WO2024045153A1 - 一种提高重组人白蛋白表达量的方法和细胞和蛋白 - Google Patents

一种提高重组人白蛋白表达量的方法和细胞和蛋白 Download PDF

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WO2024045153A1
WO2024045153A1 PCT/CN2022/116713 CN2022116713W WO2024045153A1 WO 2024045153 A1 WO2024045153 A1 WO 2024045153A1 CN 2022116713 W CN2022116713 W CN 2022116713W WO 2024045153 A1 WO2024045153 A1 WO 2024045153A1
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recombinant
seq
yeast
human albumin
superoxide dismutase
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French (fr)
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项炜
韩旭
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通化安睿特生物制药股份有限公司
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Priority to PCT/CN2022/116713 priority patent/WO2024045153A1/zh
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
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    • C12R2001/84—Pichia

Definitions

  • the present invention relates to a method for increasing the expression level of recombinant human albumin, and in particular, to a method, cells and protein for increasing the expression level of recombinant human albumin.
  • Pichia pastoris is genetically stable, has a powerful and strictly regulated promoter, and has an intracellular environment and sugar chain processing system suitable for the correct folding of eukaryotic gene products. It is simple to operate and can be cultured and fermented at high density on a large scale. It is a very widely used eukaryotic expression system for protein production in recent years.
  • Yeast has two modes of reproduction: asexual reproduction and sexual reproduction.
  • yeast reproduces asexually when it is stimulated by external environmental pressure, it will cause the yeast to flocculate and usually die when the environmental pressure is high.
  • sexual reproduction is the mating of two adjacent yeasts of different sexes.
  • Haploid spores fuse to produce diploids.
  • meiosis occurs, and diploids undergo meiosis to produce haploid spores.
  • Yeast flocculation is an asexual, reversible, calcium-dependent process, which is a multicellular aggregation phenomenon formed by the combination of flocculation proteins encoded by flocculation genes and mannose on adjacent cell walls. After cells flocculate prematurely, they will precipitate from the fermentation broth, which may lead to incomplete fermentation. Attenuate, disrupt or remove the function of at least one endogenous gene encoding flocculation proteins, including FLO5, FLO8 and FLO11, thereby inhibiting the formation of pseudohyphae. Mannose is added to the culture medium so that it occupies the binding site of the flocculating protein and is unable to bind to the mannose residues on the adjacent cell wall, thereby inhibiting the flocculation.
  • the redox state in yeast cells directly affects the survival, activation and proliferation of cells.
  • methanol When methanol is used as a carbon source, methanol is decomposed into formaldehyde and hydrogen peroxide under the action of alcohol oxidase, and hydrogen peroxide will be metabolized during the metabolic process.
  • Generating a large amount of reactive oxygen species (ROS) can cause direct damage to cellular components such as nucleic acids, lipids, and proteins, and in severe cases can cause cell senescence or death.
  • ROS reactive oxygen species
  • Superoxide dismutase catalyzes superoxide anion radicals to generate hydrogen peroxide and oxygen. Effectively resists the toxicity of superoxide ions to cells, protects the body from oxidative damage, maintains body balance, and extends cell life.
  • the endoplasmic reticulum is an important place for the synthesis and maturation of secreted proteins. It is rich in various molecular chaperones and folding enzymes that assist in protein folding and modification, such as protein disulfide isomerase PDI.
  • PDI can catalyze the formation and rearrangement of disulfide bonds between cysteine residues, helping the correct folding of proteins and improving their expression.
  • Human Serum Albumin is a single-chain globular protein composed of 585 amino acids.
  • proteins When proteins are blocked in processes such as disulfide bond formation and transport from the endoplasmic reticulum to the Golgi apparatus, unfolded or misfolded proteins accumulate in the endoplasmic reticulum, causing endoplasmic reticulum stress and thus affecting normal cell functions.
  • UTR unfolded protein response
  • ESD endoplasmic reticulum-associated degradation pathway
  • UPR uses a series of signaling reactions in cells to increase the expression of folding enzymes and molecular chaperones to improve protein folding ability; ERAD transports protein substrates that cannot be folded correctly from the endoplasmic reticulum to the cytoplasm through reverse transport. It is further marked by ubiquitination and ultimately degraded by the proteasome.
  • the ubiquitin-proteasome pathway consists of ubiquitin and a series of related enzymes.
  • the ubiquitin activating enzyme E1 relies on the energy supplied by ATP to activate the ubiquitin molecule and transfer it to the ubiquitin conjugating enzyme E2.
  • the ubiquitin ligase E3 recognizes the target protein to be degraded and connects the ubiquitin bound to E2 to the Lys of the target protein. This process is repeated on each residue, allowing the target protein to bind to multiple ubiquitins. Under the action of the 26S proteasome, it is degraded into small peptides composed of amino acid residues.
  • the proteasome itself is not selective for proteins; it is the E3 ubiquitin ligase that has selective specificity.
  • “Host cell” refers to a cell that receives a foreign gene during transformation or transduction.
  • “Expression cassette” refers to a gene expression system that contains all necessary elements required for the expression of foreign proteins, including promoters, foreign gene cloning sites, signal peptide sequences, mature peptide coding sequences of the target protein, terminators, screening markers, etc. .
  • Vector refers to autonomous DNA that can bring exogenous DNA into host cells for replication or ultimately expression of exogenous gene DNA. They are mainly divided into cloning vectors and expression vectors. The former is mainly used for gene replication and amplification, while the latter is mainly used for the expression of target genes.
  • Gene expression refers to the process in which the genetic information carried by the structural genes in the biological genome undergoes a series of processes such as transcription and translation to synthesize specific proteins and then exert their specific biological functions.
  • “Operably linked” means that transcriptional and translational regulatory elements are covalently linked to a coding sequence in a spatial arrangement such that the regulatory elements can direct the expression of the coding sequence.
  • Synchronization peptide refers to the N-terminal amino acid sequence in a newly synthesized polypeptide chain that is used to guide protein transmembrane transfer.
  • Molecular chaperones refer to proteins and peptides that assist in the correct folding of macromolecular structures within cells.
  • Recombinant promoter refers to a genetically modified or unmodified promoter, a promoter not naturally found upstream of a gene in the genome, or a wild-type promoter.
  • the specific DNA sequence that exists upstream of the 5' end of the coding sequence of the target gene recognizes and binds to RNA polymerase, and controls the transcription of the target gene.
  • FDH Formate dehydrogenase, formate dehydrogenase.
  • FLD Formaldehyde dehydrogenase, formaldehyde dehydrogenase.
  • GAL Galactose, galactose.
  • GAP Glyceraldehyde-3-phosphate dehydrogenase
  • glyceraldehyde-3-phosphate dehydrogenase glyceraldehyde-3-phosphate dehydrogenase
  • EERAD ER associated degradation, endoplasmic reticulum-related degradation pathway.
  • ROS Reactive Oxygen Species, reactive oxygen species.
  • HSA Human Serum Albumin, human serum albumin.
  • PKI Protein Disulfide Isomerase, protein disulfide isomerase.
  • MD Minimal Dextrose medium, culture medium.
  • BMGY Buffered Glycerol-complex Medium, medium.
  • YPD Yeast extract/peptone/dextrose-media, culture medium.
  • SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
  • Sequence Identity Document Number sequence number.
  • Purpose of the invention In order to provide a better method, cells and protein for increasing the expression of recombinant human albumin, the specific purpose is shown in the multiple substantial technical effects in the specific implementation section.
  • a method for increasing the expression of recombinant human albumin characterized by comprising weakening, destroying or removing the function of at least one endogenous gene encoding flocculating protein in a recombinant host cell, and/or editing the endogenous gene or heterologous gene Overexpression of superoxide dismutase; and/or editing of endogenous genes or heterologous genes to overexpression of E3 ubiquitin ligase.
  • a method for increasing the expression of recombinant human albumin characterized in that the steps include:
  • a further technical solution of the present invention is that the host cell is a yeast cell.
  • yeast is one or more of the following yeast genera: Hansenula, Pichia and Candida.
  • a further technical solution of the present invention is that the cells of the genus Pichia are Pichia pastoris cells.
  • a further technical solution of the present invention is that the host cell has weakened, destroyed or removed the function of at least one endogenous gene encoding flocculating protein, and the endogenous genes of flocculating protein include FLO5, FLO8 and FLO11.
  • a further technical solution of the present invention is that the gene encoding superoxide dismutase is genetically modified or not.
  • a further technical solution of the present invention is that the gene encoding superoxide dismutase is yeast superoxide dismutase, and more preferably is a human copper-zinc superoxide dismutase nucleic acid molecule.
  • a further technical solution of the present invention is that the gene encoding E3 ubiquitin ligase is genetically modified or not.
  • E3 ubiquitin ligase is a yeast E3 ubiquitin ligase, more preferably a human E3 ubiquitin ligase nucleic acid molecule.
  • a further technical solution of the present invention is that the host cell encodes at least one recombinant promoter-modified human albumin.
  • a further technical solution of the present invention is that the recombinant expression cassette encoding the human albumin gene, superoxide dismutase and E3 ubiquitin ligase genes is on one nucleic acid construct or on nucleic acid constructs with different selection markers.
  • nucleic acid constructs include but are not limited to pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPink-LC, pPink-HC, pPICZA, pPICZB, pPICZC, pPICZaA, pPICZaB and pPICZaC plasmids .
  • recombinant promoters include but are not limited to AOX1 promoter, GAP promoter, GAL promoter, FDH promoter and FLD promoter.
  • a further technical solution of the present invention is to provide a method for producing recombinant proteins, including a host cell in which the function of at least one endogenous gene encoding a flocculating protein has been weakened, destroyed or removed.
  • the endogenous genes of the flocculating protein include FLO5, FLO8 and FLO11; Encoding at least one recombinant promoter modified human albumin; culturing the host cell under conditions suitable for the production of the recombinant protein.
  • a further technical solution of the present invention is to provide a method for producing recombinant proteins, including a host cell in which the function of at least one endogenous gene encoding a flocculating protein has been weakened, destroyed or removed.
  • the endogenous genes of the flocculating protein include FLO5, FLO8 and FLO11; Encoding at least one endogenous or heterologous superoxide dismutase; encoding at least one recombinant promoter-modified human albumin; cultivating the host cell under conditions suitable for the production of the recombinant protein.
  • a further technical solution of the present invention is to provide a method for producing recombinant proteins, including a host cell in which the function of at least one endogenous gene encoding a flocculating protein has been weakened, destroyed or removed.
  • the endogenous genes of the flocculating protein include FLO5, FLO8 and FLO11; Encoding at least one endogenous or heterologous superoxide dismutase; encoding at least one endogenous or heterologous E3 ubiquitin ligase; encoding at least one recombinant promoter-modified human albumin; cultivating the host under conditions suitable for the production of recombinant proteins cell.
  • a method for increasing the expression of recombinant human albumin characterized in that the sequence is any one or more of the following sequences:
  • SEQ ID NO.2 human albumin signal peptide coding sequence
  • SEQ ID NO.3 human albumin mature peptide coding sequence
  • SEQ ID NO.4 yeast superoxide dismutase coding sequence
  • SEQ ID NO.5 human superoxide dismutase coding sequence
  • SEQ ID NO.6 Yeast E3 ubiquitin ligase coding sequence
  • SEQ ID NO.7 Yeast flocculating protein FL05 coding sequence
  • SEQ ID NO.8 Yeast flocculating protein FL08 coding sequence
  • SEQ ID NO.9 Yeast flocculating protein FL011 coding sequence
  • SEQ ID NO.12 Yeast superoxide dismutase-F sequence
  • SEQ ID NO.14 human superoxide dismutase-F sequence
  • SEQ ID NO.15 human superoxide dismutase-R sequence
  • SEQ ID NO.17 Yeast E3 ubiquitin ligase-R sequence.
  • Host cells genetically manipulated using any one or more of SEQ ID NO.1-SEQ ID NO.17.
  • the present invention adopting the above technical solution has the following beneficial effects compared to the existing technology: this patent can maximize the expression of recombinant human albumin.
  • the present invention provides a method to weaken, destroy or remove the function of at least one endogenous gene encoding flocculation protein through construction, and/or edit the endogenous gene or heterologous gene to overexpress superoxide dismutase; and/or edit the endogenous gene. Or methods to overexpress E3 ubiquitin ligase using heterologous genes and increase the expression of recombinant human albumin.
  • Figure 1 is a schematic structural diagram of a recombinant expression cassette encoding human albumin.
  • Figure 2 is a schematic structural diagram of a recombinant expression cassette encoding E3 ubiquitin ligase.
  • Figure 3 is a schematic structural diagram of a recombinant expression cassette encoding superoxide dismutase.
  • Figure 4 is a schematic structural diagram of a recombinant expression cassette encoding superoxide dismutase and E3 ubiquitin ligase.
  • Figure 5 is a schematic structural diagram of the recombinant expression cassette of the FLO gene knockout plasmid.
  • Figure 6 is a diagram showing SDS-PAGE results of recombinant human albumin expression in Example 3.
  • Lane 1 is the recombinant engineered bacteria IV
  • lane 2 is the recombinant engineered bacteria V
  • lane 3 is the recombinant engineered bacteria VI
  • lane 4 is the recombinant engineered bacteria I
  • lane 5 is the recombinant engineered bacteria II
  • lane 6 is the recombinant engineered bacteria III.
  • the expression level of recombinant human albumin was significantly increased.
  • Figure 7 is a relative expression map of recombinant human albumin of each recombinant engineering strain in Example 3.
  • This patent provides a variety of parallel solutions. Different expressions indicate that they are improved solutions based on the basic solution or parallel solutions. Each option has its own unique characteristics.
  • the object of the present invention is to provide a method for increasing the expression level of recombinant human albumin.
  • the present invention relates to methods for increasing the expression of recombinant human albumin, which include but are not limited to:
  • the yeast cell recombinant promoter includes, but is not limited to, AOX1 promoter, GAP promoter, GAL promoter, FDH promoter and FLD promoter.
  • the yeast cells include, but are not limited to, Hansenula, Pichia, Schizosacchromyces, Candida, Schizosaccharomyces, and Toruzospora Torulopsis and Aspergillus.
  • the genus Pichia is preferred, and Pichia pastoris is more preferred.
  • the phenotype of the yeast cells is preferably methanol-slow-utilizing Mut s , including but not limited to KM71 and KM71H; or methanol-fast-utilizing Mut + , including but not limited to GS115, X-33, and CBS7435.
  • the yeast cells have attenuated, disrupted or eliminated the function of at least one endogenous gene encoding a flocculin, including FLO5, FLO8 and FLO11.
  • the yeast cell comprises a recombinant promoter operably linked to at least one gene encoding human albumin, wherein the promoter is preferably a genetically modified or unmodified inducible yeast AOX1 promoter.
  • the yeast cell contains expression encoding at least one human superoxide dismutase or yeast superoxide dismutase.
  • the yeast cell comprises expression of at least one encoding a human E3 ubiquitin ligase or a yeast E3 ubiquitin ligase.
  • the expression cassettes and nucleic acid constructs include, but are not limited to, pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPink-LC, pPink-HC, pPICZA, pPICZB, pPICZC, pPICZaA, pPICZaB and pPICZaC plasmids.
  • the recombinant promoter is operably linked to a signal peptide sequence, the Saccharomyces cerevisiae mating factor signal peptide coding sequence (as set forth in SEQ ID NO. 1) or the human albumin signal peptide coding sequence (as set forth in SEQ ID NO. 2), and Human albumin mature peptide coding sequence (shown in SEQ ID NO.3).
  • the expression cassettes and nucleic acid constructs include, but are not limited to, pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPink-LC, pPink-HC, pPICZA, pPICZB, pPICZC, pPICZaA, pPICZaB and pPICZaC plasmids.
  • the recombinant promoter is operably linked to the yeast superoxide dismutase coding sequence.
  • the yeast superoxide dismutase coding sequence is shown in SEQ ID NO. 4.
  • the recombinant promoter is operably linked to the human superoxide dismutase coding sequence.
  • the human superoxide dismutase coding sequence is shown in SEQ ID NO.5.
  • the expression cassettes and nucleic acid constructs include, but are not limited to, pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPink-LC, pPink-HC, pPICZA, pPICZB, pPICZC, pPICZaA, pPICZaB and pPICZaC plasmids.
  • the recombinant promoter is operably linked to the yeast E3 ubiquitin ligase coding sequence.
  • the yeast E3 ubiquitin ligase coding sequence is shown in SEQ ID NO. 6.
  • yeast cells are cultured under conditions suitable for the production of recombinant human albumin, the expression of foreign proteins is induced, and the expression level of recombinant human albumin is increased.
  • E. coli DH5a competent cells were used for all E. coli cloning experiments.
  • the recombinant human albumin host cell is a yeast cell, preferably Pichia pastoris cell CBS7435, more preferably GS115 (Mut + Pichia pastoris strain with histidine dehydrogenase His4 gene mutation) and KM71 (histidine dehydrogenase Mut s Pichia pastoris strain with mutated His4 gene and disrupted AOX1 gene).
  • the expression vector needs to be integrated at a specific site in the host Pichia pastoris cell genome in the form of single copy or multiple copies, and homologous recombination occurs with the chromosome to achieve the expression of foreign genes.
  • StuI restriction endonuclease (NEB), PmeI restriction endonuclease (NEB), SacI restriction endonuclease (NEB), Gold Medal Super Endotoxin-free Plasmid Extraction Kit (Kangwei Century), Bleomyces Zeocin (Invitrogen), geneticin G418 (Gibco), ampicillin sodium (Shanghai Sangon Bioengineering Co., Ltd.).
  • MD medium MD medium, LB medium, YPD medium, BMGY medium, BMMY medium.
  • E3 ubiquitin ligase plasmid yeast E3 ubiquitin ligase or human E3 ubiquitin ligase
  • Figure 2 The schematic structural diagram of the recombinant E3 ubiquitin ligase plasmid (yeast E3 ubiquitin ligase or human E3 ubiquitin ligase) is shown in Figure 2.
  • Recombinant superoxide dismutase (yeast E3 ubiquitin ligase or human E3 ubiquitin ligase) and E3 ubiquitin ligase (yeast superoxide dismutase or human superoxide dismutase) plasmid expression cassette, its structural diagram is as follows As shown in Figure 4.
  • HSA-F primer sequence SEQ ID NO.10
  • HSA-R primer sequence SEQ ID NO.11
  • Yeast superoxide dismutase-F sequence SEQ ID NO.12
  • yeast superoxide dismutase-R sequence SEQ ID NO.13
  • human superoxide dismutase-F sequence SEQ ID NO.14
  • human superoxide dismutase-R sequence SEQ ID NO.15
  • yeast E3 ubiquitin ligase-F sequence SEQ ID NO.16
  • yeast E3 ubiquitin ligase-R sequence SEQ ID NO.17
  • Recombinant engineered bacterium I transformed recombinant human albumin expression cassette, its structural diagram is shown in Figure 1.
  • the signal peptide coding sequence is shown in SEQ ID NO.1
  • the human albumin mature peptide coding sequence is shown in SEQ ID NO.3.
  • engineering bacteria were constructed to increase the expression of recombinant human albumin.
  • Recombinant engineered strain II Use recombinant engineered strain I as the host cell to transform the recombinant yeast E3 ubiquitin ligase expression cassette (its structural diagram is shown in Figure 2). The coding sequence of yeast E3 ubiquitin ligase is shown in SEQ ID NO.6.
  • Recombinant engineering bacteria III Transform recombinant human albumin expression cassette and FLO gene knockout plasmid respectively. Construct a FLO gene knockout plasmid, which contains the homology arm sequences on both sides of the gene FLO to be knocked out. A selective G418 marker is inserted between the two homology arms.
  • the schematic diagram of its structure is shown in Figure 5.
  • the coding sequence of yeast flocculating protein FL05 is shown in SEQ ID NO.7
  • the coding sequence of yeast flocculating protein FL08 is shown in SEQ ID NO.8
  • the coding sequence of yeast flocculating protein FL011 is shown in SEQ ID NO.9.
  • Recombinant engineered bacteria IV Transform recombinant human albumin expression cassette and recombinant yeast superoxide dismutase expression cassette respectively (its structural diagram is shown in Figure 3). The coding sequence of yeast superoxide dismutase is shown in SEQ ID NO.4.
  • Recombinant engineered strain V Use recombinant engineered strain III as the host cell to transform recombinant yeast E3 ubiquitin ligase and recombinant yeast superoxide dismutase expression cassette.
  • the schematic structural diagram is shown in Figure 4.
  • Recombinant engineered strain VI Use recombinant engineered strain III as the host cell to transform the recombinant yeast E3 ubiquitin ligase and human superoxide dismutase expression cassette.
  • the schematic structural diagram is shown in Figure 4.
  • the human superoxide dismutase coding sequence is shown in SEQ ID NO.5.
  • the invention relates to a method for increasing the expression level of recombinant human albumin.
  • the term “recombinant human albumin” may also be referred to as “recombinant human serum albumin” and/or “recombinant human serum albumin” and/or “rHA” and/or “rHSA”.
  • human serum albumin refers to human albumin extracted from human serum, which may also be called “human serum albumin” and/or "HSA” and/or "HA” and/or "pdHSA”.
  • the most commonly used method for expressing human albumin by genetically recombinant microorganisms that can achieve large-scale production is mainly the yeast expression system.
  • yeast cells due to the growth and metabolism of yeast cells, premature flocculation may occur and precipitation from the fermentation broth may result. Incomplete fermentation.
  • recombinant proteins are blocked in processes such as disulfide bond formation and transport from the endoplasmic reticulum to the Golgi apparatus, unfolded or misfolded proteins accumulate in the endoplasmic reticulum, causing endoplasmic reticulum stress and affecting normal cell functions.
  • methanol metabolism will produce a large amount of reactive oxygen species, which can directly damage cellular components such as nucleic acids, lipids, and proteins.
  • the expression level of recombinant human albumin can be greatly improved.

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Abstract

提供一种提高重组人白蛋白表达量的方法，包括在表达重组人白蛋白的同时，减弱、破坏或去除至少一个编码絮凝蛋白的内源基因的功能，和/或编辑内源基因或异源基因使得超氧化物歧化酶过表达；和/或编辑内源基因或异源基因使得E3泛素连接酶过表达；还提供了相应的细胞和白蛋白。

Description

一种提高重组人白蛋白表达量的方法和细胞和蛋白 技术领域
本发明涉及提高重组人白蛋白表达量的方法，尤其涉及一种提高重组人白蛋白表达量的方法和细胞和蛋白。
背景技术
巴斯德毕赤酵母遗传稳定，拥有强有力的受严格调控的启动子，有适合真核生物基因产物正确折叠的细胞内环境和糖链加工系统。操作简单，可大规模高密度培养发酵，是近年来使用非常广泛的一种生产蛋白质的真核表达系统。
酵母具有无性繁殖和有性繁殖两种繁殖方式。酵母无性繁殖时，当受到外界环境压力刺激时，会导致酵母的絮凝现象，环境压力较大时通常死亡。而有性繁殖是相邻的两个性别不同的酵母交配，单倍体孢子融合产生双倍体，在环境恶劣时，减数分裂，双倍体减数分裂产生单倍体孢子。
酵母絮凝是一个无性的、可逆的、钙依赖的过程，是由絮凝基因编码的絮凝蛋白与邻近细胞壁上的甘露糖结合而形成的多细胞聚集现象。细胞过早絮凝后，会从发酵液中沉淀，从而可能导致发酵的不完全。减弱、破坏或去除至少一个编码絮凝蛋白的内源基因的功能，包含FLO5、FLO8和FLO11，从而抑制假菌丝的形成。在培养基中加入甘露糖，使其占据絮凝蛋白的结合位点，无法与邻近细胞壁上的甘露糖残基结合，使得絮凝作用被抑制。
酵母细胞内的氧化还原状态直接影响细胞的生存、活化和增殖，当利用甲醇作为碳源时，甲醇在醇氧化酶作用下被分解为甲醛以及过氧化氢，而过氧化氢在代谢过程中会产生大量的活性氧(ROS)，会导致对核酸、脂质、蛋白质等细胞组分直接损伤，严重时还会引起细胞衰老或死亡。超氧化物歧化酶能够催化超氧阴离子自由基生成过氧化氢和氧气。有效抵御超氧根离子对细胞的毒性，保护机体免遭氧化损伤，维持机体平衡，延长细胞寿命。
在酵母细胞中，内质网是分泌蛋白质合成和成熟的重要场所，其中富含协助蛋白质折叠和修饰的各种分子伴侣、折叠酶等，如蛋白二硫键异构酶PDI。PDI能够催化半胱氨酸残基间二硫键的形成和重排，帮助蛋白质的正确折叠，提 高其表达量。
人血清白蛋白(Human Serum Albumin，HSA)是一种单链球形蛋白质，由585个氨基酸构成。蛋白质在二硫键形成以及由内质网向高尔基体转运等过程受阻时，未折叠或错误折叠的蛋白质聚集在内质网中，引起内质网压力，从而影响细胞正常功能。调控内质网压力的途径主要有两种，即未折叠蛋白反应(UPR)和内质网相关降解途径(ERAD)。UPR借助细胞内的一系列信号传导反应，上调折叠酶和分子伴侣的表达量来提高蛋白质的折叠能力；ERAD则通过逆转运方式将不能正确折叠的蛋白底物从内质网运至细胞质中，进一步通过泛素化标记最终被蛋白酶体降解。
泛素蛋白酶体途径由泛素以及一系列相关的酶组成。泛素激活酶E1依赖ATP供给的能量，将泛素分子活化，并传递给泛素结合酶E2，泛素连接酶E3识别待降解的靶蛋白，将结合E2的泛素连接到靶蛋白的Lys残基上，重复此过程，使得靶蛋白与多个泛素结合。在26S蛋白酶体作用下，将其降解成氨基酸残基组成的小肽。蛋白酶体本身对蛋白质没有选择性，具有选择特异性的是E3泛素连接酶。
定义术语
“宿主细胞”指在转化或转导过程中接受外源基因的细胞。
“表达盒”指包含外源蛋白表达所需的所有必要元件的基因表达系统，包括启动子、外源基因克隆位点、信号肽序列、目的蛋白的成熟肽编码序列、终止子、筛选标记等。
“载体”指能将外源DNA带入宿主细胞，进行复制或最终使外源基因DNA得以表达的自主DNA。主要分为克隆载体和表达载体，前者主要用于基因的复制、扩增等，后者主要用于目的基因的表达。
“DNA”Deoxyribonucleic acid，脱氧核糖核酸。
“基因表达”指生物基因组中结构基因所携带的遗传信息经过转录、翻译等一系列过程，合成特定的蛋白质，进而发挥其特定生物学功能的过程。
“可操作的连接”指转录和翻译调控元件共价连接至编码序列，其空间排布使得调控元件可以指导编码序列的表达。
“信号肽”指新合成的多肽链中用于指导蛋白质跨膜转移的N端氨基酸 序列。
“分子伴侣”指协助细胞内大分子结构正确折叠的蛋白质和多肽。
“重组启动子”指经遗传修饰的或未经修饰的启动子，不天然存在于基因组中基因上游的启动子或野生型启动子。存在于目的基因编码序列5’端上游，与RNA聚合酶识别、结合的特异DNA序列，控制目的基因的转录。
“FDH”Formate dehydrogenase，甲酸脱氢酶。
“FLD”Formaldehyde dehydrogenase，甲醛脱氢酶。
“GAL”Galactose，半乳糖。
“GAP”Glyceraldehyde-3-phosphate dehydrogenase，三磷酸甘油醛脱氢酶。
“UPR”unfolded protein response，未折叠蛋白反应。
“ERAD”ER associated degradation，内质网相关降解途径。
“ROS”Reactive Oxygen Species，活性氧。
“HSA”Human Serum Albumin，人血清白蛋白。
“PDI”Protein Disulfide Isomerase，蛋白二硫键异构酶。
“LB”Luria bertani medium，培养基。
“MD”Minimal Dextrose medium，培养基。
“BMGY”Buffered Glycerol-complex Medium，培养基。
“BMMY”Buffered Methanol-complex Mdeium，培养基。
“YPD”Yeast extract/peptone/dextrose-media，培养基。
“Zeo”zeocin，博来霉素。
“His”Histidinol dehydrogenase，组氨酸脱氢酶。
“Da”Dalton，道尔顿。
“ml”milliliter，毫升。
“Mut s”Methanol utilization slow，甲醇利用慢表型。
“Mut +”Methanol utilization plus，甲醇利用快表型。
“PCR”Polymerase chain reaction，聚合酶链式反应。
“rpm”Rounds per minute，转/每分。
“SDS-PAGE”Sodium dodecyl sulphate polyacrylamide gel electrophoresis，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳。
“SEQ ID NO.”Sequence Identity Document Number，序列编号。
发明内容
发明的目的：为了提供效果更好的一种提高重组人白蛋白表达量的方法和细胞和蛋白，具体目的见具体实施部分的多个实质技术效果。
为了达到如上目的，本发明采取如下技术方案：
方案一：
一种提高重组人白蛋白表达量的方法，其特征在于，在重组宿主细胞中包含减弱、破坏或去除至少一个编码絮凝蛋白的内源基因的功能，和/或编辑内源基因或异源基因使得超氧化物歧化酶过表达；和/或编辑内源基因或异源基因使得E3泛素连接酶过表达。
方案二：
一种提高重组人白蛋白表达量的方法，其特征在于，实现步骤其包含：
(1)编码至少一个重组人白蛋白的宿主细胞；
(2)减弱、破坏或去除至少一个编码絮凝蛋白的内源基因的功能的宿主细胞；
(3)编码至少一个内源或异源超氧化物歧化酶的宿主细胞；
(4)编码至少一个内源或异源E3泛素连接酶的宿主细胞。
本发明进一步技术方案在于，所述的宿主细胞为酵母细胞。
本发明进一步技术方案在于，所述的酵母为如下酵母属的一种或者多种：汉逊酵母属(Hansenula)、毕赤酵母属(Pichia)和假丝酵母属(Candida)。
本发明进一步技术方案在于，所述的毕赤酵母属的细胞是巴斯德毕赤酵母细胞(Pichia pastoris)。
本发明进一步技术方案在于，所述的宿主细胞已经减弱、破坏或去除至少一个编码絮凝蛋白的内源基因的功能，絮凝蛋白的内源基因包含FLO5、FLO8和FLO11。
本发明进一步技术方案在于，编码超氧化物歧化酶的基因是经遗传修饰的或未经遗传修饰的。
本发明进一步技术方案在于，编码超氧化物歧化酶的基因是酵母超氧化物歧化酶，更优选为人铜锌超氧化物歧化酶核酸分子。
本发明进一步技术方案在于，编码E3泛素连接酶的基因是经遗传修饰的或未经遗传修饰的。
本发明进一步技术方案在于，编码E3泛素连接酶的基因是酵母E3泛素连接酶，更优选为人E3泛素连接酶核酸分子。
本发明进一步技术方案在于，宿主细胞中编码至少一个重组启动子修饰的人白蛋白。
本发明进一步技术方案在于，编码人白蛋白基因、超氧化物歧化酶和E3泛素连接酶基因的重组表达盒是在一个核酸构建体上或在不同选择标志的核酸构建体上。
本发明进一步技术方案在于，所述核酸构建体包括但不限于pHIL-D2、pPIC3.5、pHIL-S1、pPIC9、pPink-LC、pPink-HC、pPICZA、pPICZB、pPICZC、pPICZaA、pPICZaB和pPICZaC质粒。
本发明进一步技术方案在于，重组启动子包括但不限于AOX1启动子、GAP启动子、GAL启动子、FDH启动子和FLD启动子。
本发明进一步技术方案在于，提供了生产重组蛋白的方法，包含宿主细胞其中已经减弱、破坏或去除至少一个编码絮凝蛋白的内源基因的功能，絮凝蛋白的内源基因包含FLO5、FLO8和FLO11；编码至少一个重组启动子修饰的人白蛋白；在适合生产重组蛋白的条件下培养宿主细胞。
本发明进一步技术方案在于，提供了生产重组蛋白的方法，包含宿主细胞其中已经减弱、破坏或去除至少一个编码絮凝蛋白的内源基因的功能，絮凝蛋白的内源基因包含FLO5、FLO8和FLO11；编码至少一个内源或异源超氧化物歧化酶；编码至少一个重组启动子修饰的人白蛋白；在适合生产重组蛋白的条件下培养宿主细胞。
本发明进一步技术方案在于，提供了生产重组蛋白的方法，包含宿主细胞其中已经减弱、破坏或去除至少一个编码絮凝蛋白的内源基因的功能，絮凝蛋白的内源基因包含FLO5、FLO8和FLO11；编码至少一个内源或异源超氧化物歧化酶；编码至少一个内源或异源E3泛素连接酶；编码至少一个重组启动子修饰的人白蛋白；在适合生产重组蛋白的条件下培养宿主细胞。
一种提高重组人白蛋白表达量的方法，其特征在于，序列为如下任意一 种或者多种序列：
SEQ ID NO.1酿酒酵母交配因子信号肽编码序列
SEQ ID NO.2人白蛋白信号肽编码序列；
SEQ ID NO.3人白蛋白成熟肽编码序列；
SEQ ID NO.4酵母超氧化物歧化酶编码序列；
SEQ ID NO.5人超氧化物歧化酶编码序列；
SEQ ID NO.6酵母E3泛素连接酶编码序列；
SEQ ID NO.7酵母絮凝蛋白FL05编码序列；
SEQ ID NO.8酵母絮凝蛋白FL08编码序列；
SEQ ID NO.9酵母絮凝蛋白FL011编码序列；
SEQ ID NO.10HSA-F引物序列；
SEQ ID NO.11HSA-R引物序列；
SEQ ID NO.12酵母超氧化物歧化酶-F序列；
SEQ ID NO.13酵母超氧化物歧化酶-R序列；
SEQ ID NO.14人超氧化物歧化酶-F序列；
SEQ ID NO.15人超氧化物歧化酶-R序列；
SEQ ID NO.16酵母E3泛素连接酶-F序列；
SEQ ID NO.17酵母E3泛素连接酶-R序列。
利用SEQ ID NO.1-SEQ ID NO.17任意一种或者多种序列基因操作后的宿主细胞。
利用SEQ ID NO.1-SEQ ID NO.17任意一种或者多种序列基因操作后的宿主细胞表达的重组人白蛋白。
采用如上技术方案的本发明，相对于现有技术有如下有益效果：本专利能最大限度提高重组人白蛋白表达量。本发明提供了通过构建减弱、破坏或去除至少一个编码絮凝蛋白的内源基因的功能，和/或编辑内源基因或异源基因使得超氧化物歧化酶过表达；和/或编辑内源基因或异源基因使得E3泛素连接酶过表达、提高重组人白蛋白表达量的方法。
附图说明
为了进一步说明本发明，下面结合附图进一步进行说明：
图1为编码人白蛋白的重组表达盒的结构示意图。
图2为编码E3泛素连接酶的重组表达盒的结构示意图。
图3为编码超氧化物歧化酶的重组表达盒的结构示意图。
图4为编码超氧化物歧化酶和E3泛素连接酶的重组表达盒的结构示意图。
图5为FLO基因敲除质粒的重组表达盒的结构示意图。
图6为实施例3的重组人白蛋白表达SDS-PAGE结果图。泳道1为重组工程菌IV，泳道2为重组工程菌V，泳道3为重组工程菌VI，泳道4为重组工程菌I，泳道5为重组工程菌II，泳道6为重组工程菌III。重组人白蛋白表达量明显提高。
图7为实施例3的各重组工程菌的重组人白蛋白相对表达量图谱。
具体实施方式
本专利提供多种并列方案，不同表述之处，属于基于基本方案的改进型方案或者是并列型方案。每种方案都有自己的独特特点。
本发明目的在于提供提高重组人白蛋白表达量的方法。
因此，本发明涉及用于提高重组人白蛋白表达量的方法，其包含但不限于：
1)经遗传修饰的酵母细胞
所述酵母细胞重组启动子包含但不限于AOX1启动子、GAP启动子、GAL启动子、FDH启动子和FLD启动子。
所述酵母细胞包含但不限于汉逊酵母属(Hansenula)、毕赤酵母属(Pichia)、酿酒酵母属(Schizosacchromyces)、假丝酵母属(Candida)、裂殖酵母属(Schizosaccharomyces)、球拟酵母属(Torulopsis)和曲霉属(Aspergillus)。优选为毕赤酵母属，更优选为巴斯德毕赤酵母(Pichia pastoris)。
所述酵母细胞的表型优选为甲醇慢利用型Mut s，包括但不限于KM71、KM71H；或甲醇快利用型Mut +，包括但不限于GS115、X-33、CBS7435。
所述酵母细胞，已经减弱、破坏或去除至少一个编码絮凝蛋白的内源基因的功能，包含FLO5、FLO8和FLO11。
所述酵母细胞包含重组启动子可操作地连接于编码人白蛋白的至少一个 基因，其中启动子优选为经遗传修饰的或未经修饰的可诱导酵母AOX1启动子。
所述酵母细胞包含至少一个编码人超氧化物歧化酶或酵母超氧化物歧化酶的表达。
所述酵母细胞包含至少一个编码人E3泛素连接酶或酵母E3泛素连接酶的表达。
2)表达盒，其编码至少一个人白蛋白的重组核酸分子
所述表达盒，核酸构建体包括但不限于pHIL-D2、pPIC3.5、pHIL-S1、pPIC9、pPink-LC、pPink-HC、pPICZA、pPICZB、pPICZC、pPICZaA、pPICZaB和pPICZaC质粒。
重组启动子可操作地连接于信号肽序列，酿酒酵母交配因子信号肽编码序列(如SEQ ID NO.1所示)或人白蛋白信号肽编码序列(如SEQ ID NO.2所示)，和人白蛋白成熟肽编码序列(如SEQ ID NO.3所示)。
3)表达盒，其编码至少一个表达超氧化物歧化酶的重组核酸分子
所述表达盒，核酸构建体包括但不限于pHIL-D2、pPIC3.5、pHIL-S1、pPIC9、pPink-LC、pPink-HC、pPICZA、pPICZB、pPICZC、pPICZaA、pPICZaB和pPICZaC质粒。
重组启动子可操作地连接于酵母超氧化物歧化酶编码序列。所述酵母超氧化物歧化酶编码序列如SEQ ID NO.4所示。
重组启动子可操作地连接于人超氧化物歧化酶编码序列。所述人超氧化物歧化酶编码序列如SEQ ID NO.5所示。
4)表达盒，其编码至少一个表达E3泛素连接酶的重组核酸分子
所述表达盒，核酸构建体包括但不限于pHIL-D2、pPIC3.5、pHIL-S1、pPIC9、pPink-LC、pPink-HC、pPICZA、pPICZB、pPICZC、pPICZaA、pPICZaB和pPICZaC质粒。
重组启动子可操作地连接于酵母E3泛素连接酶编码序列。所述酵母E3泛素连接酶编码序列如SEQ ID NO.6所示。
依照但不限于本发明提供的方法，在适合生产重组人白蛋白的条件下，培养酵母细胞，诱导外源蛋白表达，提高重组人白蛋白表达量。
菌株
大肠杆菌DH5a感受态细胞用于所有大肠杆菌克隆实验。
重组人白蛋白宿主细胞是酵母细胞，优选巴斯德毕赤酵母细胞CBS7435，更优选GS115(组氨酸脱氢酶His4基因突变的Mut +毕赤酵母菌株)和KM71(组氨酸脱氢酶His4基因突变、AOX1基因被破坏的Mut s毕赤酵母菌株)。
表达载体
表达载体需在宿主毕赤酵母细胞基因组特定位点以单拷贝或多拷贝形式整合，与染色体发生同源重组以实现外源基因的表达。优选pPic9酵母表达载体(Invitrogen)和pPicZA酵母表达载体(Invitrogen)。
试剂和培养基
StuI限制性内切酶(NEB)、PmeI限制性内切酶(NEB)、SacI限制性内切酶(NEB)、金牌超量无内毒素质粒大提试剂盒(康为世纪)、博来霉素zeocin(Invitrogen)、遗传霉素G418(Gibco)、氨苄青霉素钠Ampicillin(上海生工生物工程股份有限公司)。
MD培养基、LB培养基、YPD培养基、BMGY培养基、BMMY培养基。
重组质粒
重组人白蛋白质粒，其结构示意图如图1所示。
重组E3泛素连接酶质粒(酵母E3泛素连接酶或人E3泛素连接酶)，其结构示意图如图2所示。
重组超氧化物歧化酶质粒(酵母超氧化物歧化酶或人超氧化物歧化酶)，其结构示意图如图3所示。
重组超氧化物歧化酶(酵母E3泛素连接酶或人E3泛素连接酶)和E3泛素连接酶(酵母超氧化物歧化酶或人超氧化物歧化酶)质粒表达盒，其结构示意图如图4所示。
FLO基因敲除质粒的重组表达盒，其结构示意图如图5所示。
转化、筛选阳性克隆
使用DNA限制性内切酶PmeI、StuI或SacI线性化质粒，再依照毕赤酵母表达试剂盒(Invitrogen)操作手册中电穿孔方法转化酵母细胞，将细胞涂布在含相应抗生素或营养缺陷的选择平板上，30℃培养48至72小时。
挑取平板上生长的单菌落至培养液中培养后，提取基因组DNA，分别采 用序列表中HSA-F引物序列(SEQ ID NO.10)和HSA-R引物序列(SEQ ID NO.11)、酵母超氧化物歧化酶-F序列(SEQ ID NO.12)和酵母超氧化物歧化酶-R序列(SEQ ID NO.13)、人超氧化物歧化酶-F序列(SEQ ID NO.14)和人超氧化物歧化酶-R序列(SEQ ID NO.15)、酵母E3泛素连接酶-F序列(SEQ ID NO.16)和酵母E3泛素连接酶-R序列(SEQ ID NO.17)等相应引物进行PCR，以筛选阳性克隆。
实施例1
本实施例构建仅含人白蛋白基因的重组工程菌。
重组工程菌I：转化的重组人白蛋白表达盒，其结构示意图如图1所示。信号肽编码序列如SEQ ID NO.1所示，人白蛋白成熟肽编码序列如SEQ ID NO.3所示。
实施例2
本实施例构建了提高重组人白蛋白表达量的工程菌。
重组工程菌II：以重组工程菌I为宿主细胞，转化重组酵母E3泛素连接酶表达盒(其结构示意图如图2所示)。酵母E3泛素连接酶编码序列如SEQ ID NO.6所示。
重组工程菌III：分别转化重组人白蛋白表达盒和FLO基因敲除质粒。构建FLO基因敲除质粒，质粒上包含待敲除基因FLO两侧的同源臂序列，两个同源臂之间插入一个选择性G418标记，其结构示意图如图5所示。酵母絮凝蛋白FL05编码序列如SEQ ID NO.7所示，酵母絮凝蛋白FL08编码序列如SEQ ID NO.8所示，酵母絮凝蛋白FL011编码序列如SEQ ID NO.9所示。
重组工程菌IV：分别转化重组人白蛋白表达盒和重组酵母超氧化物歧化酶表达盒(其结构示意图如图3所示)。酵母超氧化物歧化酶编码序列如SEQ ID NO.4所示。
重组工程菌V：以重组工程菌III作为宿主细胞，转化重组酵母E3泛素连接酶和重组酵母超氧化物歧化酶表达盒，其结构示意图如图4所示。
重组工程菌VI：以重组工程菌III作为宿主细胞，转化重组酵母E3泛素连接酶和人超氧化物歧化酶表达盒，其结构示意图如图4所示。人超氧化物歧化 酶编码序列如SEQ ID NO.5所示。
实施例3
分别取各重组工程菌I、II、III、IV、V和VI，如表1所示。接种于40ml BMGY培养液中220rpm 30℃培养过夜，取细胞液离心，将沉淀重悬于40ml BMMY培养液中，220rpm 25℃开始诱导，每24小时加入0.4％甲醇，96小时后10000rpm离心5分钟，收集上清。
表1
Figure PCTCN2022116713-appb-000001
将上清进行SDS-PAGE电泳检测(图谱见图6)，以重组工程菌I的重组人白蛋白表达量作为对照(100％)，可以观察到各重组工程菌的重组人白蛋白表达量均有所提高(图谱见图7)。其中重组工程菌VI的重组人白蛋白相对表达量高达216％。
本发明涉及一种提高重组人白蛋白表达量的方法。在本说明书中，术语“重组人白蛋白”也可以称为“重组人血清白蛋白”和/或“重组人血白蛋白”和/或“rHA”和/或“rHSA”。术语“人血清白蛋白”是指从人血清中提取出来的人白蛋白，也可以称为“人血白蛋白”和/或“HSA”和/或“HA”和/或“pdHSA”。
需要说明的是，本专利能够实现多种菌种的构建，类似的菌种也在本专利的保护范围内。
目前，最常用的可实现规模化生产的基因重组微生物表达人白蛋白的方式，主要是酵母表达系统，但由于酵母细胞生长代谢过程中，可能过早絮凝，会从发酵液中沉淀，可能导致发酵的不完全。重组蛋白质在二硫键形成以及由内质 网向高尔基体转运等过程受阻时，未折叠或错误折叠的蛋白质聚集在内质网中，引起内质网压力，可影响细胞正常功能。且甲醇代谢会产生大量的活性氧，对核酸、脂质、蛋白质等细胞组分直接损伤，严重时还会引起细胞衰老或死亡。依照但不限于本发明提供的方法，在适合生产重组人白蛋白的条件下，培养酵母细胞，诱导蛋白表达，可以使重组人白蛋白表达量大大提高。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本领域的技术人员应该了解本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的范围内。

Claims (15)

  1. 一种提高重组人白蛋白表达量的方法，其特征在于，
    所述方法的宿主细胞在表达重组人白蛋白的同时，还包含如下操作：
    减弱、破坏或去除至少一个编码絮凝蛋白的内源基因的功能；
    和/或编辑内源基因或异源基因使得超氧化物歧化酶过表达；
    和/或编辑内源基因或异源基因使得E3泛素连接酶过表达。
  2. 如权利要求1所述的一种提高重组人白蛋白表达量的方法，其特征在于，所述的宿主细胞为酵母细胞。
  3. 如权利要求1所述的一种提高重组人白蛋白表达量的方法，其特征在于，所述的编码絮凝蛋白的内源基因包含FLO5、FLO8和FLO11。
  4. 如权利要求1所述的一种提高重组人白蛋白表达量的方法，其特征在于，所述的编码超氧化物歧化酶的内源或异源基因是经遗传修饰的或未经遗传修饰的；编码E3泛素连接酶的内源或异源基因是经遗传修饰的或未经遗传修饰的。
  5. 如权利要求1所述的一种提高重组人白蛋白表达量的方法，其特征在于，编码超氧化物歧化酶的基因是酵母超氧化物歧化酶基因；或者编码超氧化物歧化酶的基因是人铜锌超氧化物歧化酶核酸分子基因。
  6. 如权利要求1所述的一种提高重组人白蛋白表达量的方法，其特征在于，编码E3泛素连接酶的基因是酵母E3泛素连接酶；或者编码E3泛素连接酶的基因为人E3泛素连接酶核酸分子。
  7. 如权利要求2所述的一种提高重组人白蛋白表达量的方法，其特征在于，所述的酵母细胞为如下酵母属的一种或者多种：汉逊酵母属(Hansenula)、毕赤酵母属(Pichia)和假丝酵母属(Candida)。
  8. 如权利要求7所述的一种提高重组人白蛋白表达量的方法，其特征在于，所述的毕赤酵母属的细胞是巴斯德毕赤酵母细胞。
  9. 如权利要求1所述的一种提高重组人白蛋白表达量的方法，其特征在于，编码人白蛋白基因、编码超氧化物歧化酶基因和编码E3泛素连接酶的基因的重组表达盒是在一个核酸构建体上或在不同选择标志的核酸构建体上。
  10. 如权利要求9所述的一种提高重组人白蛋白表达量的方法，其特征在于，所述核酸构建体包括pHIL-D2、pPIC3.5、pHIL-S1、pPIC9、pPink-LC、pPink-HC、pPICZA、pPICZB、pPICZC、pPICZaA、pPICZaB和pPICZaC质粒中的任意一种或 者多种；重组启动子包括AOX1启动子、GAP启动子、GAL启动子、FDH启动子和FLD启动子中的任意一种或者多种。
  11. 如权利要求10所述的一种提高重组人白蛋白表达量的方法，其特征在于，宿主细胞中编码至少一个重组启动子修饰的人白蛋白；宿主细胞中编码至少一个重组启动子修饰的超氧化物歧化酶；宿主细胞中编码至少一个重组启动子修饰的E3泛素连接酶。
  12. 如权利要求1-11任意一项所述的一种提高重组人白蛋白表达量的方法，其特征在于，用到的序列为如下任意一种或者多种序列：
    SEQ ID NO.1酿酒酵母交配因子信号肽编码序列
    SEQ ID NO.2人白蛋白信号肽编码序列；
    SEQ ID NO.3人白蛋白成熟肽编码序列；
    SEQ ID NO.4酵母超氧化物歧化酶编码序列；
    SEQ ID NO.5人超氧化物歧化酶编码序列；
    SEQ ID NO.6酵母E3泛素连接酶编码序列；
    SEQ ID NO.7酵母絮凝蛋白FL05编码序列；
    SEQ ID NO.8酵母絮凝蛋白FL08编码序列；
    SEQ ID NO.9酵母絮凝蛋白FL011编码序列；
    SEQ ID NO.10 HSA-F引物序列；
    SEQ ID NO.11 HSA-R引物序列；
    SEQ ID NO.12酵母超氧化物歧化酶-F序列；
    SEQ ID NO.13酵母超氧化物歧化酶-R序列；
    SEQ ID NO.14人超氧化物歧化酶-F序列；
    SEQ ID NO.15人超氧化物歧化酶-R序列；
    SEQ ID NO.16酵母E3泛素连接酶-F序列；
    SEQ ID NO.17酵母E3泛素连接酶-R序列。
  13. 如权利要求1-12任意一项所述的一种提高重组人白蛋白表达量的方法，其特征在于，重组工程菌I：转化的重组人白蛋白表达盒，信号肽编码序列如SEQ ID NO.1所示，人白蛋白成熟肽编码序列如SEQ ID NO.3所示；
    重组工程菌II：以重组工程菌I为宿主细胞，转化重组酵母E3泛素连接酶 表达盒；酵母E3泛素连接酶编码序列如SEQ ID NO.6所示；
    重组工程菌III：分别转化重组人白蛋白表达盒和FLO基因敲除质粒；构建FLO基因敲除质粒，质粒上包含待敲除基因FLO两侧的同源臂序列，两个同源臂之间插入一个选择性G418标记；酵母絮凝蛋白FL05编码序列如SEQ ID NO.7所示，酵母絮凝蛋白FL08编码序列如SEQ ID NO.8所示，酵母絮凝蛋白FL011编码序列如SEQ ID NO.9所示；
    重组工程菌IV：分别转化重组人白蛋白表达盒和重组酵母超氧化物歧化酶表达盒；酵母超氧化物歧化酶编码序列如SEQ ID NO.4所示；
    重组工程菌V：以重组工程菌III作为宿主细胞，转化重组酵母E3泛素连接酶和重组酵母超氧化物歧化酶表达盒；
    重组工程菌VI：以重组工程菌III作为宿主细胞，转化重组酵母E3泛素连接酶和人超氧化物歧化酶表达盒；人超氧化物歧化酶编码序列如SEQ ID NO.5所示；
    分别取各重组工程菌I、II、III、IV、V和VI，如表1所示；接种于40ml BMGY培养液中220rpm 30℃培养过夜，取细胞液离心，将沉淀重悬于40ml BMMY培养液中，220rpm 25℃开始诱导，每24小时加入0.4％甲醇，96小时后10000rpm离心5分钟，收集上清；
    表1
    Figure PCTCN2022116713-appb-100001
    将上清进行SDS-PAGE电泳检测，以重组工程菌I的重组人白蛋白表达量作为对照(100％)，可以观察到各重组工程菌的重组人白蛋白表达量均有所提高； 其中重组工程菌VI的重组人白蛋白相对表达量高达216％。
  14. 利用SEQ ID NO.1-SEQ ID NO.17任意一种或者多种序列基因操作后的宿主细胞。
  15. 利用SEQ ID NO.1-SEQ ID NO.17任意一种或者多种序列基因操作后的宿主细胞表达的重组人白蛋白。
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