WO2019041635A1 - 一种细胞中内源性表达rna聚合酶的核酸构建物 - Google Patents

一种细胞中内源性表达rna聚合酶的核酸构建物 Download PDF

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WO2019041635A1
WO2019041635A1 PCT/CN2017/115967 CN2017115967W WO2019041635A1 WO 2019041635 A1 WO2019041635 A1 WO 2019041635A1 CN 2017115967 W CN2017115967 W CN 2017115967W WO 2019041635 A1 WO2019041635 A1 WO 2019041635A1
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yeast
protein
nucleic acid
combination
rnap
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French (fr)
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郭敏
代田纯
薛银鸽
李海洋
王海鹏
柴智
刘帅龙
于雪
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康码（上海）生物科技有限公司
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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
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N1/145—Fungal isolates
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07006—DNA-directed RNA polymerase (2.7.7.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the present invention relates to the field of biotechnology, and preferably to a nucleic acid construct for endogenous expression of an RNA polymerase in a cell.
  • RNA polymerase is an RNA polymerase: an enzyme that catalyzes the synthesis of RNA from nucleoside-5'-triphosphate using a DNA strand or RNA as a template. It catalyzes the use of DNA as a template and ribose triphosphate.
  • a nucleoside is a substrate and an enzyme that synthesizes RNA which is polymerized by a phosphodiester bond. Since it is related to the transcription of genetic information of a gene DNA into RNA, it is also called a transcriptase.
  • T7 RNA polymerase is an RNA polymerase derived from T7 phage that recognizes a 23 nt conserved promoter sequence (pT7) and provides strong transcriptional activity.
  • T7 RNAP is widely used in the expression of foreign genes in prokaryotes such as Escherichia coli, and has been successfully applied in eukaryotes.
  • prokaryotes such as Escherichia coli
  • yeast system when T7 RNAP is expressed in Saccharomyces cerevisiae or Pichia pastoris, efficient transcription of the target gene can be achieved, although the transcription product cannot be efficiently translated into protein.
  • T7 RNAP is present in cells in the form of exogenous plasmids in both E. coli (DE3 plasmid) and yeast (2 ⁇ plasmid), and there is no cell system that is directly recombined into its genome for stable and efficient expression of T7 RNAP. .
  • Protein in vitro synthesis system refers to the addition of mRNA or DNA template, RNA polymerase, amino acid and ATP components in the lysis system of bacteria, fungi, plant cells or animal cells to complete the rapid and efficient translation of foreign proteins.
  • E. coli extract E. coli extract
  • RRL Rabbit reticulocyte Lysate
  • WGE Wheat germ extract
  • Insect cell insects
  • Kluyveromyces is an ascomycete, in which Kluyveromyces marxianus and Kluyveromyces lactis are industrially widely used yeasts.
  • Kluyveromyces cerevisiae is a yeast that uses lactic acid as its sole source of carbon and energy. Compared with other yeasts, Kluyveromyces cerevisiae has many advantages, such as superior secretion capacity, good large-scale fermentation characteristics, food safety grade, and the ability to simultaneously modify the protein, as a host system expression drug. The use of protein has also shown great potential.
  • T7 RNAP is widely used in cell in vitro expression systems to efficiently transcribe exogenous target genes, ultimately achieving In vitro translation of proteins.
  • concentration of T7 RNAP also has a significant effect on the translation efficiency of the protein.
  • T7 RNAP proteins are manually added by exogenous sources, including E. coli extract (ECE), Rabbit reticulocyte Lysate (RRL), and wheat germ. (Wash germ extract, WGE), insect (Insect cell extract, ICE) and human source systems have a negative impact on experimental efficiency, cost, complexity and stability.
  • a first aspect of the invention provides a nucleic acid construct having a structure of formula I from 5' to 3':
  • Z1 and Z2 are respectively elements for constituting the construct
  • Each "-" is independently a bond or nucleotide linkage sequence
  • Z1 is a promoter element selected from the group consisting of: RNR2, ADH1, GAPDH, TEF1, PGK1, SED1, or a combination thereof;
  • Z2 is the coding sequence of the RNP protein
  • the Z1 and Z2 are derived from yeast.
  • the promoter element is selected from the group consisting of ScRNR2, ScADH1, ScGAPDH, ScTEF1, ScPGK1, ScSED1, KlRNR2, KlADH1, KlGAPDH, KlTEF1, KlPGK1, KlSED1, or a combination thereof.
  • the promoter element has an intensity of ⁇ 20%- ⁇ 50% of the promoter ScRNR2 based on the promoter ScRNR2.
  • the strength of the promoter ScRNR2 is based on the number of transcripts.
  • the promoter strength refers to the ability of the promoter to regulate the transcriptional expression level of the downstream coding gene, and the increase in the initiation intensity increases the transcriptional expression level of the downstream coding gene, and the activation intensity decreases the transcriptional expression of the downstream coding gene. The level is lowered.
  • the RNP protein is selected from the group consisting of T7 RNAP protein, T3 RNAP, T4 RNAP, T5 RNAP, or a combination thereof.
  • the yeast is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces yeast, or a group thereof Hehe.
  • the Kluyveromyces yeast is selected from the group consisting of Kluyveromyces cerevisiae, Kluyveromyces cerevisiae, Kluyveromyces dobzhanskii, or a combination thereof.
  • sequence of the nucleic acid construct is set forth in SEQ ID NO.: 1.
  • a second aspect of the invention provides a nucleic acid construct having a structure of formula II from 5' to 3':
  • Z1, Z2, Z3 are respectively elements for constituting the construct
  • Each "-" is independently a bond or nucleotide linkage sequence
  • Z1 is a promoter element selected from the group consisting of: ScRNR2, ScADH1, ScGAPDH, ScTEF1, ScPGK1, ScSED1, KlRNR2, KlADH1, KlGAPDH, KlTEF1, KlPGK1, KlSED1, or a combination thereof;
  • Z2 is the coding sequence of the RNP protein
  • Z3 is the coding sequence of a foreign protein
  • the Z1 and Z2 are derived from yeast.
  • the coding sequence of the foreign protein is from a prokaryote, a eukaryote.
  • the coding sequence of the foreign protein is from an animal, a plant, or a pathogen.
  • the coding sequence of the foreign protein is from a mammal, preferably a primate, a rodent, including a human, a mouse, a rat.
  • the coding sequence of the foreign protein encodes a foreign protein selected from the group consisting of luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, Aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutant, alpha-amylase, enterobacterin A, C Hepatitis B virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylan Enzyme, or a combination thereof.
  • luciferin or luciferase (such as firefly luciferase)
  • green fluorescent protein yellow fluorescent protein
  • Aminoacyl tRNA synthetase
  • the exogenous protein is selected from the group consisting of luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde- 3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutation, alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor , interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, Xylanase, or a combination thereof.
  • luciferin or luciferase (such as firefly luciferase)
  • green fluorescent protein yellow fluorescent protein
  • aminoacyl tRNA synthetase aminoacyl tRNA synthetase
  • a third aspect of the invention provides a vector or combination of vectors comprising the nucleic acid construct of the first aspect of the invention or the second aspect of the invention.
  • a fourth aspect of the invention provides a genetically engineered cell, wherein one or more sites of the genome of the genetically engineered cell integrate the construct of the first aspect of the invention or the second aspect of the invention, or the gene
  • the vector or vector combination of the third aspect of the invention is contained in the engineered cells.
  • the genetically engineered cell is a yeast cell.
  • the yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces yeast, or a combination thereof.
  • the Kluyveromyces yeast is selected from the group consisting of Kluyveromyces cerevisiae, Kluyveromyces cerevisiae, Kluyveromyces dobzhanskii, or a combination thereof.
  • a fifth aspect of the invention provides a kit, the reagent contained in the kit being selected from one or more of the group consisting of:
  • the kit further comprises (d) a yeast in vitro protein synthesis system.
  • the yeast in vitro protein synthesis system is an in vitro protein synthesis system of Kluyveromyces cerevisiae (preferably an in vitro protein synthesis system of Kluyveromyces cerevisiae).
  • a sixth aspect of the invention provides a construct according to the first aspect of the invention or the second aspect of the invention, the vector or vector combination of the third aspect of the invention, the genetic engineering of the fourth aspect of the invention Use of a cell or kit of the fifth aspect of the invention for performing high throughput in vitro protein synthesis.
  • the seventh aspect of the invention provides a method for synthesizing high-throughput exogenous protein in vitro, comprising the steps of:
  • step (ii) incubating the yeast in vitro protein synthesis system of step (i) for a period of time T1 under suitable conditions to synthesize the foreign protein.
  • the method further comprises: (iii) isolating or detecting the foreign protein, optionally from the yeast in vitro protein synthesis system.
  • the yeast in vitro protein synthesis system is an in vitro protein synthesis system of Kluyveromyces cerevisiae (preferably an in vitro protein synthesis system of Kluyveromyces cerevisiae).
  • the coding sequence of the foreign protein is from a prokaryote, a eukaryote.
  • the coding sequence of the foreign protein is from an animal, a plant, or a pathogen.
  • the coding sequence of the foreign protein is from a mammal, preferably a primate, a rodent, including a human, a mouse, a rat.
  • the coding sequence of the foreign protein encodes a foreign protein selected from the group consisting of luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, Aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutant, alpha-amylase, enterobacterin A, C Hepatitis B virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylan Enzyme, or a combination thereof.
  • luciferin or luciferase (such as firefly luciferase)
  • green fluorescent protein yellow fluorescent protein
  • Aminoacyl tRNA synthetase
  • the exogenous protein is selected from the group consisting of luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde- 3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutation, alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor Interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof.
  • luciferin or luciferase (such as firefly luciferase)
  • green fluorescent protein yellow fluorescent protein
  • aminoacyl tRNA synthetase aminoacyl tRNA synthetase
  • the reaction temperature is 20 to 37 ° C, preferably 22 to 35 ° C.
  • the reaction time is from 1 to 10 h, preferably from 2 to 8 h.
  • Figure 1 shows a schematic diagram of the structure of the pKM-T7RNAP1 plasmid
  • Figure 2 shows a schematic diagram of the plasmid structure of pKM-CAS1.0-KlTDH3-1
  • Figure 3 is a schematic view showing the structure of pKM-CAS1.0-KlTDH3-2 plasmid
  • Figure 4 is a schematic view showing the structure of pKM-KlTDH3-1-T7-DD plasmid
  • Figure 5 is a schematic view showing the structure of pKM-KlTDH3-2-T7-DD plasmid
  • Figure 6 shows a schematic diagram of the plasmid structure of pKM-CAS1.0-KlXRN1
  • Figure 7 shows a schematic diagram of the structure of the pKM-KlXRN1-T7-DD plasmid
  • Figure 8 is a schematic diagram showing the in vitro translation activity assay of the transformed pKM-T7 RNAP1 plasmid strain
  • Figure 9 is a schematic diagram showing the in vitro translation activity assay of the KlTDH3-1-T7 RNAP fusion protein strain
  • Figure 10 is a schematic diagram showing the in vitro translation activity assay of the KlTDH3-2-T7 RNAP fusion protein strain
  • Figure 11 shows a schematic representation of the in vitro translational activity assay of the klxrn1 ⁇ -pScRNR2-T7 RNAP engineered strain.
  • the nucleic acid construct of the present invention is composed of a promoter having a specific priming strength (such as RNR2). , ADH1, GAPDH, TEF1, PGK1, SED1, etc.) and the coding sequence of RNAP (such as T7RNAP, T3RNAP, T4RNAP, T5RNAP, etc.) proteins, and the nucleic acid construct of the present invention is applied to the yeast in vitro protein synthesis system, and the synthesized fluorescence is synthesized.
  • the relative light unit value (RLU) of the enzyme activity is very high, and can achieve the same effect as the exogenous addition of T7 RNAP, which can be as high as 6.6 ⁇ 10 7 .
  • RLU relative light unit value
  • Yeast combines the advantages of simple, efficient protein folding, and post-translational modification. Among them, Saccharomyces cerevisiae and Pichia pastoris are model organisms that express complex eukaryotic proteins and membrane proteins. Yeast can also be used as a raw material for the preparation of in vitro translation systems.
  • Kluyveromyces is an ascomycete, in which Kluyveromyces marxianus and Kluyveromyces lactis are industrially widely used yeasts.
  • Kluyveromyces cerevisiae has many advantages over other yeasts, such as superior secretion capacity, better large-scale fermentation characteristics, food safety levels, and the ability to simultaneously modify post-translational proteins.
  • the yeast in vitro protein synthesis system is not particularly limited, and a preferred yeast in vitro protein synthesis system is the Kluyveromyces expression system (more preferably, the K. lactis expression system).
  • Kluyveromyces cerevisiae e.g., Kluyveromyces lactis
  • Kluyveromyces lactis is not particularly limited, and includes any Kluvi (e.g., Kluyveromyces lactis) strain capable of improving the efficiency of synthetic proteins.
  • the yeast in vitro protein synthesis system comprises:
  • the in vitro protein synthesis system comprises: yeast cell extract, 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenosine triphosphate (ATP) , guanosine triphosphate (GTP), cytosine triphosphate (CTP), thymidine triphosphate (TTP), amino acid mixture, creatine phosphate, dithiothreitol (DTT), creatine phosphate Kinase, RNase inhibitor, fluorescein, luciferase DNA, RNA polymerase.
  • yeast cell extract 4-hydroxyethylpiperazineethanesulfonic acid
  • potassium acetate magnesium acetate
  • adenosine triphosphate (ATP) adenosine triphosphate
  • GTP guanosine triphosphate
  • CTP cytosine triphosphate
  • TTP thymidine triphosphate
  • amino acid mixture amino acid mixture
  • creatine phosphate dithio
  • the RNA polymerase is not particularly limited and may be selected from one or more RNA polymerases, and a typical RNA polymerase is T7 RNA polymerase.
  • the ratio of the yeast cell extract in the in vitro protein synthesis system is not particularly limited, and generally the yeast cell extract accounts for 20-70% of the in vitro protein synthesis protein synthesis system, preferably Ground, 30-60%, more preferably, 40-50%.
  • the yeast cell extract does not contain intact cells, and typical yeast cell extracts include ribosomes for protein translation, transfer RNA, aminoacyl tRNA synthetase, initiation factors required for protein synthesis, and The elongation factor and the termination release factor.
  • the yeast extract contains some other proteins in the cytoplasm derived from yeast cells, especially soluble proteins.
  • the yeast cell extract contains a protein content of 20 to 100 mg/ml, preferably 50 to 100 mg/ml.
  • the method for determining protein content is a Coomassie Brilliant Blue assay.
  • the preparation method of the yeast cell extract is not limited, and a preferred preparation method comprises the following steps:
  • the solid-liquid separation method is not particularly limited, and a preferred mode is centrifugation.
  • the centrifugation is carried out in a liquid state.
  • the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5,000 to 100,000 x g, preferably 8,000 to 30,000 x g.
  • the centrifugation time is not particularly limited, and a preferred centrifugation time is 0.5 min-2 h, preferably, 20 min - 50 min.
  • the temperature of the centrifugation is not particularly limited.
  • the centrifugation is carried out at 1-10 ° C, preferably at 2-6 ° C.
  • the washing treatment method is not particularly limited, and a preferred washing treatment method is treatment with a washing liquid at a pH of 7-8 (preferably, 7.4), and the washing liquid is not particularly Typically, the wash liquor is typically selected from the group consisting of potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or combinations thereof.
  • the manner of the cell disruption treatment is not particularly limited, and a preferred cell disruption treatment includes high pressure disruption, freeze-thaw (e.g., liquid nitrogen low temperature) disruption.
  • the mixture of nucleoside triphosphates in the in vitro protein synthesis system is adenine nucleoside triphosphate, guanosine triphosphate, cytidine triphosphate, and uridine nucleoside triphosphate.
  • the concentration of each of the single nucleotides is not particularly limited, and usually the concentration of each single nucleotide is from 0.5 to 5 mM, preferably from 1.0 to 2.0 mM.
  • the mixture of amino acids in the in vitro protein synthesis system can include natural or unnatural amino acids, and can include D-form or L-form amino acids.
  • Representative amino acids include, but are not limited to, 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, serine, Tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.
  • the concentration of each amino acid is usually from 0.01 to 0.5 mM, preferably from 0.02 to 0.2 mM, such as 0.05, 0.06, 0.07, 0.08 mM.
  • the in vitro protein synthesis system further comprises polyethylene glycol or an analog thereof.
  • concentration of polyethylene glycol or the like is not particularly limited, and usually, the concentration (w/v) of polyethylene glycol or the like is from 0.1 to 8%, preferably from 0.5 to 4%, more preferably, 1-2%, based on the total weight of the protein synthesis system.
  • Representative examples of PEG include, but are not limited to, PEG3000, PEG 8000, PEG 6000, and PEG 3350. It should be understood that the system of the present invention may also include other various molecular weight polyethylene glycols (e.g., PEG 200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc.).
  • the in vitro protein synthesis system further comprises sucrose.
  • concentration of sucrose is not particularly limited, and usually, the concentration of sucrose is from 0.03 to 40% by weight, preferably from 0.08 to 10% by weight, more preferably from 0.1 to 5% by weight, based on the total weight of the protein synthesis system.
  • a particularly preferred in vitro protein synthesis system in addition to the yeast extract, further comprises the following components: 22 mM, 4-hydroxyethylpiperazineethanesulfonic acid having a pH of 7.4, 30-150 mM potassium acetate, 1.0-5.0 mM magnesium acetate, 1.5-4 mM nucleoside triphosphate mixture, 0.08-0.24 mM amino acid mixture, 25 mM phosphocreatine, 1.7 mM dithiothreitol, 0.27 mg/mL phosphocreatine kinase, 1%-4% polyethylene Glycol, 0.5% to 2% sucrose, 8-20 ng/ ⁇ L of firefly luciferase DNA, 0.027-0.054 mg/mL T7 RNA polymerase.
  • coding sequence of a foreign protein is used interchangeably with “foreign DNA” and refers to a foreign DNA molecule for directing protein synthesis.
  • the DNA molecule is linear or circular.
  • the DNA molecule contains a sequence encoding a foreign protein.
  • examples of the sequence encoding the foreign protein include, but are not limited to, a genomic sequence, a cDNA sequence.
  • the sequence encoding the foreign protein further comprises a promoter sequence, a 5' untranslated sequence, and a 3' untranslated sequence.
  • the selection of the exogenous DNA is not particularly limited.
  • the exogenous DNA is selected from the group consisting of a luciferin protein, or a luciferase (such as firefly luciferase), a green fluorescent protein, and a yellow fluorescent protein. , aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, exogenous DNA of a variable region of an antibody, DNA of a luciferase mutant, or a combination thereof.
  • the exogenous DNA may also be selected from the group consisting of alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum white. Protein, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, exogenous DNA of xylanase, or a combination thereof.
  • alpha-amylase enteromycin A
  • hepatitis C virus E2 glycoprotein insulin precursor
  • interferon alpha A interleukin-1 beta
  • lysozyme serum white.
  • Protein single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, exogenous DNA of xylanase, or a combination thereof.
  • the exogenous DNA encodes a protein selected from the group consisting of: green fluorescent protein (eGFP), yellow fluorescent protein (YFP), and Escherichia coli beta-galactosidase ( ⁇ -galactosidase, LacZ), human lysine-tRNA synthetase, human leucine-tRNA synthetase, Arabidopsis glyceraldehyde 3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate) Dehydrogenase), murine catalase (Catalase), or a combination thereof.
  • eGFP green fluorescent protein
  • YFP yellow fluorescent protein
  • Escherichia coli beta-galactosidase ⁇ -galactosidase, LacZ
  • human lysine-tRNA synthetase human leucine-tRNA synthetase
  • the invention provides a nucleic acid construct comprising a nucleic acid sequence of the formula I;
  • Z1 and Z2 are respectively elements for constituting the construct
  • Each "-" is independently a bond or nucleotide linkage sequence
  • Z1 is a promoter element selected from the group consisting of: RNR2, ADH1, GAPDH, TEF1, PGK1, SED1, or a combination thereof;
  • Z2 is the coding sequence of the RNP protein
  • the Z1 and Z2 are derived from yeast.
  • the invention also provides a nucleic acid construct having a structure of formula II from 5' to 3':
  • Z1, Z2, Z3 are respectively elements for constituting the construct
  • Each "-" is independently a bond or nucleotide linkage sequence
  • Z1 is a promoter element selected from the group consisting of: RNR2, ADH1, GAPDH, TEF1, PGK1, SED1, or a combination thereof;
  • Z2 is the coding sequence of the RNP protein
  • Z3 is the coding sequence of a foreign protein
  • the Z1 and Z2 are derived from yeast.
  • the selection of the coding sequence of the foreign protein is not particularly limited.
  • the coding sequence of the foreign protein is selected from the group consisting of luciferin, or luciferase (such as firefly luciferase), green. Fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, exogenous DNA of variable regions of antibodies, DNA of luciferase mutant Or a combination thereof.
  • the coding sequence of the foreign protein may also encode a protein selected from the group consisting of ⁇ -amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon ⁇ A, interleukin-1 ⁇ , lysobacteria Enzyme, serum albumin, single-chain antibody segment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof.
  • ⁇ -amylase enteromycin A
  • hepatitis C virus E2 glycoprotein insulin precursor
  • interferon ⁇ A interleukin-1 ⁇
  • lysobacteria Enzyme lysobacteria Enzyme
  • serum albumin serum albumin
  • scFV single-chain antibody segment
  • thyroxine transporter thyroxine transporter
  • tyrosinase tyrosinase
  • xylanase a combination thereof.
  • nucleic acid constructs of the invention may be linear or cyclic.
  • the nucleic acid constructs of the invention may be single-stranded or double-stranded.
  • the nucleic acid construct of the present invention may be DNA, RNA, or DNA/RNA hybrid.
  • sequence of the nucleic acid construct of the invention is set forth in SEQ ID NO.: 1.
  • the construct further comprises an element selected from the group consisting of a promoter, a terminator, a poly(A) element, a transport element, a gene targeting element, a selection marker gene, an enhancer , resistance gene, transposase encoding gene.
  • selectable marker genes can be used in the present invention, including but not limited to: auxotrophic markers, resistance markers, reporter gene markers.
  • the use of a selectable marker acts on the screening of recombinant cells (recombinants) such that the recipient cells are able to distinguish significantly from untransformed cells.
  • the auxotrophic marker is complementary to the recipient cell mutant gene by the transferred marker gene, thereby allowing the recipient cell to exhibit wild-type growth.
  • a resistance marker refers to the transfer of a resistance gene into a recipient cell, and the transferred gene causes the recipient cell to exhibit drug resistance at a certain drug concentration.
  • resistance markers are applied to facilitate convenient screening of recombinant cells.
  • the use of the nucleic acid construct of the present invention in the yeast in vitro protein synthesis system of the present invention can significantly improve the efficiency of protein translation, in particular, the relative luciferase activity synthesized by the nucleic acid construct of the present invention.
  • the unit value of light is as high as 6.6 ⁇ 10 7 .
  • the invention also provides a vector or combination of vectors comprising a nucleic acid construct of the invention.
  • the vector is selected from the group consisting of a bacterial plasmid, a bacteriophage, a yeast plasmid, or an animal cell vector, a shuttle vector; and the vector is a transposon vector.
  • Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. Any plasmid and vector can be employed as long as it is capable of replicating and stabilizing in the host.
  • One of ordinary skill in the art can construct expression vectors containing the promoters and/or gene sequences of interest described herein using well known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the invention also provides a genetically engineered cell comprising said construct or vector or combination of vectors, or said genetically engineered cell chromosome incorporating said construct or vector.
  • the genetically engineered cell further comprises a vector comprising a gene encoding a transposase or a gene encoding a transposase integrated into the chromosome.
  • the genetically engineered cell is a eukaryotic cell.
  • the eukaryotic cell includes, but is not limited to, a yeast cell (preferably, Kluyveromyces cells, more preferably Kluyveromyces lactis cells).
  • the construct or vector of the invention can be used to transform appropriate genetically engineered cells.
  • the genetically engineered cells may be prokaryotic cells such as Escherichia coli, Streptomyces, Agrobacterium: or lower eukaryotic cells, such as yeast cells; or higher animal cells, such as insect cells. It will be apparent to one of ordinary skill in the art how to select appropriate vectors and genetically engineered cells. Transformation of genetically engineered cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote (such as E. coli), it can be treated with the CaCl 2 method or by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.).
  • the transformed plants can also be subjected to methods such as Agrobacterium transformation or gene gun transformation, such as leaf disc method, immature embryo transformation method, flower bud soaking method and the like.
  • the invention provides an in vitro high-throughput protein synthesis method comprising the steps of:
  • step (ii) incubating the yeast in vitro protein synthesis system of step (i) for a period of time T1 under suitable conditions to synthesize the foreign protein.
  • the method further comprises: (iii) isolating or detecting the foreign protein, optionally from the yeast in vitro protein synthesis system.
  • a promoter having a specific priming strength such as RNR2, ADH1, GAPDH, TEF1, PGK1, SED1, etc.
  • RNAP such as T7RNAP
  • the present inventors have found for the first time that the relative light unit value of the luciferase activity synthesized by the nucleic acid construct of the present invention is very high, as high as 6.6 ⁇ 10 7 .
  • the present invention integrates the T7 RNAP gene into the genome of the Kluyveromyces cerevisiae by CRISPR/Cas9 combined with efficient transformation technology, thereby realizing the stable presence of T7 RNAP in the genome of the cell and the sustained expression of T7 RNAP protein. .
  • the present invention prepares the Kluyveromyces strain inserted into T7 RNAP into an in vitro expression system, thereby realizing efficient translation of the exogenous target protein, greatly simplifying the preparation steps, saving cost, and increasing the stability of protein synthesis.
  • the present invention integrates the T7 RNAP gene into the genome of the cell by DNA recombination technology, and establishes a stable cell with high endogenous expression by using a highly efficient cell transformation platform.
  • One of the applications is in an in vitro protein synthesis system.
  • the foreign protein can be efficiently translated without the need to manually add T7 RNAP.
  • the present invention integrates the T7 RNAP protein into the genome of the cell by gene editing technology, thereby creating a strain capable of stably and appropriately expressing the T7 RNAP protein. This results in a simple and efficient in vitro translation system that does not require external manual addition of T7 RNAP.
  • the present invention inserts a weaker pScRNR2 promoter in front of T7 RNAP and constructs the construct into a free plasmid to verify the role of the T7 RNAP expression cassette in an in vitro translation system.
  • the present invention integrates T7 RNAP into the genome of the cell by two protocols: a linking the T7 RNAP to the endogenous gene via a linker to form a fusion protein; and b replacing the T7 RNAP expression cassette with a certain gene of the cell.
  • the gene fused to T7 RNAP is KlTDH3
  • the gene knocked out and subjected to T7 RNAP replacement is KlXRN1.
  • the present invention first constructs a T7 RNAP free plasmid and transforms the cells.
  • the promoter of T7 RNAP in the free plasmid is ScRNR2 promoter
  • the terminator is ScCYC1 terminator
  • the resistance gene is Kan.
  • the plasmid construction and transformation methods are as follows:
  • the plasmid containing the T7 RNAP gene was used as a template, and PCR amplification was carried out with primers PF1: ATGAACACGATTAACATCGCTAAGAACG (SEQ ID NO.: 2) and PR1: TTACGCGAACGCGAAGTCCG (SEQ ID NO.: 3); Kluyveromyces lactis free plasmid was used as a template.
  • PCR amplification was carried out with primers PF2: ATCTTAGAGTCGGACTTCGCGTTCGCGTAAGAAGATGCTTCTGCTCATCATC (SEQ ID NO.: 4) and P R 2: AGTCGTTCTTAGCGATGTTAATCGTGTTCATGGTAATTGGACAAATAAATACGTGT (SEQ ID NO.: 5).
  • Kluyveromyces cerevisiae solution was streaked on YPD solid medium and single cloned, and cultured overnight in 25 mL of 2 ⁇ YPD liquid medium. 2 mL of the bacterial solution was incubated in 50 mL of liquid 2 ⁇ YPD medium. Incubate for 2-8h. The yeast cells were collected by centrifugation at 3000 g for 5 min at 20 ° C, resuspended in 500 ⁇ L of sterile water, and the cells were collected by centrifugation under the same conditions. A competent cell solution (5% v/v glycerol, 10% v/v DMSO) was prepared and the yeast cells were dissolved in 500 ⁇ L of this solution. Dispense 50 ⁇ L into a 1.5 mL centrifuge tube and store at -80 °C.
  • Transformation buffer was prepared: PEG 3350 (50% (w/v)) 260 ⁇ L, LiAc (1.0 M) 36 ⁇ L, carrier DNA (5.0 mg/mL) 20 ⁇ L, pKM-T7 RNAP1 10 ⁇ L, and sterile water was added to a final volume of 360 ⁇ L. After heat shock, the supernatant was removed by centrifugation at 13,000 g for 30 s. Add 1 mL of YPD liquid medium, incubate for 2-3 h, and pipet 200 ⁇ L of the medium in solid YPD (200 ⁇ g/mL G418) and incubate for 2-3 days until a single colony appears.
  • TDH3 exists as a tetramer and participates in the catalytic reaction in the glycolytic pathway. Its promoter pTDH3 is a persistent strong promoter widely used in genetic engineering.
  • the present invention ligates the T7 RNAP gene to the 3' end of the ORF of the K. lactis TDH3 gene.
  • TDH3 homologous gene sequence in K. lactis was determined by BLAST alignment analysis with the TDH3 gene in the NCBI database. Comparing, two TDH3 homologous genes are present in the K. lactis genome, which are classified in the present invention. Also named KlTDH3-1 (1024297...1025292 on chromosome A) and KlTDH3-2 (1960417...1961406 on chromosome F).
  • KlTDH3-1 1024297...1025292 on chromosome A
  • KlTDH3-2 (1960417...1961406 on chromosome F).
  • KlTDH3-1 CTTGTTGCTAAGAACTAAAG (SEQ ID NO.: 6)
  • KlTDH3-2 CTCTGAAAGAGTTGTCGATT (SEQ ID NO.: 7) is located at 1960378...1960397 of chromosome F).
  • PCR amplification was carried out using the primer PF3: CTTGTTGCTAAGAACTAAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT (SEQ ID NO.: 8), PR3: GCTCTAAAACCTTTAGTTCTTAGCAACAAGAAAGTCCCATTCGCCACCCG (SEQ ID NO.: 9), using the pCAS plasmid as a template. 17 ⁇ L of the amplified product was mixed, and 1 ⁇ L of DpnI, 2 ⁇ L of 10 ⁇ digestion buffer was added, and the mixture was incubated at 37° C. for 3 hours.
  • PCR amplification was carried out using the primer PF4: CTCTGAAAGAGTTGTCGATTGTTTTAGAGCTAGAAATAGCAAGTTAAAAT (SEQ ID NO.: 10), PR4: GCTCTAAAACAATCGACAACTCTTTCAGAGAAAGTCCCATTCGCCACCCG (SEQ ID NO.: 11), using the pCAS plasmid as a template. 17 ⁇ L of the amplified product was mixed, and 1 ⁇ L of DpnI, 2 ⁇ L of 10 ⁇ digestion buffer was added, and the mixture was incubated at 37° C. for 3 hours.
  • the present invention first inserts donor DNA into pMD18.
  • the linear donor DNA sequence is then amplified by PCR in a plasmid.
  • primers PF6 ATCTTAGAGTCGGACTTCGCGTTCGCGTAAAGAGGTTGATGTAATTGATATTTTCCT (SEQ ID NO.: 16) and PR6: ACCTCCTTCGACGTTTGGTCTAGATCCACCGTTCTTAGCAACAAGTTCGACC (SEQ ID NO.: 17) were amplified; using a plasmid containing a T7 RNAP sequence as a template, Amplification was carried out with primers PF7: GGTGGATCTAGACCAAACGTCGAAGGAGGTTCTAACACGATTAACATCGCTAAGAAC (SEQ ID NO.: 18) and PR7: TTACGCGAACGCGAAGTCCG (SEQ ID NO.: 19).
  • primers M13-F GTA AAACGACGGCCAGT (SEQ ID NO.: 20) and M13-R: CAGGAAACAGCTATGAC (SEQ ID NO.: 21) were amplified to obtain linearity.
  • Donor DNA was amplified using pKM-KlTDH3-1-T7-DD plasmid as a template.
  • primers PF8 GAGCTCGGTACCCGGGGATCCTCTAGAGATGAAGCTTTGATGACTACCGTTC (SEQ ID NO.: 22) and PR8: GCCAAGCTTGCATGCCTGCAGGTCGACGATGTCTATTGTATCGGAAGAACTGTCA (SEQ ID NO.: 23) PCR amplification was carried out; PCR amplification was carried out using the pMD18 plasmid as a template with primers pMD18-F: ATCGTCGACCTGCAGGCATG (SEQ ID NO.: 24) and pMD18-R: ATCTCTAGAGGATCCCCGGG (SEQ ID NO.: 25).
  • primers PF9 ATCTTAGAGTCGGACTTCGCGTTCGCGTAAATTACTCTTTTAAGTTAACGAACGCT (SEQ ID NO.: 26) and PR9: ACCTCCTTCGACGTTTGGT CTAGATCCACCAGCAAC GTGCTCAACtAAgTCa ACgACcCTTTCAGAGTAACCGTAT (SEQ ID NO.: 27) were amplified; plasmids containing T7 RNAP sequences were used.
  • amplification was carried out with primers PF10: GGTGGATCTAGACCAAACGTCGAAGGAGGTTCTAACACGATTAACATCGCTAAGAAC (SEQ ID NO.: 28) and PR10: TTACGCGAACGCGAAGTCCG (SEQ ID NO.: 29).
  • Two amplification products of 8.5 ⁇ L each, 1 ⁇ L of DpnI, 2 ⁇ L of 10 ⁇ digestion buffer were mixed, and the mixture was incubated at 37° C. for 3 hours.
  • 10 ⁇ L of DpnI-treated product was added to 100 ⁇ L of DH5 ⁇ competent cells, placed on ice for 30 min, and heat-shocked at 42 ° C for 45 s.
  • primers M13-F GTAAAACGACGGCCAGT (SEQ ID NO.: 30) and M13-R: CAGGAAACAGCTATGAC (SEQ ID NO.: 31) were amplified to obtain a linear supply. Body DNA.
  • the competent cells were thawed at 37 ° C for 15-30 s, centrifuged at 13,000 g for 2 min and the supernatant was removed.
  • T7 RNAP sequence primer ATTGGACAAAATGCCAGCACTTCCG (SEQ ID NO.: 32) / PR11 (KlTDH3-1 donor DNA 5' lateral primer): CTTCTACTGCTCCAATGTTCGTCGTT (SEQ ID NO.: 33) and primer PF12 (T7 RNAP sequence primer ): ATTGGACAAAATGCCAGCACTTCCG (SEQ ID NO.: 34)/PR12 (KlTDH3-2 donor DNA 5' lateral primer): TTAACGAAGACAAGTACAACGGTGA (SEQ ID NO.: 35) for PCR amplification, detection of CRISPR insertion at the KlTDH3 site, A positive band indicates that the T7 RNAP sequence was successfully inserted into the target site.
  • Example 4 KnXRN1 was knocked out and replaced with a T7 RNAP expression cassette by CRISPR-Cas9 technology
  • XRN1 is a 5'-3' exonuclease directed against capless structural mRNA.
  • in vitro expression of the yeast is also enhanced in the expression of T7RNAP in vivo, and the K1XRN1 gene is completely knocked out and replaced with the T7 RNAP sequence and related promoters and terminators.
  • K1XRN1 gRNA sequences identified in the present invention are AGAGTTCGACAATTTGTACT (SEQ ID NO.: 36) and CGTCGTGGCCGTAGTAATCG (SEQ ID NO.: 37).
  • Use primer PF13 AGAGTTCGACAATTTGTACTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC (SEQ ID NO.: 38), PR13: GCTCTAAAACAGTACAAATTGTCGAACTCTAAAGTCCCATTCGCCACCCG (SEQ ID NO.: 39), PCR amplification was carried out using the pCAS plasmid as a template, and the gRNA1 sequence was integrated into the CRISPR plasmid. 17 ⁇ L of the amplified product was mixed, and 1 ⁇ L of DpnI, 2 ⁇ L of 10 ⁇ digestion buffer was added, and the mixture was incubated at 37° C. for 3 hours.
  • gRNA2 was integrated into the plasmid using the primer PF14: CGTCGTGGCCGTAGTAATCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC (SEQ ID NO.: 40), PR14:GCTCTAAAACCGATTACTACGGCCACGACGAAAGTCCCATTCGCCACCCG (SEQ ID NO.: 41), and named pKM-CAS1.0-KlXRN1 (Fig. 6 ).
  • the present invention first inserts donor DNA into a pMD18 plasmid, and then obtains a linear donor DNA sequence by PCR amplification.
  • PCR amplification was carried out using clopidogrel lactic acid genomic DNA as a template, primers PF15:GAGCTCGGTACCCGGGGATCCTCTAGAGATAAAAGCTTGAACTTATGGATCCGGGTA (SEQ ID NO.:42) and PR15:GCCAAGCTTGCATGCCTGCAGGTCGACGATGTAATCCTCTTGTGCTCTAA TTGCT (SEQ ID NO.:43); pMD18 plasmid as template and primer pMD18 -F: ATCGTCGACCTGCAGGCATG (SEQ ID NO.: 44) and pMD18-R: ATCTCTAGAGGATCCCCGGG (SEQ ID NO.: 45) were subjected to PCR amplification.
  • primers PF16: ATGAATTCTATTTTGCATAATTGTACGCATGGTG (SEQ ID NO.: 46) and PR16: ttaCAAGTACAAATTGTCGAACTCTGGAATCTG (SEQ ID NO.: 47) were amplified; pKM-T7RNAP1 plasmid was used as a template, primer PF17: Amplification was carried out by ATTCCAGAGTTCGACAATTTGTACTTGtaaAGTCGAACAAGAAGCAGGCAAAG (SEQ ID NO.: 48) and PR17: ATGCGTACAATTATGCAAAATAGAATTCATCTTCGAGCGTCCCAAAACCTTC (SEQ ID NO.: 49).
  • primers M13-F GTAAAACGACGGCCAGT (SEQ ID NO.: 50) and M13-R: CAGGAAACAGCTATGAC (SEQ ID NO.: 51) were amplified to obtain linear donor DNA. .
  • Iii Draw Kluyveromyces cerevisiae solution on YPD solid medium and pick a single clone, shake culture in 25 mL 2 ⁇ YPD liquid medium overnight, and take 2 mL of the bacterial solution in 50 mL of liquid 2 ⁇ YPD medium. Incubate for 2-8 h. The yeast cells were collected by centrifugation at 3000 g for 5 min at 20 ° C, resuspended in 500 ⁇ L of sterile water, and the cells were collected by centrifugation under the same conditions. A competent cell solution (5% v/v glycerol, 10% v/v DMSO) was prepared and the yeast cells were dissolved in 500 ⁇ L of this solution. Dispense 50 ⁇ L into a 1.5 mL centrifuge tube and store at -80 °C.
  • the competent cells were thawed at 37 ° C for 15-30 s, centrifuged at 13,000 g for 2 min and the supernatant was removed.
  • the genetically modified K. lactis strain was prepared into an in vitro protein synthesis system, and a firefly Luciferase (Fluc) gene DNA template was added to determine the protein translation ability of the engineered strain.
  • the above reaction system was placed in an environment of 25-30 ° C, and allowed to stand for about 2-6 h. After the reaction, an equal volume of Fluc substrate luciferin (Luciferin) was added to a 96-well white plate or a 384-well white plate and immediately placed on an Envision 2120 multi-plate reader (Perkin Elmer). The readings were used to detect Fluc activity, relative light units.
  • the Relative Light Unit (RLU) is used as the unit of activity.
  • the strain transferred to the free plasmid pKM-T7RNAP1 showed the highest activity and was in the normal range when the concentration of T7 RNAP was 0 (10 7 ). With the increase of the concentration of T7 RNAP, the activity was inhibited, indicating that pKM-T7RNAP1
  • the T7 RNAP structure (pScRNR2+T7 RNAP) in the plasmid functions normally and has an RLU value of 1.4 ⁇ 10 7 (Fig. 8).
  • T7 RNAP In the klxrn1 ⁇ -pScRNR2-T7 RNAP structure, when the concentration of T7 RNAP is 0, it shows the highest activity and is in the normal range (10 7 ). On the surface, T7 RNAP can be expressed in an appropriate amount, which can meet the requirements of the in vitro translation system. Moreover, the nucleic acid construct of this structure was able to significantly enhance the efficiency of protein production by the yeast in vitro protein synthesis system, and its RLU value was 6.6 ⁇ 10 7 (Fig. 11).

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Abstract

提供了一种细胞中内源性表达RNA聚合酶的核酸构建物，该核酸构建物由具有特定启动强度的启动子（如RNR2、ADH1、GAPDH、TEF1、PGK1、SED1、或其组合)和RNA聚合酶 (如T7RNAP、T3RNAP、T4RNAP、T5RNAP、或其组合)的编码序列构成，用于在酵母体外蛋白质合成体系中合成外源蛋白。

Description

一种细胞中内源性表达RNA聚合酶的核酸构建物 技术领域
本发明涉及生物技术领域，较佳地，涉及一种细胞中内源性表达RNA聚合酶的核酸构建物。
背景技术
RNA聚合酶是一种RNA聚合酶(RNA polymerase)：以一条DNA链或RNA为模板催化由核苷-5′-三磷酸合成RNA的酶，是催化以DNA为模板(template)、三磷酸核糖核苷为底物、通过磷酸二酯键而聚合的合成RNA的酶，因为在细胞内与基因DNA的遗传信息转录为RNA有关，所以也称转录酶。
T7 RNA聚合酶(T7 RNAP)是来源于T7噬菌体的一种RNA聚合酶，识别23nt的保守启动子序列(pT7)并提供较强的转录活性。T7 RNAP广泛应用于大肠杆菌(Escherichia coli)等原核生物中外源基因的表达，在真核生物中也有成功应用的报。在酵母系统中，将T7 RNAP在酿酒酵母(Saccharomyces cerevisiae)或者毕赤酵母(Pichia pastoris)中表达时，均能实现靶基因的高效转录，虽然其转录产物并不能被有效翻译成蛋白质。目前，无论是大肠杆菌(DE3质粒)还是酵母(2μ质粒)中，T7 RNAP均是以外源质粒的方式存在于细胞中的，尚无直接重组至其基因组中稳定高效率表达T7 RNAP的细胞系统。
蛋白质体外合成系统是指在细菌、真菌、植物细胞或动物细胞的裂解体系中，加入mRNA或者DNA模板、RNA聚合酶及氨基酸和ATP等组分，完成外源蛋白的快速高效翻译。目前，经常实验的商业化体外蛋白表达系统包括大肠杆菌系统(E.coli extract，ECE)、兔网织红细胞(Rabbit reticulocyte Lysate，RRL)、麦胚(Wheat germ extract,WGE)、昆虫(Insect cell extract,ICE)和人源系统。
克鲁维酵母(Kluyveromyces)是一种子囊孢子酵母，其中的马克斯克鲁维酵母(Kluyveromyces marxianus)和乳酸克鲁维酵母(Kluyveromyces lactis)是工业上广泛使用的酵母。例如乳酸克鲁维酵母是一种能够以乳酸作为其唯一的碳源和能源的酵母。与其他酵母相比，乳酸克鲁维酵母具有许多优点，如超强的分泌能力，良好的大规模发酵特性、食品安全的级别及同时具有蛋白翻译后修饰的能力等，其作为宿主系统表达药用蛋白也已显示出巨大的潜力。
T7 RNAP广泛应用于细胞体外表达系统，高效转录外源目的基因，最终实现 蛋白的体外翻译。T7 RNAP的浓度，对蛋白的翻译效率也有显著影响。在现有的文献报导及商业试剂盒中，都是通过外源手动加入T7 RNAP蛋白质，包括大肠杆菌系统(E.coli extract，ECE)、兔网织红细胞(Rabbit reticulocyte Lysate，RRL)、麦胚(Wheat germ extract,WGE)、昆虫(Insect cell extract,ICE)和人源系统，对实验效率、成本、复杂性及稳定性都有不好的影响。
因此，本领域迫切需要开发一种能够增强蛋白质翻译效率的新的核酸构建物。
发明内容
本发明的目的在于提供一种能够增强蛋白质翻译效率的新的核酸构建物。
本发明第一方面提供了一种核酸构建物，所述的构建物具有从5’至3’的式I结构：
Z1-Z2   (I)
式中，
Z1、Z2分别为用于构成所述构建物的元件；
各“-”独立地为键或核苷酸连接序列；
Z1为启动子元件，所述启动子元件选自下组：RNR2、ADH1、GAPDH、TEF1、PGK1、SED1、或其组合；
Z2为RNP蛋白的编码序列；
并且，所述Z1、Z2来源于酵母。
在另一优选例中，所述启动子元件选自下组：ScRNR2、ScADH1、ScGAPDH、ScTEF1、ScPGK1、ScSED1、KlRNR2、KlADH1、KlGAPDH、KlTEF1、KlPGK1、KlSED1、或其组合。
在另一优选例中，以所述启动子ScRNR2为基准，所述启动子元件的强度为启动子ScRNR2的±20％－±50％。
在另一优选例中，所述启动子ScRNR2的强度以转录本的数量为参照。
在另一优选例中，所述启动强度是指，启动子调控其下游编码基因转录表达水平的能力，启动强度增强则下游编码基因的转录表达水平提高，启动强度减弱则下游编码基因的转录表达水平降低。
在另一优选例中，所述RNP蛋白选自下组：T7RNAP蛋白、T3RNAP、T4RNAP、T5RNAP、或其组合。
在另一优选例中，所述酵母选自下组：酿酒酵母、克鲁维酵母属酵母、或其组 合。
在另一优选例中，所述克鲁维酵母属酵母选自下组：乳酸克鲁维酵母、马克斯克鲁维酵母、多布克鲁维酵母(Kluyveromyces dobzhanskii)、或其组合。
在另一优选例中，所述核酸构建物的序列如SEQ ID NO.:1所示。
本发明第二方面提供了一种核酸构建物，所述的构建物具有从5’至3’的式II结构：
Z1-Z2-Z3   (II)
式中，
Z1、Z2、Z3分别为用于构成所述构建物的元件；
各“-”独立地为键或核苷酸连接序列；
Z1为启动子元件，所述启动子元件选自下组：ScRNR2、ScADH1、ScGAPDH、ScTEF1、ScPGK1、ScSED1、KlRNR2、KlADH1、KlGAPDH、KlTEF1、KlPGK1、KlSED1、或其组合；
Z2为RNP蛋白的编码序列；
Z3为外源蛋白的编码序列；
并且，所述Z1、Z2来源于酵母。
在另一优选例中，所述外源蛋白的编码序列来自原核生物、真核生物。
在另一优选例中，所述外源蛋白的编码序列来自动物、植物、病原体。
在另一优选例中，所述外源蛋白的编码序列来自哺乳动物，较佳地灵长动物，啮齿动物，包括人、小鼠、大鼠。
在另一优选例中，所述的外源蛋白的编码序列编码选自下组的外源蛋白：荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域、萤光素酶突变体、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、或其组合。
在另一优选例中，所述外源蛋白选自下组：荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域、萤光素酶突变、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、 木聚糖酶、或其组合。
本发明第三方面提供了一种载体或载体组合，所述的载体或载体组合含有本发明第一方面或本发明第二方面所述的核酸构建物。
本发明第四方面提供了一种基因工程细胞，所述基因工程细胞的基因组的一个或多个位点整合有本发明第一方面或本发明第二方面所述的构建物，或者所述基因工程细胞中含有本发明第三方面所述的载体或载体组合。
在另一优选例中，所述基因工程细胞为酵母细胞。
在另一优选例中，所述酵母细胞选自下组：酿酒酵母、克鲁维酵母属酵母、或其组合。
在另一优选例中，所述克鲁维酵母属酵母选自下组：乳酸克鲁维酵母、马克斯克鲁维酵母、多布克鲁维酵母(Kluyveromyces dobzhanskii)、或其组合。
本发明第五方面提供了一种试剂盒，所述试剂盒中包含的试剂选自下组中的一种或多种：
(a)本发明第一方面或本发明第二方面所述的构建物；
(b)本发明第三方面所述的载体或载体组合；和
(c)本发明第四方面所述的基因工程细胞；
在另一优选例中，所述试剂盒还包括(d)酵母体外蛋白合成体系。
在另一优选例中，所述酵母体外蛋白合成体系为克鲁维酵母体外蛋白合成体系(优选乳酸克鲁维酵母体外蛋白合成体系)。
本发明第六方面提供了一种如本发明第一方面或本发明第二方面所述的构建物、本发明第三方面所述的载体或载体组合、本发明第四方面所述的基因工程细胞或本发明第五方面所述试剂盒的用途，用于进行高通量的体外蛋白合成。
本发明第七方面提供了一种体外高通量的外源蛋白合成方法，包括步骤：
(i)在酵母体外蛋白合成体系存在下，提供本发明第一方面或本发明第二方面所述的核酸构建物；
(ii)在适合的条件下，孵育步骤(i)的酵母体外蛋白合成体系一段时间T1，从而合成所述外源蛋白。
在另一优选例中，所述方法还包括：(iii)任选地从所述酵母体外蛋白合成体系中，分离或检测所述外源蛋白。
在另一优选例中，所述酵母体外蛋白合成体系为克鲁维酵母体外蛋白合成体系(优选乳酸克鲁维酵母体外蛋白合成体系)。
在另一优选例中，所述外源蛋白的编码序列来自原核生物、真核生物。
在另一优选例中，所述外源蛋白的编码序列来自动物、植物、病原体。
在另一优选例中，所述外源蛋白的编码序列来自哺乳动物，较佳地灵长动物，啮齿动物，包括人、小鼠、大鼠。
在另一优选例中，所述的外源蛋白的编码序列编码选自下组的外源蛋白：荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域、萤光素酶突变体、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、或其组合。
在另一优选例中，所述外源蛋白选自下组：荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域、萤光素酶突变、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、或其组合。
在另一优选例中，所述步骤(ii)中，反应温度为20-37℃，较佳地，22-35℃。
在另一优选例中，所述步骤(ii)中，反应时间为1-10h，较佳地，2-8h。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了pKM-T7RNAP1质粒结构示意图；
图2显示了pKM-CAS1.0-KlTDH3-1质粒结构示意图；
图3显示了pKM-CAS1.0-KlTDH3-2质粒结构示意图；
图4显示了pKM-KlTDH3-1-T7-DD质粒结构示意图；
图5显示了pKM-KlTDH3-2-T7-DD质粒结构示意图；
图6显示了pKM-CAS1.0-KlXRN1质粒结构示意图；
图7显示了pKM-KlXRN1-T7-DD质粒结构示意图；
图8显示了转化pKM-T7RNAP1质粒菌株体外翻译活性测定示意图；
图9显示了KlTDH3-1-T7 RNAP融合蛋白菌株体外翻译活性测定示意图；
图10显示了KlTDH3-2-T7 RNAP融合蛋白菌株体外翻译活性测定示意图；
图11显示了klxrn1Δ-pScRNR2-T7 RNAP改造菌株体外翻译活性测定示意图。
具体实施方式
经过广泛而深入的研究，通过大量筛选和摸索，首次意外地发现了一种可大幅度增强蛋白质翻译效率的新型核酸构建物，本发明的核酸构建物由具有特定启动强度的启动子(如RNR2、ADH1、GAPDH、TEF1、PGK1、SED1等)和RNAP(如T7RNAP、T3RNAP、T4RNAP、T5RNAP等)蛋白的编码序列构成，在酵母体外蛋白质合成体系中应用本发明的核酸构建物，所合成的荧光素酶活性的相对光单位值(RLU)非常高，可达到与外源加入T7RNAP相同效果，可高达6.6×107。在此基础上，本发明人完成了本发明。
酵母体外蛋白质合成体系
酵母(yeast)兼具培养简单、高效蛋白质折叠、和翻译后修饰的优势。其中酿酒酵母(Saccharomyces cerevisiae)和毕氏酵母(Pichia pastoris)是表达复杂真核蛋白质和膜蛋白的模式生物，酵母也可作为制备体外翻译系统的原料。
克鲁维酵母(Kluyveromyces)是一种子囊孢子酵母，其中的马克斯克鲁维酵母(Kluyveromyces marxianus)和乳酸克鲁维酵母(Kluyveromyces lactis)是工业上广泛使用的酵母。与其他酵母相比，乳酸克鲁维酵母具有许多优点,如超强的分泌能力,更好的大规模发酵特性、食品安全的级别、以及同时具有蛋白翻译后修饰的能力等。
在本发明中，酵母体外蛋白质合成体系不受特别限制，一种优选的酵母体外蛋白质合成体系为克鲁维酵母表达系统(更佳地，乳酸克鲁维酵母表达系统)。
在本发明中，克鲁维酵母(如乳酸克鲁维酵母)不受特别限制，包括任何一种能够提高合成蛋白效率的克鲁维(如乳酸克鲁维酵母)菌株。
在本发明中，所述酵母体外蛋白质合成体系包括：
(a)酵母细胞提取物；
(b)聚乙二醇；
(c)任选的外源蔗糖；和
(d)任选的溶剂，所述溶剂为水或水性溶剂。
在一特别优选的实施方式中，本发明提供的体外蛋白合成体系包括：酵母细胞提取物，4-羟乙基哌嗪乙磺酸，醋酸钾，醋酸镁，腺嘌呤核苷三磷酸(ATP)，鸟嘌呤核苷三磷酸(GTP)，胞嘧啶核苷三磷酸(CTP)，胸腺嘧啶核苷三磷酸(TTP)，氨基酸混合物，磷酸肌酸，二硫苏糖醇(DTT)，磷酸肌酸激酶，RNA酶抑制剂，荧光素，萤光素酶DNA，RNA聚合酶。
在本发明中，RNA聚合酶没有特别限制，可以选自一种或多种RNA聚合酶，典型的RNA聚合酶为T7 RNA聚合酶。
在本发明中，所述酵母细胞提取物在体外蛋白合成体系中的比例不受特别限制，通常所述酵母细胞提取物在体外蛋白质合成蛋白合成体系中所占体系为20-70％，较佳地，30-60％，更佳地，40-50％。
在本发明中，所述的酵母细胞提取物不含完整的细胞，典型的酵母细胞提取物包括用于蛋白翻译的核糖体、转运RNA、氨酰tRNA合成酶、蛋白质合成需要的起始因子和延伸因子以及终止释放因子。此外，酵母提取物中还含有一些源自酵母细胞的细胞质中的其他蛋白，尤其是可溶性蛋白。
在本发明中，所述的酵母细胞提取物所含蛋白含量为20-100mg/ml,较佳为50-100mg/ml。所述的测定蛋白含量方法为考马斯亮蓝测定方法。
在本发明中，所述的酵母细胞提取物的制备方法不受限制，一种优选的制备方法包括以下步骤：
(i)提供酵母细胞；
(ii)对酵母细胞进行洗涤处理，获得经洗涤的酵母细胞；
(iii)对经洗涤的酵母细胞进行破细胞处理，从而获得酵母粗提物；
(iv)对所述酵母粗提物进行固液分离，获得液体部分，即为酵母细胞提取物。
在本发明中，所述的固液分离方式不受特别限制，一种优选的方式为离心。
在一优选实施方式中，所述离心在液态下进行。
在本发明中，所述离心条件不受特别限制，一种优选的离心条件为5000-100000×g，较佳地，8000-30000×g。
在本发明中，所述离心时间不受特别限制，一种优选的离心时间为0.5min－2 h，较佳地，20min－50min。
在本发明中，所述离心的温度不受特别限制，优选的，所述离心在1-10℃下进行，较佳地，在2-6℃下进行。
在本发明中，所述的洗涤处理方式不受特别限制，一种优选的洗涤处理方式为采用洗涤液在pH为7-8(较佳地，7.4)下进行处理，所述洗涤液没有特别限制，典型的所述洗涤液选自下组：4-羟乙基哌嗪乙磺酸钾、醋酸钾、醋酸镁、或其组合。
在本发明中，所述破细胞处理的方式不受特别限制，一种优选的所述的破细胞处理包括高压破碎、冻融(如液氮低温)破碎。
所述体外蛋白质合成体系中的核苷三磷酸混合物为腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸和尿嘧啶核苷三磷酸。在本发明中，各种单核苷酸的浓度没有特别限制，通常每种单核苷酸的浓度为0.5-5mM，较佳地为1.0-2.0mM。
所述体外蛋白质合成体系中的氨基酸混合物可包括天然或非天然氨基酸，可包括D型或L型氨基酸。代表性的氨基酸包括(但并不限于)20种天然氨基酸：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸。每种氨基酸的浓度通常为0.01-0.5mM，较佳地0.02-0.2mM，如0.05、0.06、0.07、0.08mM。
在优选例中，所述体外蛋白质合成体系还含有聚乙二醇或其类似物。聚乙二醇或其类似物的浓度没有特别限制，通常，聚乙二醇或其类似物的浓度(w/v)为0.1-8％，较佳地，0.5-4％，更佳地，1-2％，以所述蛋白合成体系的总重量计。代表性的PEG例子包括(但并不限于)：PEG3000，PEG8000，PEG6000和PEG3350。应理解，本发明的体系还可包括其他各种分子量的聚乙二醇(如PEG200、400、1500、2000、4000、6000、8000、10000等)。
在优选例中，所述体外蛋白质合成体系还含有蔗糖。蔗糖的浓度没有特别限制，通常，蔗糖的浓度为0.03-40wt％，较佳地，0.08-10wt％，更佳地，0.1-5wt％，以所述蛋白合成体系的总重量计。
一种特别优选的体外蛋白质合成体系，除了酵母提取物之外，还含有以下组分：22mM，pH为7.4的4-羟乙基哌嗪乙磺酸，30-150mM醋酸钾，1.0-5.0 mM醋酸镁，1.5-4mM核苷三磷酸混合物，0.08-0.24mM的氨基酸混合物，25mM磷酸肌酸，1.7mM二硫苏糖醇，0.27mg/mL磷酸肌酸激酶，1％-4％聚乙二醇，0.5％-2％蔗糖，8-20ng/μL萤火虫荧光素酶的DNA，0.027-0.054mg/mL T7 RNA聚合酶。
外源蛋白的编码序列(外源DNA)
如本文所用，术语“外源蛋白的编码序列”与“外源DNA”可互换使用，均指外源的用于指导蛋白质合成的DNA分子。通常，所述的DNA分子为线性的或环状的。所述的DNA分子含有编码外源蛋白的序列。
在本发明中，所述的编码外源蛋白的序列的例子包括(但并不限于)：基因组序列、cDNA序列。所述的编码外源蛋白的序列还含有启动子序列、5'非翻译序列、3'非翻译序列。
在本发明中，所述外源DNA的选择没有特别限制，通常，外源DNA选自下组：编码荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域的外源DNA、萤光素酶突变体的DNA、或其组合。
外源DNA还可以选自下组：编码α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶的外源DNA、或其组合。
在一优选实施方式中，所述外源DNA编码选自下组的蛋白：绿色荧光蛋白(enhanced GFP,eGFP)、黄色荧光蛋白(YFP)、大肠杆菌β-半乳糖苷酶(β-galactosidase,LacZ)、人赖氨酸-tRNA合成酶(Lysine-tRNA synthetase)、人亮氨酸-tRNA合成酶(Leucine-tRNA synthetase)、拟南芥甘油醛3-磷酸脱氢酶(Glyceraldehyde-3-phosphate dehydrogenase)、鼠过氧化氢酶(Catalase)、或其组合。
核酸构建物
本发明提供了一种核酸构建物，所述核酸构建物含有结构如式I所示的核酸序列：
Z1-Z2   (I)
式中，
Z1、Z2分别为用于构成所述构建物的元件；
各“-”独立地为键或核苷酸连接序列；
Z1为启动子元件，所述启动子元件选自下组：RNR2、ADH1、GAPDH、TEF1、PGK1、SED1、或其组合；
Z2为RNP蛋白的编码序列；
并且，所述Z1、Z2来源于酵母。
本发明还提供了一种核酸构建物，所述的构建物具有从5’至3’的式II结构：
Z1-Z2-Z3   (II)
式中，
Z1、Z2、Z3分别为用于构成所述构建物的元件；
各“-”独立地为键或核苷酸连接序列；
Z1为启动子元件，所述启动子元件选自下组：RNR2、ADH1、GAPDH、TEF1、PGK1、SED1、或其组合；
Z2为RNP蛋白的编码序列；
Z3为外源蛋白的编码序列；
并且，所述Z1、Z2来源于酵母。
在本发明中，所述外源蛋白的编码序列的选择没有特别限制，通常，外源蛋白的编码序列选自下组：编码荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域的外源DNA、萤光素酶突变体的DNA、或其组合。
外源蛋白的编码序列还可以编码选自下组的蛋白：α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、或其组合。
此外，本发明的所述核酸构建物可以是线性的，也可以是环状的。本发明的所述核酸构建物可以是单链的，也可以是双链的。本发明的所述核酸构建物可以是DNA，也可以是RNA，或DNA/RNA杂合。
在一优选实施方式中，本发明的核酸构建物的序列如SEQ ID NO.:1所示。
在另一优选例中，所述的构建物还包括选自下组的元件或其组合：启动子、终止子、poly(A)元件、转运元件、基因靶向元件、筛选标记基因、增强子、抗性基因、转座酶编码基因。
多种选择性标志基因均可应用于本发明，包括但不限于：营养缺陷型标记，抗性标记，报告基因标记。选择性标志的应用对于重组细胞(重组子)的筛选起到作用，使得受体细胞能够与未转化的细胞进行显著区分。营养缺陷型标记是通过转入的标记基因与受体细胞突变基因互补，从而使受体细胞表现野生型生长。抗性标记是指将抗性基因转入受体细胞中，转入的基因使受体细胞在一定的药物浓度下表现抗药性。作为本发明的优选方式，应用抗性标记来实现重组细胞的便捷筛选。
在本发明中，在本发明的酵母体外蛋白质合成体系中应用本发明的核酸构建物，可显著提高蛋白翻译的效率，具体地，应用本发明的核酸构建物所合成的荧光素酶活性的相对光单位值高达6.6×107。
载体，基因工程细胞
本发明还提供了一种载体或载体组合，所述载体含有本发明的核酸构建物。优选地，所述载体选自：细菌质粒、噬菌体、酵母质粒、或动物细胞载体、穿梭载体；所述的载体为转座子载体。用于制备重组载体的方法是本领域普通技术人员所熟知的。只要其能够在宿主体内复制和稳定，任何质粒和载体都是可以被采用的。
本领域普通技术人员可以使用熟知的方法构建含有本发明所述的启动子和/或目的基因序列的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。
本发明还提供了一种基因工程细胞，所述的基因工程细胞含有所述的构建物或载体或载体组合，或所述的基因工程细胞染色体整合有所述的构建物或载体。在另一优选例中，所述的基因工程细胞还包括含有编码转座酶基因的载体或其染色体上整合有转座酶基因。
优选地，所述的基因工程细胞为真核细胞。
在另一优选例中，所述真核细胞，包括(但不限于)：酵母细胞(优选，克鲁维酵母细胞，更优选乳酸克鲁维酵母细胞)。
本发明的构建物或载体，可以用于转化适当的基因工程细胞。基因工程细胞可以是原核细胞，如大肠杆菌，链霉菌属、农杆菌：或是低等真核细胞，如酵母细胞；或是高等动物细胞，如昆虫细胞。本领域一般技术人员都清楚如何选择适当的载体和基因工程细胞。用重组DNA转化基因工程细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物(如大肠杆菌)时，可以用CaCl2 法处理，也可用电穿孔法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法(如显微注射、电穿孔、脂质体包装等)。转化植物也可使用农杆菌转化或基因枪转化等方法，例如叶盘法、幼胚转化法、花芽浸泡法等。
体外高通量的蛋白合成方法
本发明提供了一种体外高通量的蛋白合成方法，包括步骤：
(i)在酵母体外蛋白合成体系存在下，提供本发明第一方面或第二方面所述的核酸构建物；
(ii)在适合的条件下，孵育步骤(i)的酵母体外蛋白合成体系一段时间T1，从而合成所述外源蛋白。
在另一优选例中，所述方法还包括：(iii)任选地从所述酵母体外蛋白合成体系中，分离或检测所述外源蛋白。
本发明的主要优点包括：
(1)本发明首次发现，将具有特定启动强度的启动子(如RNR2、ADH1、GAPDH、TEF1、PGK1、SED1等)和RNAP(如T7RNAP)作为核酸构建物，应用于本发明的酵母体外蛋白质合成体系中，可显著提高蛋白翻译的效率。
(2)本发明首次发现，本发明的核酸构建物所合成的荧光素酶活性的相对光单位值非常高，高达6.6×107。
(3)本发明首次通过CRISPR/Cas9，结合高效的转化技术，将T7 RNAP基因整合到真核细胞克鲁维酵母基因组中，实现了T7 RNAP在细胞基因组内的稳定存在和T7RNAP蛋白质的持续表达。
(4)本发明将插入T7 RNAP的克鲁维酵母菌株制备成体外表达系统，实现了外源目标蛋白的高效翻译、大大简化了制备步骤，节约了成本，并增加了蛋白质合成的稳定性。
(5)本发明通过DNA重组技术，借助高效的细胞转化平台，将T7 RNAP基因无标记整合到细胞基因组中，建立其高效内源性表达的稳定细胞，应用之一是在体外蛋白质合成系统中，不需人工添加T7 RNAP即可高效翻译外源蛋白。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说 明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
如无特别说明，则本发明实施例中所用的材料和试剂均为市售产品。
实施例1细胞基因组中插入位点的选定
为了克服现有体外翻译系统中需外源手动加入T7 RNA聚合酶蛋白的缺陷，本发明通过基因编辑技术将T7 RNAP蛋白整合到细胞基因组内，创造了一个能够稳定适量表达T7 RNAP蛋白的菌株，进而形成了一个无需外源手动加入T7 RNAP的简便高效的体外翻译系统。
体外翻译系统对T7 RNAP的含量有较高要求，过高或过少都会对系统效率产生影响。因此本发明在T7RNAP前插入较弱的pScRNR2启动子，并将该结构构建到游离质粒中，验证T7 RNAP表达盒在体外翻译系统中的作用。
经过游离质粒功能验证以后，本发明通过两种方案将T7 RNAP整合到细胞基因组中：a将T7 RNAP通过linker与内源基因连接，形成融合蛋白；b将T7 RNAP表达盒替换细胞某个基因。本发明中，与T7 RNAP融合的基因为KlTDH3，敲除并进行T7 RNAP替换的基因为KlXRN1。
实施例2 pKM-T7RNAP1质粒构建、酵母转化及活性测定
2.1 pKM-T7RNAP1质粒构建
为了验证T7 RNAP可以在细胞中有效表达且具有转录活性，本发明首先构建T7 RNAP游离质粒，并转化细胞。游离质粒中T7 RNAP的启动子为ScRNR2 promoter，终止子为ScCYC1 terminator，抗性基因为Kan。质粒构建及转化方法如下：
以含有T7 RNAP基因的质粒为模板，以引物PF1:ATGAACACGATTAACATCGCTAAGAACG(SEQ ID NO.:2)和PR1:TTACGCGAACGCGAAGTCCG(SEQ ID NO.:3)进行PCR扩增；以乳酸克鲁维酵母游离质粒为模板，以引物PF2:ATCTTAGAGTCGGACTTCGCGTTCGCGTAAGAAGATGCTTCTGCTCATCATC(SEQ ID NO.:4)和P R 2:AGTCGTTCTTAGCGATGTTAATCGTGTTCATGGTAATTGGACAAATAAATACGTGT(SEQ ID NO.:5)进行PCR扩增。将两次扩增产物各8.5μL混合， 加入1μL DpnI，2μL 10×digestion buffer，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-T7RNAP1(图1)。
2.2高效乳酸克鲁维酵母感受态制备及转化
将乳酸克鲁维酵母菌液在YPD固体培养基上划线并挑取单克隆，于25mL 2×YPD液体培养基中振荡培养过夜，取2mL菌液于50mL液体2×YPD培养基中继续振荡培养2-8h。20℃条件下3000g离心5min收集酵母细胞，加入500μL无菌水重悬，同样条件下离心收集细胞。配制感受态细胞溶液(5％v/v甘油，10％v/v DMSO)并将酵母细胞溶解于500μL该溶液中。分装50μL至1.5mL离心管中，-80℃保存。
将感受态细胞置于37℃融化15-30s，13000g离心2min并去除上清。配制转化缓冲液：PEG 3350(50％(w/v))260μL，LiAc(1.0M)36μL，carrier DNA(5.0mg/mL)20μL，pKM-T7RNAP1 10μL，加入无菌水至最终体积360μL。热激后，13000g离心30s去除上清。加入1mL YPD液体培养基，培养2-3h，吸取200μL涂布于固体YPD(200μg/mL G418)培养基，培养2-3天至单菌落出现。
实施例3通过CRISPR-Cas9技术将T7 RNAP与KlTDH3蛋白融合
3.1 KlTDH3序列检索及CRISPR gRNA序列确定
在酿酒酵母(S.cerevisiae)中，TDH3以四聚体形式存在，参与糖酵解途径中的催化反应。其启动子pTDH3是基因工程中广泛使用的一种持续型强启动子。为了实现乳酸克鲁维酵母中T7 RNAP的足量表达，并在执行转录功能时形成局部高浓度，本发明将T7 RNAP基因连接到乳酸克鲁维酵母TDH3基因ORF3’端。
i.基于S.cerevisiae酵母中的TDH3基因序列。在NCBI数据库中以TDH3基因进行BLAST比对分析，确定乳酸克鲁维酵母中TDH3同源基因序列。经比对发现，在乳酸克鲁维酵母基因组中存在两个TDH3同源基因，在本发明中分 别命名为KlTDH3-1(位于染色体A的1024297...1025292)和KlTDH3-2(位于染色体F的1960417...1961406)。在这里以此基因尾部插入一段标记DNA为例，其他目标基因或插入位置、序列均可采用类似方法操作。
ii.在KlTDH3基因终止密码子附近搜索PAM序列(NGG)，并确定KlTDH3gRNA序列(KlTDH3-1：CTTGTTGCTAAGAACTAAAG(SEQ ID NO.:6)，位于染色体A的1024272...1024291位点，KlTDH3-2：CTCTGAAAGAGTTGTCGATT(SEQ ID NO.:7)位于染色体F的1960378...1960397位点)。
3.2 KlTDH3CRISPR-Cas9质粒构建
i.对KlTDH3-1，使用引物PF3：CTTGTTGCTAAGAACTAAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT(SEQ ID NO.:8)，PR3：GCTCTAAAACCTTTAGTTCTTAGCAACAAGAAAGTCCCATTCGCCACCCG(SEQ ID NO.:9)，以pCAS质粒为模板，进行PCR扩增。将扩增产物17μL混合，加入1μL DpnI，2μL 10×digestion buffer，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-CAS1.0-KlTDH3-1(图2)。
ii.对KlTDH3-2，使用引物PF4：CTCTGAAAGAGTTGTCGATTGTTTTAGAGCTAGAAATAGCAAGTTAAAAT(SEQ ID NO.:10)，PR4：GCTCTAAAACAATCGACAACTCTTTCAGAGAAAGTCCCATTCGCCACCCG(SEQ ID NO.:11)，以pCAS质粒为模板，进行PCR扩增。将扩增产物17μL混合，加入1μL DpnI，2μL 10×digestion buffer，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-CAS1.0-KlTDH3-2(图3)。
3.3 KlTDH3-T7 RNAP供体DNA质粒构建及扩增
为了便于线性供体DNA的保存及扩增，本发明首先将供体DNA插入到pMD18 质粒中，然后通过PCR扩增得到线性供体DNA序列。
i.对KlTDH3-1，以乳酸克鲁维酵母基因组DNA为模板，以引物PF5：GAGCTCGGTACCCGGGGATCCTCTAGAGATCATCCACTCCATCACCGCTACCCAA(SEQ ID NO.:12)和PR5：GCCAAGCTTGCATGCCTGCAGGTCGACGATCAACGTCCCCATCTACAAGAGC(SEQ ID NO.:13)进行PCR扩增；以pMD18质粒为模板，以引物pMD18-F：ATCGTCGACCTGCAGGCATG(SEQ ID NO.:14)和pMD18-R：ATCTCTAGAGGATCCCCGGG(SEQ ID NO.:15)进行PCR扩增。将两次扩增产物各8.5μL混合，加入1μL DpnI，2μL 10×消化缓冲液(digestion buffer)，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-KlTDH3-1-DD。
以pKM-KlTDH3-1-DD为模板，以引物PF6:ATCTTAGAGTCGGACTTCGCGTTCGCGTAAAGAGGTTGATGTAATTGATATTTTCCT(SEQ ID NO.:16)和PR6:ACCTCCTTCGACGTTTGGTCTAGATCCACCGTTCTTAGCAACAAGTTCGACC(SEQ ID NO.:17)进行扩增；以含有T7 RNAP序列的质粒为模板，以引物PF7:GGTGGATCTAGACCAAACGTCGAAGGAGGTTCTAACACGATTAACATCGCTAAGAAC(SEQ ID NO.:18)和PR7:TTACGCGAACGCGAAGTCCG(SEQ ID NO.:19)进行扩增。将两次扩增产物各8.5μL，1μL DpnI，2μL 10×digestion buffer混合，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-KlTDH3-1-T7-DD(图4)。
以pKM-KlTDH3-1-T7-DD质粒为模板，以引物M13-F:GTA AAACGACGGCCAGT(SEQ ID NO.:20)和M13-R:CAGGAAACAGCTATGAC(SEQ ID NO.:21)进行扩增，得到线性供体DNA。
ii.对KlTDH3-2，以乳酸克鲁维酵母基因组DNA为模板，以引物PF8：GAGCTCGGTACCCGGGGATCCTCTAGAGATGAAGCTTTGATGACTACCGTTC(SEQ ID NO.:22)和PR8：GCCAAGCTTGCATGCCTGCAGGTCGACGATGTCTATTGTATCGGAAGAACTGTCA(SEQ ID  NO.:23)进行PCR扩增；以pMD18质粒为模板，以引物pMD18-F：ATCGTCGACCTGCAGGCATG(SEQ ID NO.:24)和pMD18-R：ATCTCTAGAGGATCCCCGGG(SEQ ID NO.:25)进行PCR扩增。将两次扩增产物各8.5μL混合，加入1μL DpnI，2μL 10×digestion buffer，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-KlTDH3-2-DD。
以pKM-KlTDH3-2-DD1为模板，以引物PF9:ATCTTAGAGTCGGACTTCGCGTTCGCGTAAATTACTCTTTTAAGTTAACGAACGCT(SEQ ID NO.:26)和PR9:ACCTCCTTCGACGTTTGGT CTAGATCCACCAGCAAC GTGCTCAACtAAgTCa ACgACcCTTTCAGAGTAACCGTAT(SEQ ID NO.:27)进行扩增；以含有T7 RNAP序列的质粒为模板，以引物PF10:GGTGGATCTAGACCAAACGTCGAAGGAGGTTCTAACACGATTAACATCGCTAAGAAC(SEQ ID NO.:28)和PR10:TTACGCGAACGCGAAGTCCG(SEQ ID NO.:29)进行扩增。将两次扩增产物各8.5μL，1μL DpnI，2μL 10×digestion buffer混合，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-KlTDH3-2-T7-DD(图5)。
以pKM-KlTDH3-2-T7-DD质粒为模板，以引物M13-F:GTAAAACGACGGCCAGT(SEQ ID NO.:30)和M13-R:CAGGAAACAGCTATGAC(SEQ ID NO.:31)进行扩增，得到线性供体DNA。
3.4乳酸克鲁维酵母转化及阳性鉴定
i.将乳酸克鲁维酵母菌液在YPD固体培养基上划线并挑取单克隆，于25mL2×YPD液体培养基中振荡培养过夜，取2mL菌液于50mL液体2×YPD培养基中继续振荡培养2-8h。20℃条件下3000g离心5min收集酵母细胞，加入500μL无菌水重悬，同样条件下离心收集细胞。配制感受态细胞溶液(5％v/v甘油，10％v/v DMSO)并将酵母细胞溶解于500μL该溶液中。分装50μL 至1.5mL离心管中，-80℃保存。
将感受态细胞置于37℃融化15-30s，13000g离心2min并去除上清。配制转化缓冲液：PEG 3350(50％(w/v))260μL，LiAc(1.0M)36μL，carrier DNA(5.0m g/mL)20μL，Cas9/gRNA质粒15μL，供体DNA 10μL，加入无菌水至最终体积360μL。热激后，13000g离心30s去除上清。加入1mL YPD液体培养基，培养2-3h，吸取200μL涂布于固体YPD(200μg/mL G418)培养基，培养2-3天至单菌落出现。
ii.在乳酸克鲁维酵母转化后的平板上挑取10-20个单克隆，置于1mL YPD(200μg/mL G418)液体培养基中振荡培养过夜，以菌液为模板，分别以引物PF11(T7 RNAP序列内引物):ATTGGACAAAATGCCAGCACTTCCG(SEQ ID NO.:32)/PR11(KlTDH3-1供体DNA 5’外侧引物):CTTCTACTGCTCCAATGTTCGTCGTT(SEQ ID NO.:33)和引物PF12(T7 RNAP序列内引物):ATTGGACAAAATGCCAGCACTTCCG(SEQ ID NO.:34)/PR12(KlTDH3-2供体DNA 5’外侧引物):TTAACGAAGACAAGTACAACGGTGA(SEQ ID NO.:35)进行PCR扩增，对KlTDH3位点的CRISPR插入进行检测，有阳性条带表明T7 RNAP序列插入靶位点成功。
实施例4通过CRISPR-Cas9技术将KlXRN1敲除并替换为T7 RNAP表达盒
4.1 KlXRN1序列检索及CRISPR gRNA序列确定
XRN1是一种针对无帽结构mRNA的5′-3′核酸外切酶。为了实现，在T7RNAP体内表达的同时，酵母的体外翻译活性也得到提高，本发明将KlXRN1基因完全敲除，并替换为T7 RNAP序列及相关启动子和终止子。
i.基于酿酒酵母中的XRN1基因序列。在NCBI数据库中以XRN1基因进行BLAST比对分析，确定乳酸克鲁维酵母中XRN1同源基因序列KlXRN1(位于染色体F的2091235...2095596)。在这里以此基因尾部插入一段标记DNA为例，其他目标基因或插入位置、序列均可采用类似方法操作。
ii.在KlXRN1基因两端搜索PAM序列(NGG)，并确定gRNA序列。gRNA选择的原则为：GC含量适中，本发明的标准为GC含量为40％-60％；避免poly T结构的存在。最终，本发明确定的KlXRN1gRNA序列为AGAGTTCGACAATTTGTACT(SEQ ID NO.:36)和CGTCGTGGCCGTAGTAATCG(SEQ ID NO.:37)。
4.2 KlXRN1CRISPR-Cas9质粒构建
使用引物PF13： AGAGTTCGACAATTTGTACTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC(SEQ ID NO.:38)，PR13：GCTCTAAAACAGTACAAATTGTCGAACTCTAAAGTCCCATTCGCCACCCG(SEQ ID NO.:39)，以pCAS质粒为模板，进行PCR扩增，将gRNA1序列整合到CRISPR质粒中。将扩增产物17μL混合，加入1μL DpnI，2μL 10×digestion buffer，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存。使用同样的方法，用引物PF14：CGTCGTGGCCGTAGTAATCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC(SEQ ID NO.:40)，PR14：GCTCTAAAACCGATTACTACGGCCACGACGAAAGTCCCATTCGCCACCCG(SEQ ID NO.:41)，将gRNA2整合到质粒中，命名为pKM-CAS1.0-KlXRN1(图6)。
4.3 KlXRN1-T7 RNAP供体DNA质粒构建及扩增
为了便于线性供体DNA的保存及扩增，本发明首先将供体DNA插入到pMD18质粒中，然后通过PCR扩增得到线性供体DNA序列。
以乳酸克鲁维基因组DNA为模板，以引物PF15：GAGCTCGGTACCCGGGGATCCTCTAGAGATAAAAGCTTGAACTTATGGATCCGGGTA(SEQ ID NO.:42)和PR15：GCCAAGCTTGCATGCCTGCAGGTCGACGATGTAATCCTCTTGTGCTCTAA TTGCT(SEQ ID NO.:43)进行PCR扩增；以pMD18质粒为模板，以引物pMD18-F：ATCGTCGACCTGCAGGCATG(SEQ ID NO.:44)和pMD18-R：ATCTCTAGAGGATCCCCGGG(SEQ ID NO.:45)进行PCR扩增。将两次扩增产物各8.5μL混合，加入1μL DpnI，2μL 10×digestion buffer，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-KlXRN1-DD。
以pKM-KlXRN1-DD为模板，以引物PF16:ATGAATTCTATTTTGCATAATTGTACGCATGGTG(SEQ ID NO.:46)和PR16:ttaCAAGTACAAATTGTCGAACTCTGGAATCTG(SEQ ID NO.:47)进行扩增；以pKM-T7RNAP1质粒为模板，以引物PF17： ATTCCAGAGTTCGACAATTTGTACTTGtaaAGTCGAACAAGAAGCAGGCAAAG(SEQ ID NO.:48)和PR17：ATGCGTACAATTATGCAAAATAGAATTCATCTTCGAGCGTCCCAAAACCTTC(SEQ ID NO.:49)进行扩增。将两次扩增产物各8.5μL，1μL DpnI，2μL 10×digestion buffer混合，37℃温浴3h。将DpnI处理后产物10μL加入100μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pKM-KlXRN1-T7-DD(图7)。
以pKM-KlXRN1-T7-DD质粒为模板，以引物M13-F:GTAAAACGACGGCCAGT(SEQ ID NO.:50)和M13-R:CAGGAAACAGCTATGAC(SEQ ID NO.:51)进行扩增，得到线性供体DNA。
4.4乳酸克鲁维酵母转化及阳性鉴定
iii.将乳酸克鲁维酵母菌液在YPD固体培养基上划线并挑取单克隆，于25mL2×YPD液体培养基中振荡培养过夜，取2mL菌液于50mL液体2×YPD培养基中继续振荡培养2-8h。20℃条件下3000g离心5min收集酵母细胞，加入500μL无菌水重悬，同样条件下离心收集细胞。配制感受态细胞溶液(5％v/v甘油，10％v/v DMSO)并将酵母细胞溶解于500μL该溶液中。分装50μL至1.5mL离心管中，-80℃保存。
将感受态细胞置于37℃融化15-30s，13000g离心2min并去除上清。配制转化缓冲液：PEG 3350(50％(w/v))260μL，LiAc(1.0M)36μL，carrier DNA(5.0m g/mL)20μL，Cas9/gRNA质粒15μL，供体DNA 10μL，加入无菌水至最终体积360μL。热激后，13000g离心30s去除上清。加入1mL YPD液体培养基，培养2-3h，吸取200μL涂布于固体YPD(200μg/mL G418)培养基，培养2-3天至单菌落出现。
iv.在乳酸克鲁维酵母转化后的平板上挑取10-20个单克隆，置于1mL YPD(200μg/mL G418)液体培养基中振荡培养过夜，以菌液为模板，以引物PF18(T7 RNAP序列内引物):ATTGGACAAAATGCCAGCACTTCCG(SEQ ID NO.:52)和引物PR18(供体DNA 5’外侧引物):TTTGCTGGTTGCCCGTATTCCC(SEQ ID NO.:53)进行PCR扩增，对KlXRN1位点的CRISPR插入进行检测，有阳性条带表明T7 RNAP序列插入靶位点成功。
实施例4活性测定
将基因改造后的乳酸克鲁维酵母菌株制备成体外蛋白质合成系统，并加入萤火虫荧光素酶(Firefly Luciferase,Fluc)基因DNA模板以测定改造菌株的蛋白翻译能力。将上述反应体系置于25-30℃的环境中，静置孵育约2-6h。反应结束后，在96孔白板或者384孔白板中加入等体积的Fluc底物荧光素(Luciferin)，立即放置于Envision 2120多功能酶标仪(Perkin Elmer)，读数，检测Fluc活性，相对光单位值(Relative Light Unit,RLU)作为活性单位。
转入游离质粒pKM-T7RNAP1的菌株，在外加T7 RNAP浓度为0时表现出最高活性且处于正常范围内(107)，随着外加T7 RNAP浓度增加，对活性产生抑制作用，说明pKM-T7RNAP1质粒中的T7 RNAP结构(pScRNR2+T7 RNAP)能够正常发挥功能，其RLU值为1.4×107(图8)。
在klxrn1Δ-pScRNR2-T7 RNAP结构中，在外加T7 RNAP浓度为0时表现出最高活性且处于正常范围内(107)，表面该结构中T7 RNAP可以适量表达，能够满足体外翻译系统的要求，并且该结构的核酸构建物能够显著增强酵母体外蛋白质合成体系产生蛋白质的效率，其RLU值为6.6×107(图11)。
对比例
KlTDH3-1与T7 RNAP融合蛋白的结构中，随着外加T7 RNAP浓度增加，体外翻译活性增强，表明该结构中T7 RNAP的活力不足以支持体外翻译系统的运行(图9)，其RLU值仅为4.6×105。
在KlTDH3-2与T7 RNAP融合蛋白的结构中，随着外加T7 RNAP浓度增加，体外翻译活性减弱，且整体处于受抑制状态(106)，说明该结构中T7 RNAP表达量过高，对系统产生了抑制作用，其RLU值仅为1.4×106。(图10)。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种核酸构建物，其特征在于，所述的构建物具有从5’至3’的式I结构：
    Z1-Z2   (I)
    式中，
    Z1、Z2分别为用于构成所述构建物的元件；
    各“-”独立地为键或核苷酸连接序列；
    Z1为启动子元件，所述启动子元件选自下组：RNR2、ADH1、GAPDH、TEF1、PGK1、SED1、或其组合；
    Z2为RNP蛋白的编码序列；
    并且，所述Z1、Z2来源于酵母。
  2. 如权利要求1所述的核酸构建物，其特征在于，所述启动子元件选自下组：ScRNR2、ScADH1、ScGAPDH、ScTEF1、ScPGK1、ScSED1、KlRNR2、KlADH1、KlGAPDH、KlTEF1、KlPGK1、KlSED1、或其组合。
  3. 如权利要求1所述的核酸构建物，其特征在于，所述酵母选自下组：酿酒酵母、克鲁维酵母属酵母、或其组合。
  4. 如权利要求1所述的核酸构建物，其特征在于，所述RNP蛋白选自下组：T7RNAP、T3RNAP、T4RNAP、T5RNAP、或其组合。
  5. 如权利要求3所述的核酸构建物，其特征在于，所述克鲁维酵母属酵母选自下组：乳酸克鲁维酵母、马克斯克鲁维酵母、多布克鲁维酵母(Kluyveromyces dobzhanskii)、或其组合。
  6. 一种核酸构建物，其特征在于，所述的构建物具有从5’至3’的式II结构：
    Z1-Z2-Z3   (II)
    式中，
    Z1、Z2、Z3分别为用于构成所述构建物的元件；
    各“-”独立地为键或核苷酸连接序列；
    Z1为启动子元件，所述启动子元件选自下组：ScRNR2、ScADH1、ScGAPDH、ScTEF1、ScPGK1、ScSED1、KlRNR2、KlADH1、KlGAPDH、KlTEF1、KlPGK1、KlSED1、或其组合；
    Z2为RNP蛋白的编码序列；
    Z3为外源蛋白的编码序列；
    并且，所述Z1、Z2来源于酵母。
  7. 如权利要求6所述的核酸构建物，其特征在于，所述的外源蛋白的编码序列编码选自下组的外源蛋白：荧光素蛋白、或荧光素酶、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域、萤光素酶突变体、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、或其组合。
  8. 一种载体或载体组合，其特征在于，所述的载体或载体组合含有权利要求1或权利要求6所述的核酸构建物。
  9. 一种基因工程细胞，其特征在于，所述基因工程细胞的基因组的一个或多个位点整合有权利要求1或权利要求6所述的构建物，或者所述基因工程细胞中含有权利要求8所述的载体或载体组合。
  10. 一种试剂盒，其特征在于，所述试剂盒中包含的试剂选自下组中的一种或多种：
    (a)权利要求1或权利要求6所述的构建物；
    (b)权利要求8所述的载体或载体组合；和
    (c)权利要求9所述的基因工程细胞。
  11. 如权利要求10所述的试剂盒，其特征在于，所述试剂盒还包括(d)酵母体外蛋白合成体系。
  12. 一种如权利要求1或权利要求6所述的构建物、权利要求8所述的载体或载体组合、权利要求9所述的基因工程细胞或权利要求10所述试剂盒的用途，其特征在于，用于进行高通量的体外蛋白合成。
  13. 一种体外高通量的外源蛋白合成方法，其特征在于，包括步骤：
    (i)在酵母体外蛋白合成体系存在下，提供权利要求1或6所述的核酸构建物；
    (ii)在适合的条件下，孵育步骤(i)的酵母体外蛋白合成体系一段时间T1，从而合成所述外源蛋白。
  14. 如权利要求13所述的方法，其特征在于，所述方法还包括：(iii)任选地从所述酵母体外蛋白合成体系中，分离或检测所述外源蛋白。
  15. 如权利要求13所述的方法，其特征在于，所述酵母体外蛋白合成体系为克鲁维酵母体外蛋白合成体系。
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