WO2021244255A1 - Procédé de préparation de la glycoprotéine rbd de la protéine de spicule du coronavirus, et son utilisation - Google Patents

Procédé de préparation de la glycoprotéine rbd de la protéine de spicule du coronavirus, et son utilisation Download PDF

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WO2021244255A1
WO2021244255A1 PCT/CN2021/093757 CN2021093757W WO2021244255A1 WO 2021244255 A1 WO2021244255 A1 WO 2021244255A1 CN 2021093757 W CN2021093757 W CN 2021093757W WO 2021244255 A1 WO2021244255 A1 WO 2021244255A1
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protein
amino acid
exogenous
coronavirus
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刘波
吴军
孙鹏
王甜甜
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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Definitions

  • the invention relates to the field of biomedicine, in particular to a preparation method and application of coronavirus S protein RBD glycoprotein.
  • coronaviruses In the past 20 years, a variety of coronaviruses have broken through species boundaries and spread to humans, causing about 30% of respiratory infections (Coronaviruses: drug discovery and therapeutic options) and causing huge losses. This reminds us that the coronavirus is increasingly threatening human health, and the research of related vaccines is the most urgent task.
  • the S protein is the only protein on the surface of the coronavirus. It is a type I membrane protein and is modified by N-glycosylation. Its monomer is composed of about 1300 amino acids. The monomer is folded and polymerized to form a homotrimer. The S protein monomer is composed of the N-terminal S1 subunit and the C-terminal S2 subunit. The S1 subunit is responsible for binding to the host cell receptor. After the virus is taken up by the host cell, the S2 subunit is close to the S2 site of the fusion peptide. Host protease cleavage triggers the conformational change of the S protein, and the S2 subunit mediates membrane fusion (Reference: Alexandra C Walls, et al. Glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy. Nat Struct Mol Biol .2016, Oct; 23(10):899-905.).
  • Vaccine studies based on the full-length SARS-CoV S protein have found that although the 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 tropism. The safety of acid cell immunopathology or antibody-mediated immune enhancement ADE and other adverse reactions has been widely questioned.
  • the new coronavirus SARS-CoV-2 S protein and SARS-CoV S protein have high homology, and the high-level structures of the two also have a certain degree of similarity, and SARS-CoV-2 also invades human cells through the receptor ACE2 (cryo -EM structure of the 2019-nCoV spike in the precusion conformation), which reminds us that the development experience of the SARS-CoV vaccine can provide clues for the development of the SARS-CoV-2 vaccine.
  • the RBD region of SARS-CoV S protein can form the correct conformation and contains multiple spatial structure-dependent epitopes. It is one of the main antigens investigated in several subunit vaccines, that is, RBD is in addition to the Spike protein. S1 area, S2 area, full-length S area, nucleoprotein is one of several antigens to be investigated.
  • the SARS-CoV-2 S protein RBD has two potential N-glycosylation sites, and the correct glycoform structure plays an important role in maintaining the natural conformation and immunogenicity of RBD.
  • Yeast as a microorganism that has been used in industrial scale production for a long time, has been successfully used in the production of non-glycoprotein subunit vaccines such as hepatitis B (HBV) vaccine and papilloma virus (HPV) vaccine. It has high safety , Engineering strains have short construction period, fast growth, and easy mass production, making them very suitable as an expression system for high-efficiency and large-scale vaccine production under sudden infectious diseases and other emergency conditions. However, yeast has the phenomenon of excessive glycosylation (excess mannosylation), and the masking of important epitopes by excessive glycosylation will reduce the protective effect of the vaccine.
  • HBV hepatitis B
  • HPV papilloma virus
  • yeast glycosylation modification system makes the glycosylation modification close to, or even better than, the natural glycosyl structure of the antigen, which is expected to allow the genetically modified yeast to replace chicken embryos and mammalian cells with For the rapid and efficient production of genetic engineering subunit vaccines.
  • the purpose of the present invention is to provide a method and application for preparing a coronavirus S protein RBD with mammalian glycoform structure N-sugar chain modification by using Pichia pastoris genetically modified by glycosylation modification pathway.
  • the present invention claims a method for preparing a coronavirus S protein receptor binding domain (RBD) with a mammalian glycoform structure N-sugar chain modification.
  • the method for preparing the N-sugar chain modified coronavirus S protein receptor binding domain (RBD) with mammalian glycoform structure as claimed in the present invention may include 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.
  • N-glycosylation modification will occur at its conservative N-glycosylation modification site (NXS/T), but because O-glycosylation modification does not have a conservative glycosylation site, it is generally considered O-glycosylation can occur on amino acids that are enriched in serine or threonine. Whether O-glycosylation occurs in different proteins, and on which amino acid, the degree of O-glycosylation is different.
  • Protein serine or threonine may be a potential site for O-glycosylation, but not every serine or threonine will undergo O-glycosylation modification, and not every serine or threonine containing serine or threonine Proteins will undergo O-glycosylation modification, and different proteins have different glycosylation modifications in different expression systems. If O-glycosylation is modified, most of the sugar groups on the sugar chains are mannose. Although the sugar chains are relatively short, due to the large number of sugar chains, there may be a large amount of exposed mannose on the surface of the yeast expressed protein. This glycoprotein with mannosylation has a short half-life, high immunogenicity, and is easy to be eliminated in the human body. Due to this defect, the application of Pichia pastoris in the production of most protein drugs is limited.
  • O-glycosyltransferase family members are divided into three subfamilies: PMT1 subfamily, PMT2 subfamily and PMT4 subfamily.
  • the number of members of PMT1 subfamily and PMT2 subfamily may be different in different species.
  • the PMT1 subfamily of Saccharomyces cerevisiae includes PMT1 ⁇ PMT5 ⁇ PMT7
  • the PMT2 subfamily includes PMT2 ⁇ PMT3 ⁇ PMT638.
  • Pmt1p subfamily Pmt1p, Pmt5p
  • Pmt2p subfamily Pmt2p, Pmt3p
  • Pmt4p will form homoduplexes
  • Pmt6p can neither form heteroduplexes with other members of the Pmtp family, nor can it It forms a homologous duplex with itself.
  • the complexes formed by members of the Pmt1p subfamily and Pmt2p subfamily are mainly Pmt1p–Pmt2p and Pmt5p–Pmt3p complexes, and there are also a small amount of Pmt1p–Pmt3p and Pmt2p–Pmt5p complexes.
  • step (A3) may be included after step (A2):
  • (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.
  • Step (A3) further reduces the yeast O glycosylation modification.
  • the N-sugar chain of the mammalian glycoform structure can be Gal 2 GlcNAc 2 Man 3 GlcNAc 2 , GalGlcNAcMan 5 GlcNAc 2 or Man 5 GlcNAc 2 .
  • Gal galactose
  • GlcNAc N-acetylglucosamine
  • Man mannose.
  • the foreign protein expressed in the recombinant yeast 1 in step (A2) can be exogenous mannosidase I, exogenous N-acetylglucosamine transferase I, exogenous galactose isomerase and exogenous galactose transferase, exogenous mannosidase II, and exogenous N-acetyl glucose Aminotransferase II.
  • the exogenous protein expressed in the recombinant yeast 1 in step (A2) may also be exogenous mannosidase I, exogenous N- Acetyl glucosamine transferase I, as well as exogenous galactose isomerase and exogenous galactose transferase.
  • the exogenous protein expressed in the recombinant yeast 1 in step (A2) may also be 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.
  • the gene encoding Omannose transferase I in the genomic DNA of the recombinant yeast 2 is inserted and inactivated (disrupting its corresponding nucleotide sequence by inserting inactivation).
  • 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.
  • the next technical problem is to construct an engineered Pichia strain with mammalian cell glycoform modification ability in yeast chassis cells.
  • glycosyl modification enzymes involved in the glycosyl modification of mammalian cells. What kind of enzyme modification will be obtained? What kind of sugar type? As well as the ratio and combination of sugar types obtained are not known before research.
  • the present invention is realized by the following technical methods:
  • step (A2) the exogenous mannosidase I is localized in the endoplasmic reticulum after expression.
  • 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 can be N-acetylglucosamine transferase I derived from mammals, etc., such as human N-acetylglucosamine transferase I (GenBank NO NM 002406), Candida albicans N -Acetyl glucosamine transferase I (GenBank NO NW_139513.1), Dictyostelium discoideum N-acetylglucosamine transferase I (GenBank NO NC_007088.5), etc., can be fused to the N-terminus or C-terminus Net or medial Golgi positioning signal, such as ScGLS, ScMNS1, PpSEC12, ScMNN9, etc.; preferably it is derived from humans and contains mnn9 positioning signal.
  • human N-acetylglucosamine transferase I GenBank NO NM 002406
  • step (A2) the exogenous mannosidase II is expressed and localized in the endoplasmic reticulum or medial Golgi apparatus.
  • the exogenous mannosidase II may be mannosidase II derived from filamentous fungi, plants, insects, Java, mammals, etc., Drosophila mannosidase II (GenBank NOX77652), nematode mannosidase II (GenBank NO NM 0735941), human mannosidase II (GenBank NO U31520), etc.; the expressed mannosidase II can be fused with the endoplasmic reticulum or medial Golgi localization signal at the N-terminus or C-terminus, such as ScGLS, ScMNS1, PpSEC12, ScMNN9, etc., are preferably derived from nematodes and contain mnn2 localization signal.
  • 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 may be N-acetylglucosamine transferase II derived from mammals, etc., such as human N-acetylglucosamine transferase II (GenBank NO Q10469), mouse N-acetyl Glucosamine transferase II (GenBank NO Q09326), etc.; the expressed N-acetylglucosamine transferase II can be fused with the endoplasmic reticulum or medial Golgi localization signal at the N-terminus or C-terminus, such as ScGLS, ScMNS1, PpSEC12, ScMNN9, etc., are preferably derived from humans and contain mnn2 positioning signals.
  • 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 an endoplasmic reticulum or medial Golgi localization signal at the N-terminal or C-terminal.
  • exogenous galactose isomerase and the exogenous galactose transferase are fusion proteins, both of which are selected from humans, and share a kre2 localization signal.
  • Galactosyltransferase can be a galactosyltransferase derived from mammals, such as human ⁇ -1,4-galactosyltransferase (GenBank NO gi:13929461), murine ⁇ -1,4-galactosyltransferase GenBank NO NC_000081.6) and so on.
  • the expressed galactosyltransferase can be fused with the endoplasmic reticulum or medial Golgi localization signal at the N-terminal or C-terminal, such as ScKRE2, ScGLS, ScMNS1, PpSEC12, ScMNN9, etc.
  • the galactosyltransferase in the embodiment of the present invention is derived from human , And there is a kre2 positioning 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 recombinant yeast cell can introduce the coding gene of the coronavirus S protein receptor binding region (RBD) into the genetically modified Pichia pastoris through the glycosylation modification pathway After getting.
  • 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 recombinant vector is specifically a recombinant vector obtained by cloning the coding gene of the coronavirus S protein receptor binding region (RBD) into a pPICZ ⁇ A vector (such as restriction sites XhoI and NotI).
  • coronavirus S protein receptor binding domain can be any of the following:
  • amino acid sequence defined in any one of the proteins has 99% or more, 95% or more, 90% or more, 85% or more than 80% homology and has the same function.
  • the coding gene of the coronavirus S protein receptor binding region may be the one encoding the coronavirus S protein receptor binding region shown in any one of (a1) to (a5). DNA molecule.
  • the coding gene of the coronavirus S protein receptor binding region can be any of the following:
  • (b5) 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. 25 to SEQ ID No. 28 and encodes
  • the DNA molecule of the coronavirus S protein receptor binding region (RBD) may hybridize with any of the DNA molecules shown in SEQ ID No. 25 to SEQ ID No. 28 under stringent conditions and encode the coronavirus S Protein receptor binding domain (RBD) DNA molecule.
  • SEQ ID No. 21 to SEQ ID No. 24 are all part of the S protein of 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-K537 region (RBD219) of the S protein
  • SEQ ID No. 23 is the R319-V534 region of the S protein (RBD216)
  • SEQ ID No. 24 is the R319-K528 region of the S protein (RBD210).
  • the nucleotide sequences of SEQ ID No. 25 to SEQ ID No. 28 are obtained by codon optimization according to the amino acid sequences of SEQ ID No. 21 to SEQ ID No. 24, 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
  • 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.
  • the mammalian glycoform structure N-sugar chain modified coronavirus S protein receptor binding region is purified from the culture supernatant according to a method including the following steps:
  • the culture supernatant is passed through a CaptoMMC chromatography column to capture the target protein, and then eluted with a buffer containing 1M NaCl to obtain a crude sample containing the target protein; then the crude sample is purified by a hydrophobic chromatography column Phenyl HP ,
  • 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.
  • the flow-through fluid is the target protein;
  • the target protein is the Coronavirus S protein receptor binding region modified by N-sugar chain with mammalian glycoform structure.
  • D3 A drug used to prevent and/or treat diseases caused by coronavirus infection, the active ingredient of which is D1) the mammalian glycoform structure N-sugar chain modified coronavirus S protein receptor binding region.
  • D4) A drug capable of inhibiting coronavirus, the active ingredient of which is D1) the N-sugar chain modified coronavirus S protein receptor binding domain with mammalian glycoform structure.
  • Coronavirus vaccine the active ingredient of which is D1) Coronavirus S protein receptor binding region with mammalian glycoform structure N-sugar chain modification;
  • the coronavirus vaccine contains an antigen and an adjuvant;
  • the antigen is D1) the N-sugar chain modified coronavirus S protein receptor binding region with a mammalian glycoform structure;
  • the adjuvant may be aluminum Adjuvant.
  • the adjuvant is specifically aluminum hydroxide.
  • the coronavirus vaccine is prepared by mixing the N-sugar chain-modified coronavirus S protein receptor binding region with the mammalian glycoform structure described in D1) and aluminum hydroxide in a mass ratio of 1:10.
  • D7 A product that can cause the production of specific antibodies against the coronavirus S protein receptor binding region in animals, the active ingredient of which is D1)
  • D8 D2) Application of the recombinant yeast cell in preparing D1) the N-sugar chain modified coronavirus S protein receptor binding region with mammalian glycoform structure.
  • D9) D1) The N-sugar chain modified coronavirus S protein receptor binding region of the mammalian glycoform structure is prepared in D3) or D4) the drug, D5) the reagent or kit, D6) the Coronavirus vaccine or D7) application in the product.
  • the present invention claims a method for preparing the drugs described in D3) or D4), the reagents or kits described in D5), the coronavirus vaccine described in D6), or the products described in D7).
  • the above-mentioned coronavirus S protein receptor binding region with mammalian glycoform structure N-sugar chain modification is used as a raw material for preparation.
  • the coronaviruses are all SARS-CoV-2.
  • the coronavirus is specifically SARS-CoV-2
  • 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 result of DSA-FACE glycotype analysis 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/pPICZ ⁇ -SARS2 S-RBD (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/pPICZ ⁇ -S-RBD (RBD223) at different induction times.
  • Figure 18 is an SDS-PAGE chart of SARS-CoV-2 S-RBD (RBD223) purified sample.
  • Figure 19 is a WB comparison diagram of CGMCC19488/pPICZ ⁇ -S-RBD (RBD223) and X33/pPICZ ⁇ -S-RBD (RBD223) expressing RBD (RBD223) glycoprotein.
  • the upper part is SDS-PAGE electrophoresis analysis, and the lower part is Western Blotting analysis; 1-3 are 3 different X33/pPICZ ⁇ -S-RBD clones.
  • Figure 20 shows the electrophoresis diagram of SARS-CoV-2 S-RBD (RBD223) digested by PNGF and Endo H.
  • Figure 21 is the DSA-FACE sugar chain analysis result of SARS-CoV-2 S-RBD (RBD223) glycoprotein expressed by CGMCC19488.
  • Figure 22 shows the mouse serum anti-RBD antibody titer (RBD223 glycoprotein) 14 days after the second immunization.
  • Figure 23 shows the results of virus neutralization test (RBD223 glycoprotein).
  • Figure 24 is an SDS-PAGE analysis of SARS-CoV-2 S-RBD (RBD210, RBD216, RBD219, and RBD223) expressed by CGMCC19488.
  • Figure 25 shows the glycotype analysis results of SARS-CoV-2 S-RBD (RBD210, RBD216 and RBD219) glycoproteins expressed by CGMCC19488.
  • Figure 26 shows mouse serum anti-RBD antibody titers (RBD210, RBD216, RBD219 and RBD223 glycoprotein) 14 days after the second immunization.
  • Figure 27 shows the results of virus neutralization test (RBD210, RBD216, RBD219 and RBD223 glycoprotein).
  • 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 used in the experiment 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. 35);
  • PNO1-ORF02 5'-TACAAGCCAGTTTCGCAATAA-3' (SEQ ID No. 36).
  • 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. 41) and the primer sequence inner01: 5 on the vector '-AGCGTCGATTTTTGTGATGCTCGTCA-3' (SEQ ID No. 34), the band amplified by the primer is a positive clone of about 2kb.
  • MNN4B-ORF01 5'-AAAACTATCCAATGAGGGTCTC-3' (SEQ ID No. 42);
  • MNN4B-ORF02 5'-TCTTCAATGTCTTTAACGGTGT-3' (SEQ ID No. 43).
  • 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 ARM2-5-5OUT outside the 5'homology arm of the ARM2 gene: 5'-TTTTCCTCAAGCCTTCAAAGACAG-3' (SEQ ID No. 48) and the primer sequence inner01: 5 on the vector '-AGCGTCGATTTTTGTGATGCTCGTCA-3' (SEQ ID No. 34), the band amplified by the primer is a positive clone of about 0.8kb.
  • Arm2-ORF-09 5'-gggcagaagatcctagag-3' (SEQ ID No.49);
  • Arm2-ORF-10 5'-tcgtctccattgctatctacgact-3' (SEQ ID No. 50).
  • 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. 51, 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:
  • ARM1-3-5 5'-ttggtatcttccttgctgctg GCGGCCGC acggagaaaggagaacggagaa-3' (SEQ ID No. 53, underlined part is NotI recognition site);
  • ARM1-3-3 5'-TCA ACGCGT TGGCTGGAGGTGACAGAGGAA-3' (SEQ ID No. 54, 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. 55, the underlined part is the MluI recognition site);
  • ARM3-5-3 5'-tcctactttgcttatcatctgcc GCGGCCGC ggtcaggccctcttatggttgtg-3' (SEQ ID No. 56, underlined part is NotI 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:
  • ARM3-3-5 5'-cacaaccataagagggcctgacc GCGGCCGC ggcagatgataagcaaagtagga-3' (SEQ ID No. 57, the underlined part is the NotI recognition site);
  • ARM3-3-3 5'-TCA ACGCGT CATAGGTAATGGCACAGGGATAG-3' (SEQ ID No. 58, 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. 59, the underlined part is the recognition site of MluI);
  • ARM4-5-3 5'-gcatagggctgaagcatactgt GCGGCCGC aatgatatgtacgttcccaaga-3' (SEQ ID No. 60, the underlined part is the NotI recognition site).
  • 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. 62, 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 primers 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.66);
  • Arm1-ORF-10 5'-AGATTGAGCATAGGAGTGGC-3' (SEQ ID No. 67).
  • Arm3-ORF-09 5'-AAACGGAGTCCAGTTCTTCT-3' (SEQ ID No. 68);
  • Arm3-ORF-10 5'-CAACTTTGCCTGTCATTTCC-3' (SEQ ID No. 69).
  • Arm4-ORF-09 5'-CGCTTCAGTTCACGGACATA-3' (SEQ ID No. 70);
  • Arm4-ORF-10 5'-GCAACCCAGACCTCCTTACC-3' (SEQ ID No. 71).
  • ⁇ -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. 72); the underlined sequence is the Xho I restriction recognition site.
  • TrmdsI-3 5'-AGG GAATTC TTACAACTCGTCGTGAGCAAGGTGGCCGCCCCGTCGTG ATG-3' (SEQ ID No. 73); 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.
  • yeast electrotransformation competent cells For the method of preparing yeast electrotransformation competent cells in the following steps, refer to the relevant manuals of Invitrogen and "Molecular Cloning, A Laboratory Manual (Fourth Edition)", 2012 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  • 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. Bacteria.
  • the cells were resuspended in 20 mL of pre-cooled sterile 1M sorbitol, and the cells were harvested by centrifugation at 1500 g for 10 minutes at 4°C, and the cells were resuspended in pre-cooled 1M 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. 74);
  • TrMDSI-1.3kb-02 5'-TGATGATGAACGGATGCTAAAG-3' (SEQ ID No. 75).
  • 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. 76) and downstream primer GnTI-02: 5'-GC GAATTC TTAGTGCTAATTCCAGCTAGGATCATAG-3' (SEQ ID No. 77, 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.
  • S.cere MNN9 Golgi localization signal: ScMNN9-03: tatAAT attATGTCACTTTCTCTTGTATCGTACCGCCTAAGAAAGAACCCGTGGGTTAACATTTTTCTACCTGTTTTGGCCATATTTCTAATATATATAATTTTTTTCCAGAGAGATCAATCTtcagtcagcgctctcgatggc78)
  • 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.79);
  • HuGnTI-0.9k-02 5'-CGGAACTGGAAGGTGACAATA-3' (SEQ ID No. 80).
  • 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.81);
  • GalE3' 5'-AG GGTACC ATCGGGATATCCCTGTGGATGGC-3' (SEQ ID No. 82, the underlined part is the recognition sequence of KpnI);
  • GalT5' 5'-AT GGTACC GGTGGTGGACGTGACCTTTCTCGTCTGCCA-3' (SEQ ID No. 83, the underlined part is the recognition sequence of KpnI).
  • GalT3' 5'-GC atttaaat ttaGCTCGGTGTCCCGATGTCCACTGTGAT-3' (SEQ ID No. 84, the underlined part is the recognition sequence of SwaI).
  • 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.87) and GalE- T(1.5k)-02 (5'-GGAGCTTAGC ACGATTGAATATAGT-3', SEQ ID No. 88) was used as primers, and PCR amplification was performed, and the PCR amplification products were 1.5kb, which proves that GalE-T has been inserted into the genome , Which is the positive engineering 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. 92).
  • the glyco-engineered yeast strain 150L2 with mammalian Gal2GlcNAc2Man3GlcNAc2 and a fucose-free 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.
  • the underline is the SspI restriction site
  • the downstream primer (GnTII-02: 5'-GCT atttaaat TTAtcactgcagtcttctataacttttac-3', SEQ ID No.94) (the underline is the SwaI restriction site)
  • 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.
  • Extract 150L2 of genomic DNA using glass bead preparation method use genomic DNA as template, and use RnGnTII-0.8k-01 and RnGnTII-0.8k-02 as primers to perform PCR amplification.
  • the PCR amplification product is 0.8kb, which proves that 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. 96).
  • 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, knocking out the light and heavy chain genes of the anti-her2 antibody in the yeast genome 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. 97); AOX1TT-3: 5'-tctacgcgtaagcttgcacaaacgaacttc-3' (SEQ ID No. 98) )).
  • 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 as follows: 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, the clone with the correct sequence was named Trans5 ⁇ -pYES2-URA3-AOX1TT, the plasmid was extracted, and the recombinant vector with AOX1TT terminator added to the end of URA3 gene was obtained. 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.
  • PMT1-IN-5 5’-tctatgcattaatgatagttaatgactaatagagtaaacaagtcctcaagaggt-3’ (SEQ ID No.99);
  • PMT1-IN-3 5'-tgacataactaattacatgatctattagtcattaactatcattagatcagagtggggacgacta agaaa gc-3' (SEQ ID No. 100).
  • the reaction conditions for PCR to catch the PMT1 gene fragment were 94°C pre-denaturation for 5 minutes; 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.
  • CYC1TT-5 5'-gctttcttagtcgtccccactctgatctaatgatagttaatgactaatagatcatgtaattagttatgtca-3' (SEQ ID No.101); CYC1TT-3' (SEQ ID No.101); CYC1TT-3'(SEQ ID No.101); '(SEQ ID No.102)) PCR amplification was performed 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 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. 104).
  • 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.
  • Pichia pastoris prefers codon-optimized DNA sequence and inserts it between the XhoI and NotI restriction sites of pPICZ ⁇ A vector to obtain the recombinant expression vector pPICZ ⁇ -S-RBD, namely RBD223 expression vector.
  • the structure of the recombinant expression vector pPICZ ⁇ -S-RBD 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 541 (R319-F541) of the S protein of SARS-CoV-2 "Wuhan-Hu-1" isolate, encoding SEQ The SARS-CoV-2 S-RBD (RBD223) protein indicated by ID No.21.
  • Recombinant expression vector pPICZ ⁇ -S-RBD transforms yeast CGMCC No. 19488
  • 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 pPICZ ⁇ -S-RBD 43 ⁇ L, BglII2 ⁇ 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 linearized complete 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.
  • the coated plate After the coated plate has grown out of single clones, randomly pick 8 single clones and inoculate them on a new YPD/Zeocin plate, and invert 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.
  • Example 2 Pick the positive clone identified in Example 2 (ie the recombinant strain CGMCC19488/pPICZ ⁇ -S-RBD) and inoculate it into YPD/Zeocin liquid medium, culture it at 25°C and 200 rpm until the OD 600 is 15-20, with 5% (V /V) was transferred to BMGY medium, cultured at 25°C, 200rpm for 24 hours, and 0.5% methanol was added to induce the expression of S-RBD. Induced every 12 hours, and samples were taken to detect the expression. 48 After hours, 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:
  • SDS-PAGE electrophoresis found that SARS-CoV-2 S-RBD protein can be captured by Capto MMC; after desalting, the sample is purified with SOURCE30Q, and the target protein flows through, and almost all the contaminant protein is adsorbed on the SOURCE30Q chromatography medium.
  • Wild-type Pichia pastoris expresses SARS-CoV-2 S-RBD
  • Example 2 the expression plasmid pPICZ ⁇ -S-RBD was click-transformed into wild Pichia pastoris X33. After cloning and screening, it was verified by SDS-PAGE and WB (for the method, see Example 2), the results are as follows Shown in Figure 19.
  • the N-sugar chain of wild yeast is an excessively mannose glycoform. It can be seen from the figure that the SARS-CoV-2 S-RBD electrophoresis band expressed by X33 is a diffuse area, while the genetically modified CGMCC19488 expressed by the glycosylation modification pathway is expressed.
  • the SARS-CoV-2 S-RBD is a single stripe.
  • PNGase F can cleave high-mannose, hybrid and complex N-sugar chains.
  • the cutting site is the glycosidic bond between the innermost N-acetylglucosamine (GlcNAc) of the sugar chain and asparagine.
  • Endo H only cuts high mannose and hybrid N-sugar chains, and the cutting site is the glycosidic bond between the first and second N-acetylglucosamine (GlcNAc) on the innermost side of the sugar chain.
  • the glycoform structure of SARS-CoV-2 S-RBD glycoprotein can be preliminarily judged by PNGase F and Endo H digestion.
  • the SARS-CoV-2 S-RBD glycoprotein expressed by the purified CGMCC19488 was digested according to the method described in the manual, and the digestion was performed by SDS-PAGE electrophoresis. The result is shown in Figure 20.
  • the RBD glycoform is Gal 2 GlcNAc 2 Man 3 GlcNAc 2 , GalGlcNAcMan 5 GlcNAc 2 or Man 5 GlcNAc 2 .
  • Immunization methods have been published in many documents, such as "Reproduction of Animal Models of Human Diseases, edited by Li Cai, published by People's Medical Publishing House”. The details are as follows: Take 20 female Balb/c mice aged 6-8 weeks and randomly divide them into the following two groups: a normal saline group and an immunization group, where the immunization group is 10 ⁇ g RBD+100 ⁇ g Al(OH) 3 .
  • RBD is the SARS-CoV-2 S-RBD glycoprotein expressed by CGMCC19488 prepared above, and contains 10 ⁇ g RBD and 100 ⁇ g Al(OH) 3 in a volume of 100 ⁇ l as a vaccine 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 indirect ELISA method was used to measure the anti-RBD antibody titers in the serum of each group of mice.
  • Example 5 the two groups of mice were taken serum 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 was performed according to conventional methods (reference: Feng Cai Zhu, et al. Safety, tolerance, and immunogenicity of a recombinant adenovirus 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 3 group of 10 ⁇ g RBD + 100 ⁇ g Al(OH) was 1:25, which was significantly higher than that of the negative control group.
  • the expression and purification of the recombinant SARS-CoV-2 S-RBD glycoprotein in Example 3 Glycotype analysis of 2S-RBD glycoprotein, mouse immunization experiment in Example 5 and virus neutralization experiment in Example 6, respectively construct RBD210, RBD216, RBD219 expression vectors, construct RBD210, RBD216, RBD219 yeast expression Strains were expressed and purified to obtain RBD210, RBD216, and RBD219 glycoproteins, and were subjected to glycotype analysis, mouse immune test and virus neutralization test.
  • the amino acid sequence of RBD219 is shown in SEQ ID No. 22, which is the R319-K537 region of the S protein of the SARS-CoV-2 "Wuhan-Hu-1" isolate (the corresponding coding gene sequence used in this example is shown in SEQ ID No. 26).
  • the amino acid sequence of RBD216 is shown in SEQ ID No. 23, which is the R319-V534 region of the S protein of SARS-CoV-2 "Wuhan-Hu-1" isolate (the corresponding coding gene sequence used in this example is shown in SEQ ID No. .27).
  • the amino acid sequence of RBD210 is shown in SEQ ID No. 24 (the corresponding coding gene sequence used in this example is shown in SEQ ID No. 28), which is the S protein of SARS-CoV-2 "Wuhan-Hu-1” isolate The R319-K528 area.
  • SEQ ID No. 26 to SEQ ID No. 28 are also codon-optimized nucleotide sequences.
  • the glycoform analysis results of RBD210, RBD216, RBD219, and RBD223 glycoproteins are shown in Figure 25. It can be seen that the main glycoforms are: Gal 2 GlcNAc 2 Man 3 GlcNAc 2 , GalGlcNAcMan 5 GlcNAc 2 or Man 5 GlcNAc 2 .
  • mice showed that the antibody titers produced by RBD210, RBD216, RBD219 and RBD223 proteins induced by mice could reach about 1:10000, and there was no difference between the groups, which was significantly higher than the negative control group (Figure 26).
  • the present invention utilizes the coronavirus S protein RBD expressed by Pichia pastoris genetically modified through glycosylation modification pathway to have mammalian glycoform structure N-sugar chain modification, which avoids problems such as fungal glycosylation modification that may cause allergies. .
  • the coronavirus S protein RBD expressed in the present invention can produce high-titer anti-RBD antibodies after immunizing mice, and can neutralize SARS-CoV-2 virus.
  • the engineered Pichia pastoris strain of the present invention has the characteristics of short construction period, fast growth, easy large-scale production, and high safety, which is beneficial to the efficient research and development of new coronavirus vaccines under emergency conditions such as sudden new coronavirus infection. And mass production.

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Abstract

Procédé de préparation de la glycoprotéine RBD de la protéine de spicule du coronavirus, et son utilisation. La présente invention concerne en outre un procédé de préparation d'une protéine RBD de spicule de coronavirus ayant une structure de glycoforme de mammifère modifiant le N-glycane, le procédé comprenant les étapes suivantes : expression d'une protéine RBD de spicule de coronavirus dans Pichia pastoris (mutants cellulaires de Pichia pastoris qui sont défectueux dans une voie de modification de la mannosylation et reconstruisent la voie de modification de la N-glycosylation dans les cellules de mammifères) qui est génétiquement modifiée au moyen d'une voie de modification de la glycosylation, obtenant ainsi des cellules de levure recombinées ; et culture des cellules de levure recombinées, et purification du surnageant de culture pour obtenir une protéine cible. La protéine RBD de spicule de coronavirus ayant une structure glycoforme de mammifère avec modification du N-glycane est exprimée avec succès, et après que des souris aient été immunisées avec celle-ci, des anticorps de haut niveau peuvent être produits, et ainsi, le SARS-CoV-2 peut être neutralisé. La recherche et le développement efficaces et la production à grande échelle de nouveaux vaccins contre les coronavirus sont ainsi facilités.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114957410A (zh) * 2022-06-08 2022-08-30 华兰基因工程有限公司 一种κ株2019-nCoV的表面蛋白受体结合区制备方法

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113549560B (zh) * 2020-04-24 2024-02-13 中国人民解放军军事科学院军事医学研究院 一种用于糖蛋白制备的工程化酵母构建方法及其菌株
CN114380896A (zh) * 2022-02-22 2022-04-22 河北佑仁生物科技有限公司 一种新型冠状病毒s蛋白的表达方法
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
CN115043934B (zh) * 2022-03-31 2023-06-27 深圳市人民医院 靶向新冠病毒的纳米抗体及其制备方法和应用
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 (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101195809A (zh) * 2006-12-07 2008-06-11 中国人民解放军军事医学科学院生物工程研究所 α-1,6-甘露糖转移酶缺失的毕赤酵母菌株及其构建方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013507921A (ja) * 2009-10-16 2013-03-07 メルク・シャープ・エンド・ドーム・コーポレイション 宿主細胞抗原に対する抗体に対する検出可能な交差結合活性を欠くタンパク質の、ピチア・パストリスにおける製造方法
CN105671109B (zh) * 2014-11-20 2019-08-23 中国人民解放军军事医学科学院生物工程研究所 用糖基工程酵母制备具有动物细胞糖基化修饰流感血凝素糖蛋白的方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101195809A (zh) * 2006-12-07 2008-06-11 中国人民解放军军事医学科学院生物工程研究所 α-1,6-甘露糖转移酶缺失的毕赤酵母菌株及其构建方法

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BRIAN D QUINLAN; MOU HUIHUI; ZHANG LIZHOU; GUO YAN; HE WENHUI; OJHA AMRITA; PARCELLS MARK S; LUO GUANGXIANG; LI WENHUI; ZHONG GUOC: "The SARS-CoV-2 receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement.", 11 April 2020 (2020-04-11), pages 1 - 24, XP055734440, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2020.04.10.036418v1.full.pdf> *
CHI-PANG CHUCK; CHI-HANG WONG; LARRY MING-CHEUNG CHOW; KWOK-PUI FUNG; MARY MIU-YEE WAYE; STEPHEN KWOK-WING TSUI: "Expression of SARS-coronavirus spike glycoprotein in Pichia pastoris.", VIRUS GENES, vol. 38, no. 1, 29 October 2008 (2008-10-29), pages 1 - 9, XP019690326, ISSN: 1572-994X, DOI: 10.1007/s11262-008-0292-3 *
DATABASE Protein 7 May 2020 (2020-05-07), ANONYMOUS: "PDB:6M17_E Chain E, SARS-coV-2 Receptor Binding Domain", XP009532613, retrieved from NCBI Database accession no. PDB:6M17_E *
PIETER P JACOBS, STEVEN GEYSENS, WOUTER VERVECKEN, ROLAND CONTRERAS , NICO CALLEWAERT: "Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology.", NATURE PROTOCOLS, vol. 4, no. 1, 1 January 2019 (2019-01-01), pages 58 - 70, XP002795045, ISSN: 1750-2799, DOI: 10.1038/nprot.2008.213 *
XIAOPENG YANG, BO LIU, MIAO SONG, XIN GONG, SHAOHONG CHANG, KUIJING XUE, JUN WU: "Construction of Yeast Pichia Pastoris to Produce Man5GlcNAc2 Mammalian Mannose-Type Glycoprotein", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 27, no. 1, 25 January 2011 (2011-01-25), CN, pages 108 - 117, XP055877068, ISSN: 1000-3061, DOI: 10.13345/j.cjb.2011.01.006 *
YANG TIAN-TIAN;XUE SONG;QIN MENG: "Research Progress on Diagnosis and Treatment Technology Based on Structural Protein of SARS-CoV-2", DRUGS & CLINIC, vol. 35, no. 5, 11 May 2020 (2020-05-11), pages 1015 - 1023, XP055877075, ISSN: 1674-5515, DOI: 10.7501/j.issn.1674-5515.2020.05.041 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114957410A (zh) * 2022-06-08 2022-08-30 华兰基因工程有限公司 一种κ株2019-nCoV的表面蛋白受体结合区制备方法

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