAGC

[0112] CCTTCGAGAGGGACATCTCTACAGAAATCTACCAGGCCGGCAGCAAGCCTT

[0113] GCAATGGCGTGGAGGGCTTTAACTGTTATTTCCCACTCCAGTCCTACGGCTT

[0114] CCAGCCCACAAACGGCGTGGGCTATCAGCCTTACCGCGTGGTGGTGCTGA

[0115] GCTTTGAGCTGCTGCACGCCCCAGCAACAGTGTGCGGCCCCAAGAAGTCC

[0116] ACCAAT

[0117] GAATTCGGAGGAGGCGGCAGCCCCAAATCTTGTGACAAAACTCACACATG

[0118] CCCACCGTGCCCAGCACCTGAAGCCGCTGGGGGACCGTCAGTCTTCCTCTT

[0119] CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCA

[0120] CATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC

[0121] TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGA

[0122] GGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC

[0123] ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCCGTCTCCAACAAA

[0124] GCCCTCGGAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC

[0125] CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCA

[0126] AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACA

[0127] TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC

[0128] CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT

[0129] CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCG

[0130] TGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT

[0131] CTCCGGGTAAATGA*(Stop codon)

Claims

1. An RBD-Fc fusion protein based on a delta variant of SARS-CoV-2, characterized in that: The amino acid sequence of the RBD-Fc fusion protein based on the SARS-CoV-2 delta variant is shown in SEQ ID No.

1.

2. The encoding gene of the RBD-Fc fusion protein based on the delta variant of SARS-CoV-2 as described in claim 1.

3. The encoding gene as described in claim 2, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID No.

2.

4. An expression plasmid containing the encoding gene as described in claim 2.

5. The expression plasmid as described in claim 4, characterized in that: The expression plasmid was expressed using pLVX-puro.

6. The expression plasmid as described in claim 5, characterized in that: The expression plasmid was constructed by inserting the coding gene between the Xhol restriction site and the Xbal restriction site of the vector pLVX-puro.

7. The expression cells of the RBD-Fc fusion protein based on the SARS-CoV-2 delta variant as described in claim 1.

8. The expression cell as described in claim 7, characterized in that: The host cell for the expressed cells is HEK293T cells.

9. The method for preparing the RBD-Fc fusion protein based on the delta variant of SARS-CoV-2 as described in claim 1, characterized in that... The method is as follows: The encoding gene of the RBD-Fc fusion protein based on the SARS-CoV-2 delta variant was inserted between the Xhol and Xbal restriction enzyme sites of the pLVX-puro vector to obtain the expression plasmid of the RBD-Fc fusion protein. The expression plasmid of the RBD-Fc fusion protein was transfected into HEK293T cells for expression. The resulting cell culture supernatant was purified and freeze-dried to obtain the RBD-Fc fusion protein based on the SARS-CoV-2 delta variant.

10. The application of the RBD-Fc fusion protein based on the delta variant of SARS-CoV-2 as described in claim 1 in the preparation of a subunit vaccine for SARS-CoV-2.

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