WO2021254054A1 - 一种预防冠状病毒引起疾病的疫苗 - Google Patents

一种预防冠状病毒引起疾病的疫苗 Download PDF

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WO2021254054A1
WO2021254054A1 PCT/CN2021/093756 CN2021093756W WO2021254054A1 WO 2021254054 A1 WO2021254054 A1 WO 2021254054A1 CN 2021093756 W CN2021093756 W CN 2021093756W WO 2021254054 A1 WO2021254054 A1 WO 2021254054A1
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amino acid
exogenous
protein
acid sequence
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刘波
吴军
王甜甜
孙鹏
巩新
侯旭宸
徐俊杰
殷瑛
张军
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中国人民解放军军事科学院军事医学研究院
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    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
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    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to the field of biomedicine, in particular to a vaccine for preventing diseases caused by coronavirus.
  • Coronavirus is a type of virus that exists widely in nature. It mainly infects vertebrates and infects humans to cause infectious diseases. In addition to the above-mentioned 2019-nCoV, there are also SARS-CoV that causes severe acute respiratory syndrome and the Middle East respiratory syndrome. Syndrome of MERS-CoV.
  • S protein is a trimeric glycoprotein on the surface of the virus. It guides the virus into the host cell by binding to receptors on the cell surface.
  • SARS-CoV and MERS-CoV use human angiotensinase 2 (ACE2) and human dipeptidyl peptides, respectively.
  • Enzyme DPP4, also known as CD26 serves as the main receptor.
  • S protein is a type I membrane protein, which can be hydrolyzed into S1 and S2 subunits by host protease under specific conditions. S1 binds to cell surface receptors, and S2 mediates membrane fusion through a change in its natural conformation, allowing the viral genome to enter the host cell , Complete the infection process.
  • S protein has high immunogenicity and can induce the body to produce neutralizing antibodies against SARS-CoV
  • the full-length S protein as an antigen can induce eosinophil immunopathology or antibody-mediated
  • the safety of the immune enhancement ADE and other adverse reactions has been widely questioned.
  • the RBD region of SARS-CoV S protein can form the correct conformation and contain multiple spatial structure-dependent epitopes. In animal models, it can induce high-titer neutralizing antibodies, CD8 + T cell responses, and long-term Immune protection effect.
  • the yeast strain modified by the glycosylation modification pathway not only retains the characteristics of high safety, short construction period of the engineered strain, fast growth, easy to large-scale production, etc., but also has a natural glycosyl structure that is close to the glycosylation modification and even better than that of the antigen The ability to make it very suitable as an expression system for high-efficiency and large-scale vaccine production under sudden infectious diseases and other emergency conditions.
  • the purpose of the present invention is to provide a vaccine for preventing diseases caused by coronavirus.
  • the vaccine for preventing disease caused by coronavirus as claimed in the present invention contains glycosylated coronavirus S protein receptor binding domain, aluminum hydroxide adjuvant and CpG adjuvant.
  • the coronavirus may be SARS-CoV-2.
  • the disease caused by the coronavirus may be COVID-19.
  • amino acid sequence of the glycosylated coronavirus S protein receptor binding region can be any of the following:
  • (a4) The sequence defined in any one of (a1)-(a3) has been substituted and/or deleted and/or added by one or several amino acid residues and has the same function, or a sequence with the same function as (a1)-( The amino acid sequence defined in any one of a3) has a sequence of more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% homology and has the same function.
  • SEQ ID No. 21 to SEQ ID No. 23 are all part of the S protein from the SARS-CoV-2 "Wuhan-Hu-1" isolate with GenBank number MN908947.3.
  • SEQ ID No. 21 is the R319-F541 region (RBD223) of the S protein
  • SEQ ID No. 22 is the R319-V534 region (RBD216) of the S protein
  • SEQ ID No. 23 is the R319-K528 region of the S protein (RBD210).
  • the glycosylated coronavirus S protein receptor binding region is a coronavirus S protein receptor binding region with a mammalian glycoform structure N-sugar chain modification.
  • N-sugar chain of the mammalian glycoform structure is Gal 2 GlcNAc 2 Man 3 GlcNAc 2 , GalGlcNAcMan 5 GlcNAc 2 or Man 5 GlcNAc 2 .
  • the CpG adjuvant can be any of the following:
  • the thio product is a full chain thio modified product (that is, each base is thio modified).
  • CpG adjuvant is full-chain thio-modified CpG2006, followed by full-chain thio-modified CpG684, full-chain thio-modified CpGX1 or full-chain thio-modified CpG1018.
  • the mass ratio of the glycosylated coronavirus S protein receptor binding region, the aluminum hydroxide adjuvant (in terms of aluminum content), and the CpG adjuvant may be (2.5-20) : 100: (25-50).
  • the glycosylated coronavirus S protein receptor binding domain can be expressed and prepared by mammalian cells, insect cells, yeast, or the like.
  • the glycosylated coronavirus S protein receptor binding domain (RBD) can be prepared according to the following steps:
  • the Pichia pastoris genetically modified through the glycosylation modification pathway is a Pichia pastoris cell mutant that is defective in the mannosylation modification pathway and reconstructs the N-glycosylation modification pathway of mammalian cells ;
  • Pichia pastoris genetically modified through the glycosylation modification pathway can be prepared according to a method including the following steps:
  • (A2) Express at least one of the following exogenous proteins in the recombinant yeast 1: exogenous mannosidase I, exogenous N-acetylglucosamine transferase I, exogenous mannosidase II, exogenous N- Acetyl glucosamine transferase II, exogenous galactose isomerase, and exogenous galactose transferase to obtain recombinant yeast 2; the recombinant yeast 2 is the genetically modified Pichia pastoris through the glycosylation modification pathway yeast.
  • step (A2) After step (A2), the following steps (A3) can also be included:
  • (A3) Inactivate the endogenous Omannosyltransferase I of the recombinant yeast 2 to obtain the recombinant yeast 3; the recombinant yeast 3 is also the Pichia pastoris genetically modified through the glycosylation modification pathway.
  • the mammalian glycoform N-sugar chain is Gal 2 GlcNAc 2 Man 3 GlcNAc 2 , GalGlcNAcMan 5 GlcNAc 2 or Man 5 GlcNAc 2
  • the foreign protein expressed in the recombinant yeast 1 in step (A2) Exogenous mannosidase I, exogenous N-acetylglucosamine transferase I, exogenous galactose isomerase and exogenous galactose transferase, exogenous mannosidase II, and exogenous N-acetylglucosamine transfer Enzyme II.
  • the foreign protein expressed in the recombinant yeast 1 in step (A2) is exogenous mannosidase I and exogenous N-acetylglucose Amin transferase I, as well as exogenous galactose isomerase and exogenous galactose transferase.
  • the exogenous protein expressed in the recombinant yeast 1 in step (A2) is exogenous mannosidase I.
  • the inactivation of the aforementioned glycosyl-modifying enzyme can be achieved by mutating one or more nucleotide sequences of the gene, or by deleting part or the entire gene sequence, or by inserting nucleotides Destroy the original reading frame and terminate protein synthesis in advance to inactivate the gene or the activity of the protein encoded by the gene.
  • the above-mentioned mutations, deletions, and insertion inactivation can be obtained by conventional methods such as mutagenesis and knockout. These methods have been reported in many literatures, such as J. Sambrook et al., "Molecular Cloning Experiment Guide” Second Edition, Science Press, 1995.
  • the better strain was obtained by knocking out part of the sequence of the mannose transferase gene.
  • the partial sequence is at least more than three bases, preferably more than 100 bases, and more preferably includes more than 50% of the coding sequence.
  • the strain obtained by knocking out part of the sequence of the glycosyl-modifying enzyme gene is not easy to produce back mutations, and the stability of the strain is higher than that constructed by methods such as point mutation, and is more conducive to application in the medical and industrial fields.
  • the method of knocking out part of the sequence of the glycosylation modifying enzyme gene may include: first constructing a plasmid for knocking out the gene: the plasmid contains the sequence of homology arms on both sides of the gene to be knocked out, and the two homology arms should be selected in the target gene On both sides, the length of the homology arms is at least greater than 200 bp, and the optimal size is 500 bp-2000 bp. It is also possible to use the method of insertion inactivation to obtain an amino acid sequence after one or several amino acid residue substitutions and/or deletions and/or additions, resulting in a nucleotide sequence with no functional activity, and construct it into a plasmid.
  • the plasmid also carries URA3 (orotidine-5'-phosphate decarboxylase) gene, or bleomycin, or hygromycin B, or blasticidin or G418 as selection markers.
  • URA3 orotidine-5'-phosphate decarboxylase
  • bleomycin or hygromycin B
  • blasticidin or G418 as selection markers.
  • the polynucleotide sequence encoding the homologous arm fragment of the flanking region and the nucleotide sequence of the protein whose function is to be disrupted can be obtained from the publicly available National Center for Biotechnology Information (NCBI).
  • NCBI National Center for Biotechnology Information
  • flanking homology regions required for inactivating genes are obtained, respectively including the upstream and downstream flanking homology of the gene coding region of the target gene (the sequence of which has been published in NCBI) Region, and add a suitable restriction site in the primer part.
  • polynucleotides can be obtained by methods well known in the art, such as PCR (J. Sambrook et al., "Molecular Cloning Experiment Guide” Second Edition, Science Press, 1995.), RT-PCR method, artificial The method of synthesis, genomic DNA and the method of constructing and screening cDNA library are obtained.
  • polynucleotides can be mutated, deleted, inserted, linked to other polynucleotides, etc., using methods known in the art.
  • the respectively obtained upstream (5') and downstream (3') flanking regions homologous arm fragments are fused, and various methods known in the art can be used, such as by overlapping PCR, while keeping the size of the respective fragments unchanged.
  • the standard molecular cloning process used is described in J. Sambrook et al. (J. Sambrook et al., "Molecular Cloning Experiment Guide” Second Edition, Science Press, 1995.).
  • the nucleic acid containing the fusion fragment of the homologous arm sequence of the gene to be inactivated can be cloned into various vectors suitable for yeast by methods well known in the art. Alternatively, the restriction sites on the respective homology arms can be inserted into specific regions of the vector.
  • the standard molecular cloning process used is described by J. Sambrook et al. (J. Sambrook et al., "Molecular Cloning Experiment Guide” Second Edition, Science Press, 1995.). Construct a recombinant knockout plasmid.
  • the original plasmid can be an expression vector suitable for yeast, a shuttle vector, with replication sites, selection markers, auxotrophic markers (URA3, HIS, ADE1, LEU2, ARG4), etc.
  • the construction methods of these vectors have been published in many documents Public (e.g. J. Sambrook et al., "Molecular Cloning Experiment Guide” Second Edition, Science Press, 1995), can also be purchased from various companies (e.g. Invitrogen Life Technologies, Carlsbad, California 92008, USA), priority
  • the vectors include pPICZ ⁇ A and pYES2 yeast expression vectors.
  • Inactivated vectors are shuttle plasmids, which are first replicated and amplified in E. coli, and then introduced into host yeast cells.
  • the vectors should carry resistance marker genes or auxotrophic marker genes to facilitate the selection of later transformants.
  • the homologous regions on both sides of the gene to be inactivated are respectively constructed into the yeast vector to form a recombinant knockout vector. Furthermore, the linearization site of the homology arm is used to linearize the knockout vector, and the vector is transformed into Pichia pastoris or one of its modified variants by an electrotransformation method for culture.
  • the required nucleic acid for transformation into host cells can be obtained by usual methods, such as preparing competent cells, electroporation, lithium acetate method, etc. (A. Adams et al., "Yeast Genetics Method Experimental Guide", Science Press, 2000).
  • Successfully transformed cells that is, cells containing the homologous region of the gene to be knocked out
  • the transformants that are recombined correctly once are cultured in yeast minimal medium, and then spread on a 5-fluoroorotic acid plate containing uracil and other secondary recombination screening plates.
  • the clones that grow out are then further subjected to the PCR identification of the genotype. .
  • the correct transformants lacking the coding region of the expected gene were screened separately.
  • step (A1) the endogenous ⁇ -1,6-mannose transferase, phosphomannose transferase, and mannose phosphate of the receptor Pichia pastoris Synthetase, ⁇ -mannose transferase I, ⁇ -mannose transferase II, ⁇ -mannose transferase III and ⁇ -mannose transferase IV are all knocked out by homologous recombination.
  • step (A2) the expression of the foreign protein in the recombinant yeast 1 is achieved by introducing the gene encoding the foreign protein into the recombinant yeast 1.
  • the gene encoding the foreign protein is introduced into the recombinant yeast 1 in the form of a recombinant vector.
  • the encoding gene of the exogenous mannosidase I and the encoding gene of the exogenous mannosidase II are both introduced into the recombinant yeast 1 twice.
  • step (A3) the endogenous Omannose transferase I of the recombinant yeast 2 is inactivated by processing the Omannose transferase in the genomic DNA of the recombinant yeast 2.
  • the I coding gene is inserted and inactivated.
  • the front end and the end of the target fragment of the Omannosyltransferase I encoding gene in the genomic DNA of the recombinant yeast 2 are each equipped with a different combination of stop codons, and the stop codon at the end Then install a terminator (such as the CYC1TT terminator).
  • the target fragments with different combinations of stop codons installed on the front and end are specifically obtained by PCR amplification using the genomic DNA of Pichia pastoris JC308 as a template and using primers PMT1-IN-5 and PMT1-IN-3 Fragment.
  • step (A2) the exogenous mannosidase I is localized in the endoplasmic reticulum after expression.
  • the exogenous mannosidase I is derived from Trichoderma viride, and the C-terminus is fused with the endoplasmic reticulum retention signal HDEL.
  • step (A2) the exogenous N-acetylglucosamine transferase I is localized in the endoplasmic reticulum or medial Golgi after expression.
  • the exogenous N-acetylglucosamine transferase I is derived from a mammal, and is fused with an endoplasmic reticulum or medial Golgi localization signal at the N-terminal or C-terminal.
  • exogenous N-acetylglucosamine transferase I is derived from humans and contains mnn9 localization signal.
  • step (A2) the exogenous mannosidase II is expressed and localized in the endoplasmic reticulum or medial Golgi apparatus.
  • the exogenous mannosidase II is derived from filamentous fungi, plants, insects, Java, or mammals, and is fused with endoplasmic reticulum or medial Golgi localization signals at the N-terminal or C-terminal.
  • step (A2) the exogenous N-acetylglucosamine transferase II is localized in the endoplasmic reticulum or medial Golgi after expression.
  • the exogenous N-acetylglucosamine transferase II is derived from mammals, and the endoplasmic reticulum or medial Golgi localization signal is fused at the N-terminus or C-terminus.
  • N-acetylglucosamine transferase II is derived from humans, and all contain mnn2 localization signal.
  • step (A2) the exogenous mannosidase II is expressed and localized in the endoplasmic reticulum or medial Golgi apparatus.
  • the exogenous mannosidase II is derived from nematodes and contains mnn2 localization signal.
  • step (A2) the exogenous galactose isomerase and the exogenous galactose transferase are localized in the endoplasmic reticulum or the medial Golgi after being expressed.
  • exogenous galactose isomerase and the exogenous galactose transferase are both derived from mammals, and are fused with endoplasmic reticulum or medial Golgi localization signals at the N-terminal or C-terminal.
  • exogenous galactose isomerase and the exogenous galactose transferase are fusion proteins, both of which are derived from humans, and share a kre2 localization signal.
  • the ⁇ -1,6-mannose transferase can be the following B1) or B2):
  • amino acid sequence shown in SEQ ID No. 1 has been substituted and/or deleted and/or added by one or several amino acid residues and has the same function, or the amino acid sequence shown in SEQ ID No. 1 Proteins that have homology of 99% or more, 95% or more, 90% or more, 85% or more or 80% or more, and have the same function.
  • the phosphomannose transferase can be the following B3) or B4):
  • amino acid sequence shown in SEQ ID No. 2 has been substituted and/or deleted and/or added with one or several amino acid residues and has the same function, or the amino acid sequence shown in SEQ ID No. 2 Proteins that have homology of 99% or more, 95% or more, 90% or more, 85% or more or 80% or more, and have the same function.
  • the mannose phosphate synthase can be the following B5) or B6):
  • amino acid sequence shown in SEQ ID No. 3 has been substituted and/or deleted and/or added with one or several amino acid residues and has the same function, or the amino acid sequence shown in SEQ ID No. 3 Proteins that have homology of 99% or more, 95% or more, 90% or more, 85% or more or 80% or more, and have the same function.
  • the ⁇ -mannose transferase I can be the following B7) or B8):
  • amino acid sequence shown in SEQ ID No. 4 has been substituted and/or deleted and/or added with one or several amino acid residues and has the same function, or the amino acid sequence shown in SEQ ID No. 4 Proteins that have homology of 99% or more, 95% or more, 90% or more, 85% or more or 80% or more, and have the same function.
  • the ⁇ -mannose transferase II can be the following B9) or B10):
  • amino acid sequence shown in SEQ ID No. 5 has been substituted and/or deleted and/or added with one or several amino acid residues and has the same function, or the amino acid sequence shown in SEQ ID No. 5 Proteins that have homology of 99% or more, 95% or more, 90% or more, 85% or more or 80% or more, and have the same function.
  • the ⁇ -mannose transferase III may be B11) or B12) as follows:
  • amino acid sequence shown in SEQ ID No. 6 has been substituted and/or deleted and/or added with one or several amino acid residues and has the same function, or the amino acid sequence shown in SEQ ID No. 6 Proteins that have homology of 99% or more, 95% or more, 90% or more, 85% or more or 80% or more, and have the same function.
  • the ⁇ -mannose transferase IV may be B13) or B14) as follows:
  • amino acid sequence shown in SEQ ID No. 7 has been substituted and/or deleted and/or added with one or several amino acid residues and has the same function, or the amino acid sequence shown in SEQ ID No. 7 Proteins that have homology of 99% or more, 95% or more, 90% or more, 85% or more or 80% or more, and have the same function.
  • the Omannose transferase I can be the following B15) or B16):
  • amino acid sequence shown in SEQ ID No. 8 has been substituted and/or deleted and/or added with one or several amino acid residues and has the same function, or the amino acid sequence shown in SEQ ID No. 8 Proteins that have homology of 99% or more, 95% or more, 90% or more, 85% or more or 80% or more, and have the same function.
  • the exogenous mannosidase I can be the following B17) or B18):
  • the exogenous N-acetylglucosamine transferase I can be the following B19) or B20):
  • amino acid sequence shown in SEQ ID No. 10 has been substituted and/or deleted and/or added by one or several amino acid residues and has the same function, or the amino acid sequence shown in SEQ ID No. 10 Proteins that have homology of 99% or more, 95% or more, 90% or more, 85% or more or 80% or more, and have the same function.
  • the fusion protein composed of the galactose isomerase and the galactose transferase may be the following B21) or B22):
  • amino acid sequence shown in SEQ ID No. 11 has been substituted and/or deleted and/or added with one or several amino acid residues and has the same function, or the amino acid sequence shown in SEQ ID No. 11 Proteins that have homology of 99% or more, 95% or more, 90% or more, 85% or more or 80% or more, and have the same function.
  • the mannosidase II can be the following B23) or B24):
  • amino acid sequence shown in SEQ ID No. 12 has been substituted and/or deleted and/or added with one or several amino acid residues and has the same function, or the amino acid sequence shown in SEQ ID No. 12 Proteins that have homology of 99% or more, 95% or more, 90% or more, 85% or more or 80% or more, and have the same function.
  • the N-acetylglucosamine transferase II can be the following B25) or B26):
  • amino acid sequence shown in SEQ ID No. 13 has been substituted and/or deleted and/or added with one or several amino acid residues and has the same function, or the amino acid sequence shown in SEQ ID No. 13 Proteins that have homology of 99% or more, 95% or more, 90% or more, 85% or more or 80% or more, and have the same function.
  • exogenous mannosidase I can be the following C1) or C2):
  • the nucleotide sequence is the DNA molecule of SEQ ID No. 14;
  • C2 DNA that has more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% homology with the nucleotide sequence shown in SEQ ID No. 14 and encodes the exogenous mannosidase I A molecule, or a DNA molecule that hybridizes to a DNA molecule defined by C1) under stringent conditions and encodes the exogenous mannosidase I.
  • the encoding gene of the exogenous N-acetylglucosamine transferase I can be the following C3) or C4):
  • the nucleotide sequence is the DNA molecule of SEQ ID No. 15;
  • C4 It has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the nucleotide sequence shown in SEQ ID No. 15 and encodes the exogenous N-acetylglucosamine transfer
  • the coding gene of the fusion protein composed of the galactose isomerase and the galactose transferase may be the following C5) or C6):
  • the nucleotide sequence is the DNA molecule of SEQ ID No. 16;
  • DNA molecules that have more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% homology with the nucleotide sequence shown in SEQ ID No. 16 and encode the fusion protein, or Under stringent conditions, the DNA molecule hybridizes with the DNA molecule defined by C5) and encodes the fusion protein.
  • the mannosidase II coding gene can be the following C7) or C8):
  • the nucleotide sequence is the DNA molecule of SEQ ID No. 17;
  • C8 A DNA molecule that has more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% homology with the nucleotide sequence shown in SEQ ID No. 17, and encodes the mannosidase II, Or under stringent conditions, it hybridizes with the DNA molecule defined by C7) and encodes the mannosidase II DNA molecule.
  • the N-acetylglucosamine transferase II coding gene can be the following C9) or C10):
  • the nucleotide sequence is the DNA molecule of SEQ ID No. 18;
  • C10 It has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the nucleotide sequence shown in SEQ ID No. 18 and encodes the N-acetylglucosamine transferase II DNA molecules, or DNA molecules that hybridize with the DNA molecules defined by C9) under stringent conditions and encode the N-acetylglucosamine transferase II.
  • glycosyl-modifying enzymes of the present invention can be obtained from the National Center for Biotechnology Information (NCBI) or published documents, and the functions and definitions of related enzymes can also be obtained from the documents. Even if it is the same bacteria or species, due to different sources, the amino acids of various enzymes will be slightly different, but their functions are basically the same. Therefore, the enzymes of the present invention may also include these variants.
  • NCBI National Center for Biotechnology Information
  • Pichia pastoris genetically modified through the glycosylation modification pathway is a strain with the deposit number CGMCC No. 19488 deposited in the General Microbiology Center of the China Microbial Species Collection and Management Committee.
  • the SARS-CoV-2S-RBD glycoprotein prepared by the yeast has complex and heterozygous N-glycosyl modifications without fucose side chains, and compared to mammalian cells and insect cells, the yeast is used to express SARS- CoV-2S-RBD glycoprotein has the advantages of low preparation cost, short cycle, and easy mass production.
  • step (1) the recombinant yeast cell is obtained by introducing the coding gene of the coronavirus S protein receptor binding region (RBD) into the genetically modified Pichia pastoris through a glycosylation modification pathway owned.
  • RBD coronavirus S protein receptor binding region
  • the coding gene of the coronavirus S protein receptor binding region is introduced into the Pichia pastoris genetically modified through the glycosylation modification pathway in the form of a recombinant vector.
  • the promoter in the recombinant vector that initiates transcription of the gene encoding the coronavirus S protein receptor binding region may be an AOX1 promoter.
  • the recombinant vector is specifically after the coding gene of the coronavirus S protein receptor binding region (RBD) is cloned into the downstream of the AOX1 promoter of the pPICZ ⁇ A vector (such as between the restriction sites XhoI and NotI) The resulting recombinant vector.
  • RBD coronavirus S protein receptor binding region
  • the coding gene of the coronavirus S protein receptor binding region can be any of the following:
  • (c4) It has 99% or more, 95% or more, 90% or more, 85% or more than 80% homology with the nucleotide sequence shown in SEQ ID No. 24 to SEQ ID No. 26 and encodes The DNA molecule of the coronavirus S protein receptor binding region, or hybridize with any of the DNA molecules shown in SEQ ID No. 24 to SEQ ID No. 26 under stringent conditions and encode the coronavirus S protein receptor DNA molecules in the binding zone.
  • the nucleotide sequences of SEQ ID No. 24 to SEQ ID No. 26 are obtained by codon optimization according to the amino acid sequences of SEQ ID No. 21 to SEQ ID No. 23, respectively, and DNA fragments of corresponding sequences are obtained by whole gene synthesis.
  • homology refers to the identity of amino acid sequence.
  • the homology search site on the Internet can be used to determine the identity of the amino acid sequence, such as the BLAST page of the NCBI homepage. For example, in advanced BLAST 2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set Gap existence cost, Per resistance gap cost, and Lambda ratio to 11, 1 and 0.85 (default value) and perform a search for the identity of a pair of amino acid sequences to calculate, and then the identity value (%) can be obtained.
  • homology refers to the identity of the nucleotide sequence.
  • the homology search site on the Internet can be used to determine the identity of the nucleotide sequence, such as the BLAST page of the NCBI homepage. For example, in advanced BLAST 2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set Gap existence cost, Per resistance gap cost, and Lambda ratio to 11, 1 and 0.85 (default value) and search for the identity of a pair of nucleotide sequences to calculate, and then the identity value (%) can be obtained.
  • the homology of more than 95% can be at least 96%, 97%, or 98% identity.
  • the homology of more than 90% can be at least 91%, 92%, 93%, 94% identity.
  • the homology of more than 85% can be at least 86%, 87%, 88%, 89% identity.
  • the above 80% homology can be at least 81%, 82%, 83%, 84% identity.
  • the stringent conditions can be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO 4 and 1 mM EDTA, at 50°C, 2 ⁇ SSC, 0.1 Rinse in %SDS; it can also be: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO 4 and 1mM EDTA, rinse in 50°C, 1 ⁇ SSC, 0.1% SDS; it can also be: 50°C , Hybridize in a mixed solution of 7% SDS, 0.5M NaPO 4 and 1 mM EDTA, and rinse at 50°C, 0.5 ⁇ SSC, 0.1% SDS; also: 50°C, in 7% SDS, 0.5M NaPO 4 and Hybridize in a mixed solution of 1mM EDTA, rinse at 50°C, 0.1 ⁇ SSC, 0.1% SDS; also: 50°C, hybridize in a mixed solution of 7% sodium
  • step (2) separating the components containing SARS-CoV-2 S-RBD glycoprotein may include using centrifugation, filtration and other solid-liquid separation methods to separate the culture fluid and cells (or bacteria), containing SARS-CoV-2S-
  • the component separation of RBD glycoprotein can include affinity, ion exchange, gel filtration, hydrophobic and other liquid chromatography methods.
  • the N-sugar chain modified coronavirus S protein receptor binding region of the mammalian glycoform structure can be purified from the culture supernatant according to a method including the following steps: The culture supernatant is sequentially subjected to cation exchange chromatography, hydrophobic chromatography, G25 desalting, and anion exchange chromatography to obtain the N-sugar chain modified coronavirus S protein receptor binding region with a mammalian glycoform structure.
  • step (2) is to purify from the culture supernatant to obtain the mammalian glycoform structure N-sugar chain modified coronavirus S protein receptor binding method according to the method including the following steps Regional: Pass the culture supernatant through a CaptoMMC chromatography column to capture the target protein, and then elution with a buffer containing 1M NaCl to obtain a crude sample containing the target protein; then the crude sample is subjected to hydrophobic chromatography Column Phenyl HP purification, the elution peak sample containing the target protein is desalted with a G25 chromatography column, and then anion exchange chromatography column Source30Q is used to adsorb the impurity protein, and the flow-through fluid is the target protein; the target protein That is the receptor binding region of the coronavirus S protein with mammalian glycoform structure N-sugar chain modification.
  • the present invention also claims a method for preparing the aforementioned vaccine.
  • the method for preparing the aforementioned vaccine as claimed in the present invention may include the following steps: preparing the glycosylated coronavirus S protein receptor binding region according to the aforementioned method; and then combining the glycosylated coronavirus
  • the virus S protein receptor binding region, aluminum hydroxide adjuvant (calculated by aluminum content) and the aforementioned CpG adjuvant are prepared by mixing in a mass ratio of (2.5-20): 100: (25-50). vaccine.
  • the coronavirus may be SARS-CoV-2.
  • the disease caused by the coronavirus may be COVID-19.
  • the present invention also claims a method to prevent coronavirus from causing disease, which is to use the vaccine described above to prevent coronavirus from causing disease.
  • the coronavirus may be SARS-CoV-2.
  • the disease caused by the coronavirus may be COVID-19.
  • the collection center registration number CGMCC No. 19488
  • Figure 1 shows the identification of och1 gene in GJK01 strain and the result of glycoform analysis.
  • A is the result of och1 gene identification.
  • M stands for Marker; 1: GJK01 bacteria (och1 has been knocked out); 2: X33 bacteria (och1 has not been knocked out).
  • B is the DSA-FACE glycotype analysis result of the antibody expressed by GJK01 bacteria (knockout och1).
  • Figure 2 shows the results of pno1 gene identification.
  • M stands for Marker; 1: GJK02 bacteria (pno1 has been knocked out); 2: X33 bacteria (pno1 has not been knocked out).
  • Figure 3 shows the results of mnn4b gene identification.
  • M stands for Marker; 1: GJK03 bacteria (mnn4b has been knocked out); 2: X33 bacteria (mnn4b has not been knocked out).
  • Figure 4 shows the results of DSA-FACE glycoform analysis of GJK01, GJK02, and GJK03 strains (och1, pno1, mnn4b have been knocked out).
  • Figure 5 shows the results of ARM2 gene identification.
  • M stands for Marker; 1: GJK04 bacteria (ARM2 has been knocked out); 2: X33 bacteria (ARM2 has not been knocked out).
  • Figure 6 shows the results of ARM1 gene identification.
  • M stands for Marker; 1: GJK05 strain (ARM1 has been knocked out); 2: X33 strain (ARM1 has not been knocked out).
  • Figure 7 shows the results of ARM3 gene identification.
  • M stands for Marker; 1: GJK07 bacteria (ARM3 has been knocked out); 2: X33 bacteria (ARM3 has not been knocked out).
  • Figure 8 shows the results of ARM4 gene identification.
  • M stands for Marker; 1: GJK18 bacteria (ARM4 has been knocked out); 2: X33 bacteria (ARM4 has not been knocked out).
  • Figure 9 shows the results of DSA-FACE glycoform analysis of GJK18 bacteria.
  • Figure 10 shows the TrmdsI gene identification results and DSA-FACE glycotype analysis results of W10 bacteria.
  • A is the result of TrmdsI gene identification.
  • M stands for Marker; 1: TrmdsI is introduced into W10 strain; TrmdsI is not present in X33 strain.
  • B is the result of DSA-FACE glycoform analysis of W10 bacteria.
  • Figure 11 shows the GnTI gene identification results and DSA-FACE glycotype analysis results of bacteria 1-8.
  • A is the result of GnTI gene identification.
  • M stands for Marker; 1: 1-8 bacteria introduced GnTI; 2: X33 bacteria have no GnTI.
  • B is the result of DSA-FACE glycoform analysis of 1-8 bacteria.
  • FIG. 12 shows the GalE-GalT gene identification results and DSA-FACE glycotype analysis results of 1-8-4 bacteria.
  • GalE-GalT gene identification results M stands for Marker; 1: GalE-GalT is introduced into bacteria 1-8-4; 2: GalE-GalT is not introduced into bacteria X33.
  • B is the DSA-FACE glycoform analysis result of 1-8-4 bacteria.
  • Figure 13 shows the mdsII gene and GnTII gene identification results of 52-60 and 150L2 bacteria, and the results of DSA-FACE glycoform analysis.
  • A is the result of MdsII gene identification. M stands for Marker; 1: MdsII is introduced in 52-60 bacteria; 2: MdsII is not in X33 bacteria.
  • B is the result of GnTII gene identification. M stands for Marker; 1: GnTII is introduced in 150L2 bacteria; 2: GnTII is not in X33 bacteria.
  • C is the result of DSA-FACE glycoform analysis of 52-60 bacteria.
  • Figure 14 shows the identification results of PMT1 insertion and inactivation genes.
  • M stands for Marker; 1: X33 bacteria PMT1 is not inactivated; 2: GJK30 (PMT1 is inactivated).
  • Figure 15 shows the glycoform structure analysis results of GJK30 engineering bacteria.
  • A is that the structure of Gal2GlcNAc2Man3GlcNAc2 in the early stage is less than 50%;
  • B is that the structure of Gal2GlcNAc2Man3GlcNAc2 obtained by GJK30 engineering bacteria accounts for more than 60% of the glycoform;
  • C is the glycosidase analysis of the glycoform by glycosidase (New England Biolabs, Beijing).
  • Figure 16 is a WB verification image of CGMCC19488/S-RBD223 positive clone screening.
  • the upper part is SDS-PAGE electrophoresis analysis, and the lower part is Western Blotting analysis; lanes 1-7 are different expression clones.
  • Figure 17 shows the electrophoresis detection diagram of CGMCC19488/S-RBD223 at different induction times.
  • Figure 18 is an SDS-PAGE chart of SARS-CoV-2 S-RBD223 purified sample.
  • Figure 19 is an SDS-PAGE diagram of SARS-CoV-2 S-RBD216 and SARS-CoV-2 S-RBD210 purified samples.
  • Figure 20 shows the DSA-FACE sugar chain analysis results of SARS-CoV-2 S-RBD223, SARS-CoV-2 S-RBD216 and SARS-CoV-2 S-RBD210 glycoproteins expressed by CGMCC19488.
  • Figure 21 shows the serum anti-RBD antibody titers of various mice 14 days after the second immunization.
  • A is the first batch; B is the second batch; C is the third batch.
  • ns means no significant difference, ** means there is a very significant difference at the P ⁇ 0.01 level.
  • Figure 22 shows the results of the virus neutralization test.
  • A is the first batch; B is the second batch; C is the third batch.
  • ns means no significant difference, * means extremely significant difference at P ⁇ 0.05 level, *** means extremely significant difference at P ⁇ 0.001 level.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.
  • the quantitative experiments in the following examples are all set to repeat the experiment three times, and the results are averaged.
  • pPICZ ⁇ A pYES2 vector
  • X33 GS115 Pichia pastoris are products of Invitrogen.
  • Pichia pastoris GJK01CGMCC No. 1853 in the invention patent ZL200610164912.8, the publication number is CN101195809, which is Pichia pastoris with inactivated ⁇ -1,6-mannose transferase).
  • the Pyrobest enzyme, LA Taq enzyme, dNTPs, restriction enzymes, T4 ligase, etc. used in the experiment were purchased from Dalian Bao Bioengineering Co., Ltd., and the pfu enzyme, kit, and DH5 ⁇ competent cells were from Beijing Quanshijin Co., Ltd. product.
  • Whole gene synthesis, nucleotide synthesis, primer synthesis, sequencing, etc. are provided by Shanghai Shenggong Biological Engineering Technology Service Co., Ltd.
  • SARS-CoV-2 (2019-nCoV) Spike RBD-Fc Recombinant Protein (40592-V02H) is a product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.
  • goat anti-rabbit IgG secondary antibody (SAB3700885) is a product of Sigma
  • goat anti-mouse IgG The secondary antibody (ab205719) is a product of abcam
  • BglII restriction endonuclease is a product of NEB
  • PNGaseF (P0708)
  • Endo H (P0702) are products of NEB.
  • Capto MMC chromatography media, Phenyl HP, G25, and Source30Q were all purchased from GE Healthcare.
  • the basic strain used in the present invention is the GJK01 strain constructed by us in the previous period, the preservation number is CGMCC No. 1853, and the strain authorized patent number is ZL200610164912.8.
  • the strain is a Pichia strain inactivated by ⁇ -1,6-mannosyltransferase.
  • the amino acid sequence of ⁇ -1,6-mannose transferase (OCH1) is shown in SEQ ID No. 1.
  • the yeast strain GJK02 with inactivated phosphomannose transferase gene was obtained by knocking out part of the DNA molecule encoding the phosphomannose transferase shown in SEQ ID No. 2 in Pichia pastoris GJK01, that is, knocking out the phosphate in the GJK01 yeast genome Mannose transferase gene, obtained from recombinant yeast.
  • the knockout plasmid pYES2-pno1 used to knock out the mannose transferase (PNO1) gene is to insert the gene fragment (SEQ ID No.20) corresponding to the mannose transferase (PNO1) into the KpnI and XbaI restriction sites of the vector pYES2 The vector obtained in time.
  • SEQ ID No. 20 is from the 5'end of nucleotides 7-1006, the upstream homology arm of the knockout mannose transferase (PNO1) gene fragment;
  • SEQ ID No. 20 is from the 5'end of 1015 to 2017
  • the nucleotide is the downstream homology arm of the knock-out mannose transferase (PNO1) gene fragment.
  • the genomic DNA of Pichia pastoris X33 was extracted by the glass bead preparation method (A. Adams et al., "Yeast Genetics Method Experimental Guide", Science Press, 2000), and the genomic DNA was used as a template to amplify mannose transferase (PNO1)
  • the homology arms on both sides of the gene and the homology arms on both sides of PNO1 are about 1 kb respectively, and about 1.4 kb of the coding gene is deleted in the middle.
  • the primers used to amplify the homology arm (PNO1 5'homology arm) of the upstream flanking region of pno1 are PNO-5-5 and PNO-5-3, and the primer sequences are:
  • the primers used to amplify the homology arm (PNO13' homology arm) in the downstream flanking region of PNO1 are PNO-3-5 and PNO-3-3, and the primer sequences are:
  • the PCR amplification conditions for the two homology arms are as follows: after denaturation at 94°C for 5 minutes, 30 cycles of denaturation at 94°C for 30sec, 55°C renaturation for 30sec, 72°C extension for 1min30sec, and finally 72°C for 10min; the target fragment size is 1kb about.
  • the PCR product was purified and recovered with a PCR product recovery and purification kit (purchased from Dingguo Biotechnology Co., Ltd., Beijing). Use overlap extension PCR method to fuse PNO1 5'homology arm and 3'homology arm (see J.
  • the PCR products of the source arm and the 3'homology arm were used as templates, and PNO-5-5/PNO-3-3 was used as primers.
  • the PCR amplification conditions were as follows: after denaturation at 94°C for 5 minutes, denaturation at 94°C for 1 minute and renaturation at 55°C Extend at 72°C for 1min for 3min30sec for 30 cycles, and finally extend at 72°C for 10min; the size of the target fragment is about 2kb.
  • the PCR product is purified and recovered with a PCR product recovery and purification kit.
  • Kpn I/Xba I double enzyme digestion (the restriction enzymes used in this experiment are from Bao Bioengineering Co., Ltd., Dalian) PCR product, the product after digestion is inserted into the vector pYES2 (Invitrogen Corp.USA) treated with the same double enzyme digestion
  • T4 ligase was ligated overnight at 16°C, transformed into Escherichia coli DH5 ⁇ , and positive clones were screened on LB plates containing ampicillin (100 ⁇ g/ml).
  • the plasmid of the positive clone was identified by Kpn I/Xba I double enzyme digestion, and the recombinant vector with fragments of about 4200bp and about 2000bp was named pYES2-pno1, which is a knockout plasmid for knocking out the mannose transferase (PNO1) gene.
  • PNO1 mannose transferase
  • the knockout plasmid pYES2-pno1 was transformed into Pichia pastoris GJK01 (documented in the invention patent ZL200610164912.8, publication number CN101195809) by electrotransformation.
  • the electrotransformation method is well known in the art (such as A. Adams et al. , "Yeast Genetics Method Experimental Guide", Science Press, 2000). Before electrotransformation, linearize the knock-out plasmid with the BamH I restriction site upstream of the 5'homology arm, then electrotransform it into the prepared competent cells, and spread it on MD containing arginine and histidine.
  • the enzyme used in the PCR reaction is rTaq (Bao Biological Engineering Co., Ltd.), and the PCR amplification conditions are as follows: after denaturation at 94°C for 5 minutes, 30 cycles of denaturation at 94°C for 30sec, renaturation at 55°C for 30sec, and extension at 72°C for 3 minutes, and finally 72 Extend for 10 minutes.
  • the size of the PCR product band was analyzed by gel electrophoresis, and the band amplified by the primer was about 2.3kb as a positive clone.
  • YPD medium 10g/L yeast extract, 20g/L peptone, 20g/L glucose
  • YPD medium 10g/L yeast extract, 20g/L peptone, 20g/L glucose
  • culture in a shaker at 25°C for 12 hours and spread the bacterial solution on the adenine-deficient 5 -FOA medium (YNB 1.34g/100mL, biotin 4 ⁇ 10-5g/100mL, glucose 2g/100mL, agar 1.5g/100mL, arginine 100mg/ml, histidine 100mg/ml, uracil 100mg/ ml, 5-FOA 0.1%)
  • YNB is a non-amino acid yeast nitrogen source
  • 5-FOA is 5-fluorouracil, from Sigma-aldrich POBOX14508, St. Louis , MO63178USA
  • PNO1-ORF01 5′-GGGAAAGAAAACCTTCAATTT-3′ (SEQ ID No. 37);
  • PNO1-ORF02 5'-TACAAGCCAGTTTCGCAATAA-3' (SEQ ID No. 38).
  • PCR reaction system using the genome of wild-type X33 strain (Invitrogen) as a template was set as a control.
  • the enzyme used in the PCR reaction is LA Taq (Bao Biological Engineering Co., Ltd.), and the PCR amplification conditions are as follows: after denaturation at 94°C for 5 minutes, denaturation at 94°C for 30sec, renaturation at 55°C for 30sec, and extension at 72°C for 30 cycles, and finally 72 extended for 10 minutes.
  • the present invention introduces a reporter protein after obtaining the GJK01 engineered bacteria.
  • the present invention uses anti-Her2 antibody as the reporter protein.
  • the vector transformation method has been disclosed in the patent application (publication number: CN101748145A). Using this method, the anti-Her2 antibody expression vector was transferred to the GJK01 host strain, and the GJK01-HL engineered strain expressing the anti-Her2 antibody was obtained.
  • the DSA-FACE glycoform analysis method has been publicly reported in the article "Liu Bo et al. A method for analyzing oligosaccharide chains using DSA-FACE. Biotechnology Communications. 2008.19(6).885-888".
  • Fig. 1 A is the identification result of GJK01 host strain
  • Fig. 1 B is the DSA-FACE glycotype analysis result of GJK01-HL strain (knockout och1).
  • lane 1 is PON1 deficient
  • lane 2 is wild-type
  • the size of the PCR product using wild-type X33 strain genome as template is about 490bp
  • the PON1 deficient engineered bacteria has no amplified band, which also proves the loss of PNO1 gene
  • the phosphomannose transferase knockout strain was constructed correctly and named GJK02, which is a recombinant Pichia pastoris with phosphomannose transferase knockout.
  • the yeast strain GJK03 with inactivated mannose phosphate synthase gene was obtained by knocking out part of the DNA molecule encoding the mannose phosphate synthase shown in SEQ ID No. 3 in Pichia pastoris GJK02, that is, knocking out the phosphate in the GJK02 yeast genome Mannose synthetase gene, the recombinant yeast obtained; that is, the yeast ⁇ -1,6-mannose transferase, phosphomannose phosphate transferase and phosphomannose phosphate synthase are inactivated.
  • the method of constructing the vector is the same as step one.
  • the knockout plasmid pYES2-MNN4B used to knock out the phosphomannose synthase gene is to insert the upstream and downstream homology arms of the gene fragment to be knocked out corresponding to the phosphomannose synthase into the Stu I and Spe I digestion positions of the vector pYES2 The vector obtained between the points.
  • the genomic DNA of Pichia pastoris X33 was extracted by glass bead preparation, and the genomic DNA was used as a template to amplify the knock-out mannose synthase (MNN4B) gene fragment.
  • the homology arms on both sides of MNN4B were approximately approximately It is 1 kb, and about 1 kb of the coding gene is deleted in the middle.
  • the primers used to amplify the homology arm (ARM25' homology arm) of the upstream flanking region of MNN4B are MNN4B-5-5 and MNN4B-5-3, and the primer sequences are:
  • the primers used to amplify the homology arm (MNN4B 3'homology arm) of the downstream flanking region of MNN4B are MNN4B-3-5 and MNN4B-3-3, and the primer sequences are:
  • the PCR amplification conditions, recovery methods, and enzyme digestion methods of the two homology arms are the same as in step 1.
  • the pYES2-MNN4B knockout vector was finally constructed and verified by final sequencing.
  • Knockout plasmid is transformed into the Pichia pastoris engineering strain GJK02 constructed by the electrotransformation method.
  • the electrotransformation method and identification method are the same as step 1.
  • the two pairs of primers used in the PCR reaction are: the primer sequence outside the 5'homology arm of the mnn4b gene: MNN4B-5-5OUT: 5'-TAGTCCAAGTACGAAACGACACTA-3' (SEQ ID No. 43) and the primer sequence inner01: 5 on the vector '-AGCGTCGATTTTTGTGATGCTCGTCA-3' (SEQ ID No. 36), the band amplified by the primer is a positive clone of about 2kb.
  • MNN4B-ORF01 5'-AAAACTATCCAATGAGGGTCTC-3' (SEQ ID No. 44);
  • MNN4B-ORF02 5'-TCTTCAATGTCTTTAACGGTGT-3' (SEQ ID No. 45).
  • the positive cloned genomic DNA was used as a template, and the primers MNN4B-ORF01 and MNN4B-ORF02 were used for PCR amplification.
  • the results are shown in Figure 3.
  • Lane 1 is MNN4B-deficient, and lane 2 is wild-type; the PCR product with wild-type X33 strain genome as template is about 912bp, and the MNN4-deficient engineered bacteria has no amplified band, which also proves
  • the knockout of mannose phosphate synthase named GJK03, is a recombinant Pichia pastoris knocked out of mannose phosphate transferase and mannose phosphate synthase.
  • the yeast strain GJK04 in which phosphomannose transferase, phosphate mannose synthase and ⁇ mannose transferase ARM2 (i.e. ⁇ mannose transferase II) genes are inactivated is the ⁇ mannose shown in Pichia pastoris GJK03 encoding SEQ ID No.5
  • the DNA molecule of glycotransferase ARM2 was partially knocked out, that is, the ⁇ -mannose transferase ARM2 gene in the GJK03 yeast genome was knocked out to obtain recombinant yeast; that is, the ⁇ -1,6-mannose transferase in the yeast genome,
  • the phosphomannose phosphate transferase gene, phosphomannose phosphate synthase gene and ⁇ mannose transferase ARM2 have been inactivated.
  • the vector construction method is the same as step 1, and the details are as follows:
  • the genomic DNA of Pichia pastoris X33 was extracted by glass bead preparation, and the genomic DNA was used as a template to amplify the homology arms on both sides of the ⁇ -mannose transferase (ARM2) gene.
  • the source arms were about 0.6 kb respectively, and about 0.6 kb of coding gene was deleted in the middle.
  • the primers used to amplify the homology arm (ARM2 5'homology arm) of the upstream flanking region of ARM2 are ARM2-5-5 and ARM2-5-3, and the primer sequences are:
  • primers used to amplify the homology arms (ARM23' homology arms) in the downstream flanking region of ARM2 are ARM2-3-5 and ARM2-3-3, and the primer sequences are as follows:
  • Knockout plasmids are transformed into the Pichia engineering strain GJK03 constructed in the above-mentioned construction by the electro-transformation method, and the electro-transformation method and identification method are the same as those in the above-mentioned one.
  • the two pairs of primers used in the PCR reaction are: the primer sequence outside the 5'homology arm of the ARM2 gene ARM2-5-5OUT: 5'-TTTTCCTCAAGCCTTCAAAGACAG-3' (SEQ ID No. 50) and the primer sequence inner01: 5 on the vector '-AGCGTCGATTTTTGTGATGCTCGTCA-3' (SEQ ID No. 36), the band amplified by the primers is about 0.8kb as a positive clone.
  • Arm2-ORF-09 5'-gggcagaagatcctagag-3' (SEQ ID No. 51);
  • Arm2-ORF-10 5'-tcgtctccattgctatctacgact-3' (SEQ ID No. 52).
  • PCR amplification was performed with primers Arm2-ORF-09 and Arm2-ORF-10.
  • Lane 1 is ARM2-deficient
  • Lane 2 is wild-type; the result is wild-type
  • the PCR product size of the X33 strain genome as a template is about 600bp, and the ARM2-deficient engineered bacteria has no amplified bands.
  • ARM2 knocked out is a phosphomannose transferase, Recombinant Pichia pastoris with knockout of mannose phosphate synthase and ⁇ -mannose transferase II (ARM2) genes.
  • the vector construction method is the same as step three, the differences are:
  • the primers used to amplify the homology arm (ARM1 5'homology arm) of the upstream flanking region of ARM1 are ARM1-5-5 and ARM1-5-3, and the primer sequences are:
  • ARM1-5-5 5'-TCA ACGCGT TGGCTCTGGATCGTTCTAATA-3' (SEQ ID No.53, the underlined part is the recognition site of MluI);
  • the primers used to amplify the homology arm (ARM1 3'homology arm) of the downstream flanking region of ARM1 are ARM1-3-5 and ARM1-3-3, and the primer sequences are:
  • ARM1-3-3 5'-TCA ACGCGT TGGCTGGAGGTGACAGAGGAA-3' (SEQ ID No. 56, underlined part is MluI recognition site).
  • the primers used to amplify the homology arm (ARM3 5'homology arm) of the upstream flanking region of ARM3 are ARM3-5-5 and ARM3-5-3, and the primer sequences are:
  • ARM3-5-5 5'-TCAACGCGTTAGTAGTGCCGTGCCAAGTAGCG-3' (SEQ ID No. 57, the underlined part is the MluI recognition site);
  • the primers used to amplify the homology arm (ARM3 3'homology arm) of the downstream flanking region of ARM3 are ARM3-3-5 and ARM3-3-3, and the primer sequences are:
  • ARM3-3-3 5'-TCA ACGCGT CATAGGTAATGGCACAGGGATAG-3' (SEQ ID No. 60, underlined part is MluI recognition site).
  • the primers used to amplify the homology arm (ARM4 5'homology arm) of the upstream flanking region of ARM4 are ARM4-5-5 and ARM4-5-3, and the primer sequences are:
  • ARM4-5-5 5'-TCA ACGCGT GCAGCGTTTACGAATAGTGTCC-3' (SEQ ID No. 61, the underlined part is the recognition site of MluI);
  • the primers used to amplify the homology arm (ARM4 3'homology arm) of the downstream flanking region of ARM4 are ARM4-3-5 and ARM4-3-3, and the primer sequences are:
  • ARM4-3-3 5'-TCA ACGCGT GAGGTGGACAAGAGTTCAACAAAG-3' (SEQ ID No. 64, the underlined part is the recognition site of MluI).
  • step 3 the difference is that the two pairs of primers used in the PCR reaction are:
  • the band amplified by the primer is about 3.5kb as a positive clone.
  • the band amplified by the primer is about 3.7kb as a positive clone.
  • the band amplified by the primer is about 3.7kb as a positive clone.
  • step 3 the difference is that the following primers are used to identify the engineered bacteria, and it can be found that the gene has been knocked out ( Figure 6, Figure 7 and Figure 8):
  • Arm1-ORF-09 5'-TAGTCTGGTTTGCGGTAGTGT-3' (SEQ ID No. 68);
  • Arm1-ORF-10 5'-AGATTGAGCATAGGAGTGGC-3' (SEQ ID No. 69).
  • Arm3-ORF-09 5'-AAACGGAGTCCAGTTCTTCT-3' (SEQ ID No. 70);
  • Arm3-ORF-10 5'-CAACTTTGCCTGTCATTTCC-3' (SEQ ID No. 71).
  • Arm4-ORF-09 5'-CGCTTCAGTTCACGGACATA-3' (SEQ ID No. 72);
  • Arm4-ORF-10 5'-GCAACCCAGACCTCCTTACC-3' (SEQ ID No. 73).
  • ⁇ -mannose is a potential sugar that causes immunogenicity. Therefore, for the source of drugs used in humans, There is a potential risk.
  • the present invention inactivates all ⁇ mannose, so the problem of ⁇ mannose is fundamentally solved, and the glycoform structure is not changed.
  • the present invention introduces a reporter protein into the GJK18 engineered bacteria in advance.
  • the present invention uses anti-Her2 antibody as the reporter protein, so an anti-Her2 antibody is constructed.
  • Expression vector The vector construction method and vector transformation method have been disclosed in the patent application (publication number: CN101748145A). Using this method, the anti-Her2 antibody expression vector was transferred to the GJK18 host strain, and the W2 engineered strain expressing the anti-Her2 antibody was obtained.
  • the glyco-engineered yeast strain W10 with mammalian Man5GlcNAc2 and no fucosylation structure is MDSI (TrmdsI) with the C-terminal fusion HDEL sequence.
  • the nucleotide sequence is shown in SEQ ID No. 14, which encodes SEQ ID No.
  • the MDSI protein shown in .9) is inserted into the genome of the host strain W2 to obtain an engineered strain.
  • the recombinant vector pPIC9-TrmdsI for expressing exogenous mannosidase I is a recombinant vector obtained by inserting the DNA molecule shown in SEQ ID No. 14 between the Xho I and EcoR I restriction sites of the pPIC9 vector.
  • nucleotides 1-1524 from the 5'end of SEQ ID No. 14 are the optimized mannosidase I coding gene, and the nucleotides 1525 to 1536 from the 5'end are the endoplasmic reticulum retention signal—— HDEL encoding gene.
  • MDSI Mannosidase I
  • Exogenous mannosidase I can be mannosidase I derived from filamentous fungi, plants, insects, Java, mammals, etc.
  • mannosidase I of Trichoderma viride Zhan Jie. Trichoderma viride ⁇ - Cloning, expression and activity identification of 1,2-mannosidase in Pichia pastoris [Degree Theory Master's Article].
  • the endoplasmic reticulum retention signal-HDEL was fused to the C-terminus of mannosidase I.
  • TrmdsI-5 5'-TCT CTCGAG AAAAGAGAGGCTGAAGCTTATCCAAAGCCGGGCGCCA C-3' (SEQ ID No. 74); the underlined sequence is the Xho I restriction recognition site.
  • TrmdsI-3 5'-AGG GAATTC TTACAACTCGTCGTGAGCAAGGTGGCCGCCCCGTCGTG ATG-3' (SEQ ID No. 75); the underlined sequence is the EcoR I restriction recognition site.
  • TrmdsI PCR amplification product
  • pPIC9-TrmdsI plasmid About 10 ⁇ g of pPIC9-TrmdsI plasmid was linearized with Sal I, and the linearized plasmid was precipitated with 1/10 volume of 3M sodium acetate and 3 times volume of absolute alcohol. Wash twice with a 70% ethanol aqueous solution by volume to remove the salt therein, dry, and add about 30 ⁇ L of water to resuspend the precipitate to obtain the pPIC9-TrmdsI linearized plasmid for transformation.
  • the selected host strain is the W2 engineered strain constructed above.
  • Pichia pastoris W2 was isolated on a YPD plate (yeast extract 10g/L, tryptone 20g/L, glucose 20g/L, agar 15g/L) by streaking and cultured in a 28°C incubator for 2 days. Inoculate a single clone into a 50mL Erlenmeyer flask containing 10mL YPD liquid medium (yeast extract 10g/L, tryptone 20g/L, glucose 20g/L), and incubate at 28°C overnight until the OD 600 is about 2, to obtain Bacteria.
  • the cells were resuspended with 20 mL of pre-cooled sterile 1 M sorbitol, and the cells were harvested by centrifugation at 1500 g at 4° C. for 10 minutes, and the cells were resuspended with pre-cooled 1 M sorbitol to a final volume of 1.5 mL to obtain a bacterial suspension.
  • the genomic DNA of W10 was extracted by the glass bead preparation method.
  • the genomic DNA was used as a template, and TrMDSI-1.3kb-01 and TrMDSI-1.3kb-02 were used as primers to carry out PCR amplification, and the PCR amplification product was about 1.3kb, which proved MDSI It has been inserted into the genome and is a positive engineered bacteria (A in Figure 10).
  • TrMDSI-1.3kb-01 5’-GAACACGATCCTTCAGTATGTA-3’ (SEQ ID No. 76);
  • TrMDSI-1.3kb-02 5'-TGATGATGAACGGATGCTAAAG-3' (SEQ ID No. 77).
  • the DSA-FACE glycoform analysis result of W10 bacteria (the method is the same as that described in Example 1) is shown in Figure 10 B. It can be seen that the glycoform structure of the protein expressed by W10 bacteria is Man5GlcNAc2, Man6GlcNAc2 after being transformed into TrmdsI. Mainly Man5GlcNAc2.
  • Glyco-engineered yeast strains 1-8 with mammalian GlcNAcMan5GlcNAc2 and no fucose glycosylation structure are N-acetylglucosamine transferase I (GnTI) containing mnn9 localization signal (nucleotide sequence as SEQ ID No. As shown in 15, the DNA fragment encoding the protein shown in SEQ ID No. 10) is inserted into the genome of the host bacteria W10 to obtain the engineered bacteria.
  • GnTI N-acetylglucosamine transferase I
  • SEQ ID No. 15 is the mnn9 location signal from nucleotides 1-114 from the 5'end, and nucleotides 115-1335 from the 5'end is the N-acetylglucosamine transferase I encoding gene.
  • GnTI N-acetylglucosamine transferase I
  • gnt1 gene upstream primer (mnn9-GnTI-01: 5'-tcagtcagcgctctcgatggcgaccccg-3', SEQ ID No. 78) and downstream primer GnTI-02: 5'-GC GAATTC TTAGTGCTAATTCCAGCTAGGATCATAG-3' (SEQ ID No. 79, underlined Is the EcoR I restriction site), the full-length fragment of human gnt1 gene was obtained from the human liver-fetal cDNA library (purchased from Clontech Laboratories Inc. 1290 Terra Bella Ave. Mountain View, CA94043, USA) by PCR.
  • PCR reaction conditions 94 Pre-denaturation at °C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 1 minute and 30 seconds, and 30 cycles; finally, extension at 72°C for 10 minutes.
  • the PCR amplified products were separated by 0.8% agarose gel electrophoresis, and recovered with a DNA recovery kit.
  • upstream primer ScMNN9-03 (tat AAT attATGTCACTTTCTCTTGTATCGTACCGCCTAAGAAAGAACCCGTGGGTTAACATTTTTCTACCTGTTTTTTGGCCATATTTCTAATATATCAATTTTTTTCCAGAGAGATCAATCTtcagtcagcg, downstream of the SEQ ID Gcctcagtcag, the downstream coding site of SEQ ID No. GcctcagtcagcgccaGAGATCAATCTtcagtcagtcag, the lower sequence of the SEQ ID is the coding sequence of S. cere MNN9 Golgi positioning signal coding sequence.
  • the recovered and purified 1.2 kb GnTI fragment and the S.cere MNN9 Golgi localization signal coding sequence were connected by PCR reaction, and the mnn9-gnt1 gene fragment (SEQ ID No. 15) was amplified using Pyrobest DNA polymerase.
  • PCR reaction conditions denaturation at 94°C for 2 minutes, annealing at 52°C for 30 seconds, extension at 72°C for 5 minutes, then denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 1 minute and 30 seconds, and 30 cycles; finally 72°C Extend for 10 minutes.
  • the PCR amplified products were separated by 0.8% agarose gel electrophoresis (8V/cm, 15 minutes), and the 1.3 kb band of interest was cut with a clean blade under ultraviolet light, and the DNA recovery kit was used for recovery, the method is the same as above.
  • PGE-URA3-GAP1-mnn9-GnTI is a recombinant vector obtained by inserting the DNA molecule shown in SEQ ID No. 15 between the restriction sites Ssp I and EcoR I of the PGE-URA3-GAP1 vector.
  • PGE-URA3-GAP1-mnn9-GnTI plasmid was linearized with Nhe I to obtain the PGE-URA3-GAP1-mnn9-GnTI linearized plasmid for transformation, and the method for preparing yeast electrotransformation competent cells. Step 5 above .
  • the selected host strain is the W10 engineered strain constructed in step five above.
  • the single clone formed on the MD plate after transformation is named 1-8.
  • Extract 1-8 genomic DNA using glass bead preparation method use genomic DNA as a template, HuGnTI-0.9k-01 and HuGnTI-0.9k-02 as primers, perform PCR amplification, and obtain a PCR amplification product of about 0.9kb. It proves that GnTI has been inserted into the genome, which is a positive engineering bacteria (Figure 11, A).
  • HuGnTI-0.9k-01 5’-TGGACAAGCTGCTGCATTATC-3’ (SEQ ID No.81);
  • HuGnTI-0.9k-02 5'-CGGAACTGGAAGGTGACAATA-3' (SEQ ID No. 82).
  • the glyco-engineered yeast strain 1-8-4 with mammalian GalGlcNAcMan5GlcNAc2 and no fucose glycosylation structure is the kre2-GalE-GalT gene fragment (nucleotide sequence shown in SEQ ID No. 16, encoding SEQ ID The protein shown in No. 11) is inserted into the genome of the host strain 1-8 to obtain the engineered strain 1-8-4.
  • SEQ ID No. 16 is the kre2 localization signal from nucleotides 1-294 from the 5'end, and nucleotides 29-1317 from the 5'end is the gene encoding the galactose isomerase GalE, from the 5'end Nucleotides 1325-2394 are the gene encoding the galactosyltransferase GalT.
  • the upstream primer GalE5' and downstream primer GalE3' of the human GalE gene were used respectively from the human liver fetal cDNA library (purchased from Clontech Laboratories Inc. 1290 Terra Bella Ave. Mountain View , CA94043, USA) to obtain full-length fragments of human GalE and GalT genes.
  • PCR reaction conditions 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 52°C annealing for 30 seconds, 72°C extension for 1 minute and 30 seconds, and 30 cycles; Finally, it was extended at 72°C for 10 minutes.
  • the PCR amplified products were separated by 0.8% agarose gel electrophoresis, and recovered with a DNA recovery kit.
  • GalE5 5’-ATGAGAGTTCTGGTTACCGGTGGTA-3’ (SEQ ID No. 83);
  • GalE3' 5'-AG GGTACC ATCGGGATATCCCTGTGGATGGC-3' (SEQ ID No. 84, the underlined part is the recognition sequence of KpnI);
  • GalT5' 5'-AT GGTACC GGTGGTGGACGTGACCTTTCTCGTCTGCCA-3' (SEQ ID No. 85, the underlined part is the recognition sequence of KpnI).
  • GalT3' 5'-GC atttaaat ttaGCTCGGTGTCCCGATGTCCACTGTGAT-3' (SEQ ID No. 86, the underlined part is the recognition sequence of SwaI).
  • Kre2 5' 5'-AT AATatt AAACGATGGCCCTCTTTCTCAGTAAGAG-3' (SEQ ID No. 87, the underlined part is the recognition sequence of the SspI I site);
  • the kre2 localization signal fragment was extracted from the S. cere genomic DNA of Saccharomyces cerevisiae by PCR.
  • the PCR conditions are the same as above.
  • the purified GalE, GalT fragments and the S.cere kre2 Golgi localization signal will be recovered by PCR reaction.
  • the coding sequences are connected, and the kre2-GalE-GalT gene fragment is amplified using Pyrobest DNA polymerase.
  • PCR reaction conditions denaturation at 94°C for 2 minutes, annealing at 52°C for 30 seconds, extension at 72°C for 5 minutes, then denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 4 minutes and 30 seconds, and 30 cycles; finally 72°C Extend for 10 minutes.
  • the PCR amplified products were separated by 0.8% agarose gel electrophoresis (8V/cm, 15 minutes), the 2.4kb band of interest was cut with a clean blade under ultraviolet light, and the DNA recovery kit was used for recovery, the method is the same as above.
  • PGE-URA3-GAP1-kre2-GalE-GalT is a recombinant vector obtained by inserting the DNA molecule of kre2-GalE-GalT shown in SEQ ID No. 16 into the Ssp I and SwaI restriction sites of the PGE-URA3-GAP1 vector.
  • PGE-URA3-GAP1-kre2-GalE-GalT plasmid was linearized with Nhe I to obtain the PGE-URA3-GAP1-kre2-GalE-GalT linearized plasmid for transformation to prepare yeast electrotransformation competent cells
  • the method is the same as the above step 5.
  • the selected host strain is the engineered strain 1-8 constructed in step six.
  • the single clone formed on the MD plate after transformation was named 1-8-4.
  • the genomic DNA of 1-8-4 was extracted by the glass bead preparation method, and the genomic DNA was used as the template, and GalE-T(1.5k)-01 (5'-TGATAACCTCTGTAACAGTAAGCGC-3', SEQ ID No. 89) and GalE- T(1.5k)-02 (5'-GGAGCTTAGC ACGATTGAATATAGT-3', SEQ ID No. 90) was used as primers, PCR amplification was performed, and the PCR amplification products were 1.5kb, which proved that GalE-T has been inserted into the genome , It is the positive engineered bacteria (A in Figure 12).
  • the glyco-engineered yeast strain 52-60 with mammalian GalGlcNAcMan3GlcNAc2 and no fucose glycosylation structure is an MDSII DNA molecule (nucleotide sequence is shown in SEQ ID No. 17, which encodes the protein shown in SEQ ID No. 12 ) Inserted into the genome of host strain 1-8-4 to obtain engineered strain 52-60.
  • nucleotides 1-108 from the 5'end of SEQ ID No. 17 are the mnn2 localization signal of the mannosidase II encoding gene
  • nucleotides 109-3303 from the 5'end are the mannosidase II encoding gene.
  • Mannosidase II (MDSII) expression vector containing mnn2 localization signal
  • the MDSII gene containing mnn2 (SEQ ID No. 17) was synthesized by a full-gene synthesis method, synthesized by Nanjing Jinrui Si Company and cloned into the pUC57 cloning vector to obtain pUC57-MDSII.
  • PGE-URA3-arm3-GAP-mnn2-MDSII is a recombinant vector obtained by inserting the DNA molecule shown in SEQ ID No. 17 into the Ssp I and Swa I restriction sites of the PGE-URA3-GAP1 vector.
  • PGE-URA3-arm3--GAP-mnn2-MDSII plasmid was linearized with Msc I to obtain the PGE-URA3-arm3-GAP-mnn2-MDSII linearized plasmid for transformation to prepare yeast electrotransformation competent cells
  • the method is the same as the above step 5.
  • the selected host strain is the 1-8-4 engineered strain constructed in step seven.
  • the single clone formed on the MD plate after transformation was named 52-60.
  • the genomic DNA of 52-60 was extracted by the glass bead preparation method.
  • the genomic DNA was used as a template, and CeMNSII-1.2k-01 and CeMNSII-1.2k-02 were used as primers to carry out PCR amplification.
  • the PCR amplification products were 1.2 kb, it proves that MDSII has been inserted into the genome, that is, it is a positive engineered bacteria (A in Figure 13).
  • CeMNSII-1.2k-02 5'-GACAAGAGGATAATGAAGAGAC-3' (SEQ ID No. 94).
  • the glyco-engineered yeast strain 150L2 with mammalian Gal2GlcNAc2Man3GlcNAc2 and no fucose glycosylation structure is a GnT II DNA molecule (the nucleotide sequence is shown in SEQ ID No. 18, which encodes the protein shown in SEQ ID No. 13) Inserted into the genome of the host strain 52-60, the engineered strain 150L2 was obtained.
  • SEQ ID No. 18 is the mnn2 localization signal of the gene encoding N-acetylglucosamine transferase II from nucleotides 1-108 from the 5'end, and nucleotides 109-1185 from the 5'end are N- Acetyl Glucosamine Transferase II.
  • GnTII N-acetylglucosamine transferase II
  • the GnTII gene containing mnn2 (SEQ ID No. 18) was synthesized by full-gene synthesis method, synthesized by Nanjing Jinrui Si Company and cloned into the pUC57 cloning vector to obtain pUC57-GnTII.
  • GnTII gene mnn2-GnTII-01: 5'-AT AATatt AAACCatgctgcttaccaaaa ggttttcaaagctgttc-3', SEQ ID No. 95
  • the underline is the SspI restriction site
  • the downstream primer GnTII-02: 5'-GCT atttaaat TTAtcactgcagtcttctataacttttac-3', SEQ ID No.
  • the DNA molecule of N-acetylglucosamine transferase II (GnTII) containing mnn2 localization signal was obtained from pUC57-GnTII by PCR, PCR reaction conditions: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 2 minutes and 30 seconds, 30 cycles; finally extension at 72°C for 10 minutes.
  • the PCR amplified products were separated by 0.8% agarose gel electrophoresis, and recovered with a DNA recovery kit.
  • restriction enzyme digestion and construction method are consistent with the construction method of PGE-URA3-arm3-GAP-mnn2-MDSII, and the recombinant plasmid is obtained, which is named PGE-URA3-arm3-GAP-mnn2-GnTII. Sequencing, the result is correct.
  • PGE-URA3-arm3-GAP-mnn2-GnTII is a recombinant vector obtained by inserting the DNA molecule shown in SEQ ID No. 18 into the Ssp I and Swa I restriction sites of the PGE-URA3-GAP1 vector.
  • PGE-URA3-arm3-GAP-mnn2-GnTII plasmid was linearized with Msc I to obtain the PGE-URA3-arm3-GAP-mnn2-GnTII linearized plasmid for transformation to prepare yeast electrotransformation competent cells
  • the method is the same as the above step 5.
  • the selected host strain is the 52-60 engineered strain constructed in step 8.
  • the single clone formed on the MD plate after transformation was named 150L2.
  • the genomic DNA of 150L2 was extracted by glass bead preparation method. Using genomic DNA as template, RnGnTII-0.8k-01 and RnGnTII-0.8k-02 were used as primers to carry out PCR amplification.
  • the PCR amplification product was 0.8kb, which proved GnTII It has been inserted into the genome and is a positive engineered bacteria ( Figure 13 B).
  • RnGnTII-0.8k-02 5'-AGTTCATGGTCCCTAATATCTC-3' (SEQ ID No. 98).
  • Yeast strain 3-5-11 with inactivated anti-her2 antibody gene introduced the DNA molecule shown in SEQ ID No. 19 (anti-her2 antibody light and heavy chain gene knockout sequence) into Pichia pastoris 150L2, which is the same as the 150L2 genome.
  • the source sequence undergoes homologous recombination, and the light and heavy chain genes of the anti-her2 antibody in the yeast genome are knocked out to obtain recombinant yeast.
  • the host bacteria are found to be unstable and easy to lose MDSI and MDSII genes, before the O-mannose transferase I gene is inactivated, follow the same technical method as Step 8 and Step 5 of this example, in 3-5-11
  • the medium host bacteria were successively transferred to SEQ ID No. 17 (MDSII) and SEQ ID No. 14 (MDSI), ensuring double copies of these two genes in the engineered bacteria, and 670 host bacteria were constructed.
  • Yeast strain 7b with inactivated O-mannose transferase I gene is a yeast obtained by inserting and inactivating a DNA molecule encoding O-mannose transferase I shown in SEQ ID No. 8 in Pichia pastoris 670, Named 7b, which is GJK30.
  • GJK30 has been deposited at the China Common Microbial Species Collection and Management Center on March 18, 2020, and its deposit number is CGMCC No. 19488.
  • the terminator AOXTT sequence was obtained by PCR.
  • the PCR used terminator primers AOXTT-5 and AOXTT-3 (5'-AOX1TT-5tctacgcgtccttag acatgactgttcctcagt-3', SEQ ID No. 99; AOX1TT-3: 5'-tctacgcgtaagcttgcacaaacgaacttc-3', SEQ ID No. 100) .
  • the obtained PCR product was purified and recovered with a PCR product recovery and purification kit (Dingguo Biotechnology Co., Ltd., Beijing) to obtain an AOX1TT terminator fragment.
  • the vector pYES2 (invitrogen company) used in the present invention has a yeast URA3 selection marker and can be used for subsequent screening.
  • the present invention adds an AOX1TT terminator at the end of the URA3 gene.
  • the specific construction method is: the AOX1TT terminator fragment obtained above is recovered and then digested with MluI to obtain the digested fragment; the digested fragment is ligated with the vector pYES2 that has also been treated with Mlu1, and the ligated product is transformed into E.
  • coli competent Cell Trans5 ⁇ (Beijing Quanshijin Biotechnology Co., Ltd., catalog number CD201) was amplified, and the clone with the correct sequence was named Trans5 ⁇ -pYES2-URA3-AOX1TT.
  • the plasmid was extracted to obtain a recombinant vector with AOX1TT terminator added to the end of URA3 gene. It is pYES2-URA3-AOX1TT.
  • the present invention uses PCR to catch a fragment of the ORF region of the PMT1 gene as a homologous recombination fragment.
  • this study at both the primer plus the stop codon of different combinations, at the 3 'end of the gene fragment PMT1 fished plus CYCTT terminator.
  • Pichia pastoris JC308 Using the genome of Pichia pastoris JC308 (Invitrogen) as a template, the genomic DNA of Pichia pastoris JC308 was extracted by the glass bead preparation method (A. Adams et al., "Yeast Genetics Method Experimental Guide", Science Press, 2000). Genomic DNA was used as a template, and primers PMT1-IN-5 and PMT1-IN-3 were used for PCR amplification to catch the PMT1 gene fragment.
  • the reaction conditions of PCR to catch the PMT1 gene fragment were 94°C pre-denaturation for 5min; 94°C denaturation for 30s, 55°C annealing for 30s, and 72°C extension for 1min40s. A total of 25 cycles were performed, and the final extension was at 72°C for 10 minutes.
  • the recovered PCR product is the PMT1 gene fragment.
  • the primers CYC1TT-5 and CYC1TT-3 (CYC1TT-5: 5'-gctttcttagtcgtccccactctgatctaatgatagttaatgactaatagatcatgtaattagttatgtca-3', SEQ ID No.1031: agc1TT-3', SEQ ID No.1031: agc1TT-3', , SEQ ID No. 104) Perform PCR amplification to catch the CYC1TT terminator fragment.
  • the PCR reaction conditions were pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute. A total of 25 cycles were performed, and the final extension was at 72°C for 10 minutes.
  • the recovered PCR product is the CYC1TT terminator fragment.
  • the recovered PCR product is the fusion fragment of PMT1-IN and CYC1TT terminator-PMT1-IN-CYC1TT.
  • the recovered product was digested with Nsi1 and then phosphorylated, and then ligated with the vector backbone of pYES2-URA3-AOX1TT digested with Nsi1 and Stu1.
  • the resulting recombinant vector with the correct sequence is PMT1 inserted into the inactivated vector PMT1-IN-pYES2.
  • a different combination of stop codons are installed at the front and end of the PMT1 gene fragments, and a CYC1TT terminator is installed after the end stop codon to ensure that the PMT1 gene will not be expressed if the genome is integrated correctly.
  • the pYES2 vector contains the URA3 gene of Pichia pastoris.
  • the AOX1TT terminator is inserted after the URA3 gene.
  • a CYC1TT terminator (272bp) fragment and a PMT1 (907bp) fragment were obtained, which were consistent with the theoretical size.
  • the size of the fusion fragment between the PMT1-IN fragment and CYC1TT is 1135bp.
  • the successful construction of the vector PMT1-IN-pYES2 is proved by the above PCR identification and sequencing.
  • yeast 670 competent cells To prepare yeast 670 competent cells, the preparation method is as follows:
  • this medium is a medium with a uracil concentration of 100 ⁇ g/mL obtained by adding uracil to YPD medium), and shake it at 25°C at 170r/min Cultivate for 48h; then take 500 ⁇ L of culture, inoculate it in 100mL YPD+U medium, incubate at 170r/min for 24h at 25°C, and OD 600 will reach 1.0; then centrifuge at 6000r/min for 6min at 4°C, and use 15mL of cold Resuspend the bacteria in bacterial water; centrifuge again under the same conditions, and resuspend the bacteria in 15mL of cold sterile water; centrifuge at 6000r/min for 6min at 4°C, and resuspend the bacteria in 15mL of cold 1mol/L sorbitol; the same Centrifuge again under the conditions; discard the supernatant, resuspend the
  • PMT1-ORF-OUT-3 5'-gctctgaggcaccttgggtaa-3' (SEQ ID No. 106).
  • the Pichia pastoris chromosome It is integrated into the Pichia pastoris chromosome by inserting an inactivated vector. Since the vector contains homologous fragments of the PMT1 gene, theoretically the integration of the vector is a site-directed integration, that is, insertion into the PMT1 gene, which can be carried out by designing specific primers. Identification and screening. Using the URA3 selection marker of Pichia pastoris, through pressure screening, the clones grown on the MD+RH plate were identified. The peripheral primers PMT1-ORF-OUT-5 and PMT1-ORF-OUT-3 of the PMT1 gene were used for PCR identification.
  • the above primers can be used to obtain a 8.6kb fragment; the control (ie yeast X33) is a 3kb fragment ( Figure 14); it can be seen that this PMT1-IN-
  • the pYES2 vector was correctly integrated into the PMT1 gene and was named 7b, namely GJK30. Since different stop codons and terminators are designed on the insert vector, the PMT1 gene will not be expressed if the gene is integrated correctly.
  • the present invention introduces a reporter protein after obtaining the GJK30 engineered bacteria.
  • the method is the same as the method in Example 1.
  • the anti-Her2 antibody is used as the reporter protein, and the expression vector of the anti-Her2 antibody is constructed
  • the method and vector transformation method have been disclosed in the patent application (see Example 1). Using this method, the anti-Her2 antibody expression vector was transferred to the GJK30 host strain, and the GJK30-HL engineered strain expressing the anti-Her2 antibody was obtained.
  • the glycotype and the glycotype obtained in the previous stage (the Her2 antibody expression vector is transferred to the control recombinant engineered strain obtained from the GJK08 strain constructed in Example 1 of Chinese Patent Application 201410668305.X, that is, compared with the GJK30-HL engineered strain of the present invention
  • the ⁇ -mannose transferase knocked out in the present invention is I-IV, and the control recombinant engineered bacteria only knocks out ⁇ -mannose transferase II; the present invention also inactivates O-mannose transferase I, and the control recombination Engineering bacteria do not have; the present invention introduces exogenous MDSI and MDSII is introduced twice, and the control recombinant engineering bacteria is introduced once) Although both contain the structure of Gal2GlcNAc2Man3GlcNAc2, the ratio of the two is obviously different.
  • Gal2GlcNAc2Man3GlcNAc2 in the early stage is less than 50% (Figure 15) A), while the Gal2GlcNAc2Man3GlcNAc2 structure obtained by the GJK30 engineered bacteria occupies more than 60% of the glycoform, and the overall glycoform is simpler and more uniform (Figure 15 B).
  • this Gal2GlcNAc2Man3GlcNAc2 glycoform structure will affect the biological activity of the protein, such as the ADCC and CDC activities of the antibody, so its proportion directly affects many characteristics of the protein.
  • the glycosylase was digested and analyzed by commercial glycosidase (New England Biolabs, Beijing), as shown in Figure 15 C.
  • Gal2GlcNAc2Man3GlcNAc2(G2) does not have N-acetylglucosamine at the end, it is in ⁇ -N-acetyl Under the action of glucosaminidase, the structure of Gal2GlcNAc2Man3GlcNAc2 will not change, but the exonuclease ⁇ 1,4-galactosidase can cut and remove two galactoses to form the structure of GlcNAc2Man3GlcNAc2 (G0); and at the same time Under the action of these two exonucleases, the galactose Gal and N-acetylglucosamine GlcNAc were sheared and removed successively, so the glycosyl structure was changed to the Man3GlcNAc2 structure, which proved that the expressed glycotype was correct.
  • pPICZ ⁇ A vector Recombinant expression vectors pPICZ ⁇ -S-RBD223, pPICZ ⁇ -S-RBD216, and pPICZ ⁇ -S-RBD210 were obtained between the XhoI and NotI restriction sites downstream of the daughter.
  • the structure of the recombinant expression vector pPICZ ⁇ -S-RBD223 is described as: a recombinant plasmid with the DNA fragment shown in SEQ ID No. 24 inserted between the XhoI and NotI restriction sites of the pPICZ ⁇ A vector.
  • SEQ ID No. 24 is the coding gene sequence obtained by codon-optimizing amino acids 319 to 541 (R319-F541) of the S protein of SARS-CoV-2 "Wuhan-Hu-1" isolate, encoding SEQ The SARS-CoV-2 S-RBD223 protein indicated by ID No.21.
  • the structure of the recombinant expression vector pPICZ ⁇ -S-RBD216 is described as: a recombinant plasmid with the DNA fragment shown in SEQ ID No. 25 inserted between the XhoI and NotI restriction sites of the pPICZ ⁇ A vector.
  • SEQ ID No. 25 is the coding gene sequence obtained by codon-optimizing amino acids 319 to 534 (R319-V534) of the S protein of SARS-CoV-2 "Wuhan-Hu-1" isolate, encoding SEQ The SARS-CoV-2 S-RBD216 protein indicated by ID No. 22.
  • the structure of the recombinant expression vector pPICZ ⁇ -S-RBD210 is described as: a recombinant plasmid with the DNA fragment shown in SEQ ID No. 26 inserted between the XhoI and NotI restriction sites of the pPICZ ⁇ A vector.
  • SEQ ID No. 26 is the coding gene sequence obtained after codon optimization of amino acids 319 to 528 (R319-K528) of the S protein of the SARS-CoV-2 "Wuhan-Hu-1" isolate, encoding SEQ The SARS-CoV-2 S-RBD210 protein indicated by ID No. 23.
  • Recombinant expression vectors pPICZ ⁇ -S-RBD223, pPICZ ⁇ -S-RBD216 and pPICZ ⁇ -S-RBD210 transform yeast CGMCC No. 19488
  • the yeast CGMCC No. 19488 was streaked on the YPD plate for recovery, and the single clone was isolated. Pick the recovered single clone and inoculate it into YPD liquid medium. After the test tube is cultured to its logarithmic phase, take 1ml and transfer to a 100ml YPD shake flask to culture at OD 600 to 1.3-1.5, 1500g 4°C on a 200rpm shaker at 25°C Centrifuge for 5 minutes to discard the supernatant, resuspend in an equal volume of pre-cooled distilled water and centrifuge at 1500g at 4°C for 5 minutes, discard the supernatant, repeat this step 3 times; then resuspend with an equal volume of pre-cooled 1M sorbitol and resuspend at 1500g at 4°C Centrifuge for 5 min, discard the supernatant, and repeat this step 3 times.
  • the above bacterial pellets washed with 3 times of distilled water and 3 times of sorbitol were suspended by adding 1ml of 1M sorbitol, and 100 ⁇ l each was aliquoted into sterile centrifuge tubes and stored at -80°C.
  • the restriction enzyme digestion system (50 ⁇ L) is as follows: expression plasmid 43 ⁇ L , BglII 2 ⁇ L, 10 ⁇ NEB3.1buffer 5 ⁇ L, digested at 37°C for 1h, sampled, separated by 1% agarose gel electrophoresis, and analyzed whether the plasmid is linearized completely. The separation results showed that the completely linearized digestion product was recovered with a spin column-type DNA fragment recovery kit, and the linearized plasmid was eluted with 30 ⁇ L of pure water when the linearized plasmid was finally eluted.
  • each group randomly picks 8 single clones and inoculates them on a new YPD/Zeocin plate, and inverts them in a 25°C incubator. After the colony grows, it is inoculated into 3ml YPD/Zeocin liquid medium, cultured on a shaker at 25°C and 200rpm. After the bacterial solution grows thick, transfer to 3ml BMGY according to the inoculation volume of 5% (volume percentage). The culture medium was cultured in a shaker at 200 rpm at 25°C, and induced with 0.5% (V/V) methanol every 12 hours after 48 hours. After 48 hours of induction, the culture supernatant was collected at 12000 rpm for 3 minutes.
  • the positive recombinant yeast strains were named CGMCC19488/S-RBD223, CGMCC19488/S-RBD216, CGMCC19488/S-RBD210.
  • Figure 16 is a WB verification image of CGMCC19488/S-RBD223 positive clone screening.
  • the positive clone CGMCC19488/S-RBD223 identified in Example 2 was picked and inoculated into YPD/Zeocin liquid medium, cultured at 25°C and 200 rpm to an OD 600 of 15-20, and transferred at an inoculum of 5% (V/V). Received BMGY medium, cultured at 25°C, 200rpm for 24 hours, and then added 0.5% methanol to induce the expression of S-RBD. Induced every 12 hours, and sampled for expression detection. After 48 hours of induction, the culture supernatant was collected by centrifugation .
  • the mobile phase components are:
  • the purified Phenyl HP sample was desalted with G25fine chromatographic medium, and the protein sample was collected.
  • the mobile phase composition was: 20mM pH8.5 Tris-HCl.
  • the mobile phase components are:
  • SARS-CoV-2 S-RBD223 protein can be captured by Capto MMC; after desalting, the sample was purified with SOURCE30Q, and the target protein flowed through, and almost all the impurity protein was adsorbed on the SOURCE30Q chromatography medium.
  • Recombinant SARS-CoV-2 S-RBD216 glycoprotein and recombinant SARS-CoV-2 S-RBD210 glycoprotein can be obtained by the same culture and purification method as in step 1.
  • the SDS-PAGE identification is shown in Figure 19.
  • glycoforms of SARS-CoV-2 S-RBD223, SARS-CoV-2 S-RBD216 and SARS-CoV-2S-RBD210 glycoprotein are Gal 2 GlcNAc 2 Man 3 GlcNAc 2 , GalGlcNAcMan 5 GlcNAc 2 or Man 5 GlcNAc 2 .
  • the first batch The first batch :
  • RBD is the SARS-CoV-2 S-RBD223, SARS-CoV-2 S-RBD216 or SARS-CoV-2 S-RBD210 glycoprotein expressed by CGMCC19488 prepared above.
  • the CpG adjuvant was synthesized by Dalian TaKaRa Company according to the sequence, and the whole chain thio modification was commissioned to be the agent of Beijing Liuhetong Economic and Trade Co., Ltd. (Beijing, China).
  • a 100 ⁇ l volume contains 10 ⁇ g RBD, 100 ⁇ g Al(OH) 3 (based on aluminum content, that is, 100 ⁇ g aluminum content) and 25 ⁇ g CpG adjuvant (or 100 ⁇ l volume contains 10 ⁇ g RBD and 100 ⁇ g Al(OH) 3 (based on aluminum content, that is The aluminum content is 100 ⁇ g))
  • the vaccine is compatible with physiological saline. Each group was immunized with 100 ⁇ l muscle on the 0th and 14th day, and blood was taken on the 28th day.
  • the grouping situation is shown in Table 3.
  • the method is the same as the first batch.
  • the third batch The third batch :
  • the grouping situation is shown in Table 4.
  • the method is the same as the first batch.
  • CpG-1018 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID No. 28);
  • CpG-X1 5'-TCGTTCGTTCGTTCGTTCGTT-3' (SEQ ID No. 29);
  • CpG-684 5'-TCGACGTTCGTCGTTCGTCGTTC-3' (SEQ ID No. 30).
  • the indirect ELISA method was used to determine the anti-RBD antibody titers in the serum of 3 batches of mice.
  • the corresponding SARS-CoV-2 S-RBD cladding plate expressed by CGMCC19488 prepared above is used.
  • the best CPG is full-chain thio-CpG2006, followed by full-chain thio-CpG684 or full-chain thio-CpGX1 or CpG1018.
  • Different doses of RBD or different doses of Al(OH) 3 or different doses of CpG can induce high antibody titers.
  • mice in Example 5 were sera collected 14 days after the second immunization, incubated at 56°C for 30 minutes, and diluted with normal saline at a certain dilution.
  • Virus neutralization test is performed according to conventional methods (reference: Feng Cai Zhu, et al. Safety, tolerance, and immunogenicity of a recombinantadenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first- in-human trial. Lancet. 2020 May 22; S0140-6736(20)31208-3.doi:10.1016/S0140-6736(20)31208-3.). Proceed as follows:
  • Serum dilution Dilute with normal saline according to a certain dilution, set up 3-5 multiple holes.
  • Virus dilution Dilute the virus (virus strain SARS-CoV-2/human/CHN/Wuhan_IME-BJ01/2020, described in the above reference) to 1 ⁇ 10 4 TCID50/ml.
  • the neutralizing antibody titer of the Al(OH) 3 +CpG immunization group can reach more than 1:600, and the antibody titre of the Al(OH) 3 immunization group can reach about 1:25, while the control group is only 1:3.
  • the Al(OH) 3 +CpG immunization group was significantly higher than the pure Al(OH) 3 immunization group, and the difference was extremely significant; while the Al(OH) 3 +CpG2006 immunization group was higher than the other two CpG immunization groups, indicating that even the same
  • the neutralizing antibody titers induced by CpG are also different, that is, CpG2006>CpG684/CpGX1/CpG1018. Therefore, the neutralizing antibody titers induced are measured.
  • the best CpG adjuvant is the full-chain thio-CpG2006.
  • mice After immunizing mice with the vaccine of the present invention, the mice will produce neutralizing antibodies. It is expected that humans will be injected with the vaccine to produce antibodies against the new coronavirus, thereby reducing their risk of developing new coronavirus disease (COVID-19). .

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Abstract

本发明公开了一种预防冠状病毒引起疾病的疫苗。本发明提供了一种预防冠状病毒引起疾病(COVID-19)的疫苗，含有糖基化的冠状病毒S蛋白受体结合区、氢氧化铝佐剂和CpG佐剂。

Description

一种预防冠状病毒引起疾病的疫苗 技术领域
本发明涉及生物医药领域，具体涉及一种预防冠状病毒引起疾病的疫苗。
背景技术
2019年底武汉突发病毒性肺炎疫情，该疫情是由一种以前从未在人体中发现的冠状病毒新毒株引起的，2020年1月12日世界卫生组织将该毒株命名为2019新型冠状病毒(英文简写2019-nCoV)，其所致疾病命名为COVID-19。2020年3月2日，在《自然》杂志子刊《微生物学》上，国际病毒分类学委员会冠状病毒研究小组，将该病毒命名为SARS-CoV-2。该病毒现已引起世界大流行，急需可以诱导对其免疫保护的疫苗用于疫情防控。
冠状病毒(Coronavirus)是自然界广泛存在的一类病毒，主要感染脊椎动物，感染人引起传染病的冠状病毒除上述2019-nCoV外，还有引发重症急性呼吸综合症的SARS-CoV和引发中东呼吸综合症的MERS-CoV。
已有的研究表明，SARS-CoV和MERS-CoV的正链RNA基因组编码的主要结构蛋白有表面刺突蛋白(S)、包膜蛋白(E)、外膜蛋白(E)和核衣壳蛋白(N)等。S蛋白是病毒表面三聚体糖蛋白，通过与细胞表面的受体结合，引导病毒进入宿主细胞，SARS-CoV和MERS-CoV分别以人血管紧张素酶2(ACE2)和人二肽基肽酶(DPP4，又名CD26)作为主要受体。S蛋白是I型膜蛋白，在特定条件下可以被宿主蛋白酶水解为S1和S2亚基，S1与细胞表面受体结合，S2通过天然构象的改变，介导膜融合，使病毒基因组进入宿主细胞，完成感染过程。
疫苗研究发现，S蛋白虽然具有较高的免疫原性，并可诱导机体产生针对SARS-CoV的中和抗体，但是，以全长S蛋白作为抗原可诱发嗜酸细胞免疫病理学或抗体介导的免疫增强ADE等不良反应，其安全性受到广泛质疑。SARS-CoV S蛋白RBD区作为独立的结构域，可形成正确构象，并包含多个空间结构依赖的抗原表位，在动物模型中可诱导高滴度中和抗体、CD8 +T细胞反应以及长期免疫保护效果。
糖基化修饰途径改造的酵母菌株既保留了安全性高、工程株构建周期短、生长快、易于大规模生产等特点，又具有糖基化修饰接近、甚至更优于抗原的天然糖基结构的能力，使其非常适合于在突发传染病和其它应急条件下作为高效、大规模的疫苗生产的表达系统。
发明公开
本发明的目的是提供一种预防冠状病毒引起疾病的疫苗。
本发明所要求保护的预防冠状病毒引起疾病的疫苗，含有糖基化的冠状病毒S蛋白受体结合区、氢氧化铝佐剂和CpG佐剂。
其中，所述冠状病毒可为SARS-CoV-2。相应的，所述冠状病毒引起疾病可为COVID-19。
所述糖基化的冠状病毒S蛋白受体结合区的氨基酸序列可为如下任一：
(a1)SEQ ID No.21(RBD223)；
(a2)SEQ ID No.22(RBD216)；
(a3)SEQ ID No.23(RBD210)；
(a4)将(a1)-(a3)中任一所限定的序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的序列，或与(a1)-(a3)中任一所限定的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的序列。
其中，SEQ ID No.21至SEQ ID No.23所示的3个氨基酸序列均为来源于GenBank号为MN908947.3的SARS-CoV-2"Wuhan-Hu-1"分离株S蛋白的一部分。具体的，SEQ ID No.21为S蛋白的R319-F541区域(RBD223)；SEQ ID No.22为S蛋白的R319-V534区域(RBD216)；SEQ ID No.23为S蛋白的R319-K528区域(RBD210)。
所述糖基化的冠状病毒S蛋白受体结合区为具有哺乳动物糖型结构N-糖链修饰的冠状病毒S蛋白受体结合区。
进一步地，所述哺乳动物糖型结构N-糖链为Gal 2GlcNAc 2Man 3GlcNAc 2、GalGlcNAcMan 5GlcNAc 2或Man 5GlcNAc 2。
所述CpG佐剂可为如下任一：
(b1)核苷酸序列为SEQ ID No.27的CpG2006或其硫代产物；
(b2)核苷酸序列为SEQ ID No.28的CpG-1018或其硫代产物；
(b3)核苷酸序列为SEQ ID No.29的CpGX1或其硫代产物；
(b4)核苷酸序列为SEQ ID No.30的CpG684或其硫代产物；
进一步地，所述硫代产物为全链硫代修饰产物(即每个碱基都经硫代修饰)。
所述CpG佐剂最优的是全链硫代修饰的CpG2006，次之为全链硫代修饰的CpG684、全链硫代修饰的CpGX1或全链硫代修饰的CpG1018。
在所述疫苗中，所述糖基化的冠状病毒S蛋白受体结合区、所述氢氧化铝佐剂(以铝含量计)、所述CpG佐剂的质量比可为(2.5～20)：100：(25～50)。
所述糖基化的冠状病毒S蛋白受体结合区(RBD)可以由哺乳动物细胞或昆虫细胞或酵母等表达制备。
所述糖基化的冠状病毒S蛋白受体结合区(RBD)可按照如下步骤的方法制备得到：
(1)对经过糖基化修饰途径遗传改造的巴斯德毕赤酵母进行再改造，使其能够表达冠状病毒S蛋白受体结合区(RBD)，得到重组酵母细胞；
所述经过糖基化修饰途径遗传改造的巴斯德毕赤酵母为甘露糖基化修饰途径缺陷、并重构了哺乳动物细胞N-糖基化修饰途径的巴斯德毕赤酵母细胞突变体；
(2)培养所述重组酵母细胞，从培养上清中纯化获得具有哺乳动物糖型结构N-糖链修饰的冠状病毒S蛋白受体结合区，即为所述糖基化的冠状病毒S蛋白 受体结合区。
其中，所述经过糖基化修饰途径遗传改造的巴斯德毕赤酵母可按照包括如下步骤的方法制备得到：
(A1)失活受体巴斯德毕赤酵母内源的α-1,6-甘露糖转移酶、磷酸甘露糖转移酶、磷酸甘露糖合成酶、β甘露糖转移酶I、β甘露糖转移酶II、β甘露糖转移酶III和β甘露糖转移酶IV，得到重组酵母1；
(A2)在所述重组酵母1中表达如下外源蛋白中的至少一种：外源甘露糖苷酶I、外源N-乙酰葡萄糖胺转移酶I、外源甘露糖苷酶II、外源N-乙酰葡萄糖胺转移酶II、外源半乳糖异构酶和外源半乳糖转移酶，得到重组酵母2；所述重组酵母2即为所述经过糖基化修饰途径遗传改造的巴斯德毕赤酵母。
在步骤(A2)之后还可包括如下步骤(A3)：
(A3)失活所述重组酵母2内源的O甘露糖转移酶I，得到重组酵母3；所述重组酵母3也为所述经过糖基化修饰途径遗传改造的巴斯德毕赤酵母。
当所述哺乳动物糖型结构N-糖链为Gal 2GlcNAc 2Man 3GlcNAc 2、GalGlcNAcMan 5GlcNAc 2或Man 5GlcNAc 2时，步骤(A2)中在所述重组酵母1中表达的外源蛋白为外源甘露糖苷酶I、外源N-乙酰葡萄糖胺转移酶I、外源半乳糖异构酶和外源半乳糖转移酶、外源甘露糖苷酶II，以及外源N-乙酰葡萄糖胺转移酶II。
当所述哺乳动物糖型结构N-糖链为GalGlcNAcMan 5GlcNAc 2时，步骤(A2)中在所述重组酵母1中表达的外源蛋白为外源甘露糖苷酶I、外源N-乙酰葡萄糖胺转移酶I，以及外源半乳糖异构酶和外源半乳糖转移酶。
当所述哺乳动物糖型结构N-糖链为Man 5GlcNAc 2时，步骤(A2)中在所述重组酵母1中表达的外源蛋白为外源甘露糖苷酶I。
步骤(A1)和(A3)中，失活上述糖基修饰酶，可以通过突变基因的一个或者多个核苷酸序列、或者通过缺失部分或完整基因序列来实现、也可以利用插入核苷酸破坏原有阅读框、提前终止蛋白质合成等方式来实现失活该基因或该基因编码的蛋白质活性。上述突变、缺失和插入失活等可以用常规的诱变、敲除等方法获得。这些方法已有许多文献报道，如J.萨姆布鲁克等，《分子克隆实验指南》第二版，科学出版社，1995。也可用本领域已知的其它方法来构建基因失活的酵母菌株。其中较优的菌株是通过敲除甘露糖转移酶基因的部分序列获得的。该部分序列至少大于三个碱基，较优的是大于100碱基，更优的是包括50％以上的编码序列。这种通过敲除糖基修饰酶基因的部分序列获得的菌株不易产生回复突变，菌株的稳定性比利用点突变等方法构建的稳定性更高，更有利于应用于医疗和工业范围内。
敲除糖基化修饰酶基因的部分序列的方法可以包括：首先构建敲除该基因的质粒：质粒上包括待敲除基因两侧的同源臂序列，两个同源臂应选择在目标基因两侧，所述同源臂的长度至少大于200bp，最优的大小在500bp-2000bp。 也可以利用插入灭活的方式，获得一个氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加，致使没有功能活性的核苷酸序列，并构建到质粒。质粒上还带有URA3(orotidine-5′-phosphate decarboxylase)基因、或博莱霉素、或潮霉素B、或Blasticidin或G418等作为筛选标记。编码侧翼区同源臂片段的多聚核苷酸序列、欲被破坏功能的蛋白的核苷酸序列，可以从公开的美国国立生物技术信息中心(NCBI)获得。利用PCR方法，以毕赤酵母宿主基因组为模板，获得灭活基因所需的一定长度的侧翼同源区，分别包括目的基因(其序列在NCBI中已经公开)基因编码区上游和下游侧翼同源区，并在引物部分添加合适的酶切位点。根据序列获得多聚核苷酸可以用本领域周知的方法，如PCR(J.萨姆布鲁克等，《分子克隆实验指南》第二版，科学出版社，1995.)、RT-PCR方法、人工合成的方法、基因组DNA和构建筛选cDNA文库的方法等获得。若需要可用本领域公知的方法对多聚核苷酸进行突变、缺失、插入、和与其它多聚核苷酸连接等。将分别得到的上游(5′)和下游(3′)侧翼区同源臂片段进行融合，在保持各自片段大小不变的前提下，可以用本领域周知的各种方法，如通过重叠PCR的方法，所用标准的分子克隆过程见J.萨姆布鲁克等(J.萨姆布鲁克等，《分子克隆实验指南》第二版，科学出版社，1995.)的叙述。可用本领域公知的方法分别将含欲灭活基因同源臂序列融合片段的核酸克隆到各种适用于酵母的载体中去。或者利用各自同源臂上酶切位点分别插入载体特定区域。所用标准的分子克隆过程见J.萨姆布鲁克等(J.萨姆布鲁克等，《分子克隆实验指南》第二版，科学出版社，1995.)的叙述。构建重组敲除质粒。原始质粒可以选用适于酵母的表达载体、穿梭载体，可以带有复制位点，筛选标记，营养缺陷型标记(URA3,HIS,ADE1,LEU2,ARG4)等，这些载体的构建方法已在许多文献公开(如J.萨姆布鲁克等，《分子克隆实验指南》第二版，科学出版社，1995)，也可以从各种公司购得(如Invitrogen life technologies,Carlsbad,California 92008,USA)，优先的载体有pPICZαA、pYES2酵母表达载体。灭活载体都是穿梭质粒，先在大肠杆菌中复制扩增，然后被导入宿主酵母细胞，载体应该带有抗性标记基因，或者营养缺陷型标记基因，以利于后期转化子的筛选。
将欲灭活基因两侧同源区(上游称之为5′臂，下游称之为3′臂)分别构建至酵母载体，形成重组敲除载体。进一步利用同源臂的线性化位点线性化敲除载体，通过电转化方法，转化至毕赤酵母或其改构体中的一种，进行培养。转化所需核酸至宿主细胞中去可用通常方法得到，如制备感受态细胞、电穿孔、醋酸锂法等(A.亚当斯等，《酵母遗传学方法实验指南》，科学出版社，2000)。成功转化的细胞，即含有欲敲除基因的同源区的细胞，可以通过人们熟知的技术加以鉴定，如细胞经收集并裂解，提取DNA，然后PCR方法鉴定基因型；而之前选择正确的表型可以通过营养缺陷型或者抗性标记的筛选而得以实现。一次重组正确的转化子，经过在酵母基本培养基培养后，涂布在含尿嘧啶的5-氟乳 清酸平板等二次重组筛选平板，长出的克隆，再进一步进行基因型的PCR鉴定。分别筛选到正确的缺失了预期的基因编码区的转化子。
在本发明的具体实施方式中，步骤(A1)中，所述失活受体巴斯德毕赤酵母内源的α-1,6-甘露糖转移酶、磷酸甘露糖转移酶、磷酸甘露糖合成酶、β甘露糖转移酶I、β甘露糖转移酶II、β甘露糖转移酶III和β甘露糖转移酶IV均是采用同源重组的方式进行基因敲除。
在本发明的具体实施方式中，步骤(A2)中，在所述重组酵母1中表达所述外源蛋白是通过向所述重组酵母1中导入所述外源蛋白的编码基因实现的。
进一步地，所述外源蛋白的编码基因是以重组载体的形式导入所述重组酵母1中的。
进一步地，所述外源甘露糖苷酶I的编码基因和所述外源甘露糖苷酶II的编码基因均向所述重组酵母1中导入两次。
在本发明的具体实施方式中，步骤(A3)中，失活所述重组酵母2内源的O甘露糖转移酶I，是通过对所述重组酵母2的基因组DNA中的O甘露糖转移酶I编码基因进行插入失活实现的。
在本发明中，具体是在所述重组酵母2的基因组DNA中的O甘露糖转移酶I编码基因的靶标片段的前端和末端各装上不同组合的终止密码子，并且在末端的终止密码子之后装上终止子(如CYC1TT终止子)。前端和末端各装上不同组合的终止密码子后的所述靶标片段具体为以毕赤酵母JC308的基因组DNA为模板，利用引物PMT1-IN-5和PMT1-IN-3进行PCR扩增所得的片段。
PMT1-IN-5：
5’-tctatgcattaatgatagttaatgactaatagagtaaaacaagtcctcaagaggt-3’(SEQ ID No.101)；
PMT1-IN-3：
5’-tgacataactaattacatgatctattagtcattaactatcattagatcagagtggggacgacta agaaa gc-3’(SEQ ID No.102)。
步骤(A2)中，所述外源甘露糖苷酶I表达后定位于内质网。
在本发明中，所述外源甘露糖苷酶I来源于绿色木霉，且C端融合内质网保留信号HDEL。
步骤(A2)中，所述外源N-乙酰葡萄糖胺转移酶I表达后定位于内质网或内侧高尔基体。
在本发明中，所述外源N-乙酰葡萄糖胺转移酶I来源于哺乳动物，在N-端或C-端融合内质网或内侧高尔基体定位信号。
进一步地，所述外源N-乙酰葡萄糖胺转移酶I来源于人，且含有mnn9定位信号。
步骤(A2)中，所述外源甘露糖苷酶II表达后定位于内质网或内侧高尔基体。
在本发明中，所述外源甘露糖苷酶II来源于丝状真菌、植物、昆虫、爪哇或哺乳动物，在N-端或C-端融合内质网或内侧高尔基体定位信号。
步骤(A2)中，所述外源N-乙酰葡萄糖胺转移酶II表达后定位于内质网或内侧高尔基体。
在本发明中，所述外源N-乙酰葡萄糖胺转移酶II来源于哺乳动物，在N-端或C-端融合内质网或内侧高尔基体定位信号。
进一步地，所述N-乙酰葡萄糖胺转移酶II来源于人，且均含有mnn2定位信号。
步骤(A2)中，所述外源甘露糖苷酶II表达后定位于内质网或内侧高尔基体。
所述外源甘露糖苷酶II来源于线虫，含有mnn2定位信号。
步骤(A2)中，所述外源半乳糖异构酶和所述外源半乳糖转移酶表达后定位于内质网或内侧高尔基体。
所述外源半乳糖异构酶和所述外源半乳糖转移酶均来源于哺乳动物，在N-端或C-端融合内质网或内侧高尔基体定位信号。
进一步地，所述外源半乳糖异构酶和所述外源半乳糖转移酶为融合蛋白，均来源于人，且共用一个kre2定位信号。
所述α-1,6-甘露糖转移酶可为如下B1)或B2)：
B1)氨基酸序列是SEQ ID No.1的蛋白质；
B2)将SEQ ID No.1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.1所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
所述磷酸甘露糖转移酶可为如下B3)或B4)：
B3)氨基酸序列是SEQ ID No.2的蛋白质；
B4)将SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.2所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
所述磷酸甘露糖合成酶可为如下B5)或B6)：
B5)氨基酸序列是SEQ ID No.3的蛋白质；
B6)将SEQ ID No.3所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.3所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
所述β甘露糖转移酶I可为如下B7)或B8)：
B7)氨基酸序列是SEQ ID No.4的蛋白质；
B8)将SEQ ID No.4所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.4所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
所述β甘露糖转移酶II可为如下B9)或B10)：
B9)氨基酸序列是SEQ ID No.5的蛋白质；
B10)将SEQ ID No.5所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.5所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
所述β甘露糖转移酶III可为如下B11)或B12)：
B11)氨基酸序列是SEQ ID No.6的蛋白质；
B12)将SEQ ID No.6所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.6所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
所述β甘露糖转移酶IV可为如下B13)或B14)：
B13)氨基酸序列是SEQ ID No.7的蛋白质；
B14)将SEQ ID No.7所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.7所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
所述O甘露糖转移酶I可为如下B15)或B16)：
B15)氨基酸序列是SEQ ID No.8的蛋白质；
B16)将SEQ ID No.8所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.8所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
所述外源甘露糖苷酶I可为如下B17)或B18)：
B17)氨基酸序列是SEQ ID No.9的蛋白质；
B18)将SEQ ID No.9所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.9所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
所述外源N-乙酰葡萄糖胺转移酶I可为如下B19)或B20)：
B19)氨基酸序列是SEQ ID No.10的蛋白质；
B20)将SEQ ID No.10所示的氨基酸序列经过一个或几个氨基酸残基的取代 和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.10所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
由所述半乳糖异构酶和所述半乳糖转移酶组成的所述融合蛋白可为如下B21)或B22)：
B21)氨基酸序列是SEQ ID No.11的蛋白质；
B22)将SEQ ID No.11所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.11所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
所述甘露糖苷酶II可为如下B23)或B24)：
B23)氨基酸序列是SEQ ID No.12的蛋白质；
B24)将SEQ ID No.12所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.12所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
所述N-乙酰葡萄糖胺转移酶II可为如下B25)或B26)：
B25)氨基酸序列是SEQ ID No.13的蛋白质；
B26)将SEQ ID No.13所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.13所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
所述外源甘露糖苷酶I的编码基因可为如下C1)或C2)：
C1)核苷酸序列是SEQ ID No.14的DNA分子；
C2)与SEQ ID No.14所示的核苷酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且编码所述外源甘露糖苷酶I的DNA分子，或在严格条件下与C1)限定的DNA分子杂交且编码所述外源甘露糖苷酶I的DNA分子。
所述外源N-乙酰葡萄糖胺转移酶I的编码基因可为如下C3)或C4)：
C3)核苷酸序列是SEQ ID No.15的DNA分子；
C4)与SEQ ID No.15所示的核苷酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且编码所述外源N-乙酰葡萄糖胺转移酶I的DNA分子，或在严格条件下与C3)限定的DNA分子杂交且编码所述外源N-乙酰葡萄糖胺转移酶I的DNA分子。
由所述半乳糖异构酶和所述半乳糖转移酶组成的所述融合蛋白的编码基因可为如下C5)或C6)：
C5)核苷酸序列是SEQ ID No.16的DNA分子；
C6)与SEQ ID No.16所示的核苷酸序列具有99％以上、95％以上、90％以上、 85％以上或者80％以上同源性且编码所述融合蛋白的DNA分子，或在严格条件下与C5)限定的DNA分子杂交且编码所述融合蛋白的DNA分子。
所述甘露糖苷酶II的编码基因可为如下C7)或C8)：
C7)核苷酸序列是SEQ ID No.17的DNA分子；
C8)与SEQ ID No.17所示的核苷酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且编码所述甘露糖苷酶II的DNA分子，或在严格条件下与C7)限定的DNA分子杂交且编码所述甘露糖苷酶II的DNA分子。
所述N-乙酰葡萄糖胺转移酶II的编码基因可为如下C9)或C10)：
C9)核苷酸序列是SEQ ID No.18的DNA分子；
C10)与SEQ ID No.18所示的核苷酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且编码所述N-乙酰葡萄糖胺转移酶II的DNA分子，或在严格条件下与C9)限定的DNA分子杂交且编码所述N-乙酰葡萄糖胺转移酶II的DNA分子。
本发明所有糖基修饰酶相关信息都可以在美国国立生物技术信息中心(NCBI)或者公开的文献中获得，相关酶的功能、定义也可以在文献中获得。即使是同一种菌或物种，由于来源不同等，各种酶的氨基酸会略有差别，但其功能基本相同，因此，本发明所述酶也可包括这些变异体。
进一步地，所述经过糖基化修饰途径遗传改造的巴斯德毕赤酵母为在中国微生物菌种保藏管理委员会普通微生物中心保藏的保藏编号为CGMCCNo.19488的菌株。
该酵母制备的SARS-CoV-2S-RBD糖蛋白具有无岩藻糖侧链的复杂型、杂合型N-糖基修饰，且相比于哺乳动物细胞和昆虫细胞，利用该酵母表达SARS-CoV-2S-RBD糖蛋白具有制备成本低廉、周期短、易于大规模生产等优势。
在步骤(1)中，所述重组酵母细胞是将所述冠状病毒S蛋白受体结合区(RBD)的编码基因导入所述经过糖基化修饰途径遗传改造的巴斯德毕赤酵母中后得到的。
进一步地，所述冠状病毒S蛋白受体结合区(RBD)的编码基因是通过重组载体的形式导入所述经过糖基化修饰途径遗传改造的巴斯德毕赤酵母中。
其中，所述重组载体中启动所述冠状病毒S蛋白受体结合区(RBD)的编码基因转录的启动子可为AOX1启动子。
在本发明中，所述重组载体具体为将所述冠状病毒S蛋白受体结合区(RBD)的编码基因克隆到pPICZαA载体的AOX1启动子下游(如酶切位点XhoⅠ和NotⅠ)之间后得到的重组载体。
进一步地，所述冠状病毒S蛋白受体结合区(RBD)的编码基因可为如下任一：
(c1)SEQ ID No.24所示DNA分子；
(c2)SEQ ID No.25所示DNA分子；
(c3)SEQ ID No.26所示DNA分子；
(c4)与SEQ ID No.24至SEQ ID No.26中任一所示的核苷酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且编码所述冠状病毒S蛋白受体结合区的DNA分子，或在严格条件下与SEQ ID No.24至SEQ ID No.26中任一所示的DNA分子杂交且编码所述冠状病毒S蛋白受体结合区的DNA分子。
SEQ ID No.24至SEQ ID No.26的核苷酸序列是分别根据SEQ ID No.21至SEQ ID No.23的氨基酸序列经密码子优化获得，通过全基因合成获得相应序列的DNA片段。
上述蛋白质中，同源性是指氨基酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性，如NCBI主页网站的BLAST网页。例如，可在高级BLAST2.1中，通过使用blastp作为程序，将Expect值设置为10，将所有Filter设置为OFF，使用BLOSUM62作为Matrix，将Gap existence cost，Per residue gap cost和Lambda ratio分别设置为11，1和0.85(缺省值)并进行检索一对氨基酸序列的同一性进行计算，然后即可获得同一性的值(％)。
上述基因中，同源性是指核苷酸序列的同一性。可使用国际互联网上的同源性检索站点测定核苷酸序列的同一性，如NCBI主页网站的BLAST网页。例如，可在高级BLAST2.1中，通过使用blastp作为程序，将Expect值设置为10，将所有Filter设置为OFF，使用BLOSUM62作为Matrix，将Gap existence cost，Per residue gap cost和Lambda ratio分别设置为11，1和0.85(缺省值)并进行检索一对核苷酸序列的同一性进行计算，然后即可获得同一性的值(％)。
上述蛋白质和基因中，所述95％以上的同源性可为至少96％、97％、98％的同一性。所述90％以上的同源性可为至少91％、92％、93％、94％的同一性。所述85％以上的同源性可为至少86％、87％、88％、89％的同一性。所述80％以上的同源性可为至少81％、82％、83％、84％的同一性。
上述基因中，所述严格条件可为如下：50℃，在7％十二烷基硫酸钠(SDS)、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，2×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，0.5×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，0.1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在65℃，0.1×SSC，0.1％SDS中漂洗；也可为：在6×SSC，0.5％SDS的溶液中，在65℃下杂交，然后用2×SSC，0.1％SDS和1×SSC，0.1％SDS各洗膜一次。
步骤(2)中，分离含有SARS-CoV-2 S-RBD糖蛋白的组份可以包括利用离心、过滤等固液分离方法分离培养液和细胞(或菌体)，含SARS-CoV-2S-RBD糖蛋白的组份分离可以包含亲和、离子交换、凝胶过滤、疏水等液相层析方法。
进一步地，步骤(2)中，可按照包括如下步骤的方法从所述培养上清中纯 化获得所述具有哺乳动物糖型结构N-糖链修饰的冠状病毒S蛋白受体结合区：将所述培养上清依次进行阳离子交换层析、疏水层析、G25脱盐、阴离子交换层析，获得所述具有哺乳动物糖型结构N-糖链修饰的冠状病毒S蛋白受体结合区。
在本发明的具体实施方式中，步骤(2)是按照包括如下步骤的方法从所述培养上清中纯化获得所述具有哺乳动物糖型结构N-糖链修饰的冠状病毒S蛋白受体结合区的：将所述培养上清通过CaptoMMC层析柱进行目的蛋白的捕获，然后通过含有1M NaCl的缓冲液洗脱获得含有所述目的蛋白的粗样；之后将所述粗样用疏水层析柱Phenyl HP纯化，将含有所述目的蛋白的洗脱峰样品用G25层析柱除盐，然后用阴离子交换层析柱Source30Q吸附杂蛋白，流穿液即是所述目的蛋白；所述目的蛋白即为所述具有哺乳动物糖型结构N-糖链修饰的冠状病毒S蛋白受体结合区。
另外，本发明还要求保护一种制备前文所述疫苗的方法。
本发明所要求保护的制备前文所述疫苗的方法，可包括如下步骤：按照前文所述方法制备得到所述糖基化的冠状病毒S蛋白受体结合区；然后将所述糖基化的冠状病毒S蛋白受体结合区、氢氧化铝佐剂(以铝含量计)和前文所述CpG佐剂按照质量比为(2.5～20)：100：(25～50)的比例混合制备得到所述疫苗。
再有，本发明还要求保护如下任一应用：
P1、前文所述疫苗在预防冠状病毒引起疾病中的应用；
P2、前文所述疫苗在中和冠状病毒中的应用。
其中，所述冠状病毒可为SARS-CoV-2。相应地，所述冠状病毒引起疾病可为COVID-19。
最后，本发明还要求保护一种预防冠状病毒引起疾病的方法，是利用前文所述疫苗预防冠状病毒引起疾病。
其中，所述冠状病毒可为SARS-CoV-2。相应地，所述冠状病毒引起疾病可为COVID-19。
保藏说明
菌株拉丁名：Pichia pastoris
参椐的生物材料：GJK30
建议的分类命名：巴斯德毕赤酵母
保藏机构：中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称：CGMCC
地址：北京市朝阳区北辰西路1号院3号
保藏日期：2020年03月18日
保藏中心登记入册编号：CGMCC No.19488
附图说明
图1为GJK01菌中och1基因的鉴定以及糖型分析结果。A为och1基因鉴定结果。M代表Marker；1：GJK01菌(已敲除och1)；2：X33菌(未敲除och1)。B 为GJK01菌(敲除och1)表达的抗体的DSA-FACE糖型分析结果。
图2为pno1基因鉴定结果。M代表Marker；1：GJK02菌(已敲除pno1)；2：X33菌(未敲除pno1)。
图3为mnn4b基因鉴定结果。M代表Marker；1：GJK03菌(已敲除mnn4b)；2：X33菌(未敲除mnn4b)。
图4为GJK01、GJK02、GJK03菌(已敲除och1、pno1、mnn4b)的DSA-FACE糖型分析结果。
图5为ARM2基因鉴定结果。M代表Marker；1：GJK04菌(已敲除ARM2)；2：X33菌(未敲除ARM2)。
图6为ARM1基因鉴定结果。M代表Marker；1：GJK05菌(已敲除ARM1)；2：X33菌(未敲除ARM1)。
图7为ARM3基因鉴定结果。M代表Marker；1：GJK07菌(已敲除ARM3)；2：X33菌(未敲除ARM3)。
图8为ARM4基因鉴定结果。M代表Marker；1：GJK18菌(已敲除ARM4)；2：X33菌(未敲除ARM4)。
图9为GJK18菌的DSA-FACE糖型分析结果。
图10为W10菌的TrmdsI基因鉴定结果和DSA-FACE糖型分析结果。A为TrmdsI基因鉴定结果。M代表Marker；1：W10菌中导入TrmdsI；X33菌中无TrmdsI。B为W10菌的DSA-FACE糖型分析结果。
图11为1-8菌的GnTI基因鉴定结果和DSA-FACE糖型分析结果。A为GnTI基因鉴定结果。M代表Marker；1：1-8菌中导入GnTI；2：X33菌中无GnTI。B为1-8菌的DSA-FACE糖型分析结果。
图12为1-8-4菌的GalE-GalT基因鉴定结果和DSA-FACE糖型分析结果。GalE-GalT基因鉴定结果。M代表Marker；1：1-8-4菌中导入GalE-GalT；2：X33菌中无GalE-GalT。B为1-8-4菌的DSA-FACE糖型分析结果。
图13为52-60和150L2菌的mdsII基因、GnTII基因鉴定结果和DSA-FACE糖型分析结果。A为MdsII基因鉴定结果。M代表Marker；1：52-60菌中导入MdsII；2：X33菌中无MdsII。B为GnTII基因鉴定结果。M代表Marker；1：150L2菌中导入GnTII；2：X33菌中无GnTII。C为52-60菌的DSA-FACE糖型分析结果。
图14为PMT1插入失活基因鉴定结果。M代表Marker；1：X33菌PMT1未失活；2：GJK30(PMT1失活)。
图15为GJK30工程菌的糖型结构分析结果。A为前期Gal2GlcNAc2Man3GlcNAc2结构低于50％；B为GJK30工程菌获得的Gal2GlcNAc2Man3GlcNAc2结构所占糖型比例大于60％；C为通过糖苷酶(New England Biolabs，Beijing)对该糖型进行酶切分析。
图16为CGMCC19488/S-RBD223阳性克隆筛选WB验证图。上半部分为SDS-PAGE电泳分析，下半部分为Western Blotting分析；泳道1-7为不同的表达克隆。
图17为CGMCC19488/S-RBD223不同诱导时间电泳检测图。
图18为SARS-CoV-2 S-RBD223纯化样品SDS-PAGE图。
图19为SARS-CoV-2 S-RBD216和SARS-CoV-2 S-RBD210纯化样品SDS-PAGE图。
图20为CGMCC19488表达的SARS-CoV-2 S-RBD223、SARS-CoV-2 S-RBD216和SARS-CoV-2 S-RBD210糖蛋白的DSA-FACE糖链分析结果。
图21为二免14天后各种小鼠血清抗RBD抗体滴度。A为第一批次；B为第二批次；C为第三批次。ns表示无显著差异，**表示在P<0.01水平有极显著差异。
图22为病毒中和试验结果。A为第一批次；B为第二批次；C为第三批次。ns表示无显著差异，*表示在P<0.05水平有极显著差异，***表示在P<0.001水平有极显著差异。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
除非另外说明，本文所用的所有技术和科学术语具有与本发明所属领域的普通技术人通常理解的相同的意思。示例性的方法和材料描述如下，虽然与本文描述的类似或等同的方法和材料也可以用于实施本发明，这对本领域技术人员来说是显而易见的。本文提及的所有出版物和其它参考文献都以引用的方式引入其全文。在不一致的情况下，以本说明书，包括定义，为准。材料，方法和实施例仅是举例说明而不是进行限制。
pPICZαA、pYES2载体、X33、GS115毕赤酵母为Invitrogen公司产品。
毕赤酵母GJK01CGMCC No.1853(记载发明专利ZL200610164912.8中，公开号为CN101195809，为失活了α-1,6-甘露糖转移酶的毕赤酵母)。
实验中所使用的Pyrobest酶、LA Taq酶、dNTPs、限制性内切酶、T4连接酶等购自大连宝生物工程有限公司，pfu酶、试剂盒、DH5α感受态细胞为北京全式金有限公司产品。全基因合成、核苷酸合成、引物合成、测序等由上海生工生物工程技术服务有限公司提供。
SARS-CoV-2(2019-nCoV)Spike RBD-Fc Recombinant Protein(40592-V02H)为北京义翘神州生物科技有限公司产品；羊抗兔IgG二抗(SAB3700885)为Sigma公司产品；羊抗鼠IgG二抗(ab205719)为abcam公司产品；BglⅡ限制性内切酶为NEB公司产品；PNGaseF(P0708)、Endo H(P0702)为NEB公司产品。
Capto MMC层析介质、Phenyl HP、G25、Source30Q均购自GE Healthcare公司。
下述实施例中所涉及的相关修饰酶的序列信息如表1所示。
表1 本发明所涉及的相关修饰酶
Figure PCTCN2021093756-appb-000001
Figure PCTCN2021093756-appb-000002
实施例1、构建经过糖基化修饰途径遗传改造的巴斯德毕赤酵母
一、磷酸甘露糖转移酶基因灭活的酵母菌株构建
本发明采用的基础菌株为我们前期构建的GJK01菌株，保藏号为CGMCC No.1853，菌株授权专利号：ZL200610164912.8。该菌株为α-1,6-甘露糖转移酶灭活的毕赤酵母菌株。α-1,6-甘露糖转移酶(OCH1)的氨基酸序列如SEQ ID No.1所示。
磷酸甘露糖转移酶基因灭活的酵母菌株GJK02为将毕赤酵母GJK01中编码SEQ ID No.2所示磷酸甘露糖转移酶的DNA分子部分敲除而获得，即敲除GJK01酵母基因组中的磷酸甘露糖转移酶基因，得到的重组酵母。
1、构建磷酸甘露糖转移酶基因灭活载体
用于敲除甘露糖转移酶(PNO1)基因的敲除质粒pYES2-pno1为将甘露糖转移酶(PNO1)对应的基因片段(SEQ ID No.20)插入载体pYES2的KpnI和XbaI酶切位点间得到的载体。其中SEQ ID No.20自5’末端第7-1006位核苷酸为敲除甘露糖转移酶(PNO1)基因片段的上游同源臂；SEQ ID No.20自5’末端第1015-2017位核苷酸为敲除甘露糖转移酶(PNO1)基因片段的下游同源臂。
具体如下：
用玻璃珠制备法(A.亚当斯等，《酵母遗传学方法实验指南》，科学出版社，2000)提取毕赤酵母X33的基因组DNA，以该基因组DNA为模板扩增甘露糖转移酶(PNO1)基因两侧的同源臂，PNO1两侧的同源臂分别约为1kb，中间缺失约1.4kb的编码基因。
扩增pno1上游侧翼区同源臂(PNO1 5′同源臂)所用的引物为PNO-5-5和PNO-5-3，引物序列分别为：
5′-AGT GGTACCGCAGTTTAATCATAGCCCACTGC-3′(SEQ ID No.31，划线部分为Kpn I识别位点)；
5′-ATTCCAATACCAAGAAAGTAAAGT gcggccgcAAGTGGAACTGGCGCACCGGT-3′(SEQ ID No.32，划线部分为Not I识别位点)。
扩增PNO1下游侧翼区同源臂(PNO13′同源臂)所用的引物为PNO-3-5和PNO-3-3，引物序列分别为：
5′-ACCGGTGCGCCAGTTCCACTT gcggccgcACTTTACTTTCTTGGTATTGGAAT-3′(SEQ ID No.33，划线部分为Not I识别位点)；
5′-TGT TCTAGATCCGAGATTTTGCGCTATGGAGC-3′(SEQ ID No.34，划线部分为Xba I识别位点)。
两个同源臂的PCR扩增条件如下：94℃变性5min后，按照94℃变性30sec、55℃复性30sec、72℃延伸1min30sec进行30次循环，最后72℃延伸10min；目的片段大小在1kb左右。将PCR产物用PCR产物回收纯化试剂盒纯化回收(购自鼎国生物技术有限公司，北京)。利用重叠延伸PCR的方法融合PNO1 5′同源臂和3′同源臂(参见J.萨姆布鲁克等，《分子克隆实验指南》第二版，科学出版社，1995)，以PNO1 5′同源臂和3′同源臂PCR产物为模板，以PNO-5-5/PNO-3-3为引物，PCR扩增条件如下：94℃变性5min后，按照94℃变性1min、55℃复性1min、72℃延伸3min30sec进行30次循环，最后72℃延伸10min；目的片段大小在2kb左右。PCR产物用PCR产物回收纯化试剂盒纯化回收。
Kpn I/Xba I双酶切(本试验所用的限制性内切酶均来自宝生物工程有限公司，大连)PCR产物，酶切后产物插入同样双酶切处理的载体pYES2(Invitrogen Corp.USA)中，T4连接酶16℃连接过夜，转化大肠杆菌DH5α，在含氨苄青霉素(100μg/ml)的LB平板上筛选阳性克隆。用Kpn I/Xba I双酶切鉴定阳性克隆的质粒，得到4200bp左右和2000bp左右片段的重组载体命名为pYES2-pno1，即为用于敲除甘露糖转移酶(PNO1)基因的敲除质粒，pno1基因上下游同源臂并经最终测序验证正确。
2、敲除质粒对毕赤酵母的转化
采用电转化法将敲除质粒pYES2-pno1转化入毕赤酵母GJK01(记载发明专利ZL200610164912.8中，公开号为CN101195809)中，电转化的方法为本领域所共知的(如A.亚当斯等，《酵母遗传学方法实验指南》，科学出版社，2000)。电转化前，先将敲除质粒用5’同源臂上游BamH I酶切位点线性化，然后电转入制备好的感受态细胞中，涂布于含有精氨酸和组氨酸的MD培养基(YNB1.34g/100mL，生物素4×10 -5g/100mL，葡萄糖2g/100mL，琼脂1.5g/100mL，精氨酸100mg/ml，组氨酸100mg/ml)上。待培养基上长出克隆后，随机挑取几 个克隆提取基因组，通过PCR的方法鉴定敲除质粒是否正确整合到了染色体上的目标位点，PCR反应所用的两对引物分别是：PNO1基因5’同源臂外的引物序列PNO-5-5OUT：5′-GCAGTTTAATCATAGCCCACTGCTA-3′(SEQ ID No.35)和载体上的引物序列inner01：5′-AGCGTCGATTTTTGTGATGCTCGTCA-3′(SEQ ID No.36)。PCR反应所用的酶为rTaq(宝生物工程有限公司)，PCR扩增条件如下：94℃变性5min后，按照94℃变性30sec、55℃复性30sec、72℃延伸3min进行30次循环，最后72延伸10min。通过凝胶电泳分析PCR产物条带的大小，引物所扩增的条带在2.3kb左右为阳性克隆。
3、PCR鉴定阳性工程菌株
将其中一个阳性克隆接种于YPD培养基(10g/L酵母提取物，20g/L蛋白胨，20g/L葡萄糖)中，25℃摇床培养12小时后，将菌液涂布于腺嘌呤缺陷的5-FOA培养基(YNB 1.34g/100mL，生物素4×10-5g/100mL，葡萄糖2g/100mL，琼脂1.5g/100mL，精氨酸100mg/ml,组氨酸100mg/ml，尿嘧啶100mg/ml，5-FOA0.1％)(其中，YNB，为无氨基酸酵母氮源，为北京欣经科生物技术有限公司产品，5-FOA为5-氟尿嘧啶，来自Sigma-aldrich P.O.BOX14508,St.Louis,MO63178USA)，置于25℃培养。
待5-FOA培养基上长出克隆后，提取这些克隆的基因组，进行PCR鉴定：以基因组为模板，鉴定引物为染色体上pno1基因同源臂外的序列PNO1-ORF01和PNO1-ORF02，引物序列分别为：
PNO1-ORF01：5′-GGGAAAGAAAACCTTCAATTT-3′(SEQ ID No.37)；
PNO1-ORF02：5′-TACAAGCCAGTTTCGCAATAA-3′(SEQ ID No.38)。
同时将以野生型X33菌株(Invitrogen公司)的基因组为模板的PCR反应体系设为对照。PCR反应所用的酶为LA Taq(宝生物工程有限公司)，PCR扩增条件如下：94℃变性5min后，按照94℃变性30sec、55℃复性30sec、72℃延伸3min进行30次循环，最后72延伸10min。
为了鉴定α-1,6-甘露糖转移酶是否敲除，本发明在获得GJK01工程菌后引入了一个报告蛋白，本发明以抗Her2抗体为报告蛋白，抗Her2抗体的表达载体的构建方法、载体转化方法已经在申请专利中公开(公开号：CN101748145A)。利用该方法将抗Her2抗体表达载体转入至GJK01宿主菌中，获得了表达抗Her2抗体的GJK01-HL工程菌株。DSA-FACE糖型分析方法已经公开报道于“刘波等.一种利用DSA-FACE分析寡糖链的方法.生物技术通讯.2008.19(6).885-888”一文。
将产物进行琼脂糖凝胶电泳。图1中A为GJK01宿主菌的鉴定结果；图1中B为GJK01-HL菌(敲除och1)的DSA-FACE糖型分析结果所示。图2中泳道1为PON1缺陷型，泳道2为野生型；以野生型X33菌株基因组为模板的PCR产物大小在490bp左右，PON1缺陷型工程菌无扩增条带，也证明了PNO1基因的丢失，磷酸甘露糖转移酶敲除的菌株构建正确，命名为GJK02，为磷酸甘露糖转移 酶敲除的重组毕赤酵母菌。
二、磷酸甘露糖合成酶基因灭活的酵母菌株构建
磷酸甘露糖合成酶基因灭活的酵母菌株GJK03为将毕赤酵母GJK02中编码SEQ ID No.3所示磷酸甘露糖合成酶的DNA分子部分敲除而获得，即敲除GJK02酵母基因组中的磷酸甘露糖合成酶基因，得到的重组酵母；即该酵母的α-1,6-甘露糖转移酶、磷酸甘露糖转移酶和磷酸甘露糖合成酶灭活。
构建载体的方法与步骤一相同。
1、构建磷酸甘露糖合成酶基因灭活载体
用于敲除磷酸甘露糖合成酶基因的敲除质粒pYES2-MNN4B为将磷酸甘露糖合成酶对应的欲敲除除基因片段的上下游同源臂插入载体pYES2的Stu I和Spe I酶切位点间得到的载体。
利用同上述一的方法，用玻璃珠制备法提取毕赤酵母X33的基因组DNA，以该基因组DNA为模板扩增敲除甘露糖合成酶(MNN4B)基因片段，MNN4B两侧的同源臂分别约为1kb，中间缺失约1kb的编码基因。
扩增MNN4B上游侧翼区同源臂(ARM25′同源臂)所用的引物为MNN4B-5-5和MNN4B-5-3，引物序列分别为：
5′-AGT AGGCCTTTCAACGAGTGACCAATGTAGA-3′(SEQ ID No.39，划线部分为Stu I识别位点)；
5′-TATCTCCATAGTTTCTAAGCAGG GCGGCCGCAATATGTGCGGTGTAGGGAGAAA-3′(SEQ ID No.40，划线部分为Not I识别位点)。
扩增MNN4B下游侧翼区同源臂(MNN4B 3′同源臂)所用的引物为MNN4B-3-5和MNN4B-3-3，引物序列分别为：
5′-TTTCTCCCTACACCGCACATATT GCGGCCGCCCTGCTTAGAAACTATGGAGATA-3′(SEQ ID No.41，划线部分为Not I识别位点)；
5′-TGT ACTAGTTGAAGACGTCCCCTTTGAACA-3′(SEQ ID No.42，划线部分为Spe I识别位点)。
两个同源臂的PCR扩增条件、回收方法、以及酶切方法都同步骤1，最终构建获得pYES2-MNN4B敲除载体，并经最终测序验证正确。
2、敲除质粒对毕赤酵母的转化
敲除质粒采用电转化法将敲除质粒转化入上述一构建的毕赤酵母工程菌株GJK02中，电转化的方法、鉴定方法同步骤一。
PCR反应所用的两对引物分别是：mnn4b基因5’同源臂外的引物序列MNN4B-5-5OUT：5′-TAGTCCAAGTACGAAACGACACTA-3′(SEQ ID No.43)和载体上的引物序列inner01：5′-AGCGTCGATTTTTGTGATGCTCGTCA-3′(SEQ ID No.36)，引物所扩增的条带在2kb左右为阳性克隆。
3、PCR鉴定阳性工程菌株
将其中一个阳性克隆接种于5-FOA培养基(配方同前)上长出克隆后，提 取这些克隆的基因组，进行PCR鉴定：以基因组为模板，鉴定引物为染色体上mnn4b基因同源臂外的序列MNN4B-ORF01和MNN4B-ORF02，引物序列：
MNN4B-ORF01：5'-AAAACTATCCAATGAGGGTCTC-3'(SEQ ID No.44)；
MNN4B-ORF02：5'-TCTTCAATGTCTTTAACGGTGT-3'(SEQ ID No.45)。
以阳性克隆基因组DNA为模板，利用引物MNN4B-ORF01和MNN4B-ORF02进行PCR扩增。结果如图3所示，泳道1为MNN4B缺陷型，泳道2为野生型；以野生型X33菌株基因组为模板的PCR产物大小在912bp左右，MNN4缺陷型工程菌无扩增条带，也证明了磷酸甘露糖合成酶敲除的，命名为GJK03，为磷酸甘露糖转移酶和磷酸甘露糖合成酶敲除的重组毕赤酵母菌。
GJK02、GJK03菌(已敲除och1、pno1、mnn4b)的DSA-FACE糖型分析结果(方法同实施例一中所述)如图4所示，可见pno1、mnn4b敲除后糖型中的磷酸甘露糖部分被去除了。
三、β甘露糖转移酶基因ARM2灭活的酵母菌株构建
磷酸甘露糖转移酶、磷酸甘露糖合成酶和β甘露糖转移酶ARM2(即β甘露糖转移酶II)基因灭活的酵母菌株GJK04为毕赤酵母GJK03中编码SEQ ID No.5所示β甘露糖转移酶ARM2的DNA分子部分敲除而获得，即敲除GJK03酵母基因组中的β甘露糖转移酶ARM2基因，得到的重组酵母；即酵母基因组中的α-1,6-甘露糖转移酶、磷酸甘露糖转移酶基因、磷酸甘露糖合成酶基因和β甘露糖转移酶ARM2已被灭活。
1、构建β甘露糖转移酶ARM2基因灭活载体
载体构建方法同步骤一，具体如下：
利用同上述一的方法，用玻璃珠制备法提取毕赤酵母X33的基因组DNA，以该基因组DNA为模板扩增β甘露糖转移酶(ARM2)基因两侧的同源臂，ARM2两侧的同源臂分别约为0.6kb，中间缺失约0.6kb的编码基因。
扩增ARM2上游侧翼区同源臂(ARM2 5′同源臂)所用的引物为ARM2-5-5和ARM2-5-3，引物序列分别为：
5′-ActT GGTACCACACGACTCAACTTCCTGCTGCTC-3′(SEQ ID No.46，划线部分为Kpn I识别位点)；
5′-act GCGGCCGCCACGAAACTTCTTACCTTTGACAA-3′(SEQ ID No.47，划线部分为Not I识别位点)。
扩增ARM2下游侧翼区同源臂(ARM23′同源臂)所用的引物为ARM2-3-5和ARM2-3-3，引物序列分别为：
5′-TTGTCAAAGGTAAGAAGTTTCGT GGCGGCCGCTATCTTGACATTGTCATTCAGTGA-3′(SEQ ID No.48，划线部分为Not I识别位点)；
5′-caa TCTAGAGCCTCCTTCTTTTCCGCCT-3′(SEQ ID No.49，划线部分为Xba I识别位点)。
2、敲除质粒对毕赤酵母的转化
敲除质粒采用电转化法将敲除质粒转化入上述一构建的毕赤酵母工程菌株GJK03中，电转化的方法、鉴定方法同上述一。
PCR反应所用的两对引物分别是：ARM2基因5’同源臂外的引物序列ARM2-5-5OUT：5′-TTTTCCTCAAGCCTTCAAAGACAG-3′(SEQ ID No.50)和载体上的引物序列inner01：5′-AGCGTCGATTTTTGTGATGCTCGTCA-3′(SEQ ID No.36)，引物所扩增的条带在0.8kb左右为阳性克隆。
3、PCR鉴定阳性工程菌株
将其中一个阳性克隆接种于5-FOA培养基(配方同前)上长出克隆后，提取这些克隆的基因组，进行PCR鉴定：以基因组为模板，鉴定引物为染色体上ARM2基因同源臂外的序列Arm-ORF01和Arm-ORF02，引物序列：
Arm2-ORF-09：5'-gggcagaagatcctagag-3'(SEQ ID No.51)；
Arm2-ORF-10：5'-tcgtctccattgctatctacgact-3'(SEQ ID No.52)。
以阳性克隆基因组DNA为模板，用引物Arm2-ORF-09和Arm2-ORF-10进行PCR扩增，结果如图5所示，泳道1为ARM2缺陷型，泳道2为野生型；结果以野生型X33菌株基因组为模板的PCR产物大小在600bp左右，ARM2缺陷型工程菌无扩增条带，也证明了β甘露糖转移酶(ARM2)敲除的，命名为GJK04，为磷酸甘露糖转移酶、磷酸甘露糖合成酶和β甘露糖转移酶II(ARM2)基因敲除的重组毕赤酵母菌。
四、β甘露糖转移酶ARM1、ARM3、ARM4基因灭活的酵母菌株构建
根据上述步骤一至三，同β甘露糖转移酶基因ARM2灭活的酵母菌株构建的设计方法和构建过程，在GJK04工程菌的基础上先后敲除β甘露糖转移酶ARM1、ARM3、ARM4(即β甘露糖转移酶I、III和IV，氨基酸序列分别为SEQ ID No.4、SEQ ID No.6和SEQ ID No.7)，分别构建获得GJK05、GJK07、GJK18工程菌株。
1、构建β甘露糖转移酶ARM1、ARM3、ARM4基因灭活载体
载体构建方法同步骤三，差别之处在于：
扩增ARM1上游侧翼区同源臂(ARM1 5′同源臂)所用的引物为ARM1-5-5和ARM1-5-3，引物序列分别为：
ARM1-5-5：5'-TCA ACGCGTTGGCTCTGGATCGTTCTAATA-3'(SEQ ID No.53，划线部分为MluI识别位点)；
ARM1-5-3：
5'-ttctccgttctcctttctccgt GCGGCCGCcagcagcaaggaagataccaa-3'(SEQ ID No.54，划线部分为NotI识别位点)。
扩增ARM1下游侧翼区同源臂(ARM1 3′同源臂)所用的引物为ARM1-3-5和ARM1-3-3，引物序列分别为：
ARM1-3-5：
5'-ttggtatcttccttgctgctg GCGGCCGCacggagaaaggagaacggagaa-3'(SEQ ID  No.55，划线部分为NotI识别位点)；
ARM1-3-3：5'-TCA ACGCGTTGGCTGGAGGTGACAGAGGAA-3'(SEQ ID No.56，划线部分为MluI识别位点)。
扩增ARM3上游侧翼区同源臂(ARM3 5′同源臂)所用的引物为ARM3-5-5和ARM3-5-3，引物序列分别为：
ARM3-5-5：5'-TCAACGCGTTAGTAGTGCCGTGCCAAGTAGCG-3'(SEQ ID No.57，划线部分为MluI识别位点)；
ARM3-5-3：5'-
tcctactttgcttatcatctgcc GCGGCCGCggtcaggccctcttatggttgtg-3'(SEQ ID No.58，划线部分为NotI识别位点)。
扩增ARM3下游侧翼区同源臂(ARM3 3′同源臂)所用的引物为ARM3-3-5和ARM3-3-3，引物序列分别为：
ARM3-3-5：
5'-cacaaccataagagggcctgacc GCGGCCGCggcagatgataagcaaagtagga-3'(SEQ ID No.59，划线部分为NotI识别位点)；
ARM3-3-3：5'-TCA ACGCGTCATAGGTAATGGCACAGGGATAG-3'(SEQ ID No.60，划线部分为MluI识别位点)。
扩增ARM4上游侧翼区同源臂(ARM4 5′同源臂)所用的引物为ARM4-5-5和ARM4-5-3，引物序列分别为：
ARM4-5-5：5'-TCA ACGCGTGCAGCGTTTACGAATAGTGTCC-3'(SEQ ID No.61，划线部分为MluI识别位点)；
ARM4-5-3：
5'-gcatagggctgaagcatactgt GCGGCCGCaatgatatgtacgttcccaaga-3'(SEQ ID No.62，划线部分为NotI识别位点)。
扩增ARM4下游侧翼区同源臂(ARM4 3′同源臂)所用的引物为ARM4-3-5和ARM4-3-3，引物序列分别为：
ARM4-3-5：
5'-tcttgggaacgtacatatcatt GCGGCCGCacagtatgcttcagccctatgc-3'(SEQ ID No.63，划线部分为NotI识别位点)；
ARM4-3-3：5'-TCA ACGCGTGAGGTGGACAAGAGTTCAACAAAG-3'(SEQ ID No.64，划线部分为MluI识别位点)。
2、敲除质粒对毕赤酵母的转化
同步骤三，差别之处在于，PCR反应所用的两对引物分别是：
ARM1基因5’同源臂外的引物序列ARM1-5-5OUT：5′-GTTCTGGTATGCGTTCTATTCTTC-3′(SEQ ID No.65)和载体上的引物序列inner01：5′-AGCGTCGATTTTTGTGATGCTCGTCA-3′(SEQ ID No.36)，引物所扩增的条带在3.5kb左右为阳性克隆。
ARM3基因5’同源臂外的引物序列ARM3-5-5OUT：5′-TATTTGCCTTCTTCACCGT TAT-3′(SEQ ID No.66)和载体上的引物序列inner01：5′-AGCGTCGATTTTTGTGATGCTCGTCA-3′(SEQ ID No.36)，引物所扩增的条带在3.7kb左右为阳性克隆。
ARM4基因5’同源臂外的引物序列ARM4-5-5OUT：5′-TCCGTTGAGGGTGCTAATGGTA-3′(SEQ ID No.67)和载体上的引物序列inner01：5′-AGCGTCGATTTTTGTGATGCTCGTCA-3′(SEQ ID No.36)，引物所扩增的条带在3.7kb左右为阳性克隆。
3、PCR鉴定阳性工程菌株
同步骤三，差别之处在于，利用下面引物对工程菌进行鉴定，可以发现基因已被敲除(图6、图7和图8)：
Arm1-ORF-09：5'-TAGTCTGGTTTGCGGTAGTGT-3'(SEQ ID No.68)；
Arm1-ORF-10：5'-AGATTGAGCATAGGAGTGGC-3'(SEQ ID No.69)。
Arm3-ORF-09：5'-AAACGGAGTCCAGTTCTTCT-3'(SEQ ID No.70)；
Arm3-ORF-10：5'-CAACTTTGCCTGTCATTTCC-3'(SEQ ID No.71)。
Arm4-ORF-09：5'-CGCTTCAGTTCACGGACATA-3'(SEQ ID No.72)；
Arm4-ORF-10：5'-GCAACCCAGACCTCCTTACC-3'(SEQ ID No.73)。
GJK18菌的DSA-FACE糖型分析结果如图9所示。因β甘露糖的修饰仅仅添加在甘露糖的个别末端，尽管糖型分析结果并没有实质性的变化，但β甘露糖是潜在的引起免疫原性的糖，因此对于用于人体的药物来源，存在潜在的风险，本发明将所有的β甘露糖均灭活，因此从根本上解决了存在β甘露糖的问题，且糖型结构没有被改变。
五、具有哺乳动物Man5GlcNAc2且无岩藻糖糖基化结构的糖基工程酵母菌株构建
首先，为了鉴定外源甘露糖苷酶I(MDSI)是否正确地发挥了作用，本发明提前在GJK18工程菌中引入了一个报告蛋白，本发明以抗Her2抗体为报告蛋白，因此构建了抗Her2抗体的表达载体。该载体的构建方法、载体转化方法已经在申请专利中公开(公开号：CN101748145A)。利用该方法将抗Her2抗体表达载体转入至GJK18宿主菌中，获得了表达抗Her2抗体的W2工程菌株。
其次，具有哺乳动物Man5GlcNAc2且无岩藻糖糖基化结构的糖基工程酵母菌株W10为C端融合HDEL序列的MDSI(TrmdsI，核苷酸序列如SEQ ID No.14所示，编码SEQ ID No.9所示MDSI蛋白)插入宿主菌W2的基因组中，得到的工程菌。
1、外源甘露糖苷酶I(MDSI)表达载体的构建
表达外源甘露糖苷酶I重组载体pPIC9-TrmdsI为将SEQ ID No.14所示的DNA分子插入pPIC9载体的Xho I和EcoR I酶切位点间得到的重组载体。
其中，SEQ ID No.14自5’末端第1-1524位核苷酸为优化后的甘露糖苷酶 I编码基因，自5’末端第1525-1536位核苷酸为内质网保留信号——HDEL编码基因。
(1)甘露糖苷酶I(MDSI)基因
外源甘露糖苷酶I可以是来源于丝状真菌、植物、昆虫、爪哇、哺乳动物等的甘露糖苷酶I，本实施例选取绿色木霉的甘露糖苷酶I(詹洁.绿色木霉α-1,2-甘露糖苷酶在毕赤酵母中的克隆表达与活性鉴定[学位论硕士文].)，并在甘露糖苷酶I的C-端融合了内质网保留信号——HDEL。
根据詹洁.绿色木霉α-1,2-甘露糖苷酶在毕赤酵母中的克隆表达与活性鉴定[学位论硕士文].公布的绿色木霉的甘露糖苷酶I序列，根据酵母偏爱密码子和基因高表达原则优化编码基因，并在C端融合HDEL序列，得到基因片段(SEQ ID No.14)。
(2)设计并合成如下引物：
TrmdsI-5：5’-TCT CTCGAGAAAAGAGAGGCTGAAGCTTATCCAAAGCCGGGCGCCA C-3’(SEQ ID No.74)；下划线所示序列为Xho I酶切识别位点。
TrmdsI-3：5’-AGG GAATTCTTACAACTCGTCGTGAGCAAGGTGGCCGCCCCGTCGTG ATG-3’(SEQ ID No.75)；下划线所示序列为EcoR I酶切识别位点。
(3)以上述(1)得到的基因片段为模板，以TrmdsI-5和TrmdsI-3为引物，进行PCR扩增，得到PCR扩增产物，命名为TrmdsI，该产物含有SEQ ID No.14。
(4)Xho I和EcoR I双酶切上述(3)获得的PCR产物，得到基因片段；Xho I和EcoR I双酶切pPIC9载体得到载体大片段；将基因片段与载体大片段连接，得到重组质粒，将其命名为pPIC9-TrmdsI。将pPIC9-TrmdsI测序，结果正确。
2、表达外源甘露糖苷酶I的重组酵母的构建
将约10μg pPIC9-TrmdsI质粒，用Sal I线性化，用1/10体积的3M醋酸钠和3倍体积的无水酒精沉淀线性化的质粒。用体积百分含量为70％的乙醇水溶液洗两次以除去其中的盐，晾干，加入约30μL水重悬沉淀，获得用于转化的pPIC9-TrmdsI线性化质粒。
以下步骤中制备酵母电转化感受态细胞的方法参照Invitrogen公司的相关手册和“Molecular Cloning，A laboratory Manual(Fourth Edition)”,2012Cold Spring Harbor Laboratory Press,Cold Spring Harbor,New YorK。选用的宿主菌是上述构建的W2工程菌。
具体如下：
将毕赤酵母W2在YPD平板(酵母提取物10g/L，胰蛋白胨20g/L，葡萄糖20g/L，琼脂15g/L)上用划线法分离单克隆，28℃温箱培养2天。接种一个单克隆至一个装有10mL YPD液体培养基(酵母提取物10g/L，胰蛋白胨20g/L，葡萄糖20g/L)的50mL三角瓶中，28℃过夜培养至OD 600约为2，得到菌液。再将0.1-0.5mL菌液接种到含有500mL YPD液体培养基的3.5L摇瓶中，培养过夜 至OD 600至1.3-1.5之间。将菌液转移至无菌的离心瓶中，4℃，1500g离心10分钟。用500mL预冷的无菌水重悬菌体，4℃，1500g离心10分钟收获细胞，用250mL预冷的无菌水再洗一次。用20mL预冷的无菌1M山梨醇重悬菌体，4℃，1500g离心10分钟收获细胞，用预冷的1M山梨醇重悬菌体至终体积为1.5mL，得到菌悬液。
取80μL菌悬液与10μL用于转化的pPIC9-TrmdsI线性化质粒，在微量离心管中混匀，得到混合物，将其置冰上5min，将混合物转移到一个冰冷的0.2cm电转杯中，电穿孔细胞(Bio-Rad Gene Pulser，2000V，25μF，200Ω)，再立即向电转杯中加入1mL冰冷的1M山梨醇，并小心地将混合物(转化细胞)转移至15mL培养管中。
将培养管放在28℃温箱孵育1h，不要摇动。然后加入1mL YPD液体培养基后在28℃，250rpm的摇床中孵育3h。取200μL转化细胞涂布到含MD平板上(1.34g/100ml的YNB，4×10 -5g/100ml Biotin，2g/100ml的葡萄糖)。28℃温箱培养2-5天，至形成单克隆，即W2-Tr，命名为W10。
用玻璃珠制备法提取W10的基因组DNA，以基因组DNA为模板，以TrMDSI-1.3kb-01和TrMDSI-1.3kb-02为引物，进行PCR扩增，得到PCR扩增产物约1.3kb，证明MDSI已插入到基因组中，即为阳性工程菌(图10中A)。
TrMDSI-1.3kb-01：5’-GAACACGATCCTTCAGTATGTA-3’(SEQ ID No.76)；
TrMDSI-1.3kb-02：5’-TGATGATGAACGGATGCTAAAG-3’(SEQ ID No.77)。
W10菌的DSA-FACE糖型分析结果(方法同实施例一中所述方法)如图10中B所示，可见转入TrmdsI后，W10菌表达蛋白的糖型结构为Man5GlcNAc2、Man6GlcNAc2，其中以Man5GlcNAc2为主。
六、具有哺乳动物GlcNAcMan5GlcNAc2且无岩藻糖糖基化结构的糖基工程酵母菌株构建
具有哺乳动物GlcNAcMan5GlcNAc2且无岩藻糖糖基化结构的糖基工程酵母菌株1-8为将含mnn9定位信号的N-乙酰葡萄糖胺转移酶I(GnTI)(核苷酸序列如SEQ ID No.15所示，编码SEQ ID No.10所示蛋白)的DNA片段插入宿主菌W10基因组中，得到的工程菌。
其中，SEQ ID No.15自5’末端第1-114位核苷酸为mnn9定位信号，自5’末端第115-1335位核苷酸为N-乙酰葡萄糖胺转移酶I编码基因。
1、含mnn9定位信号的N-乙酰葡萄糖胺转移酶I(GnTI)表达载体的构建(1)调取人gnt1基因
用人gnt1基因上游引物(mnn9-GnTI-01：5’-tcagtcagcgctctcgatggcgaccccg-3’，SEQ ID No.78)和下游引物GnTI-02：5’-GC GAATTCTTAGTGCTAATTCCAGCTAGGATCATAG-3’(SEQ ID No.79，下划线为EcoR I酶切位点)，用PCR的方法从人肝胎cDNA文库(购自Clontech Laboratories Inc.1290Terra Bella Ave.Mountain View，CA94043,USA) 获得人gnt1基因全长片段，PCR反应条件：94℃预变性5分钟，94℃变性30秒，52℃退火30秒，72℃延伸1分钟30秒，循环30次；最后72℃延伸10分钟。PCR扩增产物用0.8％的琼脂糖凝胶电泳分离，用DNA回收试剂盒回收。
(2)含定位信号mnn9的GnTI DNA片段
S.cere MNN9高尔基体定位信号：ScMNN9-03： tatAATattATGTCACTTTCTCTTGTAT CGTACCGCCTAAGAAAGAACCCGTGGGTTAACATTTTTCTACCTGTTTTGGCCATATTTCTAATATATATAATTTTTTTCCAGAGAGATCAATCTtcagtcagcgctctcgatggcgaccccg(SEQ ID No.80)
以含有S.cere MNN9高尔基体定位信号编码序列的上游引物ScMNN9-03(tat AATattATGTCACTTTCTCTTGTATCGTACCGCCTAAGAAAGAACCCGTGGGTTAACATTTTTCTACCTGTTTTGGCCATATTTCTAATATATATAATTTTTTTCCAGAGAGATCAATCTtcagtcagcgctctcgatggcgaccccg，SEQ ID No.80，下划线为SspI酶切位点)和GnTI催化结构域编码区下游引物GnTI-02，通过PCR反应将回收纯化的1.2kb GnTI片段和S.cere MNN9高尔基体定位信号编码序列相连接，使用Pyrobest DNA聚合酶扩增mnn9-gnt1基因片段(SEQ ID No.15)。
PCR反应条件：94℃变性2分钟，52℃退火30秒、72℃延伸5分钟，之后94℃变性30秒，52℃退火30秒，72℃延伸1分钟30秒，循环30次；最后72℃延伸10分钟。
PCR扩增产物经0.8％的琼脂糖凝胶电泳(8V/cm，15分钟)分离，紫外灯下用洁净的刀片切下1.3kb的目的条带，用DNA回收试剂盒进行回收，方法同上。
(3)PGE-URA3-GAP1-mnn9-GnTI表达载体的构建
Ssp I和EcoR I双酶切上述(2)获得的mnn9-gnt1基因片段PCR产物，得到基因片段；Ssp I和EcoR I双酶切PGE-URA3-GAP1(杨晓鹏,刘波,宋淼,巩新,唱韶红,薛奎晶,吴军.Man5GlcNAc2哺乳动物甘露糖型糖蛋白的毕赤酵母表达系统构建.生物工程学报.2011；27:108-17.)载体得到载体大片段；将基因片段与载体大片段连接，得到重组质粒，将其命名为PGE-URA3-GAP1-mnn9-GnTI。测序，结果正确。
PGE-URA3-GAP1-mnn9-GnTI为将SEQ ID No.15所示的DNA分子插入PGE-URA3-GAP1载体的酶切位点Ssp I和EcoR I之间后得到的重组载体。
2、表达外源甘露糖苷酶I的重组酵母的构建
将约10μg PGE-URA3-GAP1-mnn9-GnTI质粒，用Nhe I线性化，获得用于转化的PGE-URA3-GAP1-mnn9-GnTI线性化质粒，制备酵母电转化感受态细胞的方法上述步骤五。
选用的宿主菌是上述步骤五构建的W10工程菌。转化后在MD平板上形成的单克隆，命名为1-8。
用玻璃珠制备法提取1-8的基因组DNA，以基因组DNA为模板，以HuGnTI-0.9k-01和HuGnTI-0.9k-02为引物，进行PCR扩增，得到PCR扩增产物 约0.9kb，证明GnTI已插入到基因组中，即为阳性工程菌(如图11中A)。
HuGnTI-0.9k-01：5’-TGGACAAGCTGCTGCATTATC-3’(SEQ ID No.81)；
HuGnTI-0.9k-02：5’-CGGAACTGGAAGGTGACAATA-3’(SEQ ID No.82)。
1-8菌的DSA-FACE糖型分析结果(方法同实施例一中所述方法)如图11中B所示，可见转入GnTI后，宿主菌表达蛋白的主要糖型结构为GlcNAcMan5GlcNAc2。
七、具有哺乳动物GalGlcNAcMan5GlcNAc2且无岩藻糖糖基化结构的糖基工程酵母菌构建
具有哺乳动物GalGlcNAcMan5GlcNAc2且无岩藻糖糖基化结构的糖基工程酵母菌株1-8-4为将kre2-GalE-GalT基因片段(核苷酸序列如SEQ ID No.16所示，编码SEQ ID No.11所示蛋白)插入宿主菌1-8基因组中，得到的工程菌1-8-4。
其中，SEQ ID No.16自5’末端第1-294位核苷酸为kre2定位信号，自5’末端第295-1317位核苷酸为半乳糖异构酶GalE编码基因、自5’末端第1325-2394位核苷酸为半乳糖转移酶GalT编码基因。
1、含kre2定位信号的半乳糖转移酶(GalE+T)表达载体的构建
(1)调取人GalE、GalT基因
用人GalE基因上游引物GalE5’和下游引物GalE3’，用人GalT基因上游引物GalT5’和下游引物GalT3’，用PCR的方法分别从人肝胎cDNA文库(购自Clontech Laboratories Inc.1290Terra Bella Ave.Mountain View，CA94043,USA)获得人GalE、GalT基因全长片段，PCR反应条件：94℃预变性5分钟，94℃变性30秒，52℃退火30秒，72℃延伸1分钟30秒，循环30次；最后72℃延伸10分钟。PCR扩增产物分别用0.8％的琼脂糖凝胶电泳分离，用DNA回收试剂盒分别进行回收。
GalE5’：5’-ATGAGAGTTCTGGTTACCGGTGGTA-3’(SEQ ID No.83)；
GalE3’：5’-AG GGTACCATCGGGATATCCCTGTGGATGGC-3’(SEQ ID No.84，下划线部分为KpnI的识别序列)；
GalT5’：5’-AT GGTACCGGTGGTGGACGTGACCTTTCTCGTCTGCCA-3’(SEQ ID No.85，下划线部分为KpnI的识别序列)。
GalT3’：5’-GC atttaaatttaGCTCGGTGTCCCGATGTCCACTGTGAT-3’(SEQ ID No.86，下划线部分为SwaI的识别序列)。
(2)含定位信号kre2的GalE-GalT DNA片段
Kre2 5’：5’-AT AATattAAACGATGGCCCTCTTTCTCAGTAAGAG-3’(SEQ ID No.87，下划线部分为SspI I位点的识别序列)；
Kre2 3’+GalE5’：5’-CACCGGtAACCAGaACTctCatGATCGGGGCAtctgccttttcagcggcagctttcagagccttggattc-3’(SEQ ID No.88)。
用PCR的方法从酿酒酵母S.cere基因组DNA中调取kre2定位信号片段。 PCR条件同上。
以含有S.cere kre2高尔基体定位信号编码序列的上游引物Kre2和GalE+GalT催化结构域编码区下游引物GalT3’，通过PCR反应将回收纯化的GalE、GalT片段和S.cere kre2高尔基体定位信号编码序列相连接，使用Pyrobest DNA聚合酶扩增kre2-GalE-GalT基因片段。
PCR反应条件：94℃变性2分钟，52℃退火30秒、72℃延伸5分钟，之后94℃变性30秒，52℃退火30秒，72℃延伸4分钟30秒，循环30次；最后72℃延伸10分钟。
PCR扩增产物经0.8％的琼脂糖凝胶电泳(8V/cm，15分钟)分离，紫外灯下用洁净的刀片切下2.4kb的目的条带，用DNA回收试剂盒进行回收，方法同上。
(3)PGE-URA3-GAP1-kre2-GalE-GalT载体的构建
先用SwaI酶切上述kre2-GalE-GalT的DNA分子，再用T4PNK酶(大连宝生物有限公司)磷酸化该基因片段；Ssp I和SwaI双酶切PGE-URA3-GAP1载体得到载体大片段；将基因片段与载体大片段连接，得到重组质粒，将其命名为PGE-URA3-GAP1-kre2-GalE-GalT。测序，结果正确。
PGE-URA3-GAP1-kre2-GalE-GalT为将SEQ ID No.16所示的kre2-GalE-GalT的DNA分子插入PGE-URA3-GAP1载体的Ssp I和SwaI酶切位点得到的重组载体。
2、表达外源UDP-Gal和乳糖转移酶的重组酵母的构建
将约10μg PGE-URA3-GAP1-kre2-GalE-GalT质粒，用Nhe I线性化，获得用于转化的PGE-URA3-GAP1-kre2-GalE-GalT线性化质粒，制备酵母电转化感受态细胞的方法同上述步骤五。
选用的宿主菌是步骤六构建的1-8工程菌。转化后在MD平板上形成的单克隆，命名为1-8-4。
用玻璃珠制备法提取1-8-4的基因组DNA，以基因组DNA为模板，分别以GalE-T(1.5k)-01(5’-TGATAACCTCTGTAACAGTAAGCGC-3’，SEQ ID No.89)和GalE-T(1.5k)-02(5’-GGAGCTTAGC ACGATTGAATATAGT-3’，SEQ ID No.90)为引物，进行PCR扩增，得到PCR扩增产物分别为1.5kb，证明GalE-T已插入到基因组中，即为阳性工程菌(如图12中A)。
1-8-4菌的DSA-FACE糖型分析结果(方法同实施例一中所述方法)如图12中B所示，可见转入半乳糖异构酶和半乳糖转移酶后，宿主菌表达蛋白的主要糖型结构为GalGlcNAcMan5GlcNAc2。
八、具有哺乳动物GalGlcNAcMan3GlcNAc2且无岩藻糖糖基化结构的糖基工程酵母菌株构建
具有哺乳动物GalGlcNAcMan3GlcNAc2且无岩藻糖糖基化结构的糖基工程酵母菌株52-60为将MDSII DNA分子(核苷酸序列如SEQ ID No.17所示，编码SEQ ID No.12所示蛋白)插入宿主菌1-8-4的基因组中，得到的工程菌52-60。
其中，SEQ ID No.17自5’末端第1-108位核苷酸为甘露糖苷酶II编码基 因的mnn2定位信号，自5’末端第109-3303位核苷酸为甘露糖苷酶II编码基因。
1、含mnn2定位信号的甘露糖苷酶II(MDSII)表达载体的构建
(1)全基因合成方式合成含mnn2定位信号的MDSII基因
根据序列利用全基因合成方式合成含mnn2的MDSII基因(SEQ ID No.17)，由南京金瑞斯公司合成并克隆至pUC57克隆载体中，获得pUC57-MDSII。
设计MDSII基因上游引物(mnn2-MDSII-01：5’-AT AATattAAACCatgctgcttaccaaaaggttttcaa agctgttc-3’，SEQ ID No.91)(下划线为SspI酶切位点)和下游引物(MDSII-02：5’-GCT ATTTAAATctattaCCT CAACTGGATTCGGAATGTGCTGATTTCCATTG-3’，SEQ ID No.92)(下划线为SwaI酶切位点)，用PCR的方法从pUC57-MDSII获得人MDSII基因全长片段PCR产物，PCR反应条件：94℃预变性5分钟，94℃变性30秒，52℃退火30秒，72℃延伸4分钟30秒，循环30次；最后72℃延伸10分钟。PCR扩增产物(SEQ ID No.17)用0.8％的琼脂糖凝胶电泳分离，用DNA回收试剂盒进行回收。
(2)PGE-URA3-arm3-GAP-mnn2-MDSII表达载体的构建
先用SwaI酶切上述PCR产物，再用T4PNK酶(大连宝生物有限公司)磷酸化该基因片段；Ssp I和SwaI双酶切PGE-URA3-GAP1载体得到载体大片段；将基因片段与载体大片段连接，得到重组质粒，将其命名为PGE-URA3-arm3-GAP-mnn2-MDSII。测序，结果正确。
PGE-URA3-arm3-GAP-mnn2-MDSII为将SEQ ID No.17所示DNA分子插入PGE-URA3-GAP1载体的Ssp I和Swa I酶切位点得到的重组载体。
2、表达外源甘露糖苷酶II的重组酵母的构建
将约10μg PGE-URA3-arm3--GAP-mnn2-MDSII质粒，用Msc I线性化，获得用于转化的PGE-URA3-arm3-GAP-mnn2-MDSII线性化质粒，制备酵母电转化感受态细胞的方法同上述步骤五。
选用的宿主菌是步骤七构建的1-8-4工程菌。转化后在MD平板上形成的单克隆，命名为52-60。
用玻璃珠制备法提取52-60的基因组DNA，以基因组DNA为模板，分别以CeMNSII-1.2k-01和CeMNSII-1.2k-02为引物，进行PCR扩增，得到PCR扩增产物分别为1.2kb，证明MDSII已插入到基因组中，即为阳性工程菌(图13中A)。
CeMNSII-1.2k-01：5’-CAGATGGATGAGCATAGAGTTA-3’(SEQ ID No.93)；
CeMNSII-1.2k-02：5’-GACAAGAGGATAATGAAGAGAC-3’(SEQ ID No.94)。
52-60菌的DSA-FACE糖型分析结果如图13中C所示。可见，转入后外源甘露糖苷酶II，宿主菌表达蛋白的主要糖型结构为GalGlcNAcMan3GlcNAc2。
九、具有哺乳动物Gal2GlcNAc2Man3GlcNAc2且无岩藻糖糖基化结构的糖基工程酵母菌株构建
具有哺乳动物Gal2GlcNAc2Man3GlcNAc2且无岩藻糖糖基化结构的糖基工程 酵母菌株150L2为将GnT II DNA分子(核苷酸序列如SEQ ID No.18所示，编码SEQ ID No.13所示蛋白)插入宿主菌52-60的基因组中，得到的工程菌150L2。
其中，SEQ ID No.18自5’末端第1-108位核苷酸为N-乙酰葡萄糖胺转移酶II编码基因的mnn2定位信号，自5’末端第109-1185位核苷酸为N-乙酰葡萄糖胺转移酶II。
1、mnn2定位信号的N-乙酰葡萄糖胺转移酶II(GnTII)表达载体的构建
(1)全基因合成方式合成GnTII基因
根据序列利用全基因合成方式合成含mnn2的GnTII基因(SEQ ID No.18)，由南京金瑞斯公司合成并克隆至pUC57克隆载体中，获得pUC57-GnTII。
设计GnTII基因上游引物(mnn2-GnTII-01：5’-AT AATattAAACCatgctgcttaccaaaa ggttttcaaagctgttc-3’，SEQ ID No.95)(下划线为SspI酶切位点)和下游引物(GnTII-02：5’-GCT atttaaatTTAtcactgcagtcttctataacttttac-3’，SEQ ID No.96)(下划线为SwaI酶切位点)，用PCR的方法从pUC57-GnTII获得含mnn2定位信号的N-乙酰葡萄糖胺转移酶II(GnTII)DNA分子，PCR反应条件：94℃预变性5分钟，94℃变性30秒，52℃退火30秒，72℃延伸2分钟30秒，循环30次；最后72℃延伸10分钟。PCR扩增产物用0.8％的琼脂糖凝胶电泳分离，用DNA回收试剂盒回收。
(2)PGE-URA3-arm3-GAP-mnn2-GnTII表达载体的构建
酶切及构建方法与PGE-URA3-arm3-GAP-mnn2-MDSII构建方法一致，得到重组质粒，将其命名为PGE-URA3-arm3-GAP-mnn2-GnTII。测序，结果正确。
PGE-URA3-arm3-GAP-mnn2-GnTII为将SEQ ID No.18所示DNA分子插入PGE-URA3-GAP1载体的Ssp I和Swa I酶切位点得到的重组载体。
2、表达外源N-乙酰葡萄糖胺转移酶II的重组酵母的构建
将约10μg PGE-URA3-arm3-GAP-mnn2-GnTII质粒，用Msc I线性化，获得用于转化的PGE-URA3-arm3-GAP-mnn2-GnTII线性化质粒，制备酵母电转化感受态细胞的方法同上述步骤五。
选用的宿主菌是步骤八构建的52-60工程菌。转化后在MD平板上形成的单克隆，命名为150L2。
用玻璃珠制备法提取150L2的基因组DNA，以基因组DNA为模板，分别RnGnTII-0.8k-01和RnGnTII-0.8k-02为引物，进行PCR扩增，得到PCR扩增产物为0.8kb，证明GnTII已插入到基因组中，即为阳性工程菌(图13中B)。
RnGnTII-0.8k-01：5’-ATCAACAGTCTGATCTCTAGTG-3’(SEQ ID No.97)；
RnGnTII-0.8k-02：5’-AGTTCATGGTCCCTAATATCTC-3’(SEQ ID No.98)。
十、工程化菌株中抗her2抗体基因的敲除
抗her2抗体基因灭活的酵母菌株3-5-11为将SEQ ID No.19所示的DNA分子(抗her2抗体轻重链基因敲除序列)导入毕赤酵母150L2中，与150L2基因组中的同源序列发生同源重组，敲除酵母基因组中的抗her2抗体轻重链基因， 得到的重组酵母。
构建抗her2抗体轻重链基因灭活载体、敲除质粒对毕赤酵母的转化、PCR鉴定阳性工程菌株与前述步骤方法相同，抗her2抗体基因灭活的酵母菌株命名为3-5-11。
十一、工程化菌株中灭活O-甘露糖转移酶I基因
因发现宿主菌存在不稳定性，容易丢失MDSI和MDSII基因，因此在O-甘露糖转移酶I基因灭活之前，按照本实施例步骤八和步骤五的同样技术方法，在3-5-11中宿主菌先后转入SEQ ID No.17(MDSII)和SEQ ID No.14(MDSI)，保证了工程菌内这两个基因的双拷贝，构建获得了670宿主菌。
O-甘露糖转移酶I基因灭活的酵母菌株7b为将编码SEQ ID No.8所示的O-甘露糖转移酶I的DNA分子在毕赤酵母670中进行插入灭活，得到的酵母，命名为7b，即GJK30。GJK30已经于2020年03月18日保藏于中国普通微生物菌种保藏管理中心，其保藏编号为CGMCC No.19488。
1、O-甘露糖转移酶基因灭活载体的构建
以质粒pPIC9(invitrogen公司)为模板，通过PCR方法获取终止子AOXTT序列。所用PCR钓取终止子引物AOXTT-5和AOXTT-3(5’-AOX1TT-5tctacgcgtccttag acatgactgttcctcagt-3’，SEQ ID No.99；AOX1TT-3：5’-tctacgcgtaagcttgcacaaacgaacttc-3’，SEQ ID No.100)。将得到的PCR产物用PCR产物回收纯化试剂盒纯化回收(鼎国生物技术有限公司，北京)，得到AOX1TT终止子片段。
本发明所用的载体pYES2(invitrogen公司)具有酵母的URA3筛选标记，可用于后续筛选工作。为了防止载体上的URA3基因的启动子对载体上其他基因的影响，本发明在URA3基因末端添加AOX1TT终止子。具体构建方法为：将上述获得的AOX1TT终止子片段回收后用MluI酶切，得到酶切片段；将该酶切片段与用同样用Mlu1处理过的载体pYES2连接，将连接产物转化大肠杆菌感受态细胞Trans5α(北京全式金生物技术有限公司，目录号CD201)扩增，将序列正确的克隆命名为Trans5α-pYES2-URA3-AOX1TT，提取质粒，得到URA3基因末端添加AOX1TT终止子的重组载体，记为pYES2-URA3-AOX1TT。
为了使构建的载体能够定点整合到毕赤酵母PMT1基因中，本发明利用PCR钓取PMT1基因中ORF区的一个片段作为同源重组片段。为了确保失活载体整合到PMT1基因上能够引起PMT1基因的失活，本研究在引物两端加上不同组合的终止密码子，在钓取的PMT1基因片段3 ′末端加上CYCTT终止子。
以毕赤酵母JC308(Invitrogen公司)基因组为模板，用玻璃珠制备法(A.亚当斯等，《酵母遗传学方法实验指南》，科学出版社，2000)提取毕赤酵母JC308的基因组DNA，以该基因组DNA为模板，利用引物PMT1-IN-5和PMT1-IN-3进行PCR扩增钓取PMT1基因片段。
PMT1-IN-5：
5’-tctatgcattaatgatagttaatgactaatagagtaaaacaagtcctcaagaggt-3’(SEQ ID No.101)；
PMT1-IN-3：
5’-tgacataactaattacatgatctattagtcattaactatcattagatcagagtggggacgacta agaaa gc-3’(SEQ ID No.102)。
钓取的PMT1基因片段两端加入具有不同组合的终止密码子，命名为PMT1-IN。
PCR钓取PMT1基因片段反应条件为94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸1min40s。共进行25个循环，最后72℃延伸10min。回收PCR产物即为钓取的PMT1基因片段。
以含有CYCTT终止子的质粒pYES2为模板，利用引物CYC1TT-5和CYC1TT-3(CYC1TT-5：5’-gctttcttagtcgtccccactctgatctaatgatagttaatgactaatagatcatgtaattagttatgtca-3’，SEQ ID No.1031；CYC1TT-3：5’-gcaaattaaagccttcgagcgtc-3’，SEQ ID No.104)进行PCR扩增钓取CYC1TT终止子片段。PCR反应条件为94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸1min。共进行25个循环，最后72℃延伸10min。回收PCR产物，即为CYC1TT终止子片段。
再以回收的PCR产物CYC1TT终止子片段和PMT1-IN片段(钓取的PMT1基因片段)为模板，利用引物PMT1-IN-5和CYC1TT-3进行PCR扩增，连接PMT1-IN和CYC1TT片段，构建PMT1-IN-CYC1TT融合片段。PCR反应条件为94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸2.4min。共进行25个循环，最后72℃延伸10min。回收PCR产物，即为PMT1-IN和CYC1TT终止子的连接片段——PMT1-IN-CYC1TT融合片段。回收后的产物用Nsi1酶切后磷酸化，然后与pYES2-URA3-AOX1TT经Nsi1和Stu1酶切得到的载体骨架连接，得到的序列正确的重组载体为PMT1插入失活载体PMT1-IN-pYES2。
在钓取的PMT1基因片段的前端和末端各装上不同组合的终止密码子，并且在末端的终止密码子之后又装了CYC1TT终止子，即保证如果基因组整合正确PMT1基因便不会表达。pYES2载体上含有毕赤酵母的URA3基因，为防止URA3基因启动子对PMT1基因的启动，在URA3基因后插入AOX1TT终止子。根据设计的引物，获得CYC1TT终止子(272bp)片段和PMT1(907bp)片段，与理论大小一致。PMT1-IN片段与CYC1TT融合片段大小是1135bp，通过以上PCR鉴定和测序等证明载体PMT1-IN-pYES2构建成功。
2、PMT1基因灭活菌株的构建
制备酵母670感受态细胞，制备方法为：
挑取670单菌落接种于2mL YPD+U培养基(该培养基为向YPD培养基中添加尿嘧啶得到的尿嘧啶浓度为100μg/mL的培养基)中，在25℃摇床以170r/min培养48h；然后取500μL培养物，接种于100mL YPD+U培养基中，25℃下以 170r/min培养24h，OD 600到达1.0；然后在4℃以6000r/min离心6min，用15mL的冷无菌水重悬菌体；相同条件下再次离心，用15mL的冷无菌水重悬菌体；4℃下以6000r/min离心6min，用15mL冷的1mol/L山梨醇重悬菌体；相同条件下再次离心；倒掉上清，用1mL冷的1mol/L山梨醇重悬菌体，体积约1.5mL，即酵母670感受态细胞，置于冰上备用。
PMT1插入失活载体PMT1-IN-pYES2的电击转化：将PMT1插入失活载体PMT1-IN-pYES2利用EcoRV酶切线性化后回收，终产物溶于20μL ddH 2O，即为线性化质粒；将85μL的670感受态细胞与线性化质粒混合于电转杯中，冰上放置5min，按毕赤酵母电转化手册上的条件进行电转化(2kV)，电击后立即加入700μL的1M的山梨醇，转移至1.5mL离心管中，25℃下放置1h，涂布于MD+RH平板(该平板为向MD培养基中添加组氨酸和精氨酸得到的组氨酸和精氨酸浓度分别为100μg/mL和100μg/mL的固体培养基)，置于25℃下培养，待平板上长出的克隆提取基因组DNA，利用PMT1基因组外围引物PMT1-ORF-OUT-5和PMT1-ORF-OUT-3做PCR鉴定，基因组鉴定正确的克隆命名为7b，即GJK30。
PMT1-ORF-OUT-5：5’-aagacccatgccgaacacgac-3’(SEQ ID No.105)；
PMT1-ORF-OUT-3：5’-gctctgaggcaccttgggtaa-3’(SEQ ID No.106)。
利用插入失活载体插入整合的方式整合到毕赤酵母染色体中，由于载体中含有PMT1基因同源片段，理论上载体的整合属于定点整合，即插入在PMT1基因上，可以通过设计的特定引物进行鉴定和筛选。利用毕赤酵母的URA3筛选标记，通过压力筛选，鉴定MD+RH平板上长出的克隆。通过PMT1基因外围引物PMT1-ORF-OUT-5和PMT1-ORF-OUT-3做PCR鉴定。如果PMT1-IN-pYES2载体正确整合到PMT1基因中，利用上面的引物可以得到8.6kb大小的片段；对照(即酵母X33)为3kb大小的片段(图14)；由可知，此PMT1-IN-pYES2载体正确整合到PMT1基因中，命名为7b，即GJK30。由于插入载体上设计了不同的终止密码子和终止子，因此，基因整合正确，PMT1基因便不会表达。
十二、GJK30工程菌的糖型结构分析
为了观察最终获得的GJK30的糖型结构是否正确，本发明在获得GJK30工程菌后引入了一个报告蛋白，同实施例一的方法，以抗Her2抗体为报告蛋白，抗Her2抗体的表达载体的构建方法、载体转化方法已经在申请专利中公开(见实施例1)。利用该方法将抗Her2抗体表达载体转入至GJK30宿主菌中，获得了表达抗Her2抗体的GJK30-HL工程菌株。糖型与前期获得的糖型(将Her2抗体表达载体转入至中国专利申请201410668305.X的实施例1构建的GJK08菌株中获得的对照重组工程菌，即与本发明GJK30-HL工程菌株相比，差别之处有三：本发明敲除的β甘露糖转移酶是I-IV，对照重组工程菌仅敲除了β甘露糖转移酶II；本发明还失活了O甘露糖转移酶I，对照重组工程菌没有；本发明导入外源MDSI和MDSII是导入两次，对照重组工程菌是导入一次)尽管均含有 Gal2GlcNAc2Man3GlcNAc2结构，但两者的比例明显不同，前期Gal2GlcNAc2Man3GlcNAc2结构低于50％(图15中A)，而GJK30工程菌获得的Gal2GlcNAc2Man3GlcNAc2结构所占糖型比例大于60％，且整体糖型更为简单且均一(图15中B)。据众多文献报道，这种Gal2GlcNAc2Man3GlcNAc2糖型结构会影响蛋白的生物活性，如抗体的ADCC、CDC活性，因此它所占的比重就直接影响到蛋白的很多特性。通过商业化购买的糖苷酶(New England Biolabs，Beijing)对该糖型进行酶切分析，如图15中C示，由于Gal2GlcNAc2Man3GlcNAc2(G2)末端没有N-乙酰葡萄糖胺，所以在β-N-乙酰氨基葡糖苷酶的作用下，Gal2GlcNAc2Man3GlcNAc2结构不会发生改变，而可以在外切酶β1,4-半乳糖苷酶的作用，剪切去除两个半乳糖，而形成GlcNAc2Man3GlcNAc2(G0)的结构；而同时在这两个外切酶的作用下，即先后剪切去除了半乳糖Gal和N-乙酰葡萄糖胺GlcNAc，因而糖基结构变为Man3GlcNAc2结构，证明表达的糖型正确。
实施例2、SARS-CoV-2 S-RBD重组酵母菌株的构建
一、SARS-CoV-2 S蛋白RBD基因的获取及酵母表达载体的构建
根据公布的"Wuhan-Hu-1"分离株的序列(GenBank:MN908947.3)，分别选取S蛋白的第319位至第541位氨基酸(R319-F541)、第319位至第534位氨基酸(R319-V534)、第319位至第528位氨基酸(R319-K528)，委托北京诺赛基因组研究中心有限公司按照巴斯德毕赤酵母偏爱密码子优化DNA序列，并插入到pPICZαA载体的AOX1启动子下游的XhoI和NotI酶切位点之间，经获得重组表达载体pPICZα-S-RBD223、pPICZα-S-RBD216、pPICZα-S-RBD210。
重组表达载体pPICZα-S-RBD223的结构描述为：在pPICZαA载体的XhoI和NotI酶切位点之间插入SEQ ID No.24所示DNA片段后的重组质粒。SEQ ID No.24为根据SARS-CoV-2"Wuhan-Hu-1"分离株S蛋白的第319位至第541位氨基酸(R319-F541)进行密码子优化后得到的编码基因序列，编码SEQ ID No.21所示的SARS-CoV-2 S-RBD223蛋白。
重组表达载体pPICZα-S-RBD216的结构描述为：在pPICZαA载体的XhoI和NotI酶切位点之间插入SEQ ID No.25所示DNA片段后的重组质粒。SEQ ID No.25为根据SARS-CoV-2"Wuhan-Hu-1"分离株S蛋白的第319位至第534位氨基酸(R319-V534)进行密码子优化后得到的编码基因序列，编码SEQ ID No.22所示的SARS-CoV-2 S-RBD216蛋白。
重组表达载体pPICZα-S-RBD210的结构描述为：在pPICZαA载体的XhoI和NotI酶切位点之间插入SEQ ID No.26所示DNA片段后的重组质粒。SEQ ID No.26为根据SARS-CoV-2"Wuhan-Hu-1"分离株S蛋白的第319位至第528位氨基酸(R319-K528)进行密码子优化后得到的编码基因序列，编码SEQ ID No.23所示的SARS-CoV-2 S-RBD210蛋白。
二、重组表达载体pPICZα-S-RBD223、pPICZα-S-RBD216和pPICZα-S-RBD210转化酵母菌CGMCC No.19488
将酵母菌CGMCC No.19488划线于YPD平板上复苏，分离单克隆。挑取复苏的单克隆，接种到YPD液体培养基中，试管培养至其对数期后取1ml转接到100ml YPD摇瓶中25℃200rpm摇床培养至OD 600至1.3-1.5，1500g 4℃离心5min弃上清，用等体积的预冷的蒸馏水重悬后1500g 4℃离心5min，弃上清，重复此步骤3次；再用等体积的预冷的1M山梨醇重悬后1500g 4℃离心5min，弃上清，重复此步骤3次。以上经3次蒸馏水和3次山梨醇洗涤的菌体沉淀，添加1ml 1M山梨醇悬起，100μl每支分装到无菌离心管中，-80℃保存。
分别将构建好的表达质粒pPICZα-S-RBD223、pPICZα-S-RBD216和pPICZα-S-RBD210约10μg用限制性内切酶BglII进行单点线性化，酶切体系(50μL)如下：表达质粒43μL、BglII 2μL、10×NEB3.1buffer 5μL，37℃酶切1h后取样，经1％的琼脂糖凝胶电泳分离，分析质粒是否线性化完全。分离结果显示线性化完全的酶切产物用离心柱型的DNA片段回收试剂盒进行片段回收，最后洗脱线性化的质粒时用30μL纯水洗脱。
取线性化的表达质粒pPICZα-S-RBD223、pPICZα-S-RBD216和pPICZα-S-RBD210各15μl，分别加入到100μl实施例1所得经过糖基化修饰途径遗传改造的巴斯德毕赤酵母(保藏编号为CGMCC No.19488)电击转化感受态细胞，轻轻混匀，转入预冷的0.2cm电转杯中，冰上放置5min。按照酵母电转手册要求，2kV电压电击后迅速加入900μL预冷的1M山梨醇，转入一只洁净试管中，置25℃培养箱中静置2小时。之后再加入1ml无抗生素添加的YPD液体培养基，置25℃，200rpm摇床培养3-4小时。将以上摇床培养得到的菌液，取300μL涂布于筛选抗性为Zeocin的YPD平板，25℃温箱倒置培养60-72h。
三、重组表达菌株的筛选
待所涂平板长出单克隆后，每组随机挑取8个单克隆接种至新的YPD/Zeocin的平板上，25℃温箱倒置培养。待菌落长出后，接种至3ml的YPD/Zeocin液体培养基中，25℃，200rpm摇床培养，待菌液长浓后，按照5％(体积百分含量)的接种量转接到3ml BMGY培养基，培养基中25℃200rpm摇床培养，48小时后每12小时补加0.5％(V/V)甲醇诱导。诱导48h后，12000rpm，3min收集培养上清。
以上经48小时甲醇诱导后所收集的培养上清，经WB筛选，Western Blot步骤大致如下：(1)12％的SDS-PAGE胶分离样品；(2)将SDS-PAGE胶上的样品转印到PVDF膜上；(3)5％的牛奶封闭液封闭转印有目的蛋白的PVDF膜，室温封闭1小时；(4)转到用5％的牛奶以1:1000的稀释度稀释一抗(Anti-CoV spike Antibody，义翘神州40150-T62)孵育2小时；(5)PBST洗涤5min，清洗5次；(6)转到用5％的牛奶以1:4000的稀释度稀释二抗(Sigma SAB3700885)孵育1小时；(7)PBST洗涤5min，清洗5次；(8)用Pro-light HRP Chemiluminescent显色液(天根生化，PA112-02)显色。
将表达阳性的重组酵母菌株命分别名为CGMCC19488/S-RBD223、 CGMCC19488/S-RBD216、CGMCC19488/S-RBD210。图16为CGMCC19488/S-RBD223阳性克隆筛选WB验证图。
实施例3、重组SARS-CoV-2 S-RBD糖蛋白的表达与纯化
一、重组SARS-CoV-2 S-RBD223糖蛋白的表达与纯化
1、重组菌株CGMCC19488/S-RBD223培养
挑取实施例2鉴定得到的阳性克隆CGMCC19488/S-RBD223接种到YPD/Zeocin液体培养基中，25℃，200rpm培养至OD 600为15～20，以5％(V/V)的接种量转接到BMGY培养基，25℃，200rpm培养24小时后加入体积百分比为0.5％的甲醇诱导S-RBD的表达，每12小时诱导一次，并取样检测表达情况，诱导48小时后离心收集培养上清。
不同诱导时间SDS-PAGE检测如图17所示。由图可见，目的蛋白随着诱导时间的增加，表达水平也在提高。
2、SARS-CoV-2 S-RBD的纯化
(1)阳离子交换层析
将步骤一诱导表达48小时的培养上清用水稀释2倍，调pH至6.5，用Capto MMC层析介质纯化，流动相成分为：
A：20mM pH6.5 PB(磷酸盐缓冲液)；
B：100mM pH8.5 Tris-HCl+1M NaCl。
上样结束用A平衡，然后用B洗脱。
(2)疏水层析
将用Capto MMC纯化样品用Phenyl HP纯化，先用40％(体积百分含量)B洗脱杂蛋白，再用20％(体积百分含量)B洗脱目的蛋白，流动相成分为：
A：20mM pH7.5Tris-HCl+1M硫酸铵；
B：20mM pH7.5 Tris-HCl
(3)G25脱盐
将Phenyl HP纯化样品用G25fine层析介质脱盐，收集蛋白样品，流动相成分为：20mM pH8.5 Tris-HCl。
(4)阴离子交换层析
将脱盐的样品用SOURCE30Q层析介质纯化，流动相成分为：
A：20mM pH8.5 Tris-HCl；
B：20mM pH8.5 Tris-HCl+1M NaCl。
上样结束用A平衡，然后用B洗脱。
SDS-PAGE检测，结果如图18所示。
通过SDS-PAGE电泳发现，通过Capto MMC可将SARS-CoV-2 S-RBD223蛋白捕获；脱盐后样品用SOURCE30Q纯化，目的蛋白流穿，几乎所有杂蛋白被吸附在SOURCE30Q层析介质。
二、重组SARS-CoV-2 S-RBD216糖蛋白和重组SARS-CoV-2 S-RBD210糖蛋 白的表达与纯化
利用与步骤一相同的培养和纯化方法可获得重组SARS-CoV-2 S-RBD216糖蛋白和重组SARS-CoV-2 S-RBD210糖蛋白，SDS-PAGE鉴定如图19所示。
实施例4、SARS-CoV-2 S-RBD糖蛋白的糖型分析
DSA-FACE分析SARS-CoV-2 S-RBD糖蛋白糖型结构
1、SARS-CoV-2 S-RBD糖蛋白N-糖链样品的制备
方法请参考文献“.一种利用DSA-FACE分析寡糖链的方法.生物技术通讯,2008,19(6):885-888.”将经PNGaseF酶切后的糖链样品用Carbograph柱纯化，Carbograph柱先用流动相A(80％乙腈，0.1％TFA，％表示体积百分含量)活化，水洗后上样，上样后再水洗，然后用流动相B(25％乙腈，0.05％TFA，％表示体积百分含量)洗脱，收集洗脱峰，样品冷冻抽干，沉淀物-20℃保存备用。
2、N-糖链样品的APTS标记
取糖链沉淀物加入1μL 20mM的APTS溶液和1μL 1M的NaBH 3CN溶液(溶于DMSO)，混匀，封管，置于37℃水浴反应18h。
3、Sephadex G10纯化APTS标记的糖链
有文献报道此方法能够保留70％以上的标记复合物，可除去90％的单体APTS，同时能够除去一定的盐份。标记样品经Sephadex G10两次纯化，每次用30μL的ddH 2O洗脱真空冷冻抽干。借助3100DNA测序仪辅助毛细管电泳(DSA-FACE)对标记的糖链进行分析，以商业化牛核糖核酸酶B(RNaseB)五种标准N-糖型结构Man 5-9GlcNAc 2为标准品，对CGMCC19488表达的SARS-CoV-2 S-RBD223、SARS-CoV-2 S-RBD216和SARS-CoV-2 S-RBD210糖蛋白的糖链结构进行分析，结果如图20所示。
由图可知：SARS-CoV-2 S-RBD223、SARS-CoV-2 S-RBD216和SARS-CoV-2S-RBD210糖蛋白的糖型为Gal 2GlcNAc 2Man 3GlcNAc 2、GalGlcNAcMan 5GlcNAc 2或Man 5GlcNAc 2。
实施例5、小鼠免疫实验
免疫方法已经在多篇文献中公开，如《人类疾病动物模型的复制，李才主编，人民卫生出版社出版》。具体如下：
第一批次：
取100只6～8周龄大的雌性Balb/c小鼠，随机平均分为表2中的10组。其中，生理盐水组为阴性对照组。RBD即为前文制备得到的CGMCC19488表达的SARS-CoV-2 S-RBD223、SARS-CoV-2 S-RBD216或SARS-CoV-2 S-RBD210糖蛋白。CpG佐剂根据序列由大连TaKaRa公司合成，并做全链硫代修饰委托北京六合通经贸有限公司代理(中国，北京)完成。按100μl体积含有10μg RBD、100μg Al(OH) 3(以铝含量计，即铝含量100μg)和25μg CpG佐剂(或100μl体积含有10μg RBD和100μg Al(OH) 3(以铝含量计，即铝含量100μg))用生理盐水配伍疫苗。各组均在第0、14天肌肉免疫100μl，第28天取血。
表2、RBD与佐剂各实验组设计安排(第一批次)
序号 RBD Al(OH) 3佐剂 CPG佐剂 免疫剂量
1 RBD223，10μg 100μg CpG2006，25μg 100μl
2 RBD223，10μg 100μg CpG684，25μg 100μl
3 RBD223，10μg 100μg CpGX1，25μg 100μl
4 RBD216，10μg 100μg CpG2006，25μg 100μl
5 RBD216，10μg 100μg CpG684，25μg 100μl
6 RBD216，10μg 100μg CpGX1，25μg 100μl
7 RBD223，10μg 100μg   100μl
8 RBD216，10μg 100μg   100μl
9 RBD210，10μg 100μg   100μl
10 生理盐水     100μl
第二批次：
分组情况如表3所示。方法同第一批次。
表3、RBD与佐剂各实验组设计安排(第二批次)
序号 RBD Al(OH) 3佐剂 CPG佐剂 免疫剂量
1 RBD223，10μg 100μg CpG2006，25μg 100μl
2 RBD223，10μg 100μg CpG1018，25μg 100μl
3 RBD216，10μg 100μg CpG2006，25μg 100μl
4 RBD216，10μg 100μg CpG1018，25μg 100μl
5 RBD210，10μg 100μg CpG2006，25μg 100μl
6 RBD210，10μg 100μg CpG1018，25μg 100μl
7 RBD223，10μg 100μg   100μl
8 RBD216，10μg 100μg   100μl
9 RBD210，10μg 100μg   100μl
10 生理盐水     100μl
第三批次：
分组情况如表4所示。方法同第一批次。
表4、不同剂量RBD与佐剂各实验组设计安排(第三批次)
序号 RBD Al(OH) 3佐剂 CPG佐剂 免疫剂量
1 RBD216，10μg 100μg CpG2006，50μg 100μl
2 RBD216，5μg 100μg CpG2006，25μg 100μl
3 RBD216，2.5μg 100μg CpG2006，25μg 100μl
4 RBD216，10μg 50μg CpG2006，25μg 100μl
5 RBD216，5μg 50μg CpG2006，25μg 100μl
6 RBD216，2.5μg 50μg CpG2006，25μg 100μl
7 RBD216，10μg 50μg   100μl
8 RBD216，5μg 50μg   100μl
9 RBD216，2.5μg 50μg   100μl
10 生理盐水     100μl
在表2、表3和表4中，各CpG2006的核苷酸序列如下：
CpG-2006：5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'(SEQ ID No.27)；
CpG-1018：5'-TGACTGTGAACGTTCGAGATGA-3'(SEQ ID No.28)；
CpG-X1：5'-TCGTTCGTTCGTTCGTTCGTT-3’(SEQ ID No.29)；
CpG-684：5'-TCGACGTTCGTCGTTCGTCGTTC-3'(SEQ ID No.30)。
用间接ELISA法测定3个批次各组小鼠血清中抗RBD的抗体滴度。用前文制备得到的CGMCC19488表达的相应的SARS-CoV-2 S-RBD包板，其他操作步骤参见精编分子生物学实验指南[M].科学出版社,2008.。
结果如图21所示。由图可知：Al(OH) 3+CpG免疫组抗体滴度可达1：1000000以上，Al(OH) 3免疫组抗体滴度可达1：10000，而对照组仅1：10。Al(OH) 3+CpG免疫组明显高于单纯的Al(OH) 3免疫组；而Al(OH) 3+CpG2006免疫组又高于其他两种CpG免疫组，说明即使同样是CpG，其辅助诱导的抗体滴度也有不同，即CpG2006>CpG684/CpGX1，因此作为佐剂，CPG最优的是全链硫代CpG2006，次之为全链硫代CpG684或者全链硫代CpGX1或者CpG1018。不同剂量RBD或者不同剂量Al(OH) 3或者不同剂量CpG下均能诱导高抗体滴度。
实施例6、病毒中和试验
实施例5中3批次小鼠在第二次免疫后14天取血清，56℃孵育30min，用生理盐水按一定稀释度稀释。按照常规方法进行病毒中和试验(参考文献：Feng Cai Zhu,et al.Safety,tolerability,and immunogenicity of a recombinantadenovirus type-5vectored COVID-19vaccine:a dose-escalation,open-label,non-randomised,first-in-human trial.Lancet.2020May 22；S0140-6736(20)31208-3.doi:10.1016/S0140-6736(20)31208-3.)。步骤如下：
1、准备细胞：将293T-ACE2细胞(Sino Biological，beijing，货号：OEC001)消化，稀释到3×10 4/mL，接种96孔板各100μl/孔。
2、血清稀释：用生理盐水按一定稀释度稀释，设3-5个复孔。
3、病毒稀释：将病毒(virus strain SARS-CoV-2/human/CHN/Wuhan_IME-BJ01/2020，在上述参考文献中有记载)稀释到1×10 4TCID50/ml。
4、中和：一般将血清稀释液与病毒稀释液等体积混合，于5％CO 2培养箱37℃孵育1h。
5、侵染：将温育后的混合液按100μl/孔添加至细胞中。
6、检测：置于5％CO 2培养箱37℃培养60h检测。计算50％保护效果对应的 稀释度。
如图22所示。由图可知：Al(OH) 3+CpG免疫组中和抗体滴度可达1：600以上，Al(OH) 3免疫组抗体滴度可达1：25左右，而对照组仅1：3。Al(OH) 3+CpG免疫组明显高于单纯的Al(OH) 3免疫组，差异极显著；而Al(OH) 3+CpG2006免疫组又高于其他两种CpG免疫组，说明即使同样是CpG，其辅助诱导的中和抗体滴度也有所不同，即CpG2006>CpG684/CpGX1/CpG1018，因此通过诱导的中和抗体滴度进行测量，CpG佐剂最优的是全链硫代CpG2006，次之为全链硫代CpG684或者全链硫代CpGX1或者全链硫代CpG1018。不同剂量RBD、或者不同剂量Al(OH)3、或者不同剂量CpG下均能诱导高中和抗体滴度。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
工业应用
采用本发明疫苗免疫小鼠后会使小鼠产生中和抗体，有望给人注射所述疫苗，使其产生抗新型冠状病毒的抗体，从而减少其患新型冠状病毒病(COVID-19)的风险。

Claims (65)

  1. 一种预防冠状病毒引起疾病的疫苗，含有糖基化的冠状病毒S蛋白受体结合区、氢氧化铝佐剂和CpG佐剂。
  2. 根据权利要求1所述的疫苗，其特征在于：所述冠状病毒为SARS-CoV-2。
  3. 根据权利要求1或2所述的疫苗，其特征在于：所述冠状病毒引起疾病为COVID-19。
  4. 根据权利要求1-3中任一所述的疫苗，其特征在于：所述糖基化的冠状病毒S蛋白受体结合区的氨基酸序列为如下任一：
    (a1)SEQ ID No.21；
    (a2)SEQ ID No.22；
    (a3)SEQ ID No.23；
    (a4)将(a1)-(a3)中任一所限定的序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的序列，或与(a1)-(a3)中任一所限定的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的序列。
  5. 根据权利要求1-4中任一所述的疫苗，其特征在于：所述糖基化的冠状病毒S蛋白受体结合区为具有哺乳动物糖型结构N-糖链修饰的冠状病毒S蛋白受体结合区糖蛋白。
  6. 根据权利要求5所述的疫苗，其特征在于：所述哺乳动物糖型结构N-糖链为Gal 2GlcNAc 2Man 3GlcNAc 2、GalGlcNAcMan 5GlcNAc 2或Man 5GlcNAc 2。
  7. 根据权利要求1-6中任一所述的疫苗，其特征在于：所述CpG佐剂为如下任一：
    (b1)核苷酸序列为SEQ ID No.27的CpG2006或其硫代产物；
    (b2)核苷酸序列为SEQ ID No.28的CpG1018或其硫代产物；
    (b3)核苷酸序列为SEQ ID No.29的CpGX1或其硫代产物；
    (b4)核苷酸序列为SEQ ID No.30的CpG684或其硫代产物。
  8. 根据权利要求7所述的疫苗，其特征在于：所述硫代产物为全链硫代修饰产物。
  9. 根据权利要求1-8中任一所述的疫苗，其特征在于：所述糖基化的冠状病毒S蛋白受体结合区、所述氢氧化铝佐剂、所述CpG佐剂的质量比为(2.5～20)：100：(25～50)；所述氢氧化铝佐剂的量以其中铝的含量计。
  10. 根据权利要求1-9中任一所述的疫苗，其特征在于：所述糖基化的冠状病毒S蛋白受体结合区是按照如下步骤的方法制备得到的：
    (1)对经过糖基化修饰途径遗传改造的巴斯德毕赤酵母进行再改造，使其能够表达冠状病毒S蛋白受体结合区，得到重组酵母细胞；
    所述经过糖基化修饰途径遗传改造的巴斯德毕赤酵母为甘露糖基化修饰途 径缺陷、并重构了哺乳动物细胞N-糖基化修饰途径的巴斯德毕赤酵母细胞突变体；
    (2)培养所述重组酵母细胞，从培养上清中纯化获得具有哺乳动物糖型结构N-糖链修饰的冠状病毒S蛋白受体结合区，即为所述糖基化的冠状病毒S蛋白受体结合区。
  11. 根据权利要求10所述的疫苗，其特征在于：所述经过糖基化修饰途径遗传改造的巴斯德毕赤酵母是按照包括如下步骤的方法制备得到的：
    (A1)失活受体巴斯德毕赤酵母内源的α-1,6-甘露糖转移酶、磷酸甘露糖转移酶、磷酸甘露糖合成酶、β甘露糖转移酶I、β甘露糖转移酶II、β甘露糖转移酶III和β甘露糖转移酶IV，得到重组酵母1；
    (A2)在所述重组酵母1中表达如下外源蛋白中的至少一种：外源甘露糖苷酶I、外源N-乙酰葡萄糖胺转移酶I、外源甘露糖苷酶II、外源N-乙酰葡萄糖胺转移酶II、外源半乳糖异构酶和外源半乳糖转移酶，得到重组酵母2；所述重组酵母2即为所述经过糖基化修饰途径遗传改造的巴斯德毕赤酵母。
  12. 根据权利要求11所述的疫苗，其特征在于：在步骤(A2)之后还包括如下步骤(A3)：
    (A3)失活所述重组酵母2内源的O甘露糖转移酶I，得到重组酵母3；所述重组酵母3也为所述经过糖基化修饰途径遗传改造的巴斯德毕赤酵母。
  13. 根据权利要求11或12所述的疫苗，其特征在于：当所述哺乳动物糖型结构N-糖链为Gal 2GlcNAc 2Man 3GlcNAc 2、GalGlcNAcMan 5GlcNAc 2或Man 5GlcNAc 2时，步骤(A2)中在所述重组酵母1中表达的外源蛋白为外源甘露糖苷酶I、外源N-乙酰葡萄糖胺转移酶I、外源半乳糖异构酶和外源半乳糖转移酶、外源甘露糖苷酶II，以及外源N-乙酰葡萄糖胺转移酶II。
  14. 根据权利要求11或12所述的疫苗，其特征在于：当所述哺乳动物糖型结构N-糖链为GalGlcNAcMan 5GlcNAc 2时，步骤(A2)中在所述重组酵母1中表达的外源蛋白为外源甘露糖苷酶I、外源N-乙酰葡萄糖胺转移酶I，以及外源半乳糖异构酶和外源半乳糖转移酶。
  15. 根据权利要求11或12所述的疫苗，其特征在于：当所述哺乳动物糖型结构N-糖链为Man 5GlcNAc 2时，步骤(A2)中在所述重组酵母1中表达的外源蛋白为外源甘露糖苷酶I。
  16. 根据权利要求11-15中任一所述的疫苗，其特征在于：步骤(A1)中，所述失活受体巴斯德毕赤酵母内源的α-1,6-甘露糖转移酶、磷酸甘露糖转移酶、磷酸甘露糖合成酶、β甘露糖转移酶I、β甘露糖转移酶II、β甘露糖转移酶III和β甘露糖转移酶IV均是采用同源重组的方式进行基因敲除。
  17. 根据权利要求11-16中任一所述的疫苗，其特征在于：步骤(A2)中，在所述重组酵母1中表达所述外源蛋白是通过向所述重组酵母1中导入所述外源蛋白的编码基因实现的。
  18. 根据权利要求17所述的疫苗，其特征在于：所述外源蛋白的编码基因是以重组载体的形式导入所述重组酵母1中的。
  19. 根据权利要求18所述的疫苗，其特征在于：进一步地，所述外源甘露糖苷酶I的编码基因和所述外源甘露糖苷酶II的编码基因均向所述重组酵母1中导入两次。
  20. 根据权利要求12-19中任一所述的疫苗，其特征在于：步骤(A3)中，失活所述重组酵母2内源的O甘露糖转移酶I，是通过对所述重组酵母2的基因组DNA中的O甘露糖转移酶I编码基因进行插入失活实现的。
  21. 根据权利要求11-20中任一所述的疫苗，其特征在于：步骤(A2)中，所述外源甘露糖苷酶I表达后定位于内质网。
  22. 根据权利要求21所述的疫苗，其特征在于：所述外源甘露糖苷酶I来源于绿色木霉，且C端融合内质网保留信号HDEL。
  23. 根据权利要求11-22中任一所述的疫苗，其特征在于：步骤(A2)中，所述外源N-乙酰葡萄糖胺转移酶I表达后定位于内质网或内侧高尔基体。
  24. 根据权利要求23所述的疫苗，其特征在于：所述外源N-乙酰葡萄糖胺转移酶I来源于哺乳动物，在N-端或C-端融合内质网或内侧高尔基体定位信号。
  25. 根据权利要求24所述的疫苗，其特征在于：所述外源N-乙酰葡萄糖胺转移酶I来源于人，且含有mnn9定位信号。
  26. 根据权利要求11-25中任一所述的疫苗，其特征在于：步骤(A2)中，所述外源甘露糖苷酶II表达后定位于内质网或内侧高尔基体。
  27. 根据权利要求26所述的疫苗，其特征在于：所述外源甘露糖苷酶II来源于丝状真菌、植物、昆虫、爪哇或哺乳动物，在N-端或C-端融合内质网或内侧高尔基体定位信号。
  28. 根据权利要求11-27中任一所述的疫苗，其特征在于：步骤(A2)中，所述外源N-乙酰葡萄糖胺转移酶II表达后定位于内质网或内侧高尔基体。
  29. 根据权利要求28所述的疫苗，其特征在于：所述外源N-乙酰葡萄糖胺转移酶II来源于哺乳动物，在N-端或C-端融合内质网或内侧高尔基体定位信号。
  30. 根据权利要求29所述的疫苗，其特征在于：所述N-乙酰葡萄糖胺转移酶II来源于人，且均含有mnn2定位信号。
  31. 根据权利要求11-30中任一所述的疫苗，其特征在于：步骤(A2)中，所述外源甘露糖苷酶II表达后定位于内质网或内侧高尔基体。
  32. 根据权利要求31所述的疫苗，其特征在于：所述外源甘露糖苷酶II来源于线虫，含有mnn2定位信号。
  33. 根据权利要求11-32中任一所述的疫苗，其特征在于：步骤(A2)中，所述外源半乳糖异构酶和所述外源半乳糖转移酶表达后定位于内质网或内侧高尔基体。
  34. 根据权利要求33所述的疫苗，其特征在于：所述外源半乳糖异构酶和所述外源半乳糖转移酶均来源于哺乳动物，在N-端或C-端融合内质网或内侧高尔基体定位信号。
  35. 根据权利要求34所述的疫苗，其特征在于：所述外源半乳糖异构酶和所述外源半乳糖转移酶为融合蛋白，均来源于人，且共用一个kre2定位信号。
  36. 根据权利要求11-35中任一所述的疫苗，其特征在于：所述α-1,6-甘露糖转移酶为如下B1)或B2)：
    B1)氨基酸序列是SEQ ID No.1的蛋白质；
    B2)将SEQ ID No.1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.1所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
  37. 根据权利要求11-36中任一所述的疫苗，其特征在于：所述磷酸甘露糖转移酶为如下B3)或B4)：
    B3)氨基酸序列是SEQ ID No.2的蛋白质；
    B4)将SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.2所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
  38. 根据权利要求11-37中任一所述的疫苗，其特征在于：所述磷酸甘露糖合成酶为如下B5)或B6)：
    B5)氨基酸序列是SEQ ID No.3的蛋白质；
    B6)将SEQ ID No.3所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.3所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
  39. 根据权利要求11-38中任一所述的疫苗，其特征在于：所述β甘露糖转移酶I为如下B7)或B8)：
    B7)氨基酸序列是SEQ ID No.4的蛋白质；
    B8)将SEQ ID No.4所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.4所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
  40. 根据权利要求11-39中任一所述的疫苗，其特征在于：所述β甘露糖转移酶II为如下B9)或B10)：
    B9)氨基酸序列是SEQ ID No.5的蛋白质；
    B10)将SEQ ID No.5所示的氨基酸序列经过一个或几个氨基酸残基的取代 和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.5所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
  41. 根据权利要求11-40中任一所述的疫苗，其特征在于：所述β甘露糖转移酶III为如下B11)或B12)：
    B11)氨基酸序列是SEQ ID No.6的蛋白质；
    B12)将SEQ ID No.6所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.6所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
  42. 根据权利要求11-41中任一所述的疫苗，其特征在于：所述β甘露糖转移酶IV为如下B13)或B14)：
    B13)氨基酸序列是SEQ ID No.7的蛋白质；
    B14)将SEQ ID No.7所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.7所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
  43. 根据权利要求11-42中任一所述的疫苗，其特征在于：所述O甘露糖转移酶I为如下B15)或B16)：
    B15)氨基酸序列是SEQ ID No.8的蛋白质；
    B16)将SEQ ID No.8所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.8所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
  44. 根据权利要求11-43中任一所述的疫苗，其特征在于：所述外源甘露糖苷酶I为如下B17)或B18)：
    B17)氨基酸序列是SEQ ID No.9的蛋白质；
    B18)将SEQ ID No.9所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.9所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
  45. 根据权利要求11-44中任一所述的疫苗，其特征在于：所述外源N-乙酰葡萄糖胺转移酶I为如下B19)或B20)：
    B19)氨基酸序列是SEQ ID No.10的蛋白质；
    B20)将SEQ ID No.10所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.10所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具 有相同功能的蛋白质。
  46. 根据权利要求11-45中任一所述的疫苗，其特征在于：由所述半乳糖异构酶和所述半乳糖转移酶组成的所述融合蛋白为如下B21)或B22)：
    B21)氨基酸序列是SEQ ID No.11的蛋白质；
    B22)将SEQ ID No.11所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.11所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
  47. 根据权利要求11-46中任一所述的疫苗，其特征在于：所述甘露糖苷酶II为如下B23)或B24)：
    B23)氨基酸序列是SEQ ID No.12的蛋白质；
    B24)将SEQ ID No.12所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.12所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
  48. 根据权利要求11-47中任一所述的疫苗，其特征在于：所述N-乙酰葡萄糖胺转移酶II为如下B25)或B26)：
    B25)氨基酸序列是SEQ ID No.13的蛋白质；
    B26)将SEQ ID No.13所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质，或与SEQ ID No.13所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质。
  49. 根据权利要求11-48中任一所述的疫苗，其特征在于：所述外源甘露糖苷酶I的编码基因为如下C1)或C2)：
    C1)核苷酸序列是SEQ ID No.14的DNA分子；
    C2)与SEQ ID No.14所示的核苷酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且编码所述外源甘露糖苷酶I的DNA分子，或在严格条件下与C1)限定的DNA分子杂交且编码所述外源甘露糖苷酶I的DNA分子。
  50. 根据权利要求11-49中任一所述的疫苗，其特征在于：所述外源N-乙酰葡萄糖胺转移酶I的编码基因为如下C3)或C4)：
    C3)核苷酸序列是SEQ ID No.15的DNA分子；
    C4)与SEQ ID No.15所示的核苷酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且编码所述外源N-乙酰葡萄糖胺转移酶I的DNA分子，或在严格条件下与C3)限定的DNA分子杂交且编码所述外源N-乙酰葡萄糖胺转移酶I的DNA分子。
  51. 根据权利要求11-50中任一所述的疫苗，其特征在于：由所述半乳糖异构酶和所述半乳糖转移酶组成的所述融合蛋白的编码基因为如下C5)或C6)：
    C5)核苷酸序列是SEQ ID No.16的DNA分子；
    C6)与SEQ ID No.16所示的核苷酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且编码所述融合蛋白的DNA分子，或在严格条件下与C5)限定的DNA分子杂交且编码所述融合蛋白的DNA分子。
  52. 根据权利要求11-51中任一所述的疫苗，其特征在于：所述甘露糖苷酶II的编码基因为如下C7)或C8)：
    C7)核苷酸序列是SEQ ID No.17的DNA分子；
    C8)与SEQ ID No.17所示的核苷酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且编码所述甘露糖苷酶II的DNA分子，或在严格条件下与C7)限定的DNA分子杂交且编码所述甘露糖苷酶II的DNA分子。
  53. 根据权利要求11-52中任一所述的疫苗，其特征在于：所述N-乙酰葡萄糖胺转移酶II的编码基因为如下C9)或C10)：
    C9)核苷酸序列是SEQ ID No.18的DNA分子；
    C10)与SEQ ID No.18所示的核苷酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且编码所述N-乙酰葡萄糖胺转移酶II的DNA分子，或在严格条件下与C9)限定的DNA分子杂交且编码所述N-乙酰葡萄糖胺转移酶II的DNA分子。
  54. 根据权利要求1-53中任一所述的疫苗，其特征在于：所述经过糖基化修饰途径遗传改造的巴斯德毕赤酵母为在中国微生物菌种保藏管理委员会普通微生物中心保藏的保藏编号为CGMCCNo.19488的菌株。
  55. 根据权利要求10-54中任一所述的疫苗，其特征在于：步骤(1)中，所述重组酵母细胞是将所述冠状病毒S蛋白受体结合区的编码基因导入所述经过糖基化修饰途径遗传改造的巴斯德毕赤酵母中后得到的。
  56. 根据权利要求1-55中任一所述的疫苗，其特征在于：所述冠状病毒S蛋白受体结合区的编码基因为如下任一：
    (c1)SEQ ID No.24所示DNA分子；
    (c2)SEQ ID No.25所示DNA分子；
    (c3)SEQ ID No.26所示DNA分子；
    (c4)与SEQ ID No.24至SEQ ID No.26中任一所示的核苷酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且编码所述冠状病毒S蛋白受体结合区的DNA分子，或在严格条件下与SEQ ID No.24至SEQ ID No.26中任一所示的DNA分子杂交且编码所述冠状病毒S蛋白受体结合区的DNA分子。
  57. 根据权利要求10-56中任一所述的疫苗，其特征在于：步骤(2)中，是按照包括如下步骤的方法从所述培养上清中纯化获得所述具有哺乳动物糖型结构N-糖链修饰的冠状病毒S蛋白受体结合区的：将所述培养上清依次进行阳离子交换层析、疏水层析、G25脱盐、阴离子交换层析，获得所述具有哺乳动物糖型结构N-糖链修饰的冠状病毒S蛋白受体结合区。
  58. 根据权利要求57所述的疫苗，其特征在于：步骤(2)中，是按照包括如下步骤的方法从所述培养上清中纯化获得所述具有哺乳动物糖型结构N-糖链修饰的冠状病毒S蛋白受体结合区的：将所述培养上清通过CaptoMMC层析柱进行目的蛋白的捕获，然后通过含有1M NaCl的缓冲液洗脱获得含有所述目的蛋白的粗样；之后将所述粗样用疏水层析柱Phenyl HP纯化，将含有所述目的蛋白的洗脱峰样品用G25层析柱除盐，然后用阴离子交换层析柱Source30Q吸附杂蛋白，流穿液即是所述目的蛋白；所述目的蛋白即为所述具有哺乳动物糖型结构N-糖链修饰的冠状病毒S蛋白受体结合区。
  59. 制备权利要求1-58中任一所述疫苗的方法，包括如下步骤：按照权利要求10-58任一中所述的方法制备得到所述糖基化的冠状病毒S蛋白受体结合区；然后将所述糖基化的冠状病毒S蛋白受体结合区、氢氧化铝佐剂和权利要求1-8任一中所述的CpG佐剂按照质量比为(2.5～20)：100：(25～50)的比例混合制备得到所述疫苗；所述氢氧化铝佐剂的量以其中铝的含量计。
  60. 如下任一应用：
    P1、权利要求1-58中任一所述疫苗在预防冠状病毒引起疾病中的应用；
    P2、权利要求1-58中任一所述疫苗在中和冠状病毒中的应用。
  61. 根据权利要求60所述的应用，其特征在于：所述冠状病毒为SARS-CoV-2。
  62. 根据权利要求60或61所述的应用，其特征在于：所述冠状病毒引起疾病为COVID-19。
  63. 一种预防冠状病毒引起疾病的方法，是利用权利要求1-58中任一所述疫苗预防冠状病毒引起疾病。
  64. 根据权利要求63所述的方法，其特征在于：所述冠状病毒为SARS-CoV-2。
  65. 根据权利要求63或64所述的方法，其特征在于：所述冠状病毒引起疾病为COVID-19。
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Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114717205A (zh) * 2022-03-29 2022-07-08 中国人民解放军军事科学院军事医学研究院 一种冠状病毒RBDdm变异体及其应用
CN114989308B (zh) * 2022-05-12 2023-04-04 中国科学院微生物研究所 新冠病毒嵌合核酸疫苗及其用途

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101022827A (zh) * 2004-06-30 2007-08-22 魁北克益得生物医学公司 用于治疗冠状病毒感染的疫苗组合物
CN101100680A (zh) * 2007-06-15 2008-01-09 中国科学院武汉病毒研究所 高效表达sars冠状病毒s蛋白的重组杆状病毒及构建
CN108697778A (zh) * 2016-02-16 2018-10-23 哈佛学院院长等 病原体疫苗及其生产和使用方法
CN110974950A (zh) * 2020-03-05 2020-04-10 广州恩宝生物医药科技有限公司 一种用于预防SARS-CoV-2感染的腺病毒载体疫苗
CN111088283A (zh) * 2020-03-20 2020-05-01 苏州奥特铭医药科技有限公司 mVSV病毒载体及其病毒载体疫苗、一种基于mVSV介导的新冠肺炎疫苗
CN111218458A (zh) * 2020-02-27 2020-06-02 珠海丽凡达生物技术有限公司 编码SARS-CoV-2病毒抗原的mRNA和疫苗及疫苗的制备方法
CN111217919A (zh) * 2020-03-04 2020-06-02 中山大学 一种基于火球菌铁蛋白的新型冠状病毒s蛋白双区域亚单位纳米疫苗
CN111218459A (zh) * 2020-03-18 2020-06-02 中国人民解放军军事科学院军事医学研究院 一种以人复制缺陷腺病毒为载体的重组新型冠状病毒疫苗
CN111217918A (zh) * 2020-03-04 2020-06-02 中山大学 一种基于2,4-二氧四氢喋啶合酶的新型冠状病毒s蛋白双区域亚单位纳米疫苗

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW200510450A (en) * 2003-07-21 2005-03-16 Nat Inst Health Soluble fragments of the SARS-CoV spike glycoprotein
CN101098710A (zh) * 2004-06-02 2008-01-02 纽约血液中心 产生高度有效抗体的sars疫苗和方法
EP1773388A4 (en) * 2004-06-02 2008-08-20 New York Blood Ct Inc SARS VACCINES AND METHODS OF PRODUCING VERY POWERFUL ANTIBODIES
CN101402961A (zh) * 2008-11-17 2009-04-08 中国人民解放军军事医学科学院生物工程研究所 一种生产糖蛋白疫苗的方法
US10676511B2 (en) * 2015-09-17 2020-06-09 Ramot At Tel-Aviv University Ltd. Coronaviruses epitope-based vaccines
CN106928326B (zh) * 2015-12-31 2019-12-24 中国科学院微生物研究所 一种基于二聚化的受体结合区亚单位的冠状病毒疫苗
US11141476B2 (en) * 2016-12-23 2021-10-12 Curevac Ag MERS coronavirus vaccine
CN111217917B (zh) * 2020-02-26 2020-10-23 康希诺生物股份公司 一种新型冠状病毒SARS-CoV-2疫苗及其制备方法

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101022827A (zh) * 2004-06-30 2007-08-22 魁北克益得生物医学公司 用于治疗冠状病毒感染的疫苗组合物
CN101100680A (zh) * 2007-06-15 2008-01-09 中国科学院武汉病毒研究所 高效表达sars冠状病毒s蛋白的重组杆状病毒及构建
CN108697778A (zh) * 2016-02-16 2018-10-23 哈佛学院院长等 病原体疫苗及其生产和使用方法
CN111218458A (zh) * 2020-02-27 2020-06-02 珠海丽凡达生物技术有限公司 编码SARS-CoV-2病毒抗原的mRNA和疫苗及疫苗的制备方法
CN111217919A (zh) * 2020-03-04 2020-06-02 中山大学 一种基于火球菌铁蛋白的新型冠状病毒s蛋白双区域亚单位纳米疫苗
CN111217918A (zh) * 2020-03-04 2020-06-02 中山大学 一种基于2,4-二氧四氢喋啶合酶的新型冠状病毒s蛋白双区域亚单位纳米疫苗
CN110974950A (zh) * 2020-03-05 2020-04-10 广州恩宝生物医药科技有限公司 一种用于预防SARS-CoV-2感染的腺病毒载体疫苗
CN111218459A (zh) * 2020-03-18 2020-06-02 中国人民解放军军事科学院军事医学研究院 一种以人复制缺陷腺病毒为载体的重组新型冠状病毒疫苗
CN111088283A (zh) * 2020-03-20 2020-05-01 苏州奥特铭医药科技有限公司 mVSV病毒载体及其病毒载体疫苗、一种基于mVSV介导的新冠肺炎疫苗

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHU, FENGCAI ET AL.: "Safety, Tolerability, and Immunogenicity of a Recombinant Adenovirus Type-5 Vectored COVID-19 Vaccine: A Dose-escalation, Open-label, Non-randomised, First- in-human Trial", LANCET, vol. 395, no. 10240, 13 June 2020 (2020-06-13), pages 1845 - 1854, XP086181839, DOI: 10.1016/S0140-6736(20)31208-3 *

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