WO2021159648A1 - 一种β冠状病毒抗原、其制备方法和应用 - Google Patents

一种β冠状病毒抗原、其制备方法和应用 Download PDF

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WO2021159648A1
WO2021159648A1 PCT/CN2020/097775 CN2020097775W WO2021159648A1 WO 2021159648 A1 WO2021159648 A1 WO 2021159648A1 CN 2020097775 W CN2020097775 W CN 2020097775W WO 2021159648 A1 WO2021159648 A1 WO 2021159648A1
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amino acid
seq
acid sequence
coronavirus
series
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戴连攀
严景华
高福
李燕
郑天依
徐坤
刘梅
安亚玲
施一
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中国科学院微生物研究所
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Priority to EP20919172.5A priority patent/EP4050035A4/en
Publication of WO2021159648A1 publication Critical patent/WO2021159648A1/zh
Priority to US17/827,256 priority patent/US11590220B2/en

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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

本发明提供一种β冠状病毒抗原、其制备方法和应用。β冠状病毒抗原的氨基酸序列包括：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，β冠状病毒抗原为单链二聚体结构。本发明实施例所表达的单链二聚体含量稳定，作为β冠状病毒抗原具有好的免疫原性，该单链二聚体作为β冠状病毒抗原制备成的疫苗能够激发小鼠产生很高滴度的中和抗体。

Description

一种β冠状病毒抗原、其制备方法和应用
交叉引用
本发明要求在中国专利局提交的、申请号为CN202010085038.9、发明名称为“一种β冠状病毒抗原、其制备方法和应用”的中国专利申请的优先权，该申请的全部内容通过引用结合在本发明中。
技术领域
本发明涉及生物医药技术领域，具体涉及一种β冠状病毒抗原、其制备方法和应用。
背景技术
冠状病毒属于冠状病毒科冠状病毒属，是有囊膜的正链RNA病毒，在所有RNA病毒中其基因组最大，动物和人类都是冠状病毒的宿主。冠状病毒主要感染哺乳动物和鸟类的呼吸道和消化道，目前已知的有7种冠状病毒感染人，其中四种引起不严重的感冒(HCoV-229E,HCoV-NL63,OC43和HKU1)，对全球公共卫生威胁最大的有三种，分别是2002-2003年爆发的严重呼吸综合征冠状病毒(SARS-CoV)；2012年爆发并持续至今的中东呼吸综合征冠状病毒(MERS-CoV)；以及2019年暴发的新型冠状病毒(2019-nCoV)，这三种都属于β冠状病毒。
中东呼吸综合征(MERS)是由中东呼吸综合征冠状病毒(MERS-CoV)感染所致的疾病。2012年6月沙特阿拉伯发现第一例MERS病例，并从病例痰液标本中分离到一种新的冠状病毒。这种病毒随后被国际病毒分类委员会冠状病毒小组命名为MERS-CoV。这种病毒在中东地区传播开来，并传至亚洲，非洲，欧洲和北美洲等地。据WHO统计，截止到2015年10月6日，在世界范围内，感染人数1589例，死亡人数567例，死亡率为35.6％(http://www.who.int/entity/emergencies/mers-cov/en/)。尤其是2015年5、6月间由中东输入到韩国的MERS疫情，造成了186人感染，36人死亡的结果。甚至有1例MERS病例输入到我国，给世界公共卫生体系带来了严重威胁。MERS-CoV病毒和2003年爆发的SARS病毒同属于Beta-冠状病毒亚属，却有着比SARS-CoV更高的致死率。MERS-CoV可能通过气溶胶的形式传播，因此很难防控。在中东许多国家的单峰驼的血清里能检测出MERS-CoV的中和抗体，提示单峰驼是MERS-CoV的中间宿主，而单峰驼在中东国家是重要的交通工具。因此，从2012年MERS-CoV被发现以来，在中东地区偶发性的MERS-CoV感染人事件一直没有中断。由此，随着国际交流的日益频繁，MERS在全球传播的风险一直存在。目前世界上仍然没有疫苗和有效的治疗手段。因此，开发 一种安全、有效的针对MERS-CoV的疫苗十分紧迫和重要。
2019年，出现了不明原因的肺炎病例，经过病毒的电镜图鉴定，确定是冠状病毒，暂时命名为2019新型冠状病毒(2019 novel coronavirus，2019-nCoV)，后又被命名为SARS-CoV-2。新型冠状病毒可以通过呼吸道和飞沫途径在人与人之间传播，也存在通过空气和消化道传播的可能。传染源主要是感染了新型冠状病毒的患者，但是不排除无症状的感染者也是传播源的可能，感染该病毒后可能不会立即发病，病毒的潜伏期较长，1-14天，这对疾病的防控造成困难。新型冠状病毒进入人体后，和SARS类似，通过血管紧张素转酶2(ACE2)进入细胞，感染人体，导致病人出现发烧，干咳以及肌肉疼等临床症状，少数人还会出现鼻塞，咽痛，腹泻等症状，严重的患者快速进展为急性呼吸窘迫综合征、脓毒症休克、难以纠正的代谢性酸中毒和出凝血功能障碍，造成生命危险。暂时还没有特异性药物，也没有疫苗可以预防，只能使用对症支持治疗。
此外，一些其他的冠状病毒也引发许多严重的动物疾病，尤其对农业牲畜和宠物带来严重威胁。比如，猪传染性胃肠炎病毒(TGEV)可以引发猪的严重腹泻，死亡率极高；其缺失病毒猪呼吸道冠状病毒(PRCV)可以引起猪严重的呼吸道疾病；猫腹膜炎病毒(FIPV)可引起猫腹膜炎和腹水聚集，致死率很高；犬冠状病毒(CCoV)则可使犬发生不同程度的肠胃炎症状，传播快，难控制；猪流行性腹泻病毒(PEDV)引起猪流行性腹泻等肠道疾病，容易在猪群里传播致死率很高。此外还有鼠、牛等冠状病毒。这些冠状病毒对人和动物的健康造成了严重的威胁。因此，开发针对冠状病毒的疫苗有着重要的意义。
表面刺突蛋白(S蛋白)是冠状病毒的主要中和抗原。MERS-CoV、SARS-CoV、2019-nCoV的S蛋白的受体结合区(Receptor Binding Domain,RBD)被认为是诱导机体产生中和抗体的最主要的抗原靶区域。RBD作为疫苗能够将机体刺激产生的中和抗体更加聚焦在针对病毒的受体结合，可以提高疫苗的免疫原性和免疫效率。MERS-CoV通过RBD与宿主细胞的受体(CD26，又名DPP4)结合而侵入细胞。此外，SARS-CoV和2019-nCoV都发现通过其RBD与宿主细胞受体hACE2结合而进入细胞。
公开于该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不应当被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。
发明内容
发明目的
本发明的目的在于提供一种β冠状病毒抗原、其制备方法和应用。本发明实施例中基于MERS二聚体RBD蛋白比单体RBD蛋白能更好的的激发中和抗体的结论，尝试将两段编码相同或基本相同的单体RBD蛋白的核苷酸序列直接串联或通过连接段串联，所表达 的两个相同或基本相同的单体RBD蛋白通过N端和C端的柔性区串联起来，结果显示该种方法可以很好地实现单链RBD二聚体的表达。相对于2个RBD单体简单通过其中的半胱氨酸以二硫键结合所形成的非单链形式的二聚体RBD蛋白而言，本发明实施例所得单链二聚体RBD蛋白不会因为二硫键的形成不稳定而导致生产过程中二聚体RBD蛋白含量不稳定，即避免以单体RBD形式为主，少有二聚体RBD形成的表达情况，从而使二聚体RBD表达稳定，形式均一，产量大大提高。相对于2个RBD单体简单通过其中的半胱氨酸以二硫键结合所形成的二聚体RBD蛋白而言，本发明实施例所表达的单链二聚体作为β冠状病毒抗原具有相当的免疫原性，该单链二聚体作为β冠状病毒抗原制备成的疫苗能够激发小鼠产生很高滴度的中和抗体。
解决方案
为实现本发明目的，本发明实施例提供了以下技术方案：
一种β冠状病毒抗原，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。可选地，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％、95％、99％。
上述β冠状病毒抗原在一种可能的实现方式中，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(2019新型冠状病毒也可称为2019-nCoV或者SARS-CoV-2)。
上述β冠状病毒抗原在一种可能的实现方式中，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。GGS三个字母分别表示氨基酸G、G、S。
上述β冠状病毒抗原在一种可能的实现方式中，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。
其中：SEQ ID NO：1，SEQ ID NO：2，或SEQ ID NO：3序列均来源于MERS-CoV S蛋白(NCBI上的GenBank：AFS88936.1)的一部分，分别是MERS-CoV S蛋白的RBD的E367-Y606区域、E367-N602区域、V381-L588区域。
上述β冠状病毒抗原在一种可能的实现方式中，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，即E367-Y606-GGSGGS-E367-Y606；
(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，即E367-Y606-GGS-E367-Y606；
(3)直接串联的2个重复SEQ ID NO：1氨基酸序列，即E367-Y606-E367-Y606。
(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，即E367-N602-GGS-E367-N602；
(5)直接串联的2个重复SEQ ID NO：2氨基酸序列，即E367-N602-E367-N602；
(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，即V381-L588-GGSGGSGGSGGSGGS-V381-L588；
(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，即V381-L588-GGSGGSGGSGGS-V381-L588；
(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，即V381-L588-GGSGGSGGS-V381-L588；
(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，即V381-L588-GGS-V381-L588；
(10)直接串联的2个重复SEQ ID NO：3氨基酸序列，即V381-L588-V381-L588；
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，即E367-N602-E367-N602。
上述β冠状病毒抗原在一种可能的实现方式中，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：
(1)SEQ ID NO：5，SEQ ID NO：6，或SEQ ID NO：7；
(2)在(1)中的氨基酸序列经取代、缺失或添加一个或几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：6。
其中：SEQ ID NO：5，SEQ ID NO：6，或SEQ ID NO：7序列均来源于2019-nCoV的WH01株的S蛋白序列(NCBI上的GenBank：QHR63250)的一部分，分别是2019-nCoV S蛋白的RBD的R319-S530区域、R319-K537区域、R319-F541区域。
上述β冠状病毒抗原在一种可能的实现方式中，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：
直接串联的2个重复SEQ ID NO：5氨基酸序列，即R319-S530-R319-S530；
直接串联的2个重复SEQ ID NO：6氨基酸序列，即R319-K537-R319-K537；
直接串联的2个重复SEQ ID NO：7氨基酸序列，即R319-F541-R319-F541；
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，即R319-K537-R319-K537。
上述β冠状病毒抗原在一种可能的实现方式中，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：
(1)SEQ ID NO：8；
(2)在(1)中的氨基酸序列经取代、缺失或添加一个或几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。
其中：SEQ ID NO：8序列来源于SARS-CoV的S蛋白序列(NCBI上的GenBank：AAR07630)的一部分，是SARS-CoV S蛋白的RBD的R306-Q523区域。
上述β冠状病毒抗原在一种可能的实现方式中，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，即R306-Q523-R306-Q523。
上述β冠状病毒抗原在一种可能的实现方式中，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。
本发明还提供了一种制备上述β冠状病毒抗原的方法，包括以下步骤：在编码上述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。
上述方法在一种可能的实现方式中，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。
本发明还提供了一种编码上述β冠状病毒抗原的核苷酸序列、一种包括上述核苷酸序列的重组载体、一种包括上述重组载体的表达系统细胞。
本发明还提供了一种上述β冠状病毒抗原、编码上述β冠状病毒抗原的核苷酸序列、包括上述核苷酸序列的重组载体、包括上述重组载体的表达系统细胞在制备β冠状病毒疫苗中的应用。
本发明还提供了一种β冠状病毒疫苗，包括上述β冠状病毒抗原和佐剂。
上述β冠状病毒疫苗在一种可能的实现方式中，所述佐剂选自铝佐剂、MF59佐剂或类MF59佐剂。
本发明还提供了一种β冠状病毒DNA疫苗，其包括有：包含编码上述β冠状病毒 抗原的DNA序列的重组载体。
本发明还提供了一种β冠状病毒mRNA疫苗，其包括有：包含编码上述β冠状病毒抗原的mRNA序列的重组载体。
本发明还提供了一种β冠状病毒病毒载体疫苗，其包括有：包含编码上述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。
有益效果
(1)本发明实施例β冠状病毒抗原中，基于MERS二聚体RBD蛋白比单体RBD蛋白能更好的的激发中和抗体的结论，进一步通过解析MERS-CoV二聚体RBD蛋白的晶体结构，发现了MERS二聚体RBD蛋白能够形成头并头的二聚体，因此发明人尝试将两段编码相同或基本相同的单体RBD蛋白的核苷酸序列直接串联或通过连接段串联，所得两个相同或基本相同的单体RBD蛋白通过N端和C端的柔性区串联起来，结果显示该种方法可以实现很好地实现单链二聚体的表达。相对于2个RBD单体简单通过其中的半胱氨酸以二硫键结合所形成的非单链形式的二聚体RBD蛋白而言，本发明实施例所得单链二聚体RBD蛋白不会因为二硫键的形成不稳定而导致生产过程中二聚体RBD蛋白含量不稳定，即避免以单体RBD形式为主，少有二聚体形成的表达情况，从而使二聚体RBD表达稳定，形式均一，产量大大提高。相对于2个RBD单体简单通过其中的半胱氨酸以二硫键结合所形成的非单链形式的二聚体RBD蛋白而言，本发明实施例所表达的单链二聚体作为β冠状病毒抗原具有相当的免疫原性，该单链二聚体作为β冠状病毒抗原制备成的β冠状病毒疫苗能够激发小鼠产生很高滴度的中和抗体。
(2)本发明实施例β冠状病毒抗原中，通过对所包含的RBD不同区域氨基酸的选择，发现从如图14A START的第一个氨基酸开始，到图14B STOP最后一个半胱氨酸之前的一个氨基酸为止是表达最优的构建，这样可以尽量避免末端未配对的半胱氨酸对蛋白表达和其稳定性的影响。
(3)本发明实施例β冠状病毒抗原中，通过对两段编码相同或基本相同的单体RBD蛋白的核苷酸序列直接串联或通过连接段串联的选择，发现在不引入任何外源连接序列的情况下，即将两段编码相同或基本相同的单体RBD蛋白的核苷酸序列直接串联的情况下，表达量最高，且由于没有外源序列的加入，也最为安全。由于本发明实施例中获得各种单链二聚体RBD作为β冠状病毒抗原都有很好的免疫效果，所以关键在于产量。
(4)本发明实施例β冠状病毒抗原中，所涉及的头并头单链二聚体结构适用于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。
附图说明
图1是实施例1中通过构建的载体pFastBac-SP-MERS-RBD(E367-Y606)所获得的RBD蛋白进行Superdex200 Hiload 16/60柱子(GE)分子筛层析时的紫外吸收图，以及收集紫外吸收图中的二聚体(Dimer)峰和单体(Monomer)峰获得的蛋白质在还原条件下(+DTT)或非还原条件下(-DTT)进行SDS-PAGE的电泳图。
图2是实施例2到实施例7中的免疫和MERS-CoV攻毒策略图。
图3是实施例3结果，为实施例2中小鼠第三次免疫后按照图2中的免疫策略取血的血清中MERS-CoV RBD特异性抗体IgG滴度。其中：Dimer表示使用的免疫源为MERS-CoV RBD二聚体，RBD monomer表示使用的免疫源为MERS-CoV RBD单体，AddaVax表示使用了AddaVax佐剂，Alum表示使用了铝佐剂，未表示佐剂的表示没有使用佐剂，3μg、10μg、30μg表示每一次免疫使用的免疫源的免疫量。显著性差异分析：ns，p>0.05；*,p<0.05；**,p<0.01；***,p<0.001；****,p<0.0001。
图4是实施例5结果，为实施例2中小鼠第三次免疫后按照图2中的免疫策略取血的血清中MERS-CoV假病毒90％中和抗体滴度。其中：Dimer表示使用的免疫源为MERS-CoV RBD二聚体，RBD monomer表示使用的免疫源为MERS-CoV RBD单体，AddaVax表示使用了AddaVax佐剂，Alum表示使用了铝佐剂，未表示佐剂的表示没有使用佐剂，3μg、10μg、30μg表示每一次免疫使用的免疫源的免疫量。显著性差异分析：ns，p>0.05；***,p<0.001。
图5是实施例6结果，为实施例2中小鼠第三次免疫后按照图2中的免疫策略取血的血清中对MERS-CoV真病毒(EMC株)的50％中和抗体滴度结果图。其中：Dimer表示使用的免疫源为MERS-CoV RBD二聚体，AddaVax表示使用了Addavax佐剂，Alum表示使用了铝佐剂，未表示佐剂的表示没有使用佐剂，3μg、10μg、30μg表示每一次免疫使用的免疫源的免疫量。显著性差异分析：ns，p>0.05；***,p<0.001；****,p<0.0001。
图6是实施例7中按照图2中的免疫策略对第三次免疫后的小鼠滴鼻感染表达hCD26(hDPP4)的腺病毒，5天后用MERS-CoV攻毒，攻毒之后3天，取出小鼠的肺脏，组织匀浆用来检测病毒滴度(TCID 50)的结果图。Dimer表示使用的免疫源为MERS-CoV RBD二聚体，AddaVax表示使用了AddaVax佐剂，Alum表示使用了铝佐剂，未表示佐剂的表示没有使用佐剂，3μg、10μg、30μg表示每一次免疫使用的免疫源的免疫量。显著性差异分析：ns，p>0.05；*,p<0.05；**,p<0.01；***,p<0.001；****,p<0.0001。
图7是实施例8检测疫苗对小鼠肺组织保护效果的病理结果。将实施例7中攻毒的小鼠在解剖后取出肺经过4％多聚甲醛固定、石蜡包埋、苏木精-伊红染色和切片观察病理变化。其中AddaVax表示使用了AddaVax佐剂，Alum表示使用了铝佐剂，3μg、10μg、 30μg表示每一次免疫使用的免疫源的免疫量。Slight、Mild和Severe分别说明肺组织病变程度为轻微、中等和严重。
图8是实施例9中解析出的MERS-CoV-RBD dimer(E367-Y606)的结构。
图9A、图9B、图9C是实施例10中基于MERS-CoV RBD-dimer二聚体结构设计的单链RBD二聚体设计方案。
图10是实施例10中表达的MERS-RBD-C1至MERS-RBD-C10单链二聚体在还原(+DTT)或非还原(-DTT)的条件下进行Western blot的结果图。其中RBD Monomer为MERS-CoV RBD单体蛋白。
图11是实施例11中表达的MERS-RBD-C5单链二聚体进行进行Superdex200Hiload16/60柱子(GE)分子筛层析时的紫外吸收图，以及纯化所得单链二聚体在还原(+DTT)或非还原(-DTT)的条件下下进行SDS-PAGE的结果图。
图12是实施例12中小鼠免疫MERS-CoV-RBD单链二聚体和二硫键连接的非单链二聚体蛋白后，诱导产生的MERS-CoV-RBD特异性IgG抗体滴度。其中sc-dimer是单链二聚体，Dimer是二硫键连接的非单链二聚体。显著性差异分析：ns，p>0.05；*,p<0.05；***,p<0.001；****,p<0.0001。
图13是实施例12中小鼠免疫MERS-CoV-RBD单链二聚体和二硫键连接的非单链二聚体蛋白后，诱导产生的MERS-CoV假病毒90％中和抗体滴度。其中sc-dimer是单链二聚体，Dimer是二硫键连接的非单链二聚体。显著性差异分析：ns，p>0.05；*,p<0.05；****,p<0.0001。
图14A和图14B是实施例13中Beta冠状病毒的受体结合区(RBD)的比对图，两张图中的序列连续，比对了以下几种β冠状病毒：
MERS-CoV(AFS88936),SARS-CoV(AAS00003),SARS-CoV-2(QHR63290),Bat-CoV_HKU5(ABN10875),Rousettus_bat-CoV(AOG30822),Bat-CoV_BM48-31(ADK66841),Bat-CoV_HKU9(ABN10911),Bat_Hp-betaCoV(AIL94216),SARS-related-CoV(APO40579),BtRs-Beta-CoV(QDF43825),Bat-SARS-like-CoV(ATO98231),SARS-like-CoV_WIV16(ALK02457),Bat-CoV(ARI44804),BtR1-Beta-CoV(QDF43815),HCoV_HKU1(AZS52618),MCoV_MHV1(ACN89742),BetaCoV_HKU24(AJA91217),HCoV_OC43(AAR01015),BetaCoV_Erinaceus(AGX27810)。
图15是实施例13中SARS-CoV-RBD二聚体或2019-nCoV-RBD二聚体的结构模拟图以及设计的表达2019-nCoV-RBD二聚体、2019-nCoV-RBD单体和SARS-CoV-RBD二聚体的构建。
图16是实施例13中表达的几种关于SARS-CoV-RBD和2019-nCoV-RBD的单链二聚体在还原(+DTT)或非还原(-DTT)的条件下进行Western blot的结果图。
图17是实施例14中2019-nCoV-RBD-C2抗原进行纯化时的紫外280nm吸光度图，以及纯化所得单链二聚体在还原(+DTT)或非还原(-DTT)的条件下进行SDS-PAGE的结果图。
图18是实施例14中SARS-CoV-RBD-C1抗原进行纯化时的紫外280nm吸光度图，以及纯化所得单链二聚体在还原(+DTT)或非还原(-DTT)的条件下进行SDS-PAGE的结果图。
图19为实施例15中小鼠三次免疫后(1免后19天，2免后14天，3免后14天)分别收集的血清的2019-nCoV-RBD特异性IgG抗体滴度。其中：sc-dimer表示使用的免疫源为nCoV-RBD单链二聚体，Monomer表示使用的免疫源为nCoV-RBD单体。显著性差异分析：****,p<0.0001。
图20是实施例15中小鼠三次免疫后(1免后19天，2免后14天，3免后14天)分别收集的血清的2019-nCoV假病毒90％中和抗体滴度。其中：sc-dimer表示使用的免疫源为nCoV-RBD单链二聚体，Monomer表示使用的免疫源为nCoV-RBD单体。显著性差异分析：ns，p>0.05；**,p<0.01；****,p<0.0001。
图21是实施例15中小鼠第二次免疫后(2免后14天)收集的血清的2019-nCoV真病毒(2020XN4276株)50％中和抗体滴度。其中：sc-dimer表示使用的免疫源为nCoV-RBD单链二聚体，Monomer表示使用的免疫源为nCoV-RBD单体。
图22为实施例16中小鼠三次免疫后(1免后19天，2免后14天，3免后14天)分别收集的血清的SARS-RBD特异性IgG抗体滴度。其中：sc-dimer表示使用的免疫源为SARS-CoV-RBD单链二聚体，Monomer表示使用的免疫源为SARS-CoV-RBD单体。显著性差异分析：ns，p>0.05；*,p<0.05；**,p<0.01；****,p<0.0001。
图23是实施例16中小鼠三次免疫后(1免后19天，2免后14天，3免后14天)分别收集的血清的SARS-CoV假病毒90％中和抗体滴度。其中：sc-dimer表示使用的免疫源为SARS-CoV-RBD单链二聚体，Monomer表示使用的免疫源为SARS-RBD单体。显著性差异分析：ns，p>0.05；*,p<0.05；**,p<0.01；***,p<0.001；****,p<0.0001。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
另外，为了更好的说明本发明，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本发明同样可以实施。在一些实施例中， 对于本领域技术人员熟知的原料、元件、方法、手段等未作详细描述，以便于凸显本发明的主旨。
除非另有其它明确表示，否则在整个说明书和权利要求书中，术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的元件或组成部分，而并未排除其它元件或其它组成部分。
名词解释
二硫键连接的非单链形式的RBD二聚体和RBD单体：将编码RBD单体的核苷酸序列插入载体，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RBD单体和二硫键连接的非单链形式的RBD二聚体，其中：二硫键连接的非单链形式的RBD二聚体中的2个RBD单体简单通过其中的半胱氨酸以二硫键结合所形成的二聚体RBD。二硫键连接的非单链形式的RBD二聚体、非单链形式的二聚体RBD蛋白均具有相同的含义；RBD单体、单体RBD、单体RBD蛋白均具有相同的含义。
单链形式的RBD二聚体：将两段编码相同或基本相同的单体RBD的核苷酸序列直接串联或通过连接段串联，在该核苷酸序列的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，纯化得到重组蛋白，该蛋白含有两个RBD单体，两个RBD单体相同或基本相同，可以直接通过肽键连接在一起也可以通过一段连接序列(比如GGS、GGSGGS等)连接在一起，即为单链形式的RBD二聚体。本申请中单链RBD二聚体、RBD单链二聚体、单链二聚体、sc-RBD dimer、single-chain RBD dimer等均具有相同的含义。
实施例1：表达MERS-CoV抗原的重组杆状病毒制备和RBD蛋白的表达纯化
将编码MERS-CoV S蛋白(序列如GenBank:AFS88936.1)中的氨基酸RBD(E367-Y606)序列(如SEQ ID NO：1所示)的核苷酸序列(如SEQ ID NO：24所示)3’端加上翻译终止密码子后克隆到包含gp67信号肽的pFastBac载体(pFastBac-SP来自Invitrogen公司)的EcoR I和Xho I酶切位点之间，使得蛋白编码区在信号肽gp67序列的后面融合表达，用于目的蛋白的分泌，并且使目的蛋白C端带上6个组氨酸，得到载体pFastBac-SP-MERS-RBD(E367-Y606)，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。
所得RBD蛋白纯化过Superdex200 Hiload 16/60柱子(GE)进行分子筛层析之后，典型的蛋白纯化紫外吸收图如图1所示。有一个二聚体的峰(dimer)还有一个单体的峰(Monomer)。取MERS-RBD蛋白在洗脱体积78mL附近的洗脱峰进行SDS-PAGE分析。洗脱体积78mL附近的蛋白在非还原条件下(不加DTT)的情况下，大小约为60Kd；而在还原条件下(加入DTT)，大小约为30Kd，证明该峰中所获得蛋白质是二聚体。取 洗脱体积90mL附近的洗脱峰进行SDS-PAGE分析，目的蛋白在非还原条件下(不加DTT)和还原条件的情况下大小均约为30Kd，证明该峰主要是RBD单体。以上获得为二硫键连接的非单链形式的RBD二聚体和RBD单体，以下实施例2-实施例9中使用的二聚体或单体为本实施例中获得的二硫键连接的非单链形式的RBD二聚体和RBD单体。
实施例2：MERS-RBD蛋白免疫小鼠实验
MF59(以下使用的AddaVax是一种MF59-like佐剂)和铝佐剂是SFDA批准的两种常用佐剂，我们使用这两个佐剂作为疫苗组分对于后续的临床试验有着更直接的指导作用。体外中和试验是检测疫苗保护效果的经典方法。因此，我们将不同剂量的抗原分别与AddaVax佐剂和Imject TM Alum佐剂进行混合，进行免疫。免疫分组情况、各组使用的RBD类别、每1次免疫使用的RBD免疫量和佐剂情况如表1所示，空白格部分表示“无”。
将实施例1获得的MERS-RBD抗原(二聚体或单体)于生理盐水中稀释成所需浓度，并与佐剂进行分组乳化。然后对4-6周龄的BALB/c小鼠(平均体重15-20g，下同)进行分组免疫，每组6只。
表1
Figure PCTCN2020097775-appb-000001
免疫策略如图2，即通过大腿肌肉注射的方式，每只小鼠分别在第0天，第21天，第42天接受3次疫苗免疫，每次100μl的接种体积。第56天(即三免后的第14天)，对小鼠进行尾部取血。小鼠血清通过静置后3000rpm离心10分钟获得，并与-20℃ 冰箱保存，用于特异性抗体滴度检测和假病毒中和检测。
实施例3：ELISA实验检测疫苗诱导的特异性抗体滴度
(1)将MERS-CoV的RBD单体蛋白用ELISA包被液(索莱宝,C1050)稀释至3μg/ml，96孔ELISA板(Coring，3590)每孔加入100μl，4℃放置12小时。
(2)倒掉包被液，加入PBS，洗一遍。使用PBS配制的5％脱脂牛奶作为封闭液，加入96孔板中，每孔100μl，封闭，室温放置1小时。封闭完后用PBS溶液洗一遍。
(3)封闭期间稀释小鼠血清。血清样品也用封闭液稀释。血清样品从20倍起始梯度稀释。之后在ELISA板中每孔加入100μl，阴性对照为加入封闭液，37度孵育2小时，之后使用PBST洗4遍。
(4)加入使用封闭液1:2000稀释的偶联HRP的羊抗鼠二抗(Abcam，ab6789)，37℃孵育1.5小时，之后PBST洗5-6遍。加入TMB显色液显色，反应适当时间后加入2M盐酸终止反应，在酶标仪上检测OD450读值。抗体滴度值被定义为反应值大于2.5倍阴性对照值的血清最高稀释倍数。当最低稀释倍数(检测限)的反应值仍小于2.5倍背景值时，该样品的滴度定义为最低稀释倍数的一半即1:10。
结果如图3所示，使用AddaVax佐剂3μg和10μg剂量下，RBD二聚体组和单体组诱导的抗体水平有显著差异，使用铝佐剂3μg、10μg和30μg剂量下，RBD二聚体组和单体组诱导的抗体水平有显著差异，且二聚体组诱导的抗体水平较高，显示出二聚体RBD抗原激活小鼠抗体反应的能力明显强于RBD单体疫苗。
本发明实施例中所有的ELISA实验的包被蛋白都使用的是MERS-CoV的RBD单体蛋白。
实施例4：MERS-CoV假病毒的制备
pNL43-Luci假病毒包装
(1)细胞铺盘：转染前一天，用胰酶进行消化以收获对数期生长的293T细胞，计数，重新接种细胞于10cm培养皿中培养过夜，至18-24h时细胞达到70-90％汇合时，进行转染(不含抗生素)。
(2)PEI法进行质粒共转染：将共20μg质粒(HIV pNL4-3.Luc.RE(Invitrogen)10μg，pCAGGS-MERS-S 10μg，这里pCAGGS-MERS-S是将编码MERS的Spike蛋白(M1-H1352)的DNA序列插入到pCAGGS载体的EcoRI和XhoI位点。)和40μl PEI(2mg/ml)分别溶于生理盐水或HBS中，终体积为500μL，混匀；静置5min后，将两者混合，再静置20min，将混合物逐滴加入到细胞培养皿中，4-6h后，PBS洗涤细胞2次更换新鲜的无血清培养基。
(3)收毒：转染48h后，收集细胞及上清，1000rpm低速离心10min去除细胞碎片，分装，于-80℃保存，避免反复冻融造成病毒滴度下降。
(4)感染：第一天，细胞铺板，培养过夜，至18-24h时细胞达到80-100％；
第二天，PBS洗涤易感细胞去除血清，取病毒上清感染易感细胞，4-6h换为含血清的培养基。根据实验需要，可在不同时间点测定Luciferase值，参考Promega公司Luciferase Assay System Protocol或Dual Luciferase Reporter Assay System Protocol。将收取的病毒液按5倍比稀释，加入到96孔板中的Huh7细胞(人肝癌细胞)中。感染4小时后，弃掉病毒液，PBS洗涤细胞2次，换为含10％血清的DMEM完全培养基。48小时候，弃掉培养基，PBS洗涤2次，加入细胞裂解液。-80℃冻融一次后，每孔取20μl利用GloMax 96 Microplate Luminometer(Promega)检测荧光素酶活性值。通过Reed-Muech法计算TCID 50。
实施例5：免疫血清的假病毒中和实验
实施例2中获得的血清倍比稀释，与100 TCID 50假病毒混合，37℃共孵育30分钟。将混合液加入到已铺满Huh7细胞的96孔板中。37℃孵育4小时后，弃掉病毒液，PBS洗涤细胞2次，换为含10％血清的完全培养基DMEM。48小时后，弃掉培养液，PBS洗涤细胞2次，加入细胞裂解液，检测荧光素酶活性值。假病毒表面带有刺突蛋白，假病毒感染细胞释放DNA，表达荧光素酶，但不复制。如果有中和抗体存在的情况下，假病毒就无法感染细胞，则不表达荧光素酶。以此方法来检验血清的中和滴度。
第三次免疫后免疫原性检测结果见图4。结果显示RBD二聚体(E367-Y606)在三次免疫之后，无论AddaVax佐剂组还是铝佐剂组(+Alum表示)，都产生了中和抗体。特别是AddaVax佐剂10μg组的中和抗体NT 90平均值可达1:1000以上(如图4所示)。而RBD单体(E379-E589)在三次免疫之后，除了2只小鼠有较弱的中和抗体产生，其余都检测不到(如图4所示)。假病毒中和实验证明了二聚体RBD诱导的中和抗体远比单体RBD高。
上述RBD单体(E379-E589)通过以下方法获得：将编码MERS-CoV S蛋白中的氨基酸(E379-E589)序列(如SEQ ID NO：4所示)的核酸片段(如SEQ ID NO：25所示)插入pFastBac-SP的EcoRI和XhoI酶切位点，使得蛋白编码区在信号肽gp67序列的后面融合表达，用于目的蛋白的分泌，并且使目的蛋白C端带上6个组氨酸，得到载体pFastBac-SP-MERS-RBD(E379-E589)
实施例6：免疫血清的真病毒中和(EMC株)
使用三次免疫之后的血清，进行MERS-CoV真病毒(EMC株，该病毒株已于Yao  Y,Bao L,Deng W,et al.An animal model of MERS produced by infection of rhesus macaques with MERS coronavirus.J Infect Dis.2014；209(2):236-242.doi:10.1093/infdis/jit590中公开，由北京协和医学院实验动物研究所提供)的中和实验。实验结果如图5。结果显示，无论AddaVax佐剂还是铝佐剂，RBD二聚体都能诱导小鼠产生很高的中和抗体。最高的组(Addavax佐剂10μgRBD二聚体)能达到IC50大于1:600。这结果通过MERS-CoV真病毒中和实验证明二聚体RBD可以诱导小鼠产生较高的的中和抗体。
实施例7：攻毒保护实验
实施例2中三次免疫后的小鼠，根据图2所示，第77天滴鼻感染表达hCD26(hDPP4)的腺病毒。这样在肺部能够瞬时表达MERS-CoV的受体hCD26，从而使得小鼠对MERS-CoV易感(参照Chi H等.DNA vaccine encoding Middle East respiratory syndrome coronavirus S1 protein induces protective immune responses in mice[J].Vaccine,2017,35(16):2069-2075)。5天后用MERS-CoV(EMC株)攻毒(一次攻毒剂量5X 10 5pfu)。攻毒之后3天，取出小鼠的肺脏，组织匀浆用来检测病毒滴度(TCID 50)，结果如图6所示。相比PBS对照组，疫苗组小鼠的肺组织的病毒量显著降低。其中AddaVax佐剂3μgRBD二聚体组的病毒量较PBS组下降了近1000倍，显示出了很好的保护效果。这些结果显示，RBD二聚体作为疫苗有着非常显著的针对MERS-CoV活毒攻毒的保护效果。
实施例8：检测疫苗对小鼠肺组织的保护效果
将实施例7中MERS-CoV攻毒实验的小鼠肺组织使用4％多聚甲醛固定，之后经苏木精-伊红染色，切片观察肺部的病例变化，结果如图7所示，所有对照组小鼠(即PBS组)的肺组织呈现严重的间质性肺炎，肺泡炎，炎症细胞浸润以及支气管上皮细胞坏死(如图7)。而免疫组小鼠，无论是使用AddaVax还是Alum作为佐剂都能够极大的缓解病毒攻毒引起的肺损伤，呈现出轻中度的肺损伤，肺泡清晰可见，较少有炎症细胞浸润。小量的肺部组织病变可能是由于一次攻毒剂量很大(5X 10 5pfu)导致的。这些结果显示，RBD二聚体作为疫苗能够极大的缓解MERS-CoV攻毒引起的肺部损伤。
实施例9：MERS-RBD二聚体晶体结构解析
按照实施例1的方法，表达RBD(E367-Y606)蛋白。纯化后收集二聚体蛋白峰。蛋白浓缩到10mg/ml，蛋白与结晶池液按照体积比1:1的比例进行混合，再通过
Figure PCTCN2020097775-appb-000002
蛋白结晶筛选液体工作站(TTP LabTech)进行蛋白质晶体筛选。在18℃生长，获得可用于衍射的晶体。晶体在上海同步辐射中心(SSRF)收集，最终获得
Figure PCTCN2020097775-appb-000003
的衍 射数据。数据通过HKL2000软件分析，利用MERS-RBD单体的结构为模板(PDB:4KQZ)，通过分子置换法最终解析出MERS-RBD dimer的结构。如图8所示。
实施例10：基于MERS-RBD二聚体结构设计单链RBD二聚体(sc-RBD dimer)
基于图8的MERS-RBD晶体结构，RBD两个亚基的N端(N’)和C端(C’)处于之中头对头的排列形式。N端与C端各有一段看不见的柔性序列(如图9A)。因此，我们设计通过两个亚基之间串联起来，获得单链RBD二聚体(sc-RBD dimer)。
第一种设计(如图9A)包括：
(1)在两个重复串联的(E367-Y606)序列之间加入2个GGS连接序列，得到MERS-RBD-C1(简称C1)，编码该氨基酸序列的核苷酸序列为SEQ ID NO：9；
(2)在两个重复串联的(E367-Y606)序列之间加入1个GGS连接序列，得到MERS-RBD-C2(简称C2)，编码该氨基酸序列的核苷酸序列为SEQ ID NO：10；
(3)两个重复(E367-Y606)序列直接串联，得到MERS-RBD-C3(简称C3)，编码该氨基酸序列的核苷酸序列为SEQ ID NO：11。
第二种设计(如图9B)为了避免C端603位的半胱氨酸(C603)对表达的影响，构建截到N602，包括：
(4)在两个重复的串联的(E367-N602)序列之间加入1个GGS连接序列，得到MERS-RBD-C4(简称C4)，编码该氨基酸序列的核苷酸序列为SEQ ID NO：12；
(5)两个重复的(E367-N602)序列直接串联，得到MERS-RBD-C5(简称C5)，编码该氨基酸序列的核苷酸序列为SEQ ID NO：13。
第三种设计(如图9C)直接表达结构可见序列，通过不同长度的连接序列连接起来，包括：
(6)在两个重复的串联的(V381-L588)序列之间加入5个GGS连接序列，得到MERS-RBD-C6(简称C6)，编码该氨基酸序列的核苷酸序列为SEQ ID NO：14；
(7)在两个重复的串联的(V381-L588)序列之间加入4个GGS连接序列，得到MERS-RBD-C7(简称C7)，编码该氨基酸序列的核苷酸序列为SEQ ID NO：15；
(8)在两个重复的串联的(V381-L588)序列之间加入3个GGS连接序列，得到MERS-RBD-C8(简称C8)，编码该氨基酸序列的核苷酸序列为SEQ ID NO：16；
(9)在两个重复的串联的(V381-L588)序列之间加入1个GGS连接序列，得到MERS-RBD-C9(简称C9)，编码该氨基酸序列的核苷酸序列为SEQ ID NO：17；
(10)把两个重复的(V381-L588)直接串联起来，得到MERS-RBD-C10(简称C10)，编码该氨基酸序列的核苷酸序列为SEQ ID NO：18。
将编码以上MERS-RBD-C1到C10的核苷酸序列的5’端加上编码MERS-S蛋白 自身信号肽(MIHSVFLLMFLLTPTES)的核苷酸序列，3’端加上编码6个组氨酸的核苷酸序列后，再在3’端加上终止密码子，所得核苷酸序列插入到pCAGGS载体EcoRI和XhoI酶切位点之间，其起始密码子上游含有Kozak序列gccacc。以上质粒转染293T细胞，48小时后，取上清，目的蛋白N端带有信号肽，通过蛋白印迹法(Western blot)检测目的蛋白的表达，结果如图10所示。结果显示除了C2，其余构建均有表达。无论是还原(+DTT)还是非还原(-DTT)的条件下，蛋白都在二聚体的大小左右(50-60Kda)。其中C4和C5的表达量最高。考虑到C5构建没有引入外源连接序列，完全是MERS-CoV自身的序列，因此对于临床使用更有优势、更安全。我们就进一步评价MERS-RBD-C5作为疫苗的效果。
实施例11：哺乳动物表达MERS-CoV RBD单链二聚体(sc-RBD dimer)与蛋白纯化
使用哺乳动物293T细胞表达MERS-RBD-C5。质粒转染293T细胞之后，进行表达并收获上清。细胞上清通过0.22μm的滤膜过滤，除去细胞碎片。将细胞培养上清液挂镍亲和柱(Histrap)，4度过夜。以缓冲液A(20mM Tris，150mM NaCl，pH8.0)洗涤树脂，以除去非特异结合蛋白。最后目的蛋白以缓冲液B(20mM Tris,150mM NaCl,pH 8.0,300mM咪唑)从树脂上洗脱下来，并以10K截留(10cutoff)浓缩管将洗脱液浓缩至5ml以内。再通过Superdex200 Hiload 16/60柱子(GE)进行分子筛层析进行进一步的目的蛋白纯化。分子筛层析缓冲液为20mM Tris，150mM NaCl，PH8.0。经过分子筛层析，只在洗脱体积80mL附近有1个主峰。收集进行SDS-PAGE分析。SDS-PAGE的结果可见，MERS-RBD-C5蛋白显示1条明显的蛋白带，在55-72kd之间，是RBD二聚体的大小。证明获得了MERS-RBD单链二聚体，如图11所示。同时我们也使用实施例10中的方法，即293T细胞表达纯化了非单链形式的MERS RBD二聚体，用于和sc-RBD dimer作比较。
实施例12：MERS-CoV RBD单链二聚体(sc-RBD dimer)蛋白免疫小鼠实验
将实施例11获得的MERS-RBD单链二聚体抗原于生理盐水中稀释，并与佐剂进行分组乳化。然后对4-6周龄的BALB/c小鼠进行分组免疫，每组6只小鼠，另外，一组小鼠免疫PBS作为阴性对照。一组小鼠免疫293T细胞表达的非单链形式的二聚体。通过大腿肌肉注射的方式，每只小鼠分别在第0天，第21天和第42天接受3次疫苗免疫，每次100μl的接种体积(含10μg免疫源)。1免后19天，2免14天和3免后14天，对小鼠进行眼眶取血。小鼠血清通过静置后3000rpm离心10分钟获得，并于-20℃冰箱保存，用于特异性抗体检测和假病毒中和检测。
通过ELISA实验检测小鼠血清的特异性抗体滴度，实验方法见实施例3，结果如图12所示，RBD-sc-dimer组小鼠和二硫键连接的非单链RBD-dimer组(Dimer表示)小鼠都能被诱导产生抗体反应，sc-dimer组的滴度均值比Dimer组高，在三次免疫后二者有显著差异(*，p<0.05)。此结果表明，sc-dimer和二硫键连接的非单链RBD-dimer一样具有很好的免疫原性。
假病毒中和实验参照实施例5进行。结果如图13所示，sc-dimer组小鼠和二硫键连接的非单链RBD-dimer组(Dimer表示)小鼠都能被诱导产生抗体反应，sc-dimer组的滴度均值比Dimer组高，在第一次免疫和第二次免疫后二者有显著差异(图13)。在三次免疫后sc-dimer组小鼠的假病毒中和滴度均值已经大于1:1000。此结果预示着用sc-dimer分开发的疫苗具有很大的临床开发潜力。
实施例13、单链RBD二聚体技术在其他冠状病毒疫苗中的应用
为了验证这一思路是否能应用于所有其他冠状病毒的疫苗设计，我们将19种常见的Beta冠状病毒的受体结合区(RBD)进行了比较，如图14A和图14B。所有的Beta冠状病毒RBD都在MERS-CoV的C603位置呈现保守的半胱氨酸，如图14B。我们选取了2019-nCoV(以下简称nCoV)和SARS-CoV两种冠状病毒来进行验证。根据SARS-RBD的结构(PDB：3D0G)，通过Pymol软件，我们将SARS-RBD的晶体结构建模到
Figure PCTCN2020097775-appb-000004
分辨率的MERS-RBD二聚体晶体结构中。得到如图15的模拟SARS-RBD二聚体结构。我们发现和MERS-RBD二聚体一样，SARS-RBD的二聚体也以头并头形式存在(如图15)。由于新冠状病毒2019-nCoV与SARS-CoV的RBD区域同源性高达75％以上，我们预计2019-nCoV的RBD二聚体形成这种头并头的形式。考虑到之前MERS-CoV里面二聚体较单体能诱导更高滴度的中和抗体，我们因此考虑仍然用单链二聚体(sc-dimer)的方式来设计疫苗。首先，以2019-nCoV的WH01株的S蛋白序列为基础，我们设计了3种单链二聚体(sc-dimer)的构建，如图15：(1)两个R319-S530串联，命名为nCoV-RBD-C1(编码该氨基酸序列的核苷酸序列为SEQ ID NO：19)；(2)两个R319-K537串联,命名为nCoV-RBD-C2(编码该氨基酸序列的核苷酸序列为SEQ ID NO：20)；(3)两个R319-F541串联,命名为nCoV-RBD-C3(编码该氨基酸序列的核苷酸序列为SEQ ID NO：21)；(4)另外单体构建为R319-F541,命名为nCoV-RBD-C4(编码该氨基酸序列的核苷酸序列为SEQ ID NO：22)。此外，还构建了SARS-CoV的单链二聚体,两个R306-Q523串联到一起，如图14A和图14B，命名为SARS-CoV-RBD-C1(编码该氨基酸序列的核苷酸序列为SEQ ID NO：23)。
将编码以上nCoV-RBD-C1到C4的核苷酸序列和编码SARS-CoV-RBD-C1的核苷酸序列(SEQ ID NO:23)的5’端加上编码MERS-S蛋白自身信号肽 (MIHSVFLLMFLLTPTES)的核苷酸序列；3’端加上编码6个组氨酸的核苷酸序列后，再在3’端加上终止密码子，插入到pCAGGS载体EcoRI和XhoI酶切位点，其起始密码子上游含有Kozak序列gccacc。以上质粒转染293T细胞，48小时后，取上清，通过蛋白印迹法(Western blot)检测目的蛋白的表达。表达结果如图16。结果显示在2019-nCoV的几种抗原设计中nCoV-RBD-C2的表达最高。而SARS-CoV-RBD-C1的构建蛋白表达量也很高。
以上结果说明，Beta冠状病毒的单链二聚体设计，从图14A的第一个氨基酸(标记为Start)开始，到图14B最后一个半胱氨酸(标记为Stop)之前的一个氨基酸为止是表达最优的构建。
实施例14、2019-nCoV-RBD单链二聚体抗原和SARS-CoV-RBD单链二聚体抗原的表达纯化
哺乳动物293T细胞表达nCoV-RBD-C2。质粒转染293T细胞之后，收获上清。细胞上清通过0.22μm的滤膜过滤，除去细胞碎片。将细胞培养上清液挂镍亲和柱(Histrap)，4度过夜。以缓冲液A(20mM Tris，150mM NaCl，pH8.0)洗涤树脂，以除去非特异结合蛋白。最后目的蛋白以缓冲液B(20mM Tris,150mM NaCl,pH 8.0,300mM咪唑)从树脂上洗脱下来，并以10K截留(10cutoff)浓缩管将洗脱液浓缩至5ml以内。再通过Superdex200 Hiload 16/60柱子(GE)进行分子筛层析进行进一步的目的蛋白纯化。分子筛层析缓冲液为20mM Tris，150mM NaCl，PH8.0。经过分子筛层析，只在洗脱体积80mL附近有1个主峰。收集进行SDS-PAGE分析。SDS-PAGE的结果显示，nCoV-RBD-C2蛋白显示1条明显的蛋白带，在48-63kd之间，是RBD二聚体的大小。证明获得了2019-nCoV-RBD单链二聚体，如图17。纯度在95％以上。该结果表明，这样的构建能够获得足量、高纯度的2019-nCoV单链二聚体蛋白。
以相同的方法表达和纯化了2019-nCoV单体RBD蛋白(由nCoV-RBD-C4构建表达获得)、SARS-CoV单体RBD蛋白(SARS-CoV RBD R306-F527，氨基酸序列和编码其氨基酸序列的核苷酸序列如SEQ ID NO：26和SEQ ID NO：27所示)和SARS-CoV单链二聚体蛋白(由SARS-CoV-RBD-C1构建表达获得)。
SARS-CoV单链二聚体蛋白结果如图18所示，可见经过分子筛层析，只在洗脱体积80ml附近有1个主峰。收集进行SDS-PAGE分析，由图18可见SARS-CoV-RBD-C1目的蛋白分子量在在55-72kd之间，是RBD二聚体的大小。证明获得了SARS-RBD单链二聚体，如图18，且具有很高的纯度。
实施例15：2019-nCoV-RBD单链二聚体蛋白免疫小鼠实验
将实施例14获得的2019-nCoV-RBD单链二聚体和2019-nCoV-RBD单体于PBS溶液中稀释，并与AddaVax佐剂进行分组乳化。然后对6-8周龄的BALB/c小鼠(平均体重15-20g，下同)进行分组免疫，每组8只小鼠。通过大腿肌肉注射的方式，每只小鼠分别在第0天，第21天和第42天接受3次疫苗免疫，每次100μl的接种体积(含10μg免疫源)。分别在1免19天，2免14天和3免14天，对小鼠进行采血。小鼠血清通过静置后3000rpm离心10分钟获得，并于-20℃冰箱保存，用于特异性抗体检测和假病毒中和检测。
通过ELISA实验检测小鼠血清的2019-CoV RBD特异性抗体滴度，实验方法见实施例3，结果如图19所示，单链二聚体RBD(标记sc-dimer)和单体RBD(标记Monomer)都能诱导小鼠产生抗体反应，每次免疫后单链二聚体RBD组的滴度均值都比单体RBD组高(10-100倍提高)，且每次免疫后二者都有显著差异(图19)。三次免疫后单链二聚体RBD诱导小鼠产生的抗体水平高达约1:10 6。此结果表明，单链二聚体形式的RBD抗原比单体形式的RBD抗原免疫原性更强，作为潜在的新冠病毒疫苗具有极大潜力。
2019-nCoV假病毒中和实验方法参照实施例5进行。结果如图20所示，一次免疫后，仅单链二聚体RBD(标记sc-dimer)组诱导产生了中和抗体，单体RBD(标记Monomer)和PBS组都检测不到中和抗体，单链二聚体RBD组与单体RBD组中和抗体滴度有显著差异(图20)。第二次和第三次免疫后，单链二聚体RBD和单体RBD都能诱导小鼠产生中和抗体，每次免疫后单链二聚体RBD组的中和抗体滴度均值都比单体RBD组高(10-100倍提高)，且每次免疫后二者都有显著差异(图20)。三次免疫后单链二聚体RBD诱导小鼠产生的中和抗体水平高达约1:10 4。此结果表明，单链二聚体RBD抗原相比单体RBD抗原能够诱导小鼠产生更高的中和抗体水平，在应用中单链二聚体RBD抗原具有很高的优势。
使用二次免疫之后的血清，进行2019-nCoV真病毒(2020XN4276株，该病毒株公开于Lu J,du Plessis L,Liu Z,et al.Genomic Epidemiology of SARS-CoV-2 in Guangdong Province,China.Cell.2020；181(5):997-1003.e9.doi:10.1016/j.cell.2020.04.023，由广东省疾病预防控制中心提供)的中和实验。实验结果见图21。结果显示，RBD二聚体能诱导小鼠产生很高的新冠病毒中和抗体，最高的中和NT50能大于4096，NT50最低的一只鼠数值是512。而RBD单体组的8只鼠，仅2只检测出新冠病毒中和抗体，NT50较低，分别是128和256。此结果表明二聚体RBD可以诱导小鼠产生较高的的新冠病毒中和抗体。
实施例16：SARS-RBD单链二聚体蛋白免疫小鼠实验
将实施例14获得的SARS-RBD单链二聚体和SARS-RBD单体于PBS溶液中稀释， 并与AddaVax佐剂进行分组乳化。然后对6-8周龄的BALB/c小鼠进行分组免疫，每组6只小鼠。通过大腿肌肉注射的方式，每只小鼠分别在第0天，第2天和第42天接受3次疫苗免疫，每次100μl的接种体积(含10μg免疫源)。分别在1免19天，2免14天和3免14天，对小鼠进行采血。小鼠血清通过静置后3000rpm离心10分钟获得，并与-20℃冰箱保存，用于特异性抗体检测和假病毒中和检测。
通过ELISA实验检测小鼠血清的SARS-RBD特异性抗体滴度，实验方法见实施例3，结果如图22所示，单链二聚体RBD(标记sc-dimer)和单体RBD(标记Monomer)都能诱导小鼠产生抗体反应，每次免疫后单链二聚体RBD组的滴度均值都比单体RBD组高，在第二次和第三次免疫后二者都有显著差异(图22)。三次免疫后二聚体RBD诱导小鼠产生的抗体水平高达约1:10 6。此结果表明，二聚体形式的RBD抗原比单体形式的RBD抗原免疫原性更强。
SARS-CoV假病毒中和实验方法参照实施例5进行。结果如图23所示，两次免疫后，二聚体RBD(标记sc-dimer)组和单体RBD(标记Monomer)组都诱导产生了中和抗体，但是二聚体RBD组滴度值更高，二者相比具有显著差异(图23)。第三次免疫后，二聚体RBD组的中和抗体滴度均值仍然比单体RBD组高，且具有显著差异(图23)。三次免疫后二聚体RBD诱导小鼠产生的中和抗体水平高于1:10 3。此结果表明，二聚体RBD抗原相比单体RBD抗原能够诱导小鼠产生更高的中和抗体水平，在应用中二聚体RBD抗原具有很高的优势。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
工业实用性
本发明实施例涉及一种β冠状病毒抗原、其制备方法和应用。β冠状病毒抗原的氨基酸序列包括：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。本发明实施例所表达的单链二聚体含量稳定，作为β冠状病毒抗原具有好的免疫原性，该单链二聚体作为β冠状病 毒抗原制备成的疫苗能够激发小鼠产生很高滴度的中和抗体。

Claims (18)

  1. 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。
  2. 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。
  3. 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。
  4. 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％。
  5. 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。
  6. 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。
  7. 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。
  8. 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。
  9. 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。
  10. 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。
  11. 一种包括权利要求10所述的核苷酸序列的重组载体。
  12. 一种包括权利要求11所述的重组载体的表达系统细胞。
  13. 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。
  14. 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。
  15. 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。
  16. 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。
  17. 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。
  18. 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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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
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