WO2021159648A1 - 一种β冠状病毒抗原、其制备方法和应用 - Google Patents
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Definitions
- the invention relates to the technical field of biomedicine, in particular to a beta coronavirus antigen, its preparation method and application.
- Coronaviruses belong to the genus Coronavirus, the family of Coronaviruses. They are positive-stranded RNA viruses with envelopes. Among all RNA viruses, their genomes are the largest. Animals and humans are the hosts of coronaviruses. Coronaviruses mainly infect the respiratory and digestive tracts of mammals and birds. There are currently seven known types of coronaviruses that infect humans, four of which cause mild colds (HCoV-229E, HCoV-NL63, OC43 and HKU1).
- SARS-CoV severe respiratory syndrome coronavirus
- MERS-CoV Middle East respiratory syndrome coronavirus
- 2019-nCoV 2019 The new type of coronavirus that broke out in the year, all three of them belong to ⁇ -coronavirus.
- Middle East Respiratory Syndrome is a disease caused by MERS-CoV infection.
- MERS-CoV Middle East Respiratory Syndrome
- MERS-CoV may spread through aerosols, so it is difficult to prevent and control.
- Neutralizing antibodies to MERS-CoV can be detected in the serum of dromedaries in many countries in the Middle East, suggesting that dromedaries are the intermediate hosts of MERS-CoV, and dromedaries are an important means of transportation in Middle Eastern countries. Therefore, since MERS-CoV was discovered in 2012, incidents of MERS-CoV infection in humans in the Middle East have not been interrupted. Therefore, with the increasing frequency of international exchanges, the risk of global spread of MERS has always existed. There are still no vaccines and effective treatments in the world. Therefore, it is urgent and important to develop a safe and effective vaccine against MERS-CoV.
- ACE2 angiotensin transferase 2
- ACE2 angiotensin transferase 2
- Symptoms such as diarrhea, severe patients rapidly progress to acute respiratory distress syndrome, septic shock, difficult to correct metabolic acidosis and blood clotting dysfunction, causing life-threatening.
- TGEV Porcine Transmissible Gastroenteritis Virus
- PRCV Porcine Respiratory Coronavirus
- FIPV Feline Peritonitis Virus
- CoV Canine Coronavirus
- PEDV Porcine epidemic diarrhea virus
- PEDV causes porcine epidemic diarrhea and other intestines The disease is easily spread in pigs and has a high fatality rate.
- coronaviruses such as rats and cattle. These coronaviruses pose a serious threat to the health of humans and animals. Therefore, the development of a vaccine against the coronavirus is of great significance.
- the surface spike protein (S protein) is the main neutralizing antigen of coronaviruses.
- the receptor binding domain (RBD) of the S protein of MERS-CoV, SARS-CoV, and 2019-nCoV is considered to be the most important antigen target area that induces the body to produce neutralizing antibodies.
- RBD can focus the neutralizing antibodies produced by the body's stimulation more on the receptor binding to the virus, which can improve the immunogenicity and immune efficiency of the vaccine.
- MERS-CoV invades cells through the binding of RBD to the host cell's receptor (CD26, also known as DPP4).
- CD26 also known as DPP4
- both SARS-CoV and 2019-nCoV are found to enter cells through their RBD binding to the host cell receptor hACE2.
- the purpose of the present invention is to provide a beta coronavirus antigen, its preparation method and application.
- the MERS dimer RBD protein can stimulate neutralizing antibodies better than the monomer RBD protein
- the single-chain dimeric RBD protein obtained in the embodiment of the present invention does not Because the formation of disulfide bonds is unstable, the dimer RBD protein content is unstable during the production process, that is, to avoid the monomer RBD form as the main form, and the expression of dimer RBD formation is rare, so that the dimer RBD expression Stable, uniform in form, and greatly improved in output.
- the single-chain dimer expressed in the embodiment of the present invention has equivalent performance as a ⁇ -coronavirus antigen.
- the immunogenicity of the single-chain dimer as a vaccine prepared by the ⁇ -coronavirus antigen can stimulate mice to produce high titers of neutralizing antibodies.
- a beta coronavirus antigen whose amino acid sequence includes: an amino acid sequence arranged in the pattern of (AB)-(AB) or an amino acid sequence arranged in the pattern of (AB)-C-(AB) or (AB)-(A-B' ) Amino acid sequence arranged in a pattern or an amino acid sequence arranged in a pattern of (AB)-C-(A-B'), where: AB represents a partial or all amino acid sequence of the receptor binding region of the surface spike protein of ⁇ coronavirus , C represents the linking amino acid sequence, A-B' represents the amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence in AB, and the protein encoded by A-B' has the same or substantially the same as the protein encoded by AB With the same immunogenicity, the beta coronavirus antigen has a single-chain dimer structure.
- the partial amino acid sequence of the receptor binding region of the surface spike protein of ⁇ coronavirus is at least 50%, 60%, 70%,
- the ⁇ -coronaviruses include: severe respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, and 2019 new coronavirus (2019-new coronavirus may also be referred to as 2019- nCoV or SARS-CoV-2).
- the linking amino acid sequence includes: (GGS) n linking sequence, where n represents the number of GGS, and n is an integer ⁇ 1; optionally, n is an option An integer from 1-10; further optionally, n is an integer selected from 1-5.
- the three letters GGS represent amino acids G, G, and S respectively.
- a partial or all amino acid sequence of the receptor binding region of its surface spike protein is selected from the group consisting of the following amino acids Any kind of sequence:
- SEQ ID NO: 1 SEQ ID NO: 2, or SEQ ID NO: 3;
- the partial amino acid sequence of the receptor binding region of the surface spike protein includes SEQ ID NO: 2.
- SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 are all derived from a part of the MERS-CoV S protein (GenBank on NCBI: AFS88936.1), which is the part of the MERS-CoV S protein.
- the amino acid sequence of the ⁇ -coronavirus antigen includes any one selected from the following amino acid sequences:
- the amino acid sequence of the ⁇ -coronavirus antigen includes two repeated SEQ ID NO: 2 amino acid sequences directly connected in series, that is, E367-N602-E367-N602.
- part of the amino acid sequence or the entire amino acid sequence of the receptor binding region of the surface spike protein is selected from the group consisting of the following amino acid sequences: Any one:
- SEQ ID NO: 5 SEQ ID NO: 6, or SEQ ID NO: 7;
- the partial amino acid sequence of the receptor binding region of the surface spike protein includes SEQ ID NO: 6.
- SEQ ID NO: 5 SEQ ID NO: 6, or SEQ ID NO: 7 are all part of the S protein sequence of the WH01 strain of 2019-nCoV (GenBank on NCBI: QHR63250), which are respectively 2019-nCoV The R319-S530 region, R319-K537 region, and R319-F541 region of the RBD of the S protein.
- the amino acid sequence of the ⁇ -coronavirus antigen includes any one selected from the following amino acid sequences:
- amino acid sequence of 2 repeated SEQ ID NO: 7 directly connected in series namely R319-F541-R319-F541;
- the amino acid sequence of the beta coronavirus antigen includes two repeated SEQ ID NO: 6 amino acid sequences directly connected in series, that is, R319-K537-R319-K537.
- part of the amino acid sequence or the entire amino acid sequence of the receptor binding region of the surface spike protein is selected from the group consisting of the following amino acids Any kind of sequence:
- SEQ ID NO: 8 is a part of the S protein sequence of SARS-CoV (GenBank on NCBI: AAR07630), which is the R306-Q523 region of the RBD of the SARS-CoV S protein.
- the amino acid sequence of the ⁇ -coronavirus antigen includes: 2 repeated SEQ ID NO: 8 amino acid sequences directly connected in series, Namely R306-Q523-R306-Q523.
- nucleotide sequence encoding the two repeated amino acid sequences of SEQ ID NO: 1 connected in series by the GGSGGS connection sequence is shown in SEQ ID NO: 9;
- the nucleotide sequence encoding the two repeated SEQ ID NO: 1 amino acid sequence connected in series by the GGS connection sequence is shown in SEQ ID NO: 10;
- SEQ ID NO: 12 The nucleotide sequence encoding the two repeated SEQ ID NO: 2 amino acid sequences connected in series by the GGS connection sequence is shown in SEQ ID NO: 12;
- nucleotide sequence encoding the two repeated SEQ ID NO: 3 amino acid sequences connected in series by the GGSGGSGGSGGSGGS connection sequence is shown in SEQ ID NO: 14;
- the nucleotide sequence encoding the 2 repeated SEQ ID NO: 3 amino acid sequence connected in series by the GGSGGSGGSGGS connection sequence is shown in SEQ ID NO: 15;
- nucleotide sequence encoding the two repeated SEQ ID NO: 3 amino acid sequences connected in series by the GGSGGSGGS connection sequence is shown in SEQ ID NO: 16;
- nucleotide sequence encoding the two repeated SEQ ID NO: 3 amino acid sequences connected in series by the GGS connection sequence is shown in SEQ ID NO: 17;
- SEQ ID NO: 18 The nucleotide sequence encoding the 2 repeated SEQ ID NO: 3 amino acid sequence directly in series is shown in SEQ ID NO: 18;
- SEQ ID NO: 19 The nucleotide sequence encoding the 2 repeating amino acid sequence of SEQ ID NO: 5 in direct tandem is shown in SEQ ID NO: 19;
- SEQ ID NO: 20 The nucleotide sequence encoding the 2 repeating amino acid sequence of SEQ ID NO: 6 directly in series is shown in SEQ ID NO: 20;
- SEQ ID NO: 21 The nucleotide sequence encoding the 2 repeating amino acid sequence of SEQ ID NO: 7 directly in series is shown in SEQ ID NO: 21;
- SEQ ID NO: 23 The nucleotide sequence encoding the two repeated SEQ ID NO: 8 amino acid sequence directly connected in series is shown in SEQ ID NO: 23.
- the present invention also provides a method for preparing the above-mentioned ⁇ -coronavirus antigen, which includes the following steps: a sequence encoding a signal peptide is added to the 5'end of the nucleotide sequence encoding the above-mentioned ⁇ -coronavirus antigen, and a termination code is added to the 3'end After expressing, clone and express, screen the correct recombinant, and then transfect the cells of the expression system for expression. After expression, the cell supernatant is collected and purified to obtain the ⁇ -coronavirus antigen.
- the cells of the expression system include mammalian cells, insect cells, yeast cells, or bacterial cells, optionally; the mammalian cells include 293T cells or CHO cells, and Bacterial cells include E. coli cells.
- the present invention also provides a nucleotide sequence encoding the above-mentioned beta coronavirus antigen, a recombinant vector including the above-mentioned nucleotide sequence, and an expression system cell including the above-mentioned recombinant vector.
- the present invention also provides a beta coronavirus antigen, a nucleotide sequence encoding the beta coronavirus antigen, a recombinant vector comprising the aforementioned nucleotide sequence, and an expression system cell comprising the aforementioned recombinant vector in the preparation of a beta coronavirus vaccine Applications.
- the present invention also provides a ⁇ -coronavirus vaccine, which includes the above-mentioned ⁇ -coronavirus antigen and an adjuvant.
- the adjuvant is selected from aluminum adjuvant, MF59 adjuvant or MF59-like adjuvant.
- the present invention also provides a ⁇ -coronavirus DNA vaccine, which includes: a recombinant vector containing the DNA sequence encoding the above-mentioned ⁇ -coronavirus antigen.
- the present invention also provides a ⁇ -coronavirus mRNA vaccine, which includes: a recombinant vector containing the mRNA sequence encoding the above-mentioned ⁇ -coronavirus antigen.
- the present invention also provides a beta coronavirus virus vector vaccine, which includes: a recombinant virus vector comprising a nucleotide sequence encoding the above beta coronavirus antigen; optionally, the virus vector is selected from one or more of the following : Adenovirus vector, poxvirus vector, influenza virus vector, adeno-associated virus vector.
- the crystals of the MERS-CoV dimer RBD protein are further analyzed Structure, found that the MERS dimer RBD protein can form a head-to-head dimer, so the inventors tried to connect two nucleotide sequences encoding the same or substantially the same monomeric RBD protein directly in series or through a connecting segment.
- the obtained two identical or substantially identical monomeric RBD proteins are connected in series through the flexible regions of the N-terminal and the C-terminal, and the results show that this method can realize the expression of single-chain dimers well.
- the single-chain dimeric RBD protein obtained in the embodiment of the present invention does not Due to the unstable formation of disulfide bonds, the content of dimer RBD protein in the production process is unstable, that is, the monomer RBD form is avoided as the main form, and the expression of dimer formation is rare, so that the dimer RBD expression is stable , The form is uniform, and the output is greatly improved.
- the single-chain dimer expressed in the embodiment of the present invention is used as ⁇ Coronavirus antigens have considerable immunogenicity.
- the single-chain dimer is used as a ⁇ -coronavirus antigen to prepare a ⁇ -coronavirus vaccine that can stimulate mice to produce high titers of neutralizing antibodies.
- the head-to-head single-stranded dimer structure is applicable to severe respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and 2019 new coronavirus.
- Figure 1 is the UV absorption graph of the RBD protein obtained by the vector pFastBac-SP-MERS-RBD (E367-Y606) constructed in Example 1 when subjected to Superdex200 Hiload 16/60 molecular sieve chromatography, and UV collection
- the protein obtained from the Dimer peak and Monomer peak in the absorption pattern is electrophoresed by SDS-PAGE under reducing conditions (+DTT) or non-reducing conditions (-DTT).
- Fig. 2 is a diagram of immunization and MERS-CoV challenge strategies in Example 2 to Example 7.
- Fig. 3 is the result of Example 3, showing the IgG titers of MERS-CoV and RBD specific antibodies in the blood serum of the mice obtained in accordance with the immunization strategy in Fig. 2 after the third immunization in Example 2.
- Dimer means that the immune source used is MERS-CoV RBD dimer
- RBD monomer means that the immune source used is MERS-CoV RBD monomer
- AddaVax means AddaVax adjuvant is used
- Alum means aluminum adjuvant is used
- Adjuvant means no adjuvant is used
- 3 ⁇ g, 10 ⁇ g, and 30 ⁇ g indicate the amount of immunization of the immunosource used for each immunization.
- Significant difference analysis ns, p>0.05; *,p ⁇ 0.05; **,p ⁇ 0.01; ***,p ⁇ 0.001; ****,p ⁇ 0.0001.
- FIG 4 is the result of Example 5, showing the MERS-CoV pseudovirus 90% neutralizing antibody titer in the serum of the mice obtained in accordance with the immunization strategy in Figure 2 after the third immunization in Example 2.
- Dimer means that the immune source used is MERS-CoV RBD dimer
- RBD monomer means that the immune source used is MERS-CoV RBD monomer
- AddaVax means AddaVax adjuvant is used
- Alum means aluminum adjuvant is used, not indicated
- Adjuvant means no adjuvant is used
- 3 ⁇ g, 10 ⁇ g, and 30 ⁇ g indicate the amount of immunization of the immunosource used for each immunization.
- Significant difference analysis ns, p>0.05; ***, p ⁇ 0.001.
- FIG 5 is the result of Example 6, which is the result of 50% neutralizing antibody titers against MERS-CoV true virus (EMC strain) in the blood serum collected according to the immunization strategy in Figure 2 after the third immunization of the mice in Example 2 picture.
- Dimer means that the immune source used is MERS-CoV RBD dimer
- AddaVax means that Addavax adjuvant is used
- Alum means that aluminum adjuvant is used, if no adjuvant is indicated, no adjuvant is used, 3 ⁇ g, 10 ⁇ g, 30 ⁇ g means The amount of immune source used for each immunization.
- Fig. 6 is the third immunization of the mouse after the third immunization with adenovirus expressing hCD26 (hDPP4) in the nose according to the immunization strategy in Fig. 2 in Example 7.
- hDPP4 adenovirus expressing hCD26
- Figure 7 is the pathological results of Example 8 detecting the protective effect of the vaccine on mouse lung tissue.
- the lungs of the mice challenged in Example 7 were taken out after dissection, fixed with 4% paraformaldehyde, embedded in paraffin, stained with hematoxylin and eosin, and sectioned to observe pathological changes.
- AddaVax indicates the use of AddaVax adjuvant
- Alum indicates the use of aluminum adjuvant
- 3 ⁇ g, 10 ⁇ g, and 30 ⁇ g indicate the amount of immune source used for each immunization. Slight, Mild and Severe indicated that the lung tissue lesions were mild, moderate and severe respectively.
- FIG. 8 shows the structure of MERS-CoV-RBD dimer (E367-Y606) analyzed in Example 9.
- 9A, 9B, and 9C are the design schemes of the single-chain RBD dimer based on the structure design of the MERS-CoV RBD-dimer dimer in Example 10.
- RBD Monomer is MERS-CoV RBD monomer protein.
- Figure 11 is the UV absorption graph of the MERS-RBD-C5 single-chain dimer expressed in Example 11 when subjected to Superdex200Hiload16/60 column (GE) molecular sieve chromatography, and the purified single-chain dimer is reduced (+DTT) ) Or non-reducing (-DTT) conditions of SDS-PAGE results.
- GE Superdex200Hiload16/60 column
- Fig. 12 shows the titers of MERS-CoV-RBD-specific IgG antibodies induced after mice immunized with MERS-CoV-RBD single-chain dimer and disulfide-linked non-single-chain dimer protein in Example 12.
- sc-dimer is a single-chain dimer
- Dimer is a non-single-chain dimer connected by disulfide bonds.
- Fig. 13 shows the 90% neutralizing antibody titer of MERS-CoV pseudovirus induced after mice immunized with MERS-CoV-RBD single-chain dimer and disulfide-linked non-single-chain dimer protein in Example 12.
- sc-dimer is a single-chain dimer
- Dimer is a non-single-chain dimer connected by disulfide bonds.
- FIGS. 14A and 14B are the comparison diagrams of the receptor binding domain (RBD) of the Beta coronavirus in Example 13.
- the sequences in the two diagrams are continuous, and the following types of ⁇ -coronaviruses are compared:
- Figure 15 is a structural simulation diagram of the SARS-CoV-RBD dimer or 2019-nCoV-RBD dimer in Example 13 and the designed expression 2019-nCoV-RBD dimer, 2019-nCoV-RBD monomer and SARS -Construction of CoV-RBD dimer.
- Figure 16 shows the results of Western blot of several single-chain dimers of SARS-CoV-RBD and 2019-nCoV-RBD expressed in Example 13 under reduced (+DTT) or non-reduced (-DTT) conditions picture.
- Figure 17 is the UV 280nm absorbance graph of the 2019-nCoV-RBD-C2 antigen in Example 14 when it was purified, and the purified single-chain dimer was subjected to SDS under reducing (+DTT) or non-reducing (-DTT) conditions -PAGE result graph.
- Figure 18 is a graph of the UV absorbance at 280 nm when the SARS-CoV-RBD-C1 antigen was purified in Example 14, and the purified single-chain dimer was subjected to SDS under reduced (+DTT) or non-reduced (-DTT) conditions -PAGE result graph.
- Figure 19 shows the 2019-nCoV-RBD-specific IgG antibody titers of the sera collected after three immunizations of the mice in Example 15 (19 days after 1 immunization, 14 days after 2 immunization, and 14 days after 3 immunization).
- sc-dimer means that the immunosource used is nCoV-RBD single-chain dimer
- Monomer means that the immunosource used is nCoV-RBD monomer.
- **** ****, p ⁇ 0.0001.
- Figure 20 shows the 90% neutralizing antibody titers of the 2019-nCoV pseudovirus of the sera collected after three immunizations (19 days after 1 immunization, 14 days after 2 immunization, and 14 days after 3 immunization) of the mice in Example 15 respectively.
- sc-dimer means that the immunosource used is nCoV-RBD single-chain dimer
- Monomer means that the immunosource used is nCoV-RBD monomer.
- Fig. 21 shows the 50% neutralizing antibody titer of 2019-nCoV true virus (2020XN4276 strain) in serum collected after the second immunization of mice (14 days after 2 immunization) in Example 15.
- sc-dimer means that the immunosource used is nCoV-RBD single-chain dimer
- Monomer means that the immunosource used is nCoV-RBD monomer.
- Figure 22 shows the SARS-RBD-specific IgG antibody titers of the sera collected after three immunizations of the mice in Example 16 (19 days after 1 immunization, 14 days after 2 immunization, and 14 days after 3 immunization).
- sc-dimer means that the immunosource used is SARS-CoV-RBD single-chain dimer
- Monomer means that the immunosource used is SARS-CoV-RBD monomer.
- Figure 23 shows the 90% neutralizing antibody titers of SARS-CoV pseudoviruses collected from the mice in Example 16 after three immunizations (19 days after 1 immunization, 14 days after 2 immunization, and 14 days after 3 immunization).
- sc-dimer indicates that the immune source used is SARS-CoV-RBD single-chain dimer
- Monomer indicates that the immune source used is SARS-RBD monomer.
- Non-single-chain RBD dimers and RBD monomers connected by disulfide bonds insert the nucleotide sequence encoding the RBD monomer into the vector, and then transfect the cells of the expression system for expression. After expression, the cell supernatant is collected and purified A non-single-chain RBD dimer connected by RBD monomers and disulfide bonds is obtained, wherein: the two RBD monomers in the non-single-chain RBD dimer connected by disulfide bonds simply pass through the cysteine The dimer RBD formed by the combination of acids with disulfide bonds. Disulfide bond-linked non-single-chain RBD dimers and non-single-chain dimeric RBD proteins all have the same meaning; RBD monomer, monomer RBD, and monomer RBD protein all have the same meaning.
- Single-stranded RBD dimer Two nucleotide sequences encoding the same or substantially the same monomeric RBD are directly connected in series or connected in series, and a sequence encoding a signal peptide is added to the 5'end of the nucleotide sequence , Add a stop codon to the 3'end, perform cloning and expression, select the correct recombinant, and then transfect the cells of the expression system for expression, collect the cell supernatant after expression, and purify the recombinant protein, which contains two RBD monomers .
- the two RBD monomers are the same or substantially the same, and can be directly connected by peptide bonds or by a linking sequence (such as GGS, GGSGGS, etc.), which is a single-chain RBD dimer.
- single-chain RBD dimer, RBD single-chain dimer, single-chain dimer, sc-RBD dimer, single-chain RBD dimer, etc. all have the same meaning.
- Example 1 Preparation of recombinant baculovirus expressing MERS-CoV antigen and expression and purification of RBD protein
- the nucleotide sequence (as shown in SEQ ID NO: 1) of the amino acid RBD (E367-Y606) sequence (as shown in SEQ ID NO: 1) in the MERS-CoV S protein (sequence as GenBank: AFS88936.1) (as shown in SEQ ID NO: 24) )
- After adding a translation stop codon to the 3'end cloned into the pFastBac vector containing the gp67 signal peptide (pFastBac-SP from Invitrogen) between the EcoR I and Xho I restriction sites, so that the protein coding region is in the signal peptide gp67 sequence
- the latter is fused and expressed for the secretion of the target protein, and the C-terminal of the target protein is carried with 6 histidines to obtain the vector pFastBac-SP-MERS-RBD (E367-Y606), and then transfect the cells of the expression system for expression
- the elution peak near the elution volume of 90 mL was analyzed by SDS-PAGE.
- the size of the target protein was about 30Kd under non-reducing conditions (without DTT) and reducing conditions, which proved that the peak was mainly RBD monomer.
- the RBD dimers and RBD monomers in non-single-chain form connected by disulfide bonds are obtained above.
- the dimers or monomers used in Examples 2 to 9 below are the disulfide bonds obtained in this example.
- the non-single-chain form of RBD dimers and RBD monomers are obtained in this example.
- MF59 addedaVax used below is an MF59-like adjuvant
- aluminum adjuvant are two commonly used adjuvants approved by SFDA.
- SFDA SF59-like adjuvant
- In vitro neutralization test is a classic method to detect the protective effect of vaccines. Therefore, we mixed different doses of antigens with AddaVax adjuvant and Imject TM Alum adjuvant for immunization.
- the immunization group status, the RBD type used in each group, the amount of RBD immunization used per immunization and the adjuvant status are shown in Table 1, and the blank part means "none".
- the MERS-RBD antigen (dimer or monomer) obtained in Example 1 was diluted in physiological saline to the desired concentration, and was emulsified in groups with the adjuvant. Then 4-6 weeks old BALB/c mice (average weight 15-20g, the same below) were immunized in groups, with 6 mice in each group.
- the immunization strategy is shown in Figure 2, that is, through thigh intramuscular injection, each mouse receives 3 immunizations on day 0, day 21, and day 42, each with an inoculation volume of 100 ⁇ l.
- On the 56th day that is, the 14th day after the three immunizations, blood was collected from the tail of the mice.
- Mouse serum was obtained by centrifugation at 3000 rpm for 10 minutes after standing still, and stored in a refrigerator at -20°C for specific antibody titer detection and pseudovirus neutralization detection.
- Example 3 ELISA test to detect vaccine-induced specific antibody titers
- All the coating proteins in the ELISA experiments in the examples of the present invention used the RBD monomer protein of MERS-CoV.
- Plasmid co-transfection by PEI method A total of 20 ⁇ g plasmid (HIV pNL4-3.Luc.RE (Invitrogen) 10 ⁇ g, pCAGGS-MERS-S 10 ⁇ g, where pCAGGS-MERS-S is the Spike protein that will encode MERS ( M1-H1352) DNA sequence was inserted into the EcoRI and XhoI sites of pCAGGS vector.) and 40 ⁇ l PEI (2mg/ml) were dissolved in normal saline or HBS, the final volume was 500 ⁇ L, and mixed; after standing for 5min, The two were mixed, and then allowed to stand for 20 minutes. The mixture was added dropwise to the cell culture dish. After 4-6 hours, the cells were washed twice with PBS and replaced with fresh serum-free medium.
- the susceptible cells were washed with PBS to remove the serum, the virus supernatant was taken to infect the susceptible cells, and the culture medium containing serum was changed for 4-6 hours.
- the Luciferase value can be determined at different time points, refer to Promega's Luciferase Assay System Protocol or Dual Luciferase Reporter Assay System Protocol.
- the collected virus solution was diluted by a 5-fold ratio and added to Huh7 cells (human liver cancer cells) in a 96-well plate. After 4 hours of infection, the virus solution was discarded, the cells were washed twice with PBS, and replaced with DMEM complete medium containing 10% serum.
- the serum obtained in Example 2 was diluted in multiples, mixed with 100 TCID 50 pseudovirus, and incubated at 37°C for 30 minutes. Add the mixture to a 96-well plate that has been contaminated with Huh7 cells. After 4 hours of incubation at 37°C, the virus solution was discarded, the cells were washed twice with PBS, and replaced with complete medium DMEM containing 10% serum. After 48 hours, the culture medium was discarded, the cells were washed twice with PBS, and the cell lysate was added to detect the luciferase activity value.
- the pseudovirus has a spike protein on its surface. The pseudovirus infects cells to release DNA and express luciferase, but does not replicate. If neutralizing antibodies are present, the pseudovirus cannot infect the cells and does not express luciferase. This method is used to test the neutralization titer of the serum.
- the immunogenicity test results after the third immunization are shown in Figure 4.
- the results showed that the RBD dimer (E367-Y606) produced neutralizing antibodies after three immunizations, regardless of the AddaVax adjuvant group or the aluminum adjuvant group (+Alum).
- the average value of neutralizing antibody NT 90 in the AddaVax adjuvant 10 ⁇ g group can reach more than 1:1000 (as shown in Figure 4).
- RBD monomer (E379-E589) after three immunizations, except for 2 mice that produced weaker neutralizing antibodies, the rest were undetectable (as shown in Figure 4).
- Pseudovirus neutralization experiments proved that the neutralizing antibody induced by dimer RBD was much higher than that of monomer RBD.
- the above-mentioned RBD monomer (E379-E589) is obtained by the following method: the nucleic acid fragment (as shown in SEQ ID NO: 4) encoding the amino acid (E379-E589) sequence in the MERS-CoV S protein (as shown in SEQ ID NO: 25) Shown) Insert the EcoRI and XhoI restriction sites of pFastBac-SP to make the protein coding region fused and expressed after the signal peptide gp67 sequence for the secretion of the target protein, and make the C-terminal of the target protein carry 6 histidines , Get the vector pFastBac-SP-MERS-RBD (E379-E589)
- mice after three immunizations in Example 2 were infected with adenovirus expressing hCD26 (hDPP4) by intranasal drip on the 77th day as shown in Fig. 2.
- hCD26 the receptor of MERS-CoV
- MERS-CoV Middle East respiratory syndrome coronavirus S1 protein induces protective immune responses in mice[J] .Vaccine,2017,35(16):2069-2075.
- EMC strain MERS-CoV
- the lung tissues of the mice in the MERS-CoV challenge experiment in Example 7 were fixed with 4% paraformaldehyde, then stained with hematoxylin-eosin, and sectioned to observe the changes in the lungs.
- the results are shown in Figure 7.
- the lung tissue of the mice in the control group ie, the PBS group
- the lung tissue of the mice in the control group showed severe interstitial pneumonia, alveolitis, inflammatory cell infiltration, and bronchial epithelial cell necrosis (Figure 7).
- AddaVax or Alum as an adjuvant, it can greatly alleviate the lung injury caused by the virus challenge, showing mild to moderate lung injury, with clearly visible alveoli and less inflammatory cell infiltration.
- a small amount of lung tissue lesions may be caused by a large challenge dose (5X 10 5 pfu).
- RBD E367-Y606 protein was expressed. After purification, the dimer protein peaks were collected. The protein is concentrated to 10mg/ml, the protein and the crystallization pool liquid are mixed in a volume ratio of 1:1, and then passed Protein crystal screening liquid workstation (TTP LabTech) performs protein crystal screening. Grow at 18°C to obtain crystals that can be used for diffraction. The crystal was collected at the Shanghai Synchrotron Radiation Center (SSRF) and finally obtained Diffraction data. The data was analyzed by HKL2000 software, and the structure of the MERS-RBD monomer was used as a template (PDB: 4KQZ), and the structure of the MERS-RBD dimer was finally resolved by the molecular replacement method. As shown in Figure 8.
- Example 10 Design of single-chain RBD dimer based on the structure of MERS-RBD dimer (sc-RBD dimer)
- N-terminal (N') and C-terminal (C') of the two subunits of RBD are arranged in a head-to-head arrangement.
- N-terminal and C-terminal each have an invisible flexible sequence ( Figure 9A). Therefore, we designed a single-chain RBD dimer (sc-RBD dimer) by connecting two subunits in series.
- the first design (Figure 9A) includes:
- MERS-RBD-C1 (abbreviated as C1).
- the nucleotide sequence encoding the amino acid sequence is SEQ ID NO: 9 ;
- MERS-RBD-C2 (abbreviated as C2).
- the nucleotide sequence encoding the amino acid sequence is SEQ ID NO: 10 ;
- MERS-RBD-C4 (abbreviated as C4).
- the nucleotide sequence encoding the amino acid sequence is SEQ ID NO: 12;
- the third design (as shown in Figure 9C) directly expresses the visible sequence of the structure and is connected by linking sequences of different lengths, including:
- MERS-RBD-C8 (abbreviated as C8).
- the nucleotide sequence encoding the amino acid sequence is SEQ ID NO: 16;
- the two repeats (V381-L588) are directly connected in series to obtain MERS-RBD-C10 (C10 for short).
- the nucleotide sequence encoding the amino acid sequence is SEQ ID NO: 18.
- the nucleotide sequence encoding the MERS-S protein signal peptide (MIHSVFLLMFLLTPTES) is added to the 5'end of the nucleotide sequence encoding the above MERS-RBD-C1 to C10, and the nucleotide sequence encoding 6 histidines is added to the 3'end.
- a stop codon is added to the 3'end, and the resulting nucleotide sequence is inserted between the EcoRI and XhoI restriction sites of the pCAGGS vector, and the start codon upstream of the start codon contains the Kozak sequence gccacc.
- the above plasmids were transfected into 293T cells.
- the N-terminus of the target protein contained a signal peptide.
- the expression of the target protein was detected by Western blot. The results are shown in Figure 10. The results showed that except for C2, all other constructs were expressed. Whether it is reducing (+DTT) or non-reducing (-DTT) conditions, the protein is about the size of the dimer (50-60Kda). Among them, C4 and C5 have the highest expression levels. Considering that the C5 construction does not introduce foreign connection sequences, it is entirely the sequence of MERS-CoV itself, so it is more advantageous and safer for clinical use. We will further evaluate the effectiveness of MERS-RBD-C5 as a vaccine.
- Example 11 Mammalian expression of MERS-CoV RBD single-chain dimer (sc-RBD dimer) and protein purification
- Mammalian 293T cells were used to express MERS-RBD-C5. After the plasmid was transfected into 293T cells, the expression was performed and the supernatant was harvested. The cell supernatant was filtered through a 0.22 ⁇ m filter membrane to remove cell debris. Hang the cell culture supernatant on a nickel affinity column (Histrap) at 4°C overnight. Wash the resin with buffer A (20mM Tris, 150mM NaCl, pH 8.0) to remove non-specific binding proteins. Finally, the target protein was eluted from the resin with buffer B (20mM Tris, 150mM NaCl, pH 8.0, 300mM imidazole), and the eluate was concentrated to within 5ml with a 10K cutoff tube.
- buffer A (20mM Tris, 150mM NaCl, pH 8.0
- buffer B 20mM Tris, 150mM NaCl, pH 8.0, 300mM imidazole
- the MERS-RBD single-chain dimer antigen obtained in Example 11 was diluted in physiological saline and emulsified in groups with adjuvants. Then, BALB/c mice aged 4-6 weeks were immunized in groups with 6 mice in each group. In addition, a group of mice was immunized with PBS as a negative control. A group of mice were immunized with non-single-chain dimers expressed by 293T cells. By means of thigh intramuscular injection, each mouse received 3 vaccine immunizations on day 0, day 21, and day 42, each with an inoculation volume of 100 ⁇ l (containing 10 ⁇ g of immune source).
- Mouse serum was obtained by centrifugation at 3000 rpm for 10 minutes after standing, and stored in a refrigerator at -20°C for specific antibody detection and pseudovirus neutralization detection.
- the specific antibody titer of mouse serum was detected by ELISA experiment.
- the experimental method is shown in Example 3.
- the results are shown in Figure 12.
- the RBD-sc-dimer group of mice and the disulfide-linked non-single-chain RBD-dimer group ( Dimer said) mice can be induced to produce an antibody response.
- the average titer of the sc-dimer group is higher than that of the Dimer group, and there is a significant difference between the two after three immunizations (*, p ⁇ 0.05). This result shows that sc-dimer is as immunogenic as non-single-chain RBD-dimer linked by disulfide bonds.
- the pseudovirus neutralization experiment was performed according to Example 5. The results are shown in Figure 13.
- the mice in the sc-dimer group and the disulfide-linked non-single-chain RBD-dimer group (indicated by Dimer) can be induced to produce antibody responses.
- the average titer of the sc-dimer group is higher than that of Dimer.
- the group is high, there is a significant difference between the first immunization and the second immunization ( Figure 13).
- the mean neutralization titer of pseudovirus in the sc-dimer group mice was greater than 1:1000. This result indicates that the vaccine developed with sc-dimer has great potential for clinical development.
- the simulated SARS-RBD dimer structure as shown in Figure 15 is obtained.
- the SARS-RBD dimer also exists in a head-to-head form ( Figure 15). Since the RBD region homology of the new coronavirus 2019-nCoV and SARS-CoV is as high as 75% or more, we expect the RBD dimer of 2019-nCoV to form this head-to-head form.
- the dimers in MERS-CoV can induce higher titers of neutralizing antibodies than monomers, we therefore consider still using single-chain dimers (sc-dimer) to design vaccines.
- nCoV-RBD-C1 the nucleotide sequence encoding the amino acid sequence is SEQ ID NO: 19
- nCoV-RBD-C2 the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 20)
- nCoV-RBD-C3 the nucleotide sequence encoding the amino acid sequence is SEQ ID NO: 21
- Additional monomer construction R319-F541, named nCoV-RBD-C4 the nucleotide sequence encoding the amino acid sequence is SEQ ID NO: 22).
- SARS-CoV-RBD-C1 the nucleotide sequence encoding the amino acid sequence. Is SEQ ID NO: 23).
- nucleotide sequence encoding the above nCoV-RBD-C1 to C4 and the nucleotide sequence encoding SARS-CoV-RBD-C1 (SEQ ID NO: 23) plus the signal peptide encoding the MERS-S protein itself The nucleotide sequence of (MIHSVFLLMFLLTPTES); after adding a nucleotide sequence encoding 6 histidines to the 3'end, a stop codon is added to the 3'end and inserted into the pCAGGS vector EcoRI and XhoI restriction sites, The upstream of the start codon contains the Kozak sequence gccacc.
- the above plasmids were transfected into 293T cells, 48 hours later, the supernatant was taken, and the expression of the target protein was detected by Western blot.
- the expression results are shown in Figure 16. The results showed that the expression of nCoV-RBD-C2 was the highest among several antigen designs of 2019-nCoV.
- the SARS-CoV-RBD-C1 construct protein expression is also very high.
- Example 14 Expression and purification of 2019-nCoV-RBD single-chain dimer antigen and SARS-CoV-RBD single-chain dimer antigen
- Mammalian 293T cells express nCoV-RBD-C2. After the plasmid was transfected into 293T cells, the supernatant was harvested. The cell supernatant was filtered through a 0.22 ⁇ m filter membrane to remove cell debris. Hang the cell culture supernatant on a nickel affinity column (Histrap) at 4°C overnight. Wash the resin with buffer A (20mM Tris, 150mM NaCl, pH 8.0) to remove non-specific binding proteins. Finally, the target protein was eluted from the resin with buffer B (20mM Tris, 150mM NaCl, pH 8.0, 300mM imidazole), and the eluate was concentrated to within 5ml with a 10K cutoff tube.
- the 2019-nCoV monomer RBD protein (constructed and expressed from nCoV-RBD-C4), SARS-CoV monomer RBD protein (SARS-CoV RBD R306-F527, amino acid sequence and encoding its amino acid sequence) were expressed and purified in the same way
- the nucleotide sequence of is shown in SEQ ID NO: 26 and SEQ ID NO: 27) and the SARS-CoV single-chain dimer protein (obtained from the construction and expression of SARS-CoV-RBD-C1).
- SARS-CoV single-chain dimer protein The results of SARS-CoV single-chain dimer protein are shown in Figure 18. It can be seen that after molecular sieve chromatography, there is only one main peak near the elution volume of 80 ml. Collected and analyzed by SDS-PAGE, it can be seen from Figure 18 that the molecular weight of the SARS-CoV-RBD-C1 target protein is between 55-72kd, which is the size of the RBD dimer. It is proved that the SARS-RBD single-chain dimer is obtained, as shown in Figure 18, and has a high purity.
- mice were diluted in a PBS solution and emulsified in groups with AddaVax adjuvant. Then BALB/c mice aged 6-8 weeks (average weight 15-20g, the same below) were immunized in groups, with 8 mice in each group. By means of thigh intramuscular injection, each mouse received 3 vaccine immunizations on day 0, day 21, and day 42, each with an inoculation volume of 100 ⁇ l (containing 10 ⁇ g of immune source). The mice were immunized for 19 days, 2 for 14 days, and 3 for 14 days, respectively. Mouse serum was obtained by centrifugation at 3000 rpm for 10 minutes after standing, and stored in a refrigerator at -20°C for specific antibody detection and pseudovirus neutralization detection.
- the 2019-CoV RBD specific antibody titer of mouse serum was detected by ELISA experiment.
- the experimental method is shown in Example 3.
- the results are shown in Figure 19.
- Single-chain dimer RBD (labeled sc-dimer) and monomer RBD (labeled Monomer) can induce antibody response in mice.
- the average titer of the single-chain dimer RBD group after each immunization is higher than that of the monomer RBD group (10-100 times higher), and both after each immunization Significant difference (Figure 19).
- the single-chain dimer RBD induced mice to produce antibody levels as high as about 1:10 6 . This result shows that the single-chain dimer form of RBD antigen is more immunogenic than the monomer form of RBD antigen, and it has great potential as a potential new coronavirus vaccine.
- the 2019-nCoV true virus (2020XN4276 strain, which is disclosed in Lu J, du Plessis L, Liu Z, et al. Genomic Epidemiology of SARS-CoV-2 in Guangdongzhou, China. Cell.2020;181(5):997-1003.e9.doi:10.1016/j.cell.2020.04.023, provided by Guangdong Provincial Center for Disease Control and Prevention) neutralization experiment.
- the experimental results are shown in Figure 21.
- the results show that RBD dimer can induce mice to produce high levels of neutralizing antibodies against the new coronavirus.
- the highest neutralizing NT50 can be greater than 4096, and the lowest NT50 value for a mouse is 512.
- mice in the RBD monomer group only 2 detected neutralizing antibodies to the new coronavirus, and the NT50 was lower, 128 and 256, respectively. This result indicates that the dimeric RBD can induce mice to produce higher levels of neutralizing antibodies against the new coronavirus.
- the SARS-RBD single-chain dimer and SARS-RBD monomer obtained in Example 14 were diluted in a PBS solution and emulsified in groups with AddaVax adjuvant. Then BALB/c mice aged 6-8 weeks were immunized in groups, with 6 mice in each group. By intramuscular injection of the thigh, each mouse received 3 vaccine immunizations on day 0, day 2 and day 42, each with an inoculation volume of 100 ⁇ l (containing 10 ⁇ g of immunosource). The mice were immunized for 19 days, 2 for 14 days, and 3 for 14 days, respectively.
- the mouse serum was obtained by centrifugation at 3000 rpm for 10 minutes after standing, and stored in a refrigerator at -20°C for specific antibody detection and pseudovirus neutralization detection.
- the SARS-RBD specific antibody titer in mouse serum was detected by ELISA experiment.
- the experimental method is shown in Example 3.
- the result is shown in Figure 22.
- Single-chain dimer RBD (labeled sc-dimer) and monomer RBD (labeled Monomer) ) Can induce antibody response in mice. After each immunization, the average titer of the single-chain dimer RBD group is higher than that of the monomer RBD group, and there are significant differences between the two after the second and third immunizations ( Figure 22). After three immunizations, the dimer RBD induced mice to produce antibody levels as high as about 1:10 6 . This result indicates that the RBD antigen in the dimer form is more immunogenic than the RBD antigen in the monomer form.
- the embodiment of the present invention relates to a beta coronavirus antigen, its preparation method and application.
- the amino acid sequence of the ⁇ coronavirus antigen includes: the amino acid sequence arranged in the (AB)-(AB) pattern or the amino acid sequence arranged in the (AB)-C-(AB) pattern or the (AB)-(A-B') pattern arrangement The amino acid sequence or the amino acid sequence arranged in (AB)-C-(A-B') pattern, where: AB represents part of the amino acid sequence or the entire amino acid sequence of the receptor binding region of the surface spike protein of ⁇ coronavirus, and C represents Linking the amino acid sequence, A-B' represents the amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence in AB.
- the protein encoded by A-B' has the same or substantially the same immunity as the protein encoded by AB Primarily, the beta coronavirus antigen has a single-chain dimer structure.
- the content of the single-chain dimer expressed in the examples of the present invention is stable and has good immunogenicity as a beta coronavirus antigen.
- the vaccine prepared from the single-chain dimer as a beta coronavirus antigen can stimulate mice to produce very high drops. Degree of neutralizing antibodies.
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Abstract
Description
Claims (18)
- 一种β冠状病毒抗原,其特征在于:其氨基酸序列包括:按照(A-B)-(A-B)样式排列的氨基酸序列或(A-B)-C-(A-B)样式排列的氨基酸序列或(A-B)-(A-B’)样式排列的氨基酸序列或(A-B)-C-(A-B’)样式排列的氨基酸序列,其中:A-B表示β冠状病毒的表面刺突蛋白的受体结合区的部分氨基酸序列或全部氨基酸序列,C表示连接氨基酸序列,A-B’表示A-B中的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列,A-B’编码的蛋白质具有与A-B所编码的蛋白质相同或基本相同的免疫原性,所述β冠状病毒抗原为单链二聚体结构。
- 根据权利要求1所述的β冠状病毒抗原,其特征在于:所述β冠状病毒包括:严重呼吸综合征冠状病毒、中东呼吸综合征冠状病毒和2019新型冠状病毒。
- 根据权利要求1所述的β冠状病毒抗原,其特征在于:所述连接氨基酸序列包括:(GGS) n连接序列,其中n表示GGS的个数,n为≥1的整数;可选地,n为选自1-10的整数;进一步可选地,n为选自1-5的整数。
- 根据权利要求1所述的β冠状病毒抗原,其特征在于:β冠状病毒的表面刺突蛋白的受体结合区的部分氨基酸序列为β冠状病毒的表面刺突蛋白的受体结合区的全部氨基酸序列的至少50%、60%、70%、80%、90%、99%。
- 根据权利要求1所述的β冠状病毒抗原,其特征在于:当β冠状病毒为中东呼吸综合征冠状病毒时,其表面刺突蛋白的受体结合区的部分氨基酸序列或全部氨基酸序列选自包括以下氨基酸序列的任意一种:(1)SEQ ID NO:1,SEQ ID NO:2,或SEQ ID NO:3;(2)在(1)中的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列,该氨基酸序列编码的蛋白质具有与(1)所编码的蛋白质相同或基本相同的免疫原性;可选地,其表面刺突蛋白的受体结合区的部分氨基酸序列包括SEQ ID NO:2;当β冠状病毒为2019新型冠状病毒时,其表面刺突蛋白的受体结合区的部分氨基酸序列或全部氨基酸序列选自包括以下氨基酸序列的任意一种:(3)SEQ ID NO:5,SEQ ID NO:6,或SEQ ID NO:7;(4)在(3)中的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基 酸序列,该氨基酸序列编码的蛋白质具有与(3)所编码的蛋白质相同或基本相同的免疫原性;可选地,其表面刺突蛋白的受体结合区的部分氨基酸序列包括SEQ ID NO:6;当β冠状病毒为严重呼吸综合征冠状病毒时,其表面刺突蛋白的受体结合区的部分氨基酸序列或全部氨基酸序列选自包括以下氨基酸序列的任意一种:(5)SEQ ID NO:8;(6)在(5)中的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列,该氨基酸序列编码的蛋白质具有与(5)所编码的蛋白质相同或基本相同的免疫原性。
- 根据权利要求5所述的β冠状病毒抗原,其特征在于:当β冠状病毒为中东呼吸综合征冠状病毒时,β冠状病毒抗原的氨基酸序列包括选自以下氨基酸序列的任意一种:(1)通过GGSGGS连接序列串联起来的2个重复SEQ ID NO:1氨基酸序列;(2)通过GGS连接序列串联起来的2个重复SEQ ID NO:1氨基酸序列;(3)直接串联的2个重复SEQ ID NO:1氨基酸序列;(4)通过GGS连接序列串联起来的2个重复SEQ ID NO:2氨基酸序列;(5)直接串联的2个重复SEQ ID NO:2氨基酸序列;(6)通过GGSGGSGGSGGSGGS连接序列串联起来的2个重复SEQ ID NO:3氨基酸序列;(7)通过GGSGGSGGSGGS连接序列串联起来的2个重复SEQ ID NO:3氨基酸序列;(8)通过GGSGGSGGS连接序列串联起来的2个重复SEQ ID NO:3氨基酸序列;(9)通过GGS连接序列串联起来的2个重复SEQ ID NO:3氨基酸序列;(10)直接串联的2个重复SEQ ID NO:3氨基酸序列;可选地,β冠状病毒抗原的氨基酸序列包括直接串联的2个重复SEQ ID NO:2氨基酸序列;当β冠状病毒为2019新型冠状病毒时,β冠状病毒抗原的氨基酸序列包括选自以下氨基酸序列的任意一种:(1)直接串联的2个重复SEQ ID NO:5氨基酸序列;(2)直接串联的2个重复SEQ ID NO:6氨基酸序列;(3)直接串联的2个重复SEQ ID NO:7氨基酸序列;可选地,β冠状病毒抗原的氨基酸序列包括直接串联的2个重复SEQ ID NO:6氨基酸序列;当β冠状病毒为严重呼吸综合征冠状病毒时,β冠状病毒抗原的氨基酸序列包括:直接串联的2个重复SEQ ID NO:8氨基酸序列。
- 根据权利要求6所述的β冠状病毒抗原,其特征在于:编码通过GGSGGS连接序列串联起来的2个重复SEQ ID NO:1氨基酸序列的核苷酸序列如SEQ ID NO:9所示;编码通过GGS连接序列串联起来的2个重复SEQ ID NO:1氨基酸序列的核苷酸序列如SEQ ID NO:10所示;编码直接串联的2个重复SEQ ID NO:1氨基酸序列的核苷酸序列如SEQ ID NO:11所示;编码通过GGS连接序列串联起来的2个重复SEQ ID NO:2氨基酸序列的核苷酸序列如SEQ ID NO:12所示;编码直接串联的2个重复SEQ ID NO:2氨基酸序列的核苷酸序列如SEQ ID NO:13所示;编码通过GGSGGSGGSGGSGGS连接序列串联起来的2个重复SEQ ID NO:3氨基酸序列的核苷酸序列如SEQ ID NO:14所示;编码通过GGSGGSGGSGGS连接序列串联起来的2个重复SEQ ID NO:3氨基酸序列的核苷酸序列如SEQ ID NO:15所示;编码通过GGSGGSGGS连接序列串联起来的2个重复SEQ ID NO:3氨基酸序列的核苷酸序列如SEQ ID NO:16所示;编码通过GGS连接序列串联起来的2个重复SEQ ID NO:3氨基酸序列的核苷酸序列如SEQ ID NO:17所示;编码直接串联的2个重复SEQ ID NO:3氨基酸序列的核苷酸序列如SEQ ID NO:18所示;编码直接串联的2个重复SEQ ID NO:5氨基酸序列的核苷酸序列如SEQ ID NO: 19所示;编码直接串联的2个重复SEQ ID NO:6氨基酸序列的核苷酸序列如SEQ ID NO:20所示;编码直接串联的2个重复SEQ ID NO:7氨基酸序列的核苷酸序列如SEQ ID NO:21所示;编码直接串联的2个重复SEQ ID NO:8氨基酸序列的核苷酸序列如SEQ ID NO:23所示。
- 一种制备权利要求1-7之一所述的β冠状病毒抗原的方法,其特征在于:包括以下步骤:在编码权利要求1-7之一所述的β冠状病毒抗原的核苷酸序列的5’端加入编码信号肽的序列,3’端加上终止密码子,进行克隆表达,筛选正确的重组子,然后转染表达系统的细胞进行表达,表达后收集细胞上清,纯化得到β冠状病毒抗原。
- 根据权利要求8所述的方法,其特征在于:所述表达系统的细胞包括为哺乳动物细胞、昆虫细胞、酵母细胞或细菌细胞,可选地;所述哺乳动物细胞包括293T细胞或CHO细胞,所述细菌细胞包括大肠杆菌细胞。
- 一种编码权利要求1-7之一所述的β冠状病毒抗原的核苷酸序列。
- 一种包括权利要求10所述的核苷酸序列的重组载体。
- 一种包括权利要求11所述的重组载体的表达系统细胞。
- 一种权利要求1-7之一所述的β冠状病毒抗原、权利要求10所述的核苷酸序列、权利要求11所述的重组载体、权利要求12所述的表达系统细胞在制备β冠状病毒疫苗中的应用。
- 一种β冠状病毒疫苗,其特征在于:包括权利要求1-7之一所述的β冠状病毒抗原和佐剂。
- 根据权利要求14所述的β冠状病毒疫苗,其特征在于:所述佐剂选自铝佐剂、MF59佐剂或类MF59佐剂。
- 一种β冠状病毒DNA疫苗,其特征在于包括有:包含编码权利要求1-7之一所述的β冠状病毒抗原的DNA序列的重组载体。
- 一种β冠状病毒mRNA疫苗,其特征在于包括有:包含编码权利要求1-7之一所述的β冠状病毒抗原的mRNA序列的重组载体。
- 一种β冠状病毒病毒载体疫苗,其特征在于包括有:包含编码权利要求1-7 之一所述的β冠状病毒抗原的核苷酸序列的重组病毒载体;可选地,病毒载体选自以下的一种或几种:腺病毒载体、痘病毒载体、流感病毒载体、腺相关病毒载体。
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US11547673B1 (en) | 2020-04-22 | 2023-01-10 | BioNTech SE | Coronavirus vaccine |
WO2023025257A1 (zh) * | 2021-08-26 | 2023-03-02 | 中国科学院微生物研究所 | 一种β冠状病毒异源多聚体抗原、其制备方法和应用 |
WO2023023674A3 (en) * | 2020-08-31 | 2023-09-14 | Coronavax, Llc | Coronavirus vaccine formulations incorporating prime and boost |
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WO2023023674A3 (en) * | 2020-08-31 | 2023-09-14 | Coronavax, Llc | Coronavirus vaccine formulations incorporating prime and boost |
CN114717251A (zh) * | 2021-08-24 | 2022-07-08 | 广州恩宝生物医药科技有限公司 | 一种用于预防SARS-CoV-2原始株和Beta株的腺病毒载体疫苗 |
WO2023025257A1 (zh) * | 2021-08-26 | 2023-03-02 | 中国科学院微生物研究所 | 一种β冠状病毒异源多聚体抗原、其制备方法和应用 |
US11878055B1 (en) | 2022-06-26 | 2024-01-23 | BioNTech SE | Coronavirus vaccine |
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US11590220B2 (en) | 2023-02-28 |
CN111592602B (zh) | 2021-03-02 |
CN113416259B (zh) | 2023-04-18 |
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