WO2022206222A1 - 一种新型冠状病毒s-rbd三聚体蛋白疫苗、其制备方法和应用 - Google Patents

一种新型冠状病毒s-rbd三聚体蛋白疫苗、其制备方法和应用 Download PDF

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WO2022206222A1
WO2022206222A1 PCT/CN2022/077527 CN2022077527W WO2022206222A1 WO 2022206222 A1 WO2022206222 A1 WO 2022206222A1 CN 2022077527 W CN2022077527 W CN 2022077527W WO 2022206222 A1 WO2022206222 A1 WO 2022206222A1
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novel coronavirus
protein
rbd
vaccine
host cell
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French (fr)
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李启明
梁宇
苏计国
张靖
靳玉琴
侯俊伟
杜丽芳
韩子泊
张学峰
邵帅
张�浩
唐芳
刘兆明
侯亚楠
陈实
雷泽华
马智静
郑凡
刘宁
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国药中生生物技术研究院有限公司
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Publication of WO2022206222A1 publication Critical patent/WO2022206222A1/zh

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Definitions

  • the invention relates to the field of biomedicine, in particular to a novel coronavirus S-RBD trimer protein vaccine, a preparation method and application thereof.
  • SARS-CoV-2 belonging to the order Nidovirales, the family Coronaviridae, the subfamily orthocoronavirus, the genus Betacoronavirus, the subgenus Sarbecovirus, a SARS-like virus species, a single-stranded positive-stranded RNA virus, with an envelope,
  • the full length of the genome is about 29.9kb, most of which encode non-structural proteins involved in viral replication and translation, and a small number of sequences encode structural proteins, such as spike protein (S), M protein (membrane protein), E protein (envelope protein) and N protein (nucleo protein), in addition to several accessory proteins: 3a, 3b, p6, 7a, 7b, 8b, 9b and orf14, these proteins are involved in viral assembly.
  • S spike protein
  • M protein membrane protein
  • E protein envelope protein
  • N protein nucleo protein
  • the S, M and E proteins constitute the viral envelope and are the main surface antigens for the virus to elicit an immune response.
  • the S protein is a transmembrane glycoprotein with a molecular weight of about 150kDa, which forms a prominent homotrimer on the surface of the virus.
  • S consists of two functional subunits that are cleaved at the boundary between the S1 and S2 subunits (S1/S2 cleavage point), which remain non-covalently bound in the prefusion conformation.
  • the S2 subunit is also composed of multiple domains, and its function is mainly to mediate the fusion of virus and host cells.
  • the distal S1 subunit is structurally divided into four distinct domains: NTD, RBD, CTD1, and CTD2, where RBD is the receptor-binding domain responsible for binding with the host cell surface receptor angiotensin-converting enzyme 2 ( Angiotensin converting enzyme 2, ACE2) binds to mediate virus infection of host cells. Therefore, S protein and RBD are the main targets of current genetic engineering vaccine development.
  • One aspect of the present invention provides a novel coronavirus S-RBD trimer protein in view of the lack of recombinant vaccines capable of producing high titers of protective neutralizing antibodies against novel coronaviruses in the prior art.
  • a novel coronavirus S-RBD trimer protein characterized in that the trimer protein is composed of amino acid fragments at positions 319 to 537 in the RBD region of the novel coronavirus S protein in a trimer form.
  • the present invention designs a brand-new fusion protein by using computational biology methods.
  • the S-RBD trimeric protein is recombined and purified by genetic engineering technology, and then mixed with adjuvant to prepare a vaccine, according to a certain dose and doses of immunized animals can produce high titers of protective neutralizing antibodies against novel coronavirus for the treatment and/or prevention of novel coronavirus (SARS-CoV-2) infection and/or novel coronavirus disease (COVID-19) ).
  • the trimeric protein described in the present invention can be formed by self-assembly of three polypeptide subunits of the same sequence.
  • a suitable linker (Linker) or spacer region may be included between the above-mentioned three identical sequences, which may be oligopeptide or polypeptide, and its function may be to increase flexibility.
  • the primary structure of the trimeric protein is that the three amino acid fragments are connected in the sequence from N-terminal to C-terminal.
  • the amino acid sequence of the fusion protein is as shown in SEQ ID No. 1 or a sequence having more than 95% homology with it.
  • the trimeric protein of the present invention can form a trimeric form with stable antigenic conformation without introducing any exogenous linker arm or other unrelated components.
  • the above sequence having more than 95% homology with it refers to the amino acid sequence which is 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the fusion protein.
  • Those skilled in the art can perform random or engineered point mutations in the amino acid sequence of the fusion proteins described in this specification in a suitable manner, the purpose of which may be, for example, to obtain better affinity and/or dissociation properties, and these mutations
  • the latter amino acid sequences are all included in the protection scope of the present invention.
  • Another aspect of the present invention is to provide a fusion protein comprising the above-mentioned novel coronavirus S-RBD trimer protein.
  • the fusion protein further comprises one or more selected from a signal peptide, a tag or an immune-enhancing peptide.
  • the function of the signal peptide may be more favorable for the expression of the protein;
  • the tag may be, for example, a Flag tag, enhanced green fluorescent protein (eGFP), glutathione thioltransferase (GST), etc., which The role can be for detection, purification, separation and the like.
  • the above functional sequences can be used in any combination.
  • nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned novel coronavirus S-RBD trimer protein, or encoding the above-mentioned fusion protein.
  • the inventors optimize the codons of the trimer protein, and the obtained nucleotide sequence is shown in SEQ ID No. 2 or has more than 95% homology with it sexual sequence.
  • sequence having more than 95% homology thereto refers to a nucleotide sequence that is 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence.
  • the preparation method of the above-mentioned nucleic acid molecule can be prepared by known techniques such as chemical synthesis or PCR amplification based on the above-mentioned nucleotide sequence.
  • the codons encoding the amino acids of the above domains can be optimized to optimize their expression in host cells.
  • the above-mentioned base sequence information can be obtained by searching known literature or databases such as NCBI (https://www.ncbi.nlm.nih.gov/).
  • Another aspect of the present invention provides a vector, which contains the above-mentioned nucleic acid molecule.
  • the above-mentioned carrier may be a linear carrier or a circular carrier. It may be a non-viral vector such as a plasmid, a viral vector, or a vector using a transposon.
  • the vector may contain regulatory sequences such as promoters and terminators, as well as marker sequences such as drug resistance genes and reporter genes.
  • the vector is an expression vector of the nucleic acid molecule in the present invention.
  • Another aspect of the present invention provides a host cell comprising the above-mentioned nucleic acid molecule or the above-mentioned vector.
  • the host cell is Escherichia coli, yeast cell, insect cell or mammalian cell;
  • the host cell is a CHO cell.
  • Another aspect of the present invention provides a method for preparing the above-mentioned novel coronavirus S-RBD trimer protein or the above-mentioned fusion protein, comprising the following steps:
  • Step A) preparing the nucleic acid molecule, constructing the expression vector, transforming or transfecting the expression vector into the host cell;
  • Step B) using the product of step A) for protein expression
  • Step C) purify the expression product obtained in step B) to obtain the novel coronavirus S-RBD trimer protein or fusion protein.
  • the nucleic acid molecule of step A) comprises a nucleotide sequence encoding the above-mentioned novel coronavirus S-RBD trimer protein, or encoding the above-mentioned fusion protein.
  • the nucleotide sequence is as shown in SEQ ID No. 2 or a sequence having more than 95% homology with it.
  • the nucleic acid molecules can be prepared from the nucleotide sequences described in this specification using any suitable molecular biology method.
  • the construction of the expression vector in step A) can use any suitable method to construct the above-mentioned nucleotide sequence into the corresponding expression vector of the host cell.
  • the expression vector is then transformed or transfected into the host cell.
  • the inventor transfects it into 293FT cells or CHO cells to construct a recombinant cell line.
  • the protein expression in step B) can express the recombinant protein according to the different expression systems used.
  • the inventors obtained cell lines that can stably secrete and express S-RBD trimer protein or fusion protein by limiting dilution method.
  • the purification in step C) can be any suitable method.
  • the above-mentioned S-RBD trimer protein or fusion protein is purified by the methods of ion exchange and hydrophobic chromatography.
  • the collection process of the target protein should also be included, eg. Collection of the cell culture supernatant rich in the target protein; the process of disrupting the host cells after the target protein has been expressed, for example, any suitable disruption such as ultrasonic disruption, repeated freeze-thaw disruption, chemical treatment, etc. can be used method.
  • any suitable disruption such as ultrasonic disruption, repeated freeze-thaw disruption, chemical treatment, etc. can be used method.
  • the above-described collection of host cells should also be understood to be included within the scope of the purification.
  • novel coronavirus S-RBD trimer protein, the fusion protein, the nucleic acid molecule, the vector or the host cell are prepared for treatment and/or prevention Use in medicine for novel coronavirus infection and/or disease caused by novel coronavirus.
  • the disease caused by the novel coronavirus is preferably novel coronavirus pneumonia (COVID-19).
  • Another aspect of the present invention provides a vaccine comprising the novel coronavirus S-RBD trimer protein or the fusion protein, and an adjuvant.
  • the vaccine is a recombinant protein vaccine (or a genetically engineered subunit vaccine). Further, in other embodiments of the present invention, the vaccine may also be a genetically engineered vector vaccine, or may be a nucleic acid vaccine, and the above-mentioned vaccine comprises the nucleotide sequence described in this specification.
  • any suitable adjuvant may be included.
  • the adjuvant is aluminum hydroxide, aluminum phosphate, MF59 or CpG. More preferably, the adjuvant is aluminum hydroxide.
  • Another aspect of the present invention provides a method for preparing the above vaccine, wherein the purified novel coronavirus S-RBD trimeric protein or the fusion protein is mixed with the adjuvant.
  • Another aspect of the present invention provides the use of the above vaccine in the treatment and/or prevention of novel coronavirus infection and/or diseases caused by novel coronavirus.
  • the disease caused by the novel coronavirus is preferably novel coronavirus pneumonia (COVID-19).
  • Another aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the vaccine and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can be any pharmaceutically acceptable additive, for example, physiological saline, cell culture medium, glucose, water for injection, glycerol, amino acids and their compositions, stabilizers, surfactants, preservatives, Isotonicity etc.
  • the pharmaceutical composition of the present invention can also be used in combination with other medicines for the treatment and/or prevention of novel coronavirus infection and/or diseases caused by novel coronavirus at an effective and safe dose.
  • Another aspect of the present invention is to provide a method for eliciting an immune response in a subject against a novel coronavirus or treating a subject's novel coronavirus infection, and administering an effective dose of the vaccine or the pharmaceutical composition.
  • the subject can be a human or other animal.
  • the administration can be intramuscular, intraperitoneal or subcutaneous.
  • the vaccine prepared by the present invention uses S-RBD trimer protein as antigen, and after adjuvant, immunizes the body, can produce high-titer protective neutralizing antibody against novel coronavirus, and can be used for the treatment and/or prevention of novel coronavirus Coronavirus (SARS-CoV-2) infection and/or novel coronavirus disease.
  • SARS-CoV-2 novel coronavirus Coronavirus
  • A is the S protein monomer structure (based on a coordinate file with a PDB code of 6zgg, drawn by UCSF Chimera software), and the S1 structural unit includes NTD, RBD, SD1 and SD2 domains;
  • B is the complex structure of RBD and ACE2 receptor (based on the coordinate file with PDB code 6m0j, drawn by UCSF Chimera software);
  • Fig. 2 is the S-RBD trimer protein design diagram in Example 1 of the present invention, wherein, A is the S-RBD monomer protein structure; B is the S-RBD trimer protein structure;
  • Figure 3 is the SDS-PAGE chart of the S-RBD trimer protein purified in Example 2 of the present invention, wherein, lanes 1-5 are the S-RBD trimer proteins obtained by purification; lane M1 is the protein marker (molecular weight standard are: kDa: 250, 150, 100, 70, 50, 40, 30, 20, 15, 10, 5); lane M2 is a protein marker (molecular weight standard: kDa: 250, 130, 100, 70, 55, 35 , 25, 15, 10);
  • Example 4 is a Western-blot identification diagram of the S-RBD trimer protein purified in Example 2 of the present invention, wherein, lanes 1-5 are the S-RBD trimer proteins obtained by purification; lane M2 is a protein marker;
  • Example 5 is a transmission electron microscope image of the S-RBD trimeric protein purified in Example 2 of the present invention.
  • Example 6 is a graph showing the binding curve of the S-RBD trimeric protein purified in Example 3 of the present invention and the MM43 neutralizing monoclonal antibody;
  • Example 7 is a graph showing the binding curve of the S-RBD trimeric protein purified in Example 3 of the present invention and the MM57 neutralizing monoclonal antibody;
  • Figure 8 is a graph showing the SPR detection results of S-RBD trimeric protein and ACE2 protein purified in Example 4 of the present invention.
  • Fig. 9 is the result diagram of serum-specific IgG titer detection results after immunizing Wistar rats with different doses of recombinant novel coronavirus vaccine in Example 6 of the present invention.
  • Figure 10 is a graph of the detection results of serum neutralizing antibody titers against pseudoviruses after immunizing Wistar rats with different doses of recombinant novel coronavirus vaccine in Example 6 of the present invention
  • Figure 11 is a graph showing the detection results of serum neutralizing antibody titers against wild virus after immunizing Wistar rats with different doses of recombinant novel coronavirus vaccine in Example 6 of the present invention.
  • Figure 12 is the SDS-PAGE chart of the dimeric protein and the trimeric protein in Comparative Example 1 of the present invention, wherein, lane 1 is the purified trimeric protein; lane 2 is the purified dimeric protein; lane M2 Protein marker (molecular weight standard: kDa: 250, 130, 100, 70, 55, 35, 25, 15, 10);
  • Figure 13 is a graph of the detection result of serum specific IgG titer in Comparative Example 1 of the present invention.
  • Figure 14 is a graph of the detection results of serum neutralizing antibody titers against pseudoviruses in Comparative Example 1 of the present invention.
  • Fig. 15 is a graph showing the detection result of serum neutralizing antibody titer against wild virus in Comparative Example 1 of the present invention.
  • SEQIDNo.1 is the amino acid sequence of the novel coronavirus S-RBD trimer protein in the examples of the present invention.
  • SEQIDNo.2 is the nucleotide sequence of the novel coronavirus S-RBD trimer protein in the examples of the present invention
  • SEQIDNo.3 is the amino acid sequence of the novel coronavirus S-RBD dimer protein in the comparative example of the present invention.
  • SEQ ID No.4 is the nucleotide sequence of the novel coronavirus S-RBD dimer protein in the comparative example of the present invention.
  • the present invention discloses a novel coronavirus S-RBD trimer protein vaccine, a preparation method and application thereof, and those skilled in the art can learn from the content of this article and appropriately improve process parameters to achieve. It should be specially pointed out that all similar substitutions and modifications are obvious to those skilled in the art, they are all considered to be included in the present invention, and relevant persons can obviously do so without departing from the content, spirit and scope of the present invention.
  • the content described herein can be modified or appropriately changed and combined to realize and apply the technology of the present invention.
  • new coronavirus namely SARS-CoV-2
  • SARS-CoV-2 belongs to the order Nidovirales, the family Coronaviridae, the subfamily orthocoronavirus, the genus Betacoronavirus, the subgenus Sarbecovirus, the SARS-like virus species, the single-stranded positive Stranded RNA virus, with an envelope, the total length of the genome is about 29.9kb, most of which encode non-structural proteins, which are involved in functions such as virus replication and translation, and a small number of sequences encode structural proteins, such as: spike protein (S) , M protein (membrane protein), E protein (envelope protein) and N protein (nucleo protein), in addition to several accessory proteins: 3a, 3b, p6, 7a, 7b, 8b, 9b and orf14, these proteins are involved in the virus assembled.
  • S spike protein
  • M protein membrane protein
  • E protein envelope protein
  • N protein nucleo protein
  • the S, M and E proteins constitute the viral envelope and are the main surface antigens for the virus to elicit an immune response.
  • the S protein is a transmembrane glycoprotein with a molecular weight of about 150kDa, which forms a prominent homotrimer on the surface of the virus.
  • S consists of two functional subunits that are cleaved at the boundary between the S1 and S2 subunits (S1/S2 cleavage point), which remain non-covalently bound in the prefusion conformation.
  • the S2 subunit is also composed of multiple domains, and its function is mainly to mediate the fusion of virus and host cells.
  • the distal S1 subunit is structurally divided into four distinct domains: NTD, RBD, CTD1, and CTD2, where RBD is the receptor-binding domain responsible for binding with the host cell surface receptor angiotensin-converting enzyme 2 ( Angiotensin converting enzyme 2, ACE2) binds to mediate virus infection of host cells. Therefore, S protein and RBD are the main targets of current genetic engineering vaccine development.
  • trimer form is a type of protein quaternary structure.
  • Protein quaternary structure refers to the number and arrangement of protein subunits relative to each other (Chou, Kuo-Chen; Cai, Yu-Dong. Predicting protein quaternary structure by pseudo amino acid composition. Proteins: Structure, Function, and Bioinformatics. 1 November 2003, 53(2):282–289.). It contains three protein subunits in the form of trimers.
  • primary structure is the linear sequence of amino acids in a peptide or protein. By convention, the primary structure of proteins is reported from the amino-terminal (N) terminus to the carboxy-terminus (C) terminus.
  • fusion protein refers to the expression product after one, two or more gene recombination obtained by DNA recombination technology. Fusion protein technology is a purposeful gene fusion and protein expression method to obtain a large number of standard fusion proteins. Using fusion protein technology, new target proteins with various functions can be constructed and expressed.
  • vector is a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector When the vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 derived artificial chromosomes (PACs) ; Phage such as ⁇ phage or M13 phage and animal viruses.
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PACs P1 derived artificial chromosomes
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses eg, adeno-associated viruses
  • herpesviruses eg, herpes simplex virus
  • poxviruses baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg SV40
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and
  • host cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. , these techniques include transfection of viral vectors, transformation with plasmid vectors, and accelerated introduction of naked DNA by electroporation, lipofection, and particle guns.
  • treating refers to reducing the likelihood of disease pathology, reducing the occurrence of disease symptoms, eg, to the extent that the subject has longer survival or reduced discomfort.
  • Treatment can refer to the ability of a therapy to reduce the symptoms, signs, or cause of a disease when the therapy is administered to a subject. Treating also refers to alleviating or reducing at least one clinical symptom and/or inhibiting or delaying the progression of a disorder and/or preventing or delaying the onset of a disease or disorder.
  • subject refers to any human or other animal, particularly other mammals, receiving prophylaxis, treatment, or diagnosis.
  • Other mammals can include, for example, dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, guinea pigs, mice, and the like.
  • Example 1 Design of novel coronavirus S-RBD trimer protein based on structural biology
  • sequence fragment (amino acids 319-537) of the S protein RBD region of the novel coronavirus (Genebank No.: MN908947.3) was cut, and the distance between the N-terminus and the C-terminus was The three identical RBD fragments are connected end to end to form a new fusion protein, the amino acid sequence is shown in SEQ ID NO. 1, using the multimer structure prediction software (initio domain assembly, AIDA) and molecular dynamics simulation (GROMACS) , showing that the fusion protein contains three independent RBD domains, forming a trimeric form with stable antigen conformation (as shown in Figure 2), which is the novel coronavirus S-RBD trimer protein.
  • AIDA initio domain assembly
  • GROMACS molecular dynamics simulation
  • Example 2 Expression, purification and identification of novel coronavirus S-RBD trimer protein
  • the amino acid sequence of SEQ ID NO.1 was codon-optimized, and the optimized gene sequence was shown in SEQ ID NO.2.
  • After constructing a CHO cell expression vector it was transfected into 293FT cells or The recombinant cell line was constructed in CHO cells, and the cell line that could stably secrete and express the S-RBD trimer protein was obtained by limiting dilution method.
  • the S-RBD trimeric protein with a purity of ⁇ 95% was obtained after a series of chromatographic purification.
  • SDS-PAGE detection results are shown in Figure 3, the molecular weight of the protein is about 86kD, and some product-related substances, such as dimer protein and monomer protein, can be seen.
  • the purified protein was electrophoresed on SDS-PAGE and then transferred to PVDF membrane, and identified by Western-blot using RBD-specific antibody (manufacturer: Beijing Yiqiao Shenzhou Technology Co., Ltd.; product number: 40591-T62; dilution: 2000 times). As shown in Figure 4), the purified protein can be combined with RBD-specific antibodies and has good immunogenicity.
  • the purified protein was added dropwise to the copper omentum for 1 minute of adsorption, negatively stained with phosphotungstic acid for 1 minute, and the protein morphology was observed under a transmission electron microscope (the results are shown in Figure 5). On the omentum, the particle size is smaller.
  • Example 3 Biological analysis of the binding of novel coronavirus S-RBD trimer protein to neutralizing monoclonal antibodies
  • Example 4 Biological analysis of the binding of novel coronavirus S-RBD trimer protein to ACE2 receptor
  • angiotensin converting enzyme is the receptor of the new coronavirus, so by measuring the S-RBD trimer The binding of the protein to ACE2 can be used to analyze the biological activity of the protein.
  • SPR Surface plasmon resonance
  • the purified S-RBD trimeric protein was diluted to 2 times the target antigen concentration, mixed with 1.2 mg/ml aluminum hydroxide adjuvant at a ratio of 1:1 (w/w) for adsorption, and stirred on a magnetic stirrer for 40 ⁇ 120min, the rotation speed is 200 ⁇ 300rpm, and the semi-finished product of the vaccine is obtained.
  • the residual protein content of the supernatant should be less than 10% of the total protein content.
  • Example 6 Evaluation of the immunological effect of recombinant novel coronavirus vaccine
  • the prepared recombinant novel coronavirus vaccine was immunized by intramuscular injection in Wistar rats (purchased from Speifu (Beijing) Biotechnology Co., Ltd., 6-8 weeks old, animal certificate number: No. 110324201102113753), 0.5 ml/only, the specific animal test protocol is shown in Table 1.
  • the titer of specific IgG antibody against S-RBD protein in the immunized serum was detected by ELISA, and the titer of neutralizing antibody was detected by microneutralization test of pseudovirus and wild virus.
  • the serum-specific IgG results are shown in Figure 9
  • the pseudovirus microneutralization test results are shown in Figure 10
  • the wild virus microneutralization is shown in Figure 11
  • the serum antibody GMT values are shown in Table 2.
  • the recombinant new coronavirus vaccine can stimulate the production of higher titer antibody levels in rats.
  • the levels of specific IgG antibodies and neutralizing antibodies gradually increase, and there is an obvious dose relationship.
  • Serum-specific IgG antibody detection ELISA method was used to detect the specific IgG antibody level against S-RBD protein in post-immunization serum. Dilute to 1 ⁇ g/ml, and coat 100 ⁇ l/well in an ELISA plate. After blocking, add serially diluted serum, use enzyme-labeled antibody (mouse source) to detect the signal, determine the cut-off value, and the highest dilution of positive serum The multiple is the specific IgG antibody titer of the serum sample.
  • Pseudovirus micro-neutralization test The S protein gene sequence of the pseudovirus was derived from Wuhan-Hu-1 strain (GenBank sequence number: MN908947), the reporter gene was firefly luciferase, and the pseudovirus working concentration in the neutralization test was (1 ⁇ 2) ⁇ 10 4 TCID50/ml, the serum dilution ratio when calculating the 50% infection inhibition rate using the Reed-Muench method is the neutralizing antibody titer of the serum sample.
  • Wild virus micro-neutralization test This test was completed in a BSL3 laboratory, and the virus strain was 2020XN4276.
  • the serially diluted serum and virus (100TCID50) were mixed and incubated in equal volumes, neutralized in a 37°C incubator for 2 hours, and added to a In the cell culture plate of Vero-E6 cells (2 ⁇ 10 5 /ml), the cell control and virus control were set at the same time, and cultured in a 37°C incubator for 3 to 5 days to observe the cytopathic condition, and use the Reed-Muench method to calculate 50% Infection inhibition rate is the dilution ratio of serum, which is the neutralizing antibody titer of the serum sample.
  • the new coronavirus (Genebank No.: MN908947.3) S protein RBD region sequence fragment (319-537 amino acids), connect two identical RBD region fragments end to end to form a dimer fusion protein, the amino acid sequence is as SEQ ID NO As shown in .3, according to the codon preference of the CHO cell expression system, the amino acid sequence of SEQ ID NO.3 was codon-optimized, and the optimized gene sequence was shown in SEQ ID NO.4.
  • a cell line capable of expressing the dimer protein was obtained, and the S-RBD dimer protein with a purity of ⁇ 95% was obtained after purification by a series of chromatography (as shown in Figure 12), which was mixed with 1.2mg/ml hydrogen Alumina adjuvant was mixed and adsorbed at a ratio of 1:1 (w/w) to prepare a dimer protein immune substance, and the trimeric protein immune substance prepared by the same process was intraperitoneally injected to immunize BALB/c mice, and the antigen content was 2.0 ⁇ g/dose, the content of aluminum adjuvant was 0.30mg/dose, immunized for 3 times according to 0w, 1w and 2w, the whole process was collected and separated 2w after immunization, and the ELISA method was used to detect the specificity of S-RBD protein in the serum after immunization.
  • the neutralizing antibody titer was measured by the pseudovirus and wild virus microneutralization test. The test results are shown in Figure 13, Figure 14 and Figure 15, and the GMT test results are shown in Table 3. Trimers can be seen. The level of antibody produced by the protein was higher than that of the dimeric protein, and the specific IgG antibody titer had no statistical difference. Although the titer of the pseudovirus neutralizing antibody was about 2 times higher, there was no statistical difference. The wild virus neutralizing antibody The titer was approximately 3-fold higher and the difference was statistically significant.

Abstract

本发明公开了一种新型冠状病毒S-RBD三聚体蛋白，所述三聚体蛋白由三聚体形式的新型冠状病毒S蛋白RBD区第319～537位氨基酸片段构成。本发明制备的疫苗以S-RBD三聚体蛋白为抗原，辅以佐剂后，免疫机体，可产生针对新型冠状病毒的高滴度保护性中和抗体，可以用于治疗和/或预防新型冠状病毒(SARS-CoV-2)感染和/或新型冠状病毒疾病。

Description

一种新型冠状病毒S-RBD三聚体蛋白疫苗、其制备方法和应用
交叉引用说明
本申请要求于2021年3月31日提交中国专利局、申请号为202110348881.6，发明名称为“一种新型冠状病毒S-RBD三聚体蛋白疫苗、其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医药领域，特别涉及一种新型冠状病毒S-RBD三聚体蛋白疫苗、其制备方法和应用。
背景技术
2020年由新型冠状病毒(Severe Acute Respiratory Syndrome Coronavirus 2，SARS-CoV-2)所引发的新型冠状病毒肺炎(Coronavirus Disease，COVID-19)已在全球发生大流行，其传播速度之快、蔓延地区之广、感染人数之多，数十年来实属罕见，对比SARS和MERS病毒，新冠肺炎病毒的严重性有过之而无不及。截止到目前，新冠病毒已传播至全球200多个国家和地区，WHO数据显示，截止到2021年2月23日，全球累计确诊人数超过1.1亿人，死亡人数超过246万人。虽然口罩加隔离措施能预防病毒感染，但会严重影响全球经济发展和人民日常生活，而只有疫苗才是终止这场疫情的最佳手段。有专家表示，新冠病毒将与人类长期共存，并有反复暴发的可能，而作为战胜疫情的终极武器，疫苗也将长期存在。因此，全球各大企业纷纷参与新冠疫苗研发，全球已有超过100个新冠疫苗处于研发的不同阶段。由此可见，新冠疫苗有着广阔的发展前景。
SARS-CoV-2，属巢病毒目(Nidovirales)、冠状病毒科(Coronaviridae)、正冠状病毒亚科、Betacoronavirus属、Sarbecovirus亚属、类SARS病毒种、单股正链RNA病毒，有包膜，基因组全长约为29.9kb，绝大部分编码非结构蛋白，参与病毒复制和翻译等功能，少部分序列编码结构蛋白，如：刺突蛋白(spike protein，S)、M蛋白(membrane protein)、E蛋白(envelope protein)和N蛋白 (nucleo protein)，此外还有若干附属蛋白：3a,3b,p6,7a,7b,8b,9b和orf14，这些蛋白均参与病毒组装。S、M和E蛋白构成病毒囊膜，是病毒引起免疫反应的主要表面抗原。其中S蛋白是一种跨膜糖蛋白，分子量约为150kDa，在病毒表面形成突出的同源三聚体。S由两个功能亚基组成，在S1和S2亚基之间的边界处(S1/S2裂解点)被切割，这两个亚基在融合前构象中保持非共价结合。S2亚基也由多个结构域构成，它的功能主要是介导病毒与宿主细胞的融合。远端S1亚基在结构上分为四个不同的结构域：NTD、RBD、CTD1和CTD2，其中RBD是受体结合结构域，主要负责与宿主细胞表面的受体血管紧张素转换酶2(angiotensin converting enzyme 2,ACE2)结合，从而介导病毒侵染宿主细胞，因此S蛋白及RBD均为目前基因工程疫苗研发的主要靶标。
截至目前，全球获批上市的疫苗共有7款，分别是美国批准紧急使用授权(EUA)的BNT162b2和mRNA-1273，英国批准紧急使用授权(EUA)的AZD1222，中国国药中生(北京公司和武汉公司)和北京科兴各自附条件上市的3款新冠灭活疫苗、康希诺生物腺病毒载体疫苗以及智飞生物重组蛋白疫苗，印度批准紧急使用授权(EUA)国产灭活疫苗，以及俄罗斯批准上市的“卫星V”，另外还有数十种疫苗处于临床研究不同阶段。利用基因工程技术研制重组疫苗由于其高度的安全性和有效性已被广泛证实，加之当前新冠病毒变异株不断出现并且占比持续上升，现有疫苗及中和抗体对特定突变株的保护效果大幅降低，引发各界对新冠疫情走势及疫苗和药物有效性的担忧。
因此，开发一种可产生针对新型冠状病毒高滴度的保护性中和抗体的重组疫苗已成为当务之急。
发明内容
本发明的一个方面，是针对现有技术中缺少可产生针对新型冠状病毒高滴度的保护性中和抗体的重组疫苗的缺陷，提供了一种新型冠状病毒S-RBD三聚体蛋白。
本发明提供的技术方案为：
一种新型冠状病毒S-RBD三聚体蛋白，其特征在于，所述三聚体蛋白由三聚体形式的新型冠状病毒S蛋白RBD区第319～537位氨基酸片段构成。
本发明基于新冠病毒S-RBD区结构学特征，利用计算生物学方法设计了一种全新的融合蛋白，该蛋白包含有三个RBD结构域，在可以不引入任何外源连接臂或其它无关成分情况下，形成抗原构象稳定的三聚体形式，实现S-RBD蛋 白三聚化，利用基因工程技术重组表达并纯化出S-RBD三聚体蛋白后，与佐剂混合制备成疫苗，按一定剂量和剂次免疫动物可产生针对新型冠状病毒高滴度的保护性中和抗体，用于治疗和/或预防新型冠状病毒(SARS-CoV-2)感染和/或新型冠状病毒疾病(COVID-19)。另外，由于RBD区功能明确，结构清楚，负责识别受体细胞的ACE区，同时针对RBD产生的抗体功能明确，最大程度避免诱导机体产生抗体依赖的增强作用(Antibody Dependent Enhancement，ADE)。
本发明中所述的三聚体蛋白可由三个相同序列的多肽亚基通过自组装形成。在本发明的实施方式中，上述三个相同序列之间可以包含合适的连接体(Linker)或间隔区，其可以为寡肽或多肽，其作用可以为增加柔性。
但作为优选，在本发明的一个实施方式中，所述三聚体蛋白的一级结构为三个所述氨基酸片段按照N末端至C末端的顺序连接。
更优选地，在本发明的一个实施方式中，所述融合蛋白的氨基酸序列如SEQIDNo.1所示或与其具有95％以上同源性的序列。
在上述实施方式中，本发明三聚体蛋白在可以不引入任何外源连接臂或其它无关成分情况下，形成抗原构象稳定的三聚体形式。
上述与其具有95％以上同源性的序列是指与所述融合蛋白的氨基酸序列具有95％、96％、97％、98％或99％相同的氨基酸序列。本领域技术人员可以对本说明书中所述融合蛋白的氨基酸序列以合适的方式进行随机或者工程化的点突变，其目的可以为，例如，获得更好的亲和力和/或解离性质，而这些突变后的氨基酸序列均包含在本发明的保护范围之内。
本发明的另一个方面，是提供了一种融合蛋白，所述融合蛋白包含上述新型冠状病毒S-RBD三聚体蛋白。
作为优选，在本发明的实施方式中，所述融合蛋白还包含选自信号肽、标签或免疫增强肽中的一种或几种。所述信号肽的作用可以是更有利于蛋白质的表达；所述标签可以为，例如，Flag标签、增强型绿色荧光蛋白(eGFP)、谷胱甘肽巯基转移酶(GST)，等等，其作用可以是用于检测、纯化、分离等等。上述功能性序列可任意组合使用。
本发明的另一个方面，是提供了一种核酸分子，所述核酸分子包含编码上述新型冠状病毒S-RBD三聚体蛋白，或编码上述融合蛋白的核苷酸序列。
作为优选，在本发明的一个实施方式中，发明人对所述三聚体蛋白的密码子进行了优化，得到的所述核苷酸序列如SEQIDNo.2所示或与其具有95％以上同源性的序列。
上述与其具有95％以上同源性的序列是指与所述核苷酸序列具有95％、96％、97％、98％或99％相同的核苷酸序列。
对于上述核酸分子的制备方法，可基于上述核苷酸序列，通过化学合成或PCR扩增等已知技术制备。通常，可以对编码上述结构域的氨基酸的密码子进行优化，以优化其在宿主细胞中的表达。上述碱基序列的信息可通过检索已知文献或NCBI(https://www.ncbi.nlm.nih.gov/)等数据库来获得。
本发明的另一个方面，是提供了一种载体，所述载体包含上述核酸分子。
在本发明中，上述载体可以为直链载体，也可以为环状载体。可以为质粒等非病毒载体，也可以为病毒载体，还可以为利用转座子的载体。所述载体中可含有启动子、终止子等调控序列，以及耐药基因、报告基因等标记序列。
作为优选，在本发明的一个实施方式中，所述载体为本发明中所述核酸分子的表达载体。
本发明的另一个方面，是提供了一种宿主细胞，所述宿主细胞包含上述核酸分子或上述载体。
作为优选，在本发明的实施方式中，所述宿主细胞为大肠杆菌、酵母细胞、昆虫细胞或哺乳动物细胞；
更优选地，在本发明的一个实施方式中，所述宿主细胞为CHO细胞。
本发明的另一个方面，是提供了一种上述新型冠状病毒S-RBD三聚体蛋白或上述融合蛋白的制备方法，包括以下步骤：
步骤A)制备所述核酸分子，构建所述表达载体，将表达载体转化或转染至所述宿主细胞内；
步骤B)利用步骤A)的产物进行蛋白质表达；
步骤C)纯化步骤B)中获得的表达产物，得到所述新型冠状病毒S-RBD三聚体蛋白或融合蛋白。
其中，步骤A)所述核酸分子包含编码上述新型冠状病毒S-RBD三聚体蛋白，或编码上述融合蛋白的核苷酸序列。
作为优选，在本发明的一个实施方式中，所述核苷酸序列如SEQIDNo.2所示或与其具有95％以上同源性的序列。
可使用任意合适的分子生物学方法根据本说明书中所述核苷酸序列制备所述核酸分子。
其中，步骤A)所述构建表达载体可以使用任意合适的方法将上述核苷酸序列构建在宿主细胞相应的表达载体中。
然后将表达载体转化或转染至所述宿主细胞内。作为优选，在本发明的一个实施方式中，发明人在构建CHO细胞表达载体后将其转染至293FT细胞或CHO细胞内构建重组细胞株。
其中，步骤B)所述蛋白质表达可以根据所使用的不同表达系统对重组蛋白进行表达。进一步地，在本发明的一个实施方式中，发明人通过有限稀释法筛选得到能够稳定分泌表达S-RBD三聚体蛋白或融合蛋白的细胞株。
其中，步骤C)所述纯化可以为任意合适的方法。例如，盐析法、沉淀法、透析或超滤、分子筛层析法、离子交换层析、疏水层析法、亲和层析法，等等。作为优选，在本发明的一个实施方式中，采用离子交换和疏水层析的方法纯化上述S-RBD三聚体蛋白或融合蛋白。
当然，根据现有技术，在进行纯化步骤之前，还应包含对目标蛋白质的收集过程，例如。对富含目标蛋白质的细胞培养液上清的收集；对已表达目标蛋白质后的所述宿主细胞进行破碎的过程，可以使用例如，超声波破碎、反复冻融破碎、化学处理法等任意合适的破碎方法。上述对宿主细胞的收集过程也应理解为包含在所述纯化的范围之内。
本发明的另一个方面，是提供了所述新型冠状病毒S-RBD三聚体蛋白、所述融合蛋白、所述核酸分子、所述载体或所述宿主细胞在制备用于治疗和/或预防新型冠状病毒感染和/或新型冠状病毒引起的疾病的药物中的用途。
所述新型冠状病毒引起的疾病优选为新型冠状病毒肺炎(COVID-19)。
本发明的另一个方面，是提供了一种疫苗，所述疫苗包含所述新型冠状病毒S-RBD三聚体蛋白或所述融合蛋白，以及佐剂。
在本发明的实施方式中，所述疫苗为重组蛋白疫苗(或称基因工程亚单位疫苗)。进一步地，在本发明的另外一些实施方式中，所述疫苗还可以为基因工程载体疫苗，或者可以为核酸疫苗，上述疫苗包含本说明书中所述核苷酸序列。
在本发明所述疫苗中，可以包含任意合适的佐剂。但作为优选，在本发明的实施方式中，所述佐剂为氢氧化铝、磷酸铝、MF59或CpG。更优选地，所述佐剂为氢氧化铝。
本发明的另一个方面，是提供了上述疫苗的制备方法，将纯化所得的所述新型冠状病毒S-RBD三聚体蛋白或所述融合蛋白与所述佐剂混合。
本发明的另一个方面，是提供了上述疫苗在治疗和/或预防新型冠状病毒感染和/或新型冠状病毒引起的疾病中的用途。
所述新型冠状病毒引起的疾病优选为新型冠状病毒肺炎(COVID-19)。
本发明的另一个方面，是提供了一种药物组合物，所述药物组合物包含所述疫苗，以及药学上可接受的载体。
所述药学上可接受的载体可以为任意药学上允许的添加剂，例如，生理盐水、细胞培养基、葡萄糖、注射用水、甘油、氨基酸以及它们的组合物、稳定剂、表面活性剂、防腐剂、等渗剂等。
本发明所述药物组合物也可以和其他治疗和/或预防新型冠状病毒感染和/或新型冠状病毒引起的疾病的药物在有效安全的剂量下联合使用。
本发明的另一个方面，是提供了一种引发受试者针对新型冠状病毒的免疫应答或治疗受试者的新型冠状病毒感染的方法，向所述受试者施用有效剂量的所述疫苗或所述药物组合物。
所述受试者可以为人类或者其他动物。
所述施用可以为肌肉注射、腹腔注射或皮下注射。
本发明的有益效果：
本发明制备的疫苗以S-RBD三聚体蛋白为抗原，辅以佐剂后，免疫机体，可产生针对新型冠状病毒的高滴度保护性中和抗体，可以用于治疗和/或预防新型冠状病毒(SARS-CoV-2)感染和/或新型冠状病毒疾病。
附图说明
图1为本发明实施例1中新型冠状病毒S蛋白结构分析图，其中，A为S蛋白单体结构(基于PDB代码为6zgg的坐标文件，采用UCSF Chimera软件绘制)，S1结构单元包括NTD、RBD、SD1和SD2结构域；B为RBD与ACE2受体复合物结构(基于PDB代码为6m0j的坐标文件，采用UCSF Chimera软件绘制)；
图2为本发明实施例1中S-RBD三聚体蛋白设计图，其中，A为S-RBD单体蛋白结构；B为S-RBD三聚体蛋白结构；
图3为本发明实施例2中纯化所得S-RBD三聚体蛋白的SDS-PAGE图，其中，泳道1-5为纯化获得的S-RBD三聚体蛋白；泳道M1为蛋白质marker(分子量标准为：kDa：250、150、100、70、50、40、30、20、15、10、5)；泳道M2为蛋白质marker(分子量标准为：kDa：250、130、100、70、55、35、25、15、10)；
图4为本发明实施例2中纯化所得S-RBD三聚体蛋白的Western-blot鉴定图，其中，泳道1-5为纯化获得的S-RBD三聚体蛋白；泳道M2为蛋白质marker；
图5为本发明实施例2中纯化所得S-RBD三聚体蛋白的透射电镜图；
图6为本发明实施例3中纯化所得S-RBD三聚体蛋白与MM43中和性单克隆抗体结合曲线图；
图7为本发明实施例3中纯化所得S-RBD三聚体蛋白与MM57中和性单克隆抗体结合曲线图；
图8为本发明实施例4中纯化所得S-RBD三聚体蛋白与ACE2蛋白的SPR检测结果图；
图9为本发明实施例6中重组新型冠状病毒疫苗不同剂量免疫Wistar大鼠后血清特异性IgG滴度检测结果图；
图10为本发明实施例6中重组新型冠状病毒疫苗不同剂量免疫Wistar大鼠后针对假病毒的血清中和抗体滴度检测结果图；
图11为本发明实施例6中重组新型冠状病毒疫苗不同剂量免疫Wistar大鼠后针对野病毒的血清中和抗体滴度检测结果图。图12为本发明对比例1中二聚体蛋白和三聚体蛋白的SDS-PAGE图，其中，泳道1为纯化获得的三聚体蛋白；泳道2为纯化所得的二聚体蛋白；泳道M2蛋白质marker(分子量标准为：kDa：250、130、100、70、55、35、25、15、10)；
图13为本发明对比例1中血清特异性IgG滴度检测结果图；
图14为本发明对比例1中针对假病毒的血清中和抗体滴度检测结果图；
图15为本发明对比例1中针对野病毒的血清中和抗体滴度检测结果图。
序列说明
SEQIDNo.1为本发明实施例中新型冠状病毒S-RBD三聚体蛋白的氨基酸序列；
SEQIDNo.2为本发明实施例中新型冠状病毒S-RBD三聚体蛋白的核苷酸序列；
SEQIDNo.3为本发明对比例中新型冠状病毒S-RBD二聚体蛋白的氨基酸序列；
SEQ ID No.4为本发明对比例中新型冠状病毒S-RBD二聚体蛋白的核苷酸序列。
序列表
Figure PCTCN2022077527-appb-000001
Figure PCTCN2022077527-appb-000002
Figure PCTCN2022077527-appb-000003
Figure PCTCN2022077527-appb-000004
Figure PCTCN2022077527-appb-000005
具体实施方式
本发明公开了一种新型冠状病毒S-RBD三聚体蛋白疫苗、其制备方法和应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。需要特别指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明，并且相关人员明显能在不脱离本发明内容、精神和范围的基础上对本文所述内容进行改动或适当变更与组合，来实现和应用本发明技术。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。下面就本发明中出现的部分术语作以解释。
术语“新型冠状病毒”，即SARS-CoV-2，属巢病毒目(Nidovirales)、冠状病毒科(Coronaviridae)、正冠状病毒亚科、Betacoronavirus属、Sarbecovirus亚属、类SARS病毒种、单股正链RNA病毒，有包膜，基因组全长约为29.9kb，绝大部分编码非结构蛋白，参与病毒复制和翻译等功能，少部分序列编码结构蛋白，如：刺突蛋白(spike protein，S)、M蛋白(membrane protein)、E蛋白(envelope protein)和N蛋白(nucleo protein)，此外还有若干附属蛋白：3a,3b,p6,7a,7b,8b,9b和orf14，这些蛋白均参与病毒组装。S、M和E蛋白构成病毒囊膜，是病毒引起免疫反应的主要表面抗原。其中S蛋白是一种跨膜糖蛋白，分子量约为150kDa，在病毒表面形成突出的同源三聚体。S由两个功能亚基组成，在S1和S2亚基之间的边界处(S1/S2裂解点)被切割，这两个亚基在融合前构象中保持非共价结合。S2亚基也由多个结构域构成，它的功能主要是介导病毒与宿主细胞的融合。远端S1亚基在结构上分为四个不同的结构域：NTD、RBD、CTD1和CTD2，其中RBD是受体结合结构域，主要负责与宿主细胞表面的受体血管紧张素转换酶2(angiotensin converting enzyme 2,ACE2)结合，从而介导病毒侵染宿主细胞，因此S蛋白及RBD均为目前基因工程疫苗研发的主要靶标。
术语“三聚体形式”，是蛋白质四级结构中的一种类型。蛋白质四级结构是指蛋白质亚基相对于彼此的数目和排列(Chou,Kuo-Chen；Cai,Yu-Dong.Predicting protein quaternary structure by pseudo amino acid composition.Proteins: Structure,Function,and Bioinformatics.1 November 2003,53(2):282–289.)。其中含有三个蛋白质亚基即为三聚体形式。
术语“一级结构”，是肽或蛋白质中氨基酸的线性序列。按照惯例，蛋白质的一级结构被报道从氨基末端(N)端到羧基末端(C)端。
术语“融合蛋白”，fusionprotein，是指通过DNA重组技术得到的一个、两个或多个基因重组后的表达产物。融合蛋白技术是为获得大量标准融合蛋白而进行的有目的性的基因融合和蛋白表达方法，利用融合蛋白技术，可构建和表达具有多种功能的新型目的蛋白。
术语“载体”，是可将多核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白质获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
术语“宿主细胞”，是已经通过分子生物学技术将核酸分子引入的细胞。，这些技术包括转染病毒载体，用质粒载体转化，以及通过电穿孔、脂转染、和粒子枪加速引入裸DNA。
术语“治疗”，是指减少疾病病理的可能性，减少疾病症状的发生，例如在一定程度上受试者具有更长的存活期或减少的不适。治疗可以是指当向受试者给予疗法时该疗法减少疾病症状、体征或病因的能力。治疗还指缓和或减少至少一种临床症状和/或抑制或延迟病症的进展和/或预防或延迟疾病或疾患的发作。
术语“受试者”是指接受预防、治疗、诊断的任何人或其他动物，特别是其他哺乳动物。其他哺乳动物可以包括，例如，狗、猫、牛、马、绵羊、猪、山羊、兔子、大鼠、豚鼠、小鼠等。
为了使本领域的技术人员更好地理解本发明的技术方案，下面结合具体实施例对本发明作进一步的详细说明。
实施例1：基于结构生物学设计新型冠状病毒S-RBD三聚体蛋白
通过对天然S蛋白三聚体空间结构分析(如图1所示)、并对RBD结构域的N端和C端的空间距离进行计算，发现在不引入外源载体或序列的情况下，可以利用RBD自身的结构特征实现三聚化，同时不会存在较大的空间位障，受体结合位点和主要中和抗体表位也不会被遮蔽。基于此，截取新型冠状病毒(Genebank编号：MN908947.3)S蛋白RBD区序列片段(319～537位氨基酸)，N端和C端的间距为
Figure PCTCN2022077527-appb-000006
将三个相同RBD区片段首尾串联，形成一个新的融合蛋白，氨基酸序列如SEQ ID NO.1所示，利用多聚体结构预测软件(initio domain assembly，AIDA)和分子动力学模拟(GROMACS)，显示该融合蛋白包含有三个独立RBD结构域，形成抗原构象稳定的三聚体形式(如图2所示)，即为新型冠状病毒S-RBD三聚体蛋白。
实施例2：新型冠状病毒S-RBD三聚体蛋白的表达、纯化及鉴定
按照CHO细胞表达系统的密码子偏爱性，对SEQ ID NO.1氨基酸序列进行密码子优化，优化后的基因序列如SEQ ID NO.2所示，构建CHO细胞表达载体后转染至293FT细胞或CHO细胞内构建重组细胞株，通过有限稀释法筛选得到能够稳定分泌表达S-RBD三聚体蛋白的细胞株。
经系列层析纯化后获得纯度≥95％的S-RBD三聚体蛋白。SDS-PAGE检测结果如图3所示，蛋白分子量大小约为86kD，同时可见有部分产品相关物质，如二聚体蛋白和单体蛋白。将纯化后蛋白SDS-PAGE电泳后电转至PVDF膜上，利用RBD特异性抗体(厂家：北京义翘神州科技有限公司；货号：40591-T62；稀释度：2000倍)进行Western-blot鉴定(结果如图4所示)，纯化后的蛋白可与RBD特异性抗体发生结合，具有良好的免疫原性。将纯化后蛋白滴加在铜网膜上吸附1分钟，磷钨酸负染1分钟，透射电镜下观察蛋白形态(结果如图5所示)，可见S-RBD三聚体蛋白均匀分布在铜网膜上，粒径较小。
实施例3：新型冠状病毒S-RBD三聚体蛋白与中和性单克隆抗体结合生物学分析
将纯化后的S-RBD三聚体蛋白和RBD单体蛋白(厂家：北京义翘神州科技有限公司；货号：40592-V08B)稀释至10μg/ml、1μg/ml、0.1μg/ml、0.01μg/ml、0.001μg/ml、0.0001μg/ml，100μl/孔，包被至96孔酶标板上，4℃，8～12h，以 空白孔为阴性对照；PBST溶液洗板后加入封闭液，37℃封闭3h；PBST溶液洗板后分别加入稀释后的MM43单克隆抗体(厂家：北京义翘神州科技有限公司；货号：40591-MM43，稀释度：2000倍)或MM57单克隆抗体(厂家：北京义翘神州科技有限公司；货号：40592-MM57；稀释度：2000倍)，100μl/孔，37℃孵育1h；PBST溶液洗板后加入稀释后的HEP标记的羊抗鼠IgG(厂家：北京中杉金桥生物技术有限公司；货号：ZB-2305；稀释度：10000倍)，100μl/孔，37℃孵育1h；PBST溶液洗板后先后加入显色液(厂家：万泰生物)A和B，室温下显色5～10min，加入终止液C；酶标仪上进行双波长(OD450nm和630nm)读值，确定cutoff值，并绘制蛋白浓度-吸光度值曲线，结果如图6和图7所示，可见S-RBD三聚体蛋白与MM43和MM57两种中和性单克隆抗体均具有良好的结合活性。
实施例4：新型冠状病毒S-RBD三聚体蛋白与ACE2受体结合生物学分析
随着对新型冠状病毒结构和感染机制的深入研究，越来越多研究者发现血管紧张素转换酶(Angiotensin converting enzyme,ACE2)是新型冠状病毒的受体，因此通过测定S-RBD三聚体蛋白与ACE2的结合情况，可用于分析该蛋白的生物学活性。采用表面等离子共振(Surface Plasmon Resonance,SPR)技术测定纯化后的S-RBD三聚体蛋白与ACE2蛋白(厂家：北京义翘神州科技有限公司；货号：10108-H08B)的亲和力，结果如图8所示，亲和力常熟(kD)值为3.90e -12M，S-RBD三聚体蛋白与ACE2呈现高亲和力结合，提示该蛋白具有良好的生物学活性。
实施例5：重组新型冠状病毒疫苗制备
将纯化后的S-RBD三聚体蛋白稀释至2倍目标抗原浓度，与1.2mg/ml氢氧化铝佐剂按1:1比例(w/w)混合吸附，并于磁力搅拌器上搅拌40～120min，转速为200～300rpm，获得疫苗半成品，上清残留蛋白含量应低于总蛋白含量的10％，半成品每瓶按0.5ml装量无菌分装后即为疫苗成品。
实施例6：重组新型冠状病毒疫苗免疫学效果评价
将制备的重组新型冠状病毒疫苗分别按照不同剂量经肌肉注射免疫Wistar大鼠(购自斯贝福(北京)生物技术有限公司，6-8周龄，动物合格证编号：No.110324201102113753)，0.5ml/只，具体动物试验方案如表1所示。用ELISA 方法检测免后血清中针对S-RBD蛋白的特异性IgG抗体滴度，利用假病毒和野病毒微量中和试验检测中和抗体滴度。血清特异性IgG结果如图9所示，假病毒微量中和试验结果如图10所示，野病毒微量中和如图11所示，血清抗体GMT值如表2所示。可见重组新型冠状病毒疫苗可刺激大鼠产生较高滴度的抗体水平，随着免疫针次或免疫剂量增加，特异性IgG抗体和中和抗体水平逐渐升高，具有明显的剂量关系。
血清特异性IgG抗体检测：采用ELISA方法检测免后血清中针对S-RBD蛋白的特异性IgG抗体水平，将市售RBD蛋白(购自北京义翘神州科技股份有限公司，货号为40592-V08B)稀释至1μg/ml，并以100μl/孔包被于酶标板中，封闭后，加入经系列稀释的血清，利用酶标记抗体(鼠源)检测信号，确定cut-off值，阳性血清最高稀释倍数即为该血清样品的特异性IgG抗体滴度。
假病毒微量中和试验：假病毒中S蛋白基因序列来源于Wuhan-Hu-1毒株(GenBank序列编号：MN908947)，报告基因为萤火虫荧光素酶，中和试验中假病毒工作浓度为(1～2)×10 4TCID50/ml，使用Reed-Muench法计算50％感染抑制率时血清稀释倍数，即为该血清样品的中和抗体滴度。
野病毒微量中和试验：该试验于BSL3级实验室完成，病毒株为2020XN4276株，将系列稀释后的血清与病毒(100TCID50)等体积混合孵育，37℃温箱中和2小时，加入到含有Vero-E6细胞(2×10 5/ml)的细胞培养板中，同时设置细胞对照和病毒对照，37℃温箱中培养3～5天，观察细胞病变情况，采用Reed-Muench法计算50％感染抑制率时血清稀释倍数，即为该血清样品的中和抗体滴度。
表1.重组新型冠状病毒疫苗Wistar大鼠试验方案
Figure PCTCN2022077527-appb-000007
表2.重组新型冠状病毒疫苗免疫大鼠血清GMT值
Figure PCTCN2022077527-appb-000008
Figure PCTCN2022077527-appb-000009
对比例1：S-RBD三聚体蛋白和二聚体蛋白比较
截取新型冠状病毒(Genebank编号：MN908947.3)S蛋白RBD区序列片段(319～537位氨基酸)，将两个相同RBD区片段首尾串联，形成一个二聚体融合蛋白，氨基酸序列如SEQ ID NO.3所示，按照CHO细胞表达系统的密码子偏爱性，对SEQ ID NO.3氨基酸序列进行密码子优化，优化后的基因序列如SEQ ID NO.4所示，构建CHO细胞表达载体后转染至293FT细胞后，获得能够表达二聚体蛋白的细胞株，经系列层析纯化后获得纯度≥95％的S-RBD二聚体蛋白(如图12所示)，与1.2mg/ml氢氧化铝佐剂按1:1比例(w/w)混合吸附后制备二聚体蛋白免疫物，与相同工艺制备的三聚体蛋白免疫物，经腹腔注射免疫BALB/c小鼠，抗原含量为2.0μg/剂，铝佐剂含量为0.30mg/剂，按照0w，1w和2w免疫3针次，全程免后2w采集并分离血清，利用ELISA方法检测免后血清中针对S-RBD蛋白的特异性IgG抗体滴度，利用假病毒和野病毒微量中和试验检测中和抗体滴度，检测结果如图13、图14和图15所示，GMT检测结果如表3所示，可见三聚体蛋白产生的抗体水平均高于二聚体蛋白，其中特异性IgG抗体滴度差异无统计学差异，假病毒中和抗体滴度虽然高约2倍，但统计学无差异，野病毒中和抗体滴度高约3倍，差异具有统计学意义。
表3.三聚体蛋白和二聚体蛋白免疫血清GMT值
Figure PCTCN2022077527-appb-000010
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (33)

  1. 一种新型冠状病毒S-RBD三聚体蛋白，其特征在于，所述三聚体蛋白由三聚体形式的新型冠状病毒S蛋白RBD区第319～537位氨基酸片段构成。
  2. 根据权利要求1所述的新型冠状病毒S-RBD三聚体蛋白，其特征在于，所述三聚体蛋白的一级结构为三个所述氨基酸片段按照N末端至C末端的顺序连接。
  3. 根据权利要求2所述的新型冠状病毒S-RBD三聚体蛋白，其特征在于，所述新型冠状病毒S-RBD三聚体蛋白的氨基酸序列如SEQIDNo.1所示或与其具有95％以上同源性的序列。
  4. 一种融合蛋白，其特征在于，所述融合蛋白包含如权利要求1所述的新型冠状病毒S-RBD三聚体蛋白。
  5. 根据权利要求4所述的融合蛋白，其特征在于，所述融合蛋白还包含选自信号肽、标签或免疫增强肽中的一种或几种。
  6. 一种核酸分子，其特征在于，所述核酸分子包含编码如权利要求1所述的新型冠状病毒S-RBD三聚体蛋白的核苷酸序列。
  7. 一种核酸分子，其特征在于，所述核酸分子包含编码如权利要求4所述的融合蛋白的核苷酸序列。
  8. 根据权利要求6所述的核酸分子，其特征在于，所述核苷酸序列如SEQIDNo.2所示或与其具有95％以上同源性的序列。
  9. 一种载体，其特征在于，所述载体包含如权利要求6所述的核酸分子。
  10. 一种载体，其特征在于，所述载体包含如权利要求7所述的核酸分子。
  11. 一种宿主细胞，其特征在于，所述宿主细胞包含如权利要求6所述的核酸分子。
  12. 一种宿主细胞，其特征在于，所述宿主细胞包含如权利要求9所述的载体。
  13. 一种宿主细胞，其特征在于，所述宿主细胞包含如权利要求10所述的载体。
  14. 根据权利要求11所述的宿主细胞，其特征在于，所述宿主细胞为大肠杆菌、酵母细胞、昆虫细胞或哺乳动物细胞。
  15. 根据权利要求13所述的宿主细胞，其特征在于，所述宿主细胞为CHO 细胞。
  16. 如权利要求1所述的新型冠状病毒S-RBD三聚体蛋白的制备方法，其特征在于，包括以下步骤：
    步骤A)制备如权利要求6所述的核酸分子，构建如权利要求9所述的表达载体，将表达载体转化或转染至如权利要求12所述的宿主细胞内；
    步骤B)利用步骤A)的产物进行蛋白质表达；
    步骤C)纯化步骤B)中获得的表达产物，得到所述新型冠状病毒S-RBD三聚体蛋白。
  17. 如权利要求4所述的融合蛋白的制备方法，其特征在于，包括以下步骤：
    步骤A)制备如权利要求7所述的核酸分子，构建如权利要求10所述的表达载体，将表达载体转化或转染至如权利要求13所述的宿主细胞内；
    步骤B)利用步骤A)的产物进行蛋白质表达；
    步骤C)纯化步骤B)中获得的表达产物，得到所述融合蛋白。
  18. 如权利要求1所述的新型冠状病毒S-RBD三聚体蛋白在制备用于治疗和/或预防新型冠状病毒感染和/或新型冠状病毒引起的疾病的药物中的用途。
  19. 如权利要求4所述的融合蛋白在制备用于治疗和/或预防新型冠状病毒感染和/或新型冠状病毒引起的疾病的药物中的用途。
  20. 如权利要求6所述的核酸分子在制备用于治疗和/或预防新型冠状病毒感染和/或新型冠状病毒引起的疾病的药物中的用途。
  21. 如权利要求9所述的载体在制备用于治疗和/或预防新型冠状病毒感染和/或新型冠状病毒引起的疾病的药物中的用途。
  22. 如权利要求11所述的宿主细胞在制备用于治疗和/或预防新型冠状病毒感染和/或新型冠状病毒引起的疾病的药物中的用途。
  23. 一种疫苗，其特征在于，所述疫苗包含如权利要求1所述的新型冠状病毒S-RBD三聚体蛋白，以及佐剂。
  24. 一种疫苗，其特征在于，所述疫苗包含如权利要求4所述的融合蛋白，以及佐剂。
  25. 根据权利要求23所述的疫苗，其特征在于，所述佐剂为氢氧化铝、磷酸铝、MF59或CpG。
  26. 根据权利要求25所述的疫苗，其特征在于，所述佐剂为氢氧化铝。
  27. 如权利要求23所述的疫苗的制备方法，其特征在于，将纯化所得的所述新型冠状病毒S-RBD三聚体蛋白与所述佐剂混合。
  28. 如权利要求24所述的疫苗的制备方法，其特征在于，将纯化所得的所述所述融合蛋白与所述佐剂混合。
  29. 如权利要求23所述的疫苗在治疗和/或预防新型冠状病毒感染和/或新型冠状病毒引起的疾病中的用途。
  30. 如权利要求24所述的疫苗在治疗和/或预防新型冠状病毒感染和/或新型冠状病毒引起的疾病中的用途。
  31. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求23所述的疫苗，以及药学上可接受的载体。
  32. 一种引发受试者针对新型冠状病毒的免疫应答或治疗受试者的新型冠状病毒感染的方法，其特征在于，向所述受试者施用有效剂量的如权利要求23所述的疫苗。
  33. 一种引发受试者针对新型冠状病毒的免疫应答或治疗受试者的新型冠状病毒感染的方法，其特征在于，向所述受试者施用有效剂量的如权利要求32所述的药物组合物。
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