WO2024051855A1 - 一种核酸构建物以及在ivtt体系中的应用 - Google Patents

一种核酸构建物以及在ivtt体系中的应用 Download PDF

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WO2024051855A1
WO2024051855A1 PCT/CN2023/118103 CN2023118103W WO2024051855A1 WO 2024051855 A1 WO2024051855 A1 WO 2024051855A1 CN 2023118103 W CN2023118103 W CN 2023118103W WO 2024051855 A1 WO2024051855 A1 WO 2024051855A1
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nucleic acid
sequence
acid construct
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郭敏
徐丽琼
褚军青
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
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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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N15/67—General methods for enhancing the expression
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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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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, preferably to a nucleic acid construct and its application in an IVTT system.
  • Proteins are important molecules in cells and participate in almost all functions of cells. The different sequences and structures of proteins determine their different functions. Within cells, proteins can serve as enzymes to catalyze various biochemical reactions, serve as signaling molecules to coordinate various activities of organisms, support biological forms, store energy, transport molecules, and make organisms move. In the field of biomedicine, protein antibodies, as targeted drugs, are an important means of treating diseases such as cancer.
  • Gene transcription refers to using a strand of DNA as a template, catalyzed by DNA-dependent RNA polymerase (RNP or RNAP), and using four types of NTP (ATP, CTP, GTP and UTP) as raw materials, according to the principle of complementary base pairing. , the process of synthesizing a piece of RNA. For some RNA viruses, RNA can also guide the synthesis of RNA.
  • the translation of mRNA into protein refers to the process of assembling activated amino acids into protein polypeptide chains on ribosomes (also known as ribosomes) using mRNA as a template and tRNA as a delivery vehicle under the action of relevant enzymes and cofactors.
  • the regulation of protein synthesis plays an important role in responding to external stresses such as nutritional deficiencies, cell development and differentiation, and many other processes, including transcriptional regulation and translational regulation.
  • Transcriptional regulation refers to the regulation of RNA synthesis using DNA as a template. All cells have a large number of sequence-specific DNA-binding proteins (trans-acting factors). These proteins can accurately recognize and bind to specific DNA sequences (cis-acting elements). ), functioning as a switch at the transcriptional level. Transcription level regulation is an important part of eukaryotic gene expression regulation. According to whether eukaryotic gene expression is affected by the environment, it can be divided into: developmental regulation and transient regulation. Developmental regulation refers to the regulation of gene expression by eukaryotes in order to ensure their own growth, development, differentiation, etc. according to "predetermined” and "orderly” procedures. It is an irreversible process; transient regulation refers to the regulation of gene expression by eukaryotes, including eukaryotes, Adaptive transcriptional regulation under external environmental stimulation is a reversible process.
  • the four processes of translation regulation include translation initiation, translation elongation, translation termination and ribosome recycling, among which translation initiation is the most regulated process.
  • the small ribosome subunit (40S) binds (tRNA) i Met and recognizes the 5' end of the mRNA under the action of translation initiation factors.
  • the small subunit moves downstream and combines with the large ribosome subunit (60S) at the start codon (AUG) position to form a complete ribosome and enter the translation elongation stage.
  • the in vivo biosynthetic system refers to the synthesis process of various compounds catalyzed by enzymes in the biological system, that is, the general term for assimilation reactions in the organism, including photosynthesis, gluconeogenesis, and the biosynthesis of nucleotides, nucleic acids, and proteins.
  • protein synthesis is quantitatively the most important.
  • Protein biosynthesis is also called translation, which is the process of converting the sequence of bases in the mRNA molecule into the sequence of amino acids in the protein or polypeptide chain. Protein biosynthesis is divided into five stages, activation of amino acids, initiation of polypeptide chain synthesis, extension of the peptide chain, termination and release of the peptide chain, and post-synthesis processing and modification of the protein.
  • In vitro biosynthesis system refers to the completion of specific chemical molecules or organisms by adding exogenous encoded nucleic acid DNA, RNA, substrates and energy sources into the lysis system of bacteria, fungi, plant cells or animal cells. Efficient in vitro synthesis of macromolecules (DNA, RNA, proteins).
  • a common in vitro biosynthesis system is the in vitro protein synthesis system, which is a cell-free protein synthesis system that uses exogenous mRNA or DNA templates and cell lysates to complete the rapid and efficient translation of exogenous recombinant proteins.
  • a common commercial in vitro protein synthesis system is an in vitro transcription-translation system (IVTT), which uses DNA templates and RNA polymerase to transcribe the mRNA intermediate, and then uses amino acids, ATP and other groups. to complete efficient one-step translation of foreign proteins.
  • IVTT in vitro transcription-translation system
  • EAE Escherichia coli extract
  • RRL rabbit reticuLocyte lysate
  • WGE wheat germ extract
  • insect Insect cell extract
  • ICE human source system.
  • the in vitro cell-free protein synthesis system has many advantages. For example, it can express special proteins that are toxic to cells or contain unnatural amino acids (such as D-amino acids), and can be directly used as PCR products. It can be used as a template to simultaneously synthesize multiple proteins in parallel and carry out high-throughput drug screening and proteomics research.
  • D-amino acids unnatural amino acids
  • the vector D2P1.08e currently used in the IVTT system has a LAC4 terminator sequence connected to the 3' end of its stop codon TAA for transcription termination. But I have been troubled by a problem when using it. In the LAC4 terminator sequence, the GC content is low, only 30%, and there is even a string of polyA sequences containing 12 consecutive adenine bases in the middle of the sequence. This leads to the fact that if the target gene needs to be inserted in front of the TAA during cloning PCR construction, the primer region will inevitably cover the LAC4 terminator sequence, causing the primer to need to be designed to be very long in order to achieve a Tm value above 58 degrees. This situation is not conducive to primer design. This seriously affects the overall efficiency of IVTT.
  • the purpose of the present invention is to provide a new nucleic acid construct that can significantly improve the efficiency of protein translation and synthesis in an in vitro biosynthetic system.
  • a first aspect of the present invention provides a nucleic acid construct, which contains a nucleic acid sequence with a structure shown in Formula I: Z1-Z2-Z3-Z4-Z5 (I)
  • Z1-Z5 are respectively the elements used to constitute the construct
  • Each "-" is independently a bond or nucleotide connecting sequence
  • Z1 is the coding sequence of the foreign protein
  • Z2 is none or connected sequence
  • Z3 is the stop codon
  • Z4 is the insertion sequence
  • Z5 is the terminator sequence of the lactose metabolism gene
  • the GC content of Z4 is higher than 40%.
  • Z4 includes a terminator element.
  • the terminator element is selected from the following group: T7 terminator, T3 terminator, SP6 terminator or a combination thereof.
  • the Z4 includes a T7 terminator.
  • the T7 terminator is directly connected to Z5.
  • the Z4 includes a T2 sequence, and the nucleotide sequence of the T2 is: CACTAATAAGTAAGTAAAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG.
  • sequence of Z3 is TAA.
  • lactose metabolism genes are LAC4, LAC9, LAC10, LAC12 and other genes.
  • the lactose metabolism gene is the LAC4 gene.
  • Z2 is none.
  • Z2 is a sequence containing CTCGAG.
  • the Z2 is an XhoI endonuclease cleavage sequence: CTCGAG, a NotIXhoI double enzyme cleavage sequence: GCGGCCGCACTCGAG, or a combination thereof.
  • the foreign protein sequence is from prokaryotes or eukaryotes.
  • the coding sequence of the foreign protein comes from animals, plants, and pathogens.
  • the coding sequence of the foreign protein comes from mammals, preferably primates, rodents, including humans, mice, and rats.
  • the coding sequence of the exogenous protein encodes an exogenous protein selected from the following group: 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, ⁇ -amylase, enterocin A, c Hepatitis virus E2 glycoprotein, insulin precursor, interferon ⁇ A, cytokines, interferon ⁇ 2b, interleukin-1 ⁇ , lysozyme, serum albumin, single chain antibody fragment (scFV), transthyretin, casein Aminase, xylanase, or combinations thereof.
  • luciferin or luciferase (such as firefly luciferase)
  • green fluorescent protein yellow fluorescent protein
  • the exogenous protein is selected from the following group: luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde- 3-phosphate dehydrogenase, catalase, actin, antibody variable region, luciferase mutation, alpha-amylase, enterocin A, hepatitis C virus E2 glycoprotein, insulin precursor , interferon ⁇ A, cytokine, interferon ⁇ 2b, interleukin-1 ⁇ , lysozyme, serum albumin, single chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, or its combination.
  • luciferin or luciferase (such as firefly luciferase)
  • green fluorescent protein yellow fluorescent protein
  • aminoacyl-tRNA synthetase aminoacyl-t
  • an in vitro exogenous protein synthesis system including:
  • the cell extract is a soluble cell extract.
  • the cell extract source is selected from one or more cells of the following group: Escherichia coli, bacteria, mammalian cells (such as HF9, Hela, CHO, HEK293), plant cells, yeast cells, Insect cells, or combinations thereof.
  • the cell extract source is selected from one or more cells of the following group: Hela, CHO, HF9, E ⁇ Myc, HEK293, BY-2, yeast, or a combination thereof.
  • the cell extract includes yeast cell extract.
  • the yeast cells are selected from one or more sources of yeast from the following group: Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cells include: Gram Kluyveromyces marxianus and/or Kluyveromyces lactis is more preferably Kluyveromyces marxianus.
  • the yeast cell extract is an aqueous extract of yeast cells.
  • the yeast cell extract does not contain long-chain nucleic acid molecules endogenous to yeast.
  • the yeast cell extract is prepared by a method including the following steps:
  • the solid-liquid separation includes centrifugation.
  • centrifugation is performed in a liquid state.
  • the centrifugation conditions are 5000-100000g, preferably 8000-30000g.
  • the centrifugation time is 0.5min-2h, preferably, 20-50min.
  • the centrifugation is performed at 1-10°C, preferably at 2-6°C.
  • the washing treatment is carried out using washing liquid at a pH of 7-8 (preferably, 7.4).
  • the washing liquid is selected from the following group: potassium 4-hydroxyethylpiperazine sulfonate, potassium acetate, magnesium acetate, or combinations thereof.
  • the cell disruption treatment includes high-pressure disruption and freeze-thaw (such as liquid nitrogen cryogenic) disruption.
  • the concentration of component (a) is 5-30ng/ ⁇ L, preferably, 8-20ng/ ⁇ L, more preferably, 10-16ng/ ⁇ L, Based on the total volume of the exogenous protein synthesis system.
  • the concentration (v/v) of component (b) is 20%-70%, preferably 30-60%, more preferably 40% -50%, based on the total volume of the foreign protein synthesis system.
  • the exogenous protein synthesis system further includes one or more components selected from the following group:
  • Optional solvent which is water or an aqueous solvent.
  • the magnesium ions are derived from a magnesium ion source, and the magnesium ion source is selected from the following group: magnesium acetate, magnesium glutamate, or a combination thereof.
  • the potassium ions are derived from a potassium ion source, and the potassium ion source is selected from the following group: potassium acetate, potassium glutamate, or a combination thereof.
  • the buffering agent is selected from the group consisting of: 4-hydroxyethylpiperazineethanesulfonic acid, trishydroxymethylaminomethane, or a combination thereof.
  • the energy regeneration system is selected from the following group: creatine phosphate/creatine phosphate enzyme system, glycolytic pathway and its intermediate product energy system, or a combination thereof.
  • the substrate for synthesizing RNA includes: nucleoside monophosphate, nucleoside triphosphate, or a combination thereof.
  • the substrate for protein synthesis includes: 1-20 natural amino acids and unnatural amino acids.
  • the nucleoside triphosphate is selected from the following group: adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate, uracil nucleoside triphosphate, or a combination thereof.
  • the amino acid is selected from the following group: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, Tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, or combinations thereof.
  • the amino acids include D-type amino acids and/or L-type amino acids.
  • the polyethylene glycol is selected from the following group: PEG3000, PEG8000, PEG6000, PEG3350, or combinations thereof.
  • the polyethylene glycol includes polyethylene glycol with a molecular weight (Da) of 200-10,000, preferably, polyethylene glycol with a molecular weight of 3,000-10,000.
  • the RNA polymerase is T7 RNA polymerase.
  • the exogenous protein synthesis system includes components selected from the following group: polyethylene glycol, 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, nucleoside triphosphates, amino acids , creatine phosphate, dithiothreitol (DTT), creatine phosphate kinase, sucrose, the nucleic acid construct described in the first aspect of the invention (using firefly luciferase or green fluorescent protein as the exogenous protein), T7 RNA polymerization Enzymes, yeast cell extracts, or combinations thereof.
  • the third aspect of the present invention provides a vector or vector combination, which contains the nucleic acid construct described in the first aspect of the present invention.
  • the vector is selected from: bacterial plasmid, phage, yeast plasmid, or animal cell vector, or shuttle vector. Additionally, the vector may be 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 used as long as it can replicate and be stable in the host body.
  • the fourth aspect of the present invention provides a genetically engineered cell.
  • the construct described in the first aspect of the present invention is integrated into one or more sites of the genome of the genetically engineered cell, or the genetically engineered cell contains the construct of the present invention.
  • the genome includes a nuclear genome, a cytoplasmic genome, and/or an organellar genome.
  • the organelles include mitochondria and/or chloroplasts.
  • the genetically engineered cells include prokaryotic cells and eukaryotic cells.
  • the eukaryotic cells include higher eukaryotic cells.
  • the genetically engineered cells are selected from the following group: human cells (such as Hela cells), Chinese hamster ovary cells (CHO cells), insect cells, wheat germ cells, rabbit reticulocytes, yeast cells, or combination thereof.
  • the genetically engineered cells are yeast cells.
  • 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 following group: Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces doubi, or combinations thereof.
  • a fifth aspect of the present invention provides a kit, the reagents contained in the kit are selected from one or more of the following groups:
  • the kit further includes (d) an in vitro biosynthesis system (such as a eukaryotic in vitro protein synthesis system).
  • an in vitro biosynthesis system such as a eukaryotic in vitro protein synthesis system.
  • the eukaryotic in vitro biosynthetic system is selected from the following group: a yeast in vitro biosynthetic system, a Chinese hamster ovary cell in vitro biosynthetic system, an insect cell in vitro biosynthetic system, a Hela cell in vitro biosynthetic system, or its combination.
  • the eukaryotic in vitro biosynthetic system includes a eukaryotic in vitro protein synthesis system.
  • the eukaryotic in vitro protein synthesis system is selected from the following group: yeast in vitro protein synthesis system, Chinese hamster ovary cell in vitro protein synthesis system, insect cell in vitro protein synthesis system, Hela cell in vitro protein synthesis system, or its combination.
  • the kit further includes (e) a yeast in vitro biosynthesis system (such as a yeast in vitro protein synthesis system).
  • a yeast in vitro biosynthesis system such as a yeast in vitro protein synthesis system.
  • the yeast in vitro biosynthetic system (such as the yeast in vitro protein synthesis system) is the Kluyveromyces in vitro biosynthetic system (such as the Kluyveromyces in vitro protein synthesis system) (preferably the Kluyveromyces lactis in vitro protein synthesis system).
  • Biosynthetic system (such as Kluyveromyces lactis in vitro protein synthesis system).
  • the sixth aspect of the present invention provides a construct as described in the first or second aspect of the present invention, a vector or vector combination as described in the third aspect of the present invention, a genetically engineered cell as described in the fourth aspect of the present invention, or
  • the use of the kit according to the fifth aspect of the present invention is characterized by being used for high-throughput in vitro protein synthesis.
  • the seventh aspect of the present invention provides an in vitro high-throughput exogenous protein synthesis method, including the steps:
  • step (ii) Under appropriate conditions, incubate the in vitro biosynthesis system of step (i) for a period of time T1, thereby synthesizing the exogenous protein.
  • the method further includes: (iii) optionally isolating or detecting the exogenous protein from the in vitro biosynthetic system.
  • the in vitro biosynthetic system is a eukaryotic in vitro biosynthetic system.
  • the eukaryotic in vitro biosynthetic system is a yeast in vitro biosynthetic system (such as a yeast in vitro protein synthesis system).
  • the yeast in vitro biosynthetic system (such as the yeast in vitro protein synthesis system) is the Kluyveromyces in vitro biosynthetic system (such as the Kluyveromyces in vitro protein synthesis system) (preferably the Kluyveromyces lactis in vitro protein synthesis system).
  • Biosynthetic system (such as Kluyveromyces lactis in vitro protein synthesis system).
  • the exogenous protein synthesis method includes an in vitro high-throughput exogenous protein synthesis method.
  • the coding sequence of the foreign protein comes from prokaryotes or eukaryotes.
  • the coding sequence of the foreign protein comes from animals, plants, and pathogens.
  • the coding sequence of the foreign protein comes from mammals, preferably primates, rodents, including humans, mice, and rats.
  • the coding sequence of the exogenous protein encodes an exogenous protein selected from the following group: 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, ⁇ -amylase, enterocin A, c Hepatitis virus E2 glycoprotein, insulin precursor, interferon ⁇ A, cytokines, interferon ⁇ 2b, interleukin-1 ⁇ , lysozyme, serum albumin, single chain antibody fragment (scFV), transthyretin, casein Acidase, xylanase, or combinations thereof.
  • luciferin or luciferase (such as firefly luciferase)
  • green fluorescent protein yellow fluorescent protein
  • the exogenous protein is selected from the following group: luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde- 3-phosphate dehydrogenase, catalase, actin, antibody variable region, luciferase mutation, alpha-amylase, enterocin A, hepatitis C virus E2 glycoprotein, insulin precursor , interferon ⁇ A, cytokine, interferon ⁇ 2b, interleukin-1 ⁇ , lysozyme, serum albumin, single chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, or its combination.
  • luciferin or luciferase (such as firefly luciferase)
  • green fluorescent protein yellow fluorescent protein
  • aminoacyl-tRNA synthetase aminoacyl-t
  • reaction temperature is 20-37°C, preferably 22-35°C.
  • reaction time is 1-72h, preferably 2-23h.
  • FIG. 1 shows the base sequence of D2P1.08e-T2. Based on D2P1.08e, the following base sequence is added after TAA: CACTAATAAGTAAGTAAAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTTG, where AGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTTG is the T7 terminator.
  • Figure 2 shows the base sequence of D2P1.08e-T3. Based on D2P1.08e, the following base sequence is added after TAA, aGCGGCCGCCACCAcCAcCAtCAtCAcCACTAATAAGTAAGTAAAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTTG, where AGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTTG is the T7 terminator.
  • Figure 3 shows the comparison of EGFP fluorescence after IVTT expression of three plasmids: D2P1.08e, D2P1.08e-T2, and D2P1.08e-T3.
  • the intensity of fluorescence can directly reflect the level of EGFP protein expression, as shown in Figure 3.
  • the RFU fluorescence values of D2P1.08e-T2 and D2P1.08e-T3 were higher than those of the control group D2P1.08e. Among them, the RFU fluorescence value of D2P1.08e-T2 was higher, about 1.5 times that of D2P1.08e.
  • Figure 4 shows the different modified insertion sequences.
  • the inventor gradually reduced the inserted sequences in D2P1.08e-T2 and constructed a series of truncated plasmids. According to the truncated sequences from short to long, these were sequentially
  • the plasmids were named D2P1.08e-T2-001 to D2P1.08e-T2-015.
  • Figure 5 shows the comparison of the target protein expression of the modified sequences. It can be seen that adding a T2 sequence on the basis of D2P1.08e can significantly increase the expression of the target gene on the plasmid.
  • Figure 6 shows the IVTT activity after Ampi amplification of two plasmids with enzyme cutting sequences inserted.
  • the XhoI endonuclease cutting sequence CTCGAG or NotIXhoI double enzyme cutting sequence GCGGCCGCACTCGAG was inserted at the 5' end of the TAA stop codon, which will contain this
  • the plasmids of the two sequences are labeled D2P1.08e-T2-BX and D2P1.08e-T2-BN respectively.
  • the restriction site XhoI or NotIXhoI is added.
  • the modified plasmid There is no obvious effect on the activity of D2P1.08e-T2, and the activity is higher than that of the original plasmid D2P1.08e.
  • the nucleic acid construct of the present invention is composed of a stop codon (especially TAA) and It is modified by inserting an insertion sequence with a GC content higher than 40% between the terminator sequences of the lactose metabolism gene.
  • the nucleic acid construct further includes any coding sequence for foreign proteins and with or without any connecting sequence.
  • the nucleic acid construct of the present invention is applied in an in vitro biosynthesis system (such as a yeast in vitro protein synthesis system), for example, after a sequence is inserted between the TAA codon and the LAC4 terminator of the D2P1.08e vector, it is found that the sequence can effectively enhance the vector
  • an in vitro biosynthesis system such as a yeast in vitro protein synthesis system
  • EGFP is used as the target gene for expression
  • the fluorescence value of EGFP is used to characterize the expression level of EGFP. After inserting the sequence, the fluorescence value of EGFP is increased by 50%.
  • D2P1.08e-T2 As an example, gradually reduced the inserted sequences in D2P1.08e-T2, and constructed a A series of truncated plasmids were named D2P1.08e-T2-001 to D2P1.08e-T2-015 in order from short to long truncated sequences.
  • the results showed that the group with the most obvious increase in activity was still D2P1.08e-T2. It can be proved that adding a T2 sequence on the basis of D2P1.08e can significantly increase the expression of the target gene on the plasmid.
  • the inventor inserted the XhoI endonuclease digestion sequence CTCGAG or NotIXhoI double enzyme digestion sequence GCGGCCGCACTCGAG into the 5' end of the TAA stop codon, and labeled the plasmids containing these two sequences as D2P1. .08e-T2-BX and D2P1.08e-T2-BN.
  • Protein synthesis refers to the process by which organisms synthesize proteins based on the genetic information on messenger ribonucleic acid (mRNA) transcribed from deoxyribonucleic acid (DNA). Protein biosynthesis is also called translation, which is the process of converting the sequence of bases in the mRNA molecule into the sequence of amino acids in the protein or polypeptide chain. This is the second step in gene expression and the final stage in producing the protein product of the gene. Different tissue cells have different physiological functions because they express different genes and produce proteins with special functions. There are more than 200 components involved in protein biosynthesis. The main components are mRNA, tRNA, ribonucleosomes and related proteins. It is composed of enzymes and protein factors.
  • the in vitro protein synthesis system generally refers to adding components such as mRNA or DNA template, RNA polymerase, amino acids, and ATP to 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
  • Insect cell insect extract
  • ICE human-derived systems.
  • in vitro cell-free synthesis systems have many advantages, such as the ability to express special proteins that are toxic to cells or contain unnatural amino acids (such as D-amino acids), and can directly use PCR products as templates Simultaneously synthesize multiple proteins in parallel and conduct high-throughput drug screening and proteomics research.
  • yeast has the advantages of simple culture, efficient protein folding, and post-translational modification.
  • Saccharomyces cerevisiae and Pichia pastoris are model organisms that express complex eukaryotic proteins and membrane proteins.
  • Yeast can also be used as raw materials for preparing in vitro translation systems.
  • Kluyveromyces is an ascospore yeast, among which Kluyveromyces marxianus and Kluyveromyces lactis are yeasts widely used in industry. Compared with other yeasts, Kluyveromyces lactis has many advantages, such as superior secretion ability, better large-scale fermentation characteristics, food safety level, and the ability to modify proteins post-translationally.
  • a preferred protein synthesis system is an in vitro protein synthesis system.
  • the in vitro protein synthesis system is not particularly limited.
  • a preferred in vitro protein synthesis system is the Kluyveromyces expression system (more preferably ground, Kluyveromyces lactis expression system).
  • the in vitro protein synthesis system includes:
  • the cell extract is a soluble cell extract.
  • the cell extract source is selected from one or more cells of the following group: Escherichia coli, bacteria, mammalian cells (such as HF9, Hela, CHO, HEK293), plant cells, yeast cells, Insect cells, or combinations thereof.
  • the cell extract source is selected from one or more cells of the following group: Hela, CHO, HF9, E ⁇ Myc, HEK293, BY-2, yeast, or a combination thereof.
  • the cell extract includes yeast cell extract.
  • the yeast cells are selected from one or more sources of yeast from the following group: Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cells include: Gram Kluyveromyces marxianus and/or Kluyveromyces lactis is more preferably Kluyveromyces marxianus.
  • the yeast cell extract is an aqueous extract of yeast cells.
  • the yeast cell extract does not contain long-chain nucleic acid molecules endogenous to yeast.
  • the yeast cell extract is prepared by a method including the following steps:
  • the solid-liquid separation includes centrifugation.
  • centrifugation is performed in a liquid state.
  • the centrifugation conditions are 5000-100000g, preferably 8000-30000g.
  • the centrifugation time is 0.5min-2h, preferably, 20-50min.
  • the centrifugation is performed at 1-10°C, preferably at 2-6°C.
  • the washing treatment is carried out using washing liquid at a pH of 7-8 (preferably, 7.4).
  • the washing liquid is selected from the following group: potassium 4-hydroxyethyl piperazine sulfonate, potassium acetate, magnesium acetate, or combinations thereof.
  • the cell disruption treatment includes high-pressure disruption and freeze-thaw (such as liquid nitrogen cryogenic) disruption.
  • the concentration of component (a) is 5-30ng/ ⁇ L, preferably, 8-20ng/ ⁇ L, more preferably, 10-16ng/ ⁇ L, Based on the total volume of the exogenous protein synthesis system.
  • the concentration (v/v) of component (b) is 20%-70%, preferably 30-60%, more preferably 40% -50%, based on the total volume of the foreign protein synthesis system.
  • the exogenous protein synthesis system further includes one or more components selected from the following group:
  • Optional solvent which is water or an aqueous solvent.
  • the magnesium ions are derived from a magnesium ion source, and the magnesium ion source is selected from the following group: magnesium acetate, magnesium glutamate, or a combination thereof.
  • the potassium ions are derived from a potassium ion source, and the potassium ion source is selected from the following group: potassium acetate, potassium glutamate, or a combination thereof.
  • the buffering agent is selected from the group consisting of: 4-hydroxyethylpiperazineethanesulfonic acid, trishydroxymethylaminomethane, or a combination thereof.
  • the energy regeneration system is selected from the following group: creatine phosphate/creatine phosphate enzyme system, glycolytic pathway and its intermediate product energy system, or a combination thereof.
  • the substrate for synthesizing RNA includes: nucleoside monophosphate, nucleoside triphosphate, or a combination thereof.
  • the substrate for protein synthesis includes: 1-20 natural amino acids and unnatural amino acids.
  • the nucleoside triphosphate is selected from the following group: adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate, uracil nucleoside triphosphate, or a combination thereof.
  • the amino acid is selected from the following group: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, Tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, or combinations thereof.
  • concentration of each amino acid is usually 0.01-0.5mM, preferably 0.02-0.2mM, such as 0.05, 0.06, 0.07, 0.08mM.
  • the amino acids include D-type amino acids and/or L-type amino acids.
  • the in vitro protein synthesis system also contains polyethylene glycol or its analogues.
  • concentration of polyethylene glycol or its analogs is not particularly limited. Usually, the concentration (w/v) of polyethylene glycol or its analogs is 0.1-8%, preferably, 0.5-4%, 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, PEG8000, PEG6000 and PEG3350. It should be understood that the system of the present invention can also include other polyethylene glycols of various molecular weights (such as PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc.).
  • the polyethylene glycol is selected from the following group: PEG3000, PEG8000, PEG6000, PEG3350, or combinations thereof.
  • the polyethylene glycol includes polyethylene glycol with a molecular weight (Da) of 200-10,000, preferably, polyethylene glycol with a molecular weight of 3,000-10,000.
  • the RNA polymerase is not particularly limited and can be selected from one or more RNA polymerases.
  • a typical RNA polymerase is T7 RNA polymerase.
  • the in vitro protein synthesis system also contains sucrose.
  • the concentration of sucrose is not particularly limited. Usually, the concentration of sucrose is 0.03-40wt%, preferably, 0.08-10wt%, more preferably, 0.1-5wt%, based on the total weight of the protein synthesis system.
  • the exogenous protein synthesis system includes components selected from the following group: polyethylene glycol, 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, nucleoside triphosphates, amino acids , muscle phosphate Acid, dithiothreitol (DTT), creatine phosphate kinase, sucrose, the nucleic acid construct described in the first aspect of the present invention (using firefly luciferase or green fluorescent protein as exogenous protein), T7 RNA polymerase, yeast Cell extracts, or combinations thereof.
  • components selected from the following group: polyethylene glycol, 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, nucleoside triphosphates, amino acids , muscle phosphate Acid, dithiothreitol (DTT), creatine phosphate kinase, sucrose, the nucleic acid construct described in the first aspect of the present invention (using firefly
  • the proportion of the yeast cell extract in the in vitro protein synthesis system is not particularly limited.
  • the yeast cell extract accounts for 20-70% of the system in the in vitro protein synthesis system, preferably 30-60%, preferably 40-50%.
  • yeast cell extract does not contain intact cells.
  • a typical yeast cell extract includes ribosomes for protein translation, transfer RNA, aminoacyl-tRNA synthetase, initiation factors required for protein synthesis, and elongation factors and termination release factors.
  • yeast extract also contains some other proteins derived from the cytoplasm of yeast cells, especially soluble proteins.
  • the protein content of the yeast cell extract is 20-100 mg/mL, preferably 50-100 mg/mL.
  • the method for measuring protein content is the Coomassie Brilliant Blue determination method.
  • exogenous protein coding sequence and “exogenous DNA” are used interchangeably and both refer to exogenous DNA molecules used to direct protein synthesis.
  • the DNA molecules are linear or circular.
  • the DNA molecule contains a sequence encoding a foreign protein.
  • sequences encoding foreign proteins include (but are not limited to): genomic sequences and cDNA sequences.
  • the sequence encoding the foreign protein also contains 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 following group: encoding luciferin protein, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein , aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, exogenous DNA of the 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: encoding alpha-amylase, enterocin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum albumin Protein, single-chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, foreign DNA, or combinations thereof.
  • the exogenous DNA encodes a protein selected from the following group: green fluorescent protein (enhanced GFP, eGFP), yellow fluorescent protein (YFP), Escherichia coli ⁇ -galactosidase ( ⁇ -galactosidase, LacZ), human lysine-tRNA synthetase (Lysine-tRNA synthetase), human leucine-tRNA synthetase (Leucine-tRNA synthetase), Arabidopsis thaliana Glyceraldehyde-3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate dehydrogenase), mouse catalase (Catalase), or a combination thereof.
  • green fluorescent protein enhanced GFP, eGFP
  • YFP yellow fluorescent protein
  • Escherichia coli ⁇ -galactosidase ⁇ -galactosidase, LacZ
  • a first aspect of the present invention provides a nucleic acid construct, which contains a nucleic acid sequence with a structure shown in Formula I: Z1-Z2-Z3-Z4-Z5 (I)
  • Z1-Z5 are respectively the elements used to constitute the construct
  • Each "-" is independently a bond or nucleotide connecting sequence
  • Z1 is the coding sequence of the foreign protein
  • Z2 is none or connected sequence
  • Z3 is the stop codon
  • Z4 is the insertion sequence
  • Z5 is the terminator sequence of the lactose metabolism gene
  • the GC content of Z4 is higher than 40%.
  • the selection of the coding sequence of the exogenous protein is not particularly limited.
  • the coding sequence of the exogenous protein is selected from the following group: encoding luciferin protein, or luciferase (such as firefly luciferase), green Fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, foreign DNA of variable region of antibody, 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, enterocin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon ⁇ A, interleukin-1 ⁇ , lysosomes Enzymes, serum albumin, single-chain antibody fragments (scFV), transthyretin, tyrosinase, xylanase, or combinations thereof.
  • nucleic acid construct of the present invention may be linear or circular.
  • the nucleic acid construct of the present 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 present invention is shown in SEQ ID NO.: 1-20.
  • the construct also includes elements selected from the following group or a combination thereof: promoter, terminator, poly(A) element, transport element, gene targeting element, screening marker gene, enhancer , resistance genes, transposase encoding genes.
  • selectable marker genes can be used in the present invention, including but not limited to: auxotrophic markers, resistance markers, and reporter gene markers.
  • the application of selectable markers plays a role in the selection of recombinant cells (recombinants), allowing recipient cells to be significantly distinguished from untransformed cells.
  • the auxotrophic marker is achieved by complementing the transferred marker gene with the mutated gene of the recipient cell, thereby causing the recipient cells to exhibit wild-type growth.
  • Resistance markers refer to the transfer of resistance genes into recipient cells. The transferred genes cause the recipient cells to show drug resistance at a certain drug concentration. As a preferred mode of the present invention, resistance markers are used to realize convenient screening of recombinant cells.
  • the expression and purification of foreign proteins can be completed.
  • the expression and purification of foreign proteins can be completed by applying the nucleic acid construct of the present invention.
  • the relative light unit value of the amount of enhanced green fluorescent protein synthesized by the nucleic acid construct is relatively high.
  • the invention also provides a vector or vector combination containing the nucleic acid construct of the invention.
  • the vector is selected from: bacterial plasmid, phage, yeast plasmid, or animal cell vector, shuttle vector; 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 used as long as it can replicate and be stable in the host body.
  • the invention also provides a genetically engineered cell, the genetically engineered cell containing the construct or vector or vector combination, or the genetically engineered cell having the construct or vector integrated into its chromosome.
  • the genetically engineered cells further include a vector containing a transposase gene or a transposase gene integrated into its chromosome.
  • the genetically engineered cells are eukaryotic cells.
  • the eukaryotic cells include (but are not limited to): yeast cells (preferably, Kluyveromyces lactis cells, more preferably Kluyveromyces lactis cells).
  • the construct or vector of the present invention can be used to transform appropriate genetically engineered cells.
  • Genetically engineered cells can be prokaryotic cells, such as Escherichia coli, Streptomyces, and Agrobacterium; or lower eukaryotic cells, such as plant cells, yeast cells; or higher eukaryotic cells, such as insect cells, animal cells, etc.
  • prokaryotic cells such as Escherichia coli, Streptomyces, and Agrobacterium
  • lower eukaryotic cells such as plant cells, yeast cells
  • higher eukaryotic cells such as insect cells, animal cells, etc.
  • Transformation of genetically engineered cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism (such as E. coli), it can be treated with CaCl 2 or electroporation.
  • DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.). Plants can also be transformed using methods such as Agrobacterium transformation or gene gun transformation, such as leaf disk method, immature embryo transformation method, flower bud soaking method, etc.
  • the invention provides an in vitro high-throughput protein synthesis method, which includes the steps:
  • step (ii) Under appropriate conditions, incubate the in vitro biosynthesis system of step (i) for a period of time T1, thereby synthesizing the exogenous protein.
  • the method further includes: (iii) optionally isolating or detecting the exogenous protein from the in vitro protein synthesis system.
  • the present invention discovered for the first time that the coding sequence of an optional foreign protein, or any connecting sequence, stop codon, inserted sequence with a GC content higher than 40%, and the terminator sequence of a lactose metabolism gene can be used as a nucleic acid construct. , applied in the in vitro protein synthesis system of the present invention, can significantly increase the yield of the target protein, and can be used for the expression and purification of exogenous proteins.
  • the protein expression and purification sequence of the present invention can specifically recognize and efficiently bind to beads.
  • the recognition and binding efficiency of these sequences not only exceeds that of traditional specific amino acid biotin sequences, but can also be applied to protein synthesis systems (especially in vitro protein synthesis systems).
  • the expression and purification sequence of the protein provided by the present invention is stable as long as a segment of any nucleic acid sequence with a GC content exceeding 40% (such as T2), even if there are other changes in the inserted sequence (such as adding or reducing part of the sequence, mutations, etc.), it can effectively increase the content of the target protein.
  • Kluyveromyces lactis can be applied to protein production in the food and pharmaceutical fields due to its safety and efficiency, coupled with the advantages of in vitro protein synthesis systems, such as adaptability to high-throughput protein Synthetic screening, synthesis of toxic proteins, short time and low cost, etc., so lactic acid
  • the in vitro protein synthesis system derived from Kluyveromyces cells can also be widely used in related fields.
  • the new nucleic acid construct provided by the present invention can not only improve the expression and purification effect of target foreign proteins, but more importantly, can increase the possibility of synthesizing different proteins in the Kluyveromyces lactis in vitro protein synthesis system.
  • the present invention discloses for the first time a new type of nucleic acid construct that can improve the expression and purification of target proteins.
  • the nucleic acid construct of the present invention in the protein synthesis system of the present invention (especially the in vitro protein synthesis system), external protein synthesis can be completed. Expression and purification of source proteins. Compared with nucleic acids that do not have this structure, the fluorescence value of the synthesized foreign proteins is increased by 50%.
  • the exogenous protein in the embodiment of the present invention takes eGFP as an example.
  • D2P1.08e-T2 CACTAATAAGTAAGTAAAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTG, where AGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG is the T7 terminator; based on D2P1.08e, behind TAA Add the following base sequence and label it as D2P1.08e-T3: aGCGGCCGCCACCAcCAcCAtCAtCAcCACTAATAAGTAAGTAAAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTTG, where AGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTTG is the T7 terminator (as shown in Figure 2).
  • the fluorescence intensity can directly reflect the level of EGFP protein expression.
  • the concentration of the D2P plasmid template is 1ng/uL. Place the mixture in a 37°C incubator and let it stand or shake gently at a speed of less than 30 rpm/min. Collect after reacting for 2 hours.
  • the product is the gene amplification product.
  • the reaction product can be used immediately or stored at -20 degrees.
  • the RFU fluorescence values of D2P1.08e-T2 and D2P1.08e-T3 were higher than those of the control group D2P1.08e, among which the value of D2P1.08e-T2 was higher, about 1.5 times the RFU fluorescence value of D2P1.08e ( Figure 3) .
  • results of the present invention show that inserting a new nucleic acid sequence after the TAA stop codon can improve the translation efficiency of the target protein and correspondingly increase the expression level of the target protein.
  • This sequence design can be applied to the yeast in vitro protein synthesis system, and the efficiency of initiating protein synthesis is far higher than that of a sequence directly connected to TAA and LAC4 terminator. It increases the selectivity of protein expression and purification methods in the in vitro synthesis system and greatly enhances the usability of the in vitro protein synthesis system.
  • the screened nucleotide sequences containing the T2 sequence caused the relative fluorescence unit values (Relative Fluorescence Units, RFU) emitted by the enhanced green fluorescent protein to reach high values in the in vitro protein synthesis system, among which the EGFP in D2P1.08e-T2 The fluorescence value is increased by 50%.
  • RFU Relative Fluorescence Units
  • D2P1.08e-T2 Because the activity of D2P1.08e-T2 is higher than that of D2P1.08e-T3, the inserted sequence of the former is shorter than that of the latter.
  • D2P1.08e-T2 In order to further analyze the more important sequences or regions in D2P1.08e-T2, we followed up on D2P1.08e-T2 The inserted sequences were gradually reduced and a series of truncated plasmids were constructed, as shown in Figure 4. According to the truncated sequence from short to long, these plasmids were named D2P1.08e-T2-001 to D2P1.08e-T2-015.
  • the results of the present invention show that adding a T2 sequence on the basis of D2P1.08e can significantly increase the expression of the target gene on the plasmid.
  • D2P1.08e-T2 Adding restriction site XhoI or NotIXhoI to D2P1.08e-T2 has no significant effect on the activity of D2P1.08e-T2, and both are more active than the original plasmid D2P1.08e.
  • the results of the present invention show that the nucleic acid of the present invention has excellent stability during the expression and purification process of the target protein.

Abstract

一种核酸构建物及其在IVTT体系中的应用，具体提供了一种核酸构建物，所述核酸构建物具有式(I)结构：Z1-Z2-Z3-Z4-Z5(I)，式中，Z1-Z5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为外源蛋白的编码序列；Z2为无或连接序列；Z3为终止密码子；Z4为插入序列；Z5为乳糖代谢基因的终止子序列；并且，所述Z4的GC含量高于40％。所述核酸构建物可显著提高外源蛋白合成的效率，并简化目标外源蛋白的表达与纯化过程。

Description

一种核酸构建物以及在IVTT体系中的应用 技术领域
本发明涉及生物技术领域，较佳地，涉及一种核酸构建物以及在IVTT体系中的应用。
背景技术
蛋白质是细胞中的重要分子，几乎参与了细胞所有功能的执行。蛋白的序列和结构不同，决定了其功能的不同。在细胞内，蛋白可以作为酶类催化各种生化反应，可以作为信号分子协调生物体的各种活动，可以支持生物形态，储存能量，运输分子，并使生物体运动。在生物医学领域，蛋白质抗体作为靶向药物，是治疗癌症等疾病的重要手段。
在细胞中，蛋白质的制造包括基因转录和mRNA翻译两部分。
基因转录是指以DNA的一条链为模板，在DNA依赖的RNA聚合酶(RNP或RNAP)催化作用下，以4种NTP(ATP、CTP、GTP和UTP)为原料，按照碱基互补配对原则，合成一条RNA的过程。对于有些RNA病毒，RNA也可以指导合成RNA。
mRNA翻译成蛋白质是指以mRNA为模板，tRNA为运载工具，在有关酶、辅助因子的作用下将活化的氨基酸在核糖体(亦称核蛋白体)上装配为蛋白质多肽链的过程。
蛋白质合成的调节在应对营养缺失等外界压力，细胞发育与分化等很多过程中发挥重要作用，包括转录调控和翻译调控。
转录调控是指以DNA为模板合成RNA的调控，所有的细胞都具有大量序列特异的DNA结合蛋白(反式作用因子)，这些蛋白能准确地识别并结合到特异的DNA序列(顺式作用元件)，在转录水平上起着开关的作用。转录水平调控是真核基因表达调控的重要环节。根据真核基因表达是否受环境影响可分为：发育调控和瞬时调控。其中发育调控是指真核生物为确保自身生长、发育、分化等对基因表达按“预定”和“有序”的程序进行的调控，是不可逆的过程；瞬时调控是指真核生物在内、外环境的刺激下所做出的适应性转录调控，是可逆过程。
翻译调控的四个过程包括翻译起始、翻译延伸、翻译终止和核糖体再循环，其中翻译起始是受调控最多的一个过程。在翻译起始阶段，核糖体小亚基(40S)结合(tRNA)i Met，并在翻译起始因子的作用下识别mRNA 5’末端。小亚基向下游移动，并在起始密码子(AUG)位置与核糖体大亚基(60S)结合，形成完整核糖体，并进入翻译延伸阶段。
目前常用的生物合成系统是体内生物合成系统和体外生物合成系统。体内生物合成系统是指在生物体内体系中，酶催化的各种化合物的合成过程，即生物体内进行同化反应的总称，包括光合作用，糖异生，核苷酸、核酸及蛋白质的生物合成。细胞生物合成中，蛋白质合成是数量上最重要的。蛋白质生物合成亦称为翻译，即把mRNA分子中碱基排列顺序转变为蛋白质或多肽链中的氨基酸排列顺序过程。蛋白质生物合成分为五个阶段，氨基酸的活化、多肽链合成的起始、肽链的延长、肽链的终止和释放、蛋白质合成后的加工修饰。
体外生物合成系统(in vitro biosynthesis system)是指在细菌、真菌、植物细胞或动物细胞的裂解体系中，通过加入外源编码的核酸DNA、RNA、底物和能量源，完成特定化学分子或生物大分子(DNA,RNA,蛋白质)的体外高效合成。常见的体外生物合成系统是体外蛋白质合成系统(in vitro protein synthesis system)，即无细胞蛋白质合成系统，是通过外源mRNA或者DNA模板、利用细胞裂解物，完成外源重组蛋白的快速高效翻译。
无细胞系统可以最早追溯到Buchner在1897年提出生物合成可以在体外进行，他通过酵母无细胞系统证明了生物乙醇的产生。然而，由于三磷酸腺苷(ATP)失衡，该系统不适用大规模应用。Welch和Scopes于1985年通过多种探索解决了上述问题，获得了高产量的乙醇，但是该系统也存在两大缺陷：需要额外添加高成本的酶和无法耐受温度的变化。
然而，目前该技术存在一些固有的难以解决的问题：如可逆性、不稳定性、渗漏、失活、酶的循环使用，缺乏稳定的酶、酶复合物及辅助因子等。
商业上常见的体外蛋白质合成系统是体外转录-翻译偶联的体系(in vitro transcription-translation system，简称IVTT)，通过DNA模板、经RNA聚合酶转录出mRNA中间体，再利用氨基酸和ATP等组分，完成外源蛋白的一步高效翻译。目前，常见的商业化体外蛋白表达系统包括大肠杆菌系统(Escherichia coli extract，ECE)、兔网织红细胞(Rabbit reticuLocyte  lysate，RRL)系统、麦胚(Wheat germ extract,WGE)系统、昆虫(Insect cell extract,ICE)系统和人源系统。
与传统的体内重组表达系统相比，蛋白质的体外无细胞合成系统具有多种优点，如可表达对细胞有毒害作用或含有非天然氨基酸(如D-氨基酸)的特殊蛋白质，能够直接以PCR产物作为模板同时平行合成多种蛋白质，开展高通量药物筛选和蛋白质组学的研究。
目前IVTT体系中正在使用的载体D2P1.08e，它的终止密码子TAA的3’末端连接的是LAC4终止子序列，用于转录终止。但在使用时一直受一个问题困扰。在LAC4终止子序列中，GC含量较低，只有30％，甚至序列中间有一串含有12个连续腺嘌呤碱基的polyA序列。这导致在进行克隆PCR构建时，如果需要将目的基因插入到TAA前面，那么引物区域会无法避免的覆盖在LAC4终止子序列上，导致引物需要设计很长，才能达到58度以上的Tm值。这种情况不利于引物的设计。这严重影响了IVTT的整体效率。
发明内容
本发明的目的在于提供一种新的可在体外生物合成系统中显著提高蛋白翻译合成效率的核酸构建物。
本发明第一方面提供了一种核酸构建物，所述核酸构建物含有结构如式I所示的核酸序列：
Z1-Z2-Z3-Z4-Z5   (I)
式中，
Z1－Z5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为外源蛋白的编码序列；
Z2为无或连接序列；
Z3为终止密码子；
Z4为插入序列；
Z5为乳糖代谢基因的终止子序列；
并且，所述Z4的GC含量高于40％。
在另一优选例中，所述Z4包含终止子元件。
在另一优选例中，所述终止子元件选自下组：T7终止子、T3终止子、SP6终止子或其组合。
在另一优选例中，所述Z4包含T7终止子。
在另一优选例中，T7终止子与Z5直接相连。
在另一优选例中，所述Z4包含T2序列，所述T2的核苷酸序列为：CACTAATAAGTAAGTAAAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG。
在另一优选例中，所述Z3的序列为TAA。
在另一优选例中，所述乳糖代谢基因为LAC4、LAC9、LAC10、LAC12等基因。
在另一优选例中，所述乳糖代谢基因为LAC4基因。
在另一优选例中，所述Z2为无。
在另一优选例中，所述Z2为含有CTCGAG的序列。
在另一优选例中，所述Z2为XhoI内切酶酶切序列为：CTCGAG、NotIXhoI双酶切序列为：GCGGCCGCACTCGAG，或其组合。
在另一优选例中，所述外源蛋白列来自原核生物、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、细胞因子，干扰素α2b、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、细胞因子，干扰素α2b，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。
在本发明第二方面，提供一种体外的外源蛋白合成体系，包括：
(a)本发明第一方面所述的核酸构建物；和
(b)细胞提取物。
在另一优选例中，所述细胞提取物为可溶性的细胞提取物。
在另一优选例中，所述细胞提取物来源选自下组的一种或多种细胞：大肠杆菌、细菌、哺乳动物细胞(如HF9、Hela、CHO、HEK293)、植物细胞、酵母细胞、昆虫细胞、或其组合。
在另一优选例中，所述细胞提取物来源选自下组的一种或多种细胞：Hela、CHO、HF9、EμMyc、HEK293、BY-2、酵母、或其组合。
在另一优选例中，所述细胞提取物包括酵母细胞提取物。
在另一优选例中，所述酵母细胞选自下组的一种或多种来源的酵母：毕氏酵母、克鲁维酵母、或其组合；较佳地，所述的酵母细胞包括：克鲁维酵母，更佳地为马克斯克鲁维酵母、和/或乳酸克鲁维酵母。
在另一优选例中，所述的酵母细胞提取物为对酵母细胞的水性提取物。
在另一优选例中，所述酵母细胞提取物不含酵母内源性的长链核酸分子。
在另一优选例中，所述的酵母细胞提取物是用包括以下步骤的方法制备：
(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；和
(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。
在另一优选例中，所述的固液分离包括离心。
在另一优选例中，在液态下进行离心。
在另一优选例中，所述离心条件为5000-100000g，较佳地，8000-30000g。
在另一优选例中，所述离心时间为0.5min–2h，较佳地，20–50min。
在另一优选例中，所述离心在1-10℃下进行，较佳地，在2-6℃下进行。
在另一优选例中，所述的洗涤处理采用洗涤液在pH为7-8(较佳地，7.4)下进行处
在另一优选例中，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。
在另一优选例中，所述的破细胞处理包括高压破碎、冻融(如液氮低温)破碎。
在另一优选例中，所述外源蛋白合成体系中，组分(a)的浓度为5-30ng/μL，较佳地，8-20ng/μL，更佳地，10-16ng/μL，以所述外源蛋白合成体系的总体积计。
在另一优选例中，所述外源蛋白合成体系中，组分(b)的浓度(v/v)为20％-70％，较佳地，30-60％，更佳地，40％-50％，以所述外源蛋白合成体系的总体积计。
在另一优选例中，所述外源蛋白合成体系还包括选自下组的一种或多种组分：
(c1)镁离子；
(c2)钾离子；
(c3)缓冲剂；
(c4)能量再生系统；
(c5)聚乙二醇；
(c6)用于合成RNA的底物；
(c7)用于合成蛋白的底物；
(c8)RNA聚合酶；
(c9)任选的外源蔗糖；
(c10)任选的溶剂，所述溶剂为水或水性溶剂。
在另一优选例中，所述镁离子来源于镁离子源，所述镁离子源选自下组：醋酸镁、谷氨酸镁、或其组合。
在另一优选例中，所述钾离子来源于钾离子源，所述钾离子源选自下组：醋酸钾、谷氨酸钾、或其组合。
在另一优选例中，所述缓冲剂选自下组：4-羟乙基哌嗪乙磺酸、三羟甲基氨基甲烷、或其组合。
在另一优选例中，所述能量再生系统选自下组：磷酸肌酸/磷酸肌酸酶系统、糖酵解途径及其中间产物能量系统、或其组合。
在另一优选例中，所述的合成RNA的底物包括：核苷单磷酸、核苷三磷酸、或其组合。
在另一优选例中，所述的合成蛋白的底物包括：1-20种天然氨基酸、以及非天然氨基酸。
在另一优选例中，所述核苷三磷酸选自下组：腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸、尿嘧啶核苷三磷酸、或其组合。
在另一优选例中，所述氨基酸为选自下组：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、组氨酸、或其组合。
在另一优选例中，所述氨基酸包括D型氨基酸和/或L型氨基酸。
在另一优选例中，所述聚乙二醇选自下组：PEG3000、PEG8000、PEG6000、PEG3350、或其组合。
在另一优选例中，所述聚乙二醇包括分子量(Da)为200-10000的聚乙二醇，较佳地，分子量为3000-10000的聚乙二醇。在另一优选例中，所述RNA聚合酶为T7RNA聚合酶。
在另一优选例中，所述外源蛋白合成体系包括选自下组的成分：聚乙二醇、4-羟乙基哌嗪乙磺酸、醋酸钾、醋酸镁、核苷三磷酸、氨基酸、磷酸肌酸、二硫苏糖醇(DTT)、磷酸肌酸激酶、蔗糖、本发明第一方面所述的核酸构建物(以萤火虫荧光素酶或绿色荧光蛋白为外源蛋白)、T7RNA聚合酶、酵母细胞提取物、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所述的核酸构建物。
在另一优选例中，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。此外，载体可以为转座子载体。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，所述基因组包括核基因组、细胞质基因组、和/或细胞器基因组。
在另一优选例中，所述细胞器包括线粒体、和/或叶绿体。
在另一优选例中，所述基因工程细胞包括原核细胞、真核细胞。
在另一优选例中，所述真核细胞包括高等真核细胞。
在另一优选例中，所述基因工程细胞选自下组：人源细胞(如Hela细胞)、中国仓鼠卵巢细胞(CHO细胞)、昆虫细胞、麦胚细胞、兔网织红细胞、酵母细胞、或其组合。
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、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)本发明第一方面所述的构建物；
(b)本发明第三方面所述的载体或载体组合；
(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)体外生物合成体系(如真核体外蛋白合成体系)。
在另一优选例中，所述真核体外生物合成体系选自下组：酵母体外生物合成体系、中国仓鼠卵巢细胞体外生物合成体系、昆虫细胞体外生物合成体系、Hela细胞体外生物合成体系、或其组合。
在另一优选例中，所述真核体外生物合成体系包括真核体外蛋白合成体系。
在另一优选例中，所述真核体外蛋白合成体系选自下组：酵母体外蛋白合成体系、中国仓鼠卵巢细胞体外蛋白合成体系、昆虫细胞体外蛋白合成体系、Hela细胞体外蛋白合成体系、或其组合。
在另一优选例中，所述试剂盒还包括(e)酵母体外生物合成体系(如酵母体外蛋白合成体系)。
在另一优选例中，所述酵母体外生物合成体系(如酵母体外蛋白合成体系)为克鲁维酵母体外生物合成体系(如克鲁维酵母体外蛋白合成体系)(优选乳酸克鲁维酵母体外生物合成体系)(如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的试剂盒的用途，其特征在于，用于进行高通量的体外蛋白质合成。
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)在体外生物合成体系存在下，提供本发明第一方面所述的核酸构建物；
(ii)在适合的条件下，孵育步骤(i)的体外生物合成体系一段时间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)任选地从所述体外生物合成体系中，分离或检测所述外源蛋白。
在另一优选例中，所述体外生物合成体系为真核所述体外生物合成体系。
在另一优选例中，所述真核体外生物合成体系为酵母体外生物合成体系(如酵母体外蛋白合成体系)。
在另一优选例中，所述酵母体外生物合成体系(如酵母体外蛋白合成体系)为克鲁维酵母体外生物合成体系(如克鲁维酵母体外蛋白合成体系)(优选乳酸克鲁维酵母体外生物合成体系)(如乳酸克鲁维酵母体外蛋白合成体系)。
在另一优选例中，所述外源蛋白合成方法包括体外高通量的外源蛋白合成方法。
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、细胞因子，干扰素α2b、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、细胞因子，干扰素α2b、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1-72h，较佳地，2-23h。
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D2P1.08e-T2的碱基序列，以D2P1.08e为基础，在TAA后面添加如下碱基序列：CACTAATAAGTAAGTAAAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG，其中，AGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG为T7终止子。
图2显示了D2P1.08e-T3的碱基序列，以D2P1.08e为基础，在TAA后面添加如下碱基序列，aGCGGCCGCCACCAcCAcCAtCAtCAtCAcCACTAATAAGTAAGTAAAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG，其中，AGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG为T7终止子。
图3显示了D2P1.08e，D2P1.08e-T2,D2P1.08e-T3三种质粒IVTT表达后的EGFP荧光比较，荧光强弱可以直接反应EGFP蛋白表达量的高低，如图3中所示，D2P1.08e-T2，D2P1.08e-T3的RFU荧光值均高于对照组D2P1.08e，其中D2P1.08e-T2的RFU荧光值较高，约为D2P1.08e的1.5倍。
图4显示了改造的不同的插入序列，发明人对D2P1.08e-T2中插入的序列进行了逐步减少，构建了一系列截短的质粒，根据截短的序列由短到长，依次将这些质粒命名为D2P1.08e-T2-001至D2P1.08e-T2-015。
图5显示了改造后的序列在目标蛋白表达上的比较，看见，在D2P1.08e的基础上增加一段T2序列，可以明显提升质粒上目的基因的表达量。
图6显示了插入了酶切序列的两个质粒Ampi扩增后IVTT活性，在TAA终止密码子的5’端插入了XhoI内切酶酶切序列CTCGAG或NotIXhoI双酶切序列GCGGCCGCACTCGAG，将含有这两种序列的质粒分别标记为D2P1.08e-T2-BX和D2P1.08e-T2-BN，在D2P1.08e-T2的基础上增加酶切位点XhoI或NotIXhoI，由图看见，改造后的质粒对于D2P1.08e-T2的活性没有明显影响，且都比原始质粒D2P1.08e活性高。
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是在终止密码子(特别是TAA)和乳糖代谢基因的终止子序列之间插入GC含量高于40％的插入序列改造而成，该核酸构建物还进一步包括了任意的外源蛋白的编码序列以及有或没有任意地连接序列。在体外生物合成体系(如酵母体外蛋白合成体系)中应用本发明的核酸构建物，如在D2P1.08e载体的TAA密码子和LAC4终止子之间插入一段序列后，发现该序列可以有效提升载体上目的基因在IVTT体系中的表达水平。以EGFP作为表达的目的基因，以EGFP的荧光值表征EGFP的表达水平，插入序列后，EGFP的荧光值有50％的提升。
此外，本发明人还意外的发现，为了进一步分析较为重要的序列或区域，本发明人以D2P1.08e-T2为例，对D2P1.08e-T2中插入的序列进行了逐步减少，构建了一系列截短的质粒，根据截短的序列由短到长，依次将这些质粒命名为D2P1.08e-T2-001至D2P1.08e-T2-015。结果发现，活性提升最为明显的一组仍是D2P1.08e-T2。由此可以证明，在D2P1.08e的基础上增加一段T2序列，可以明显提升质粒上目的基因的表达量。
另外，为了进一步方便克隆构建，本发明人在TAA终止密码子的5’端插入了XhoI内切酶酶切序列CTCGAG或NotIXhoI双酶切序列GCGGCCGCACTCGAG，将含有这两种序列的质粒分别标记为D2P1.08e-T2-BX和D2P1.08e-T2-BN。测试这两个质粒Ampi扩增后的IVTT活性后发现，在D2P1.08e-T2的基础上增加 酶切位点XhoI或NotIXhoI，对于D2P1.08e-T2的活性没有明显影响，且都比原始质粒D2P1.08e活性高。
在此基础上，本发明人完成了本发明。
蛋白质合成体系
蛋白质合成是指生物按照从脱氧核糖核酸(DNA)转录得到的信使核糖核酸(mRNA)上的遗传信息合成蛋白质的过程。蛋白质生物合成亦称为翻译(Translation)，即把mRNA分子中碱基排列顺序转变为蛋白质或多肽链中的氨基酸排列顺序过程。这是基因表达的第二步，产生基因产物蛋白质的最后阶段。不同的组织细胞具有不同的生理功能，是因为它们表达不同的基因，产生具有特殊功能的蛋白质，参与蛋白质生物合成的成份超过200种，其主要体是由mRNA、tRNA、核糖核蛋白体以及有关的酶和蛋白质因子共同组成。
蛋白质体外合成系统一般是指在细菌、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。与传统的体内重组表达系统相比,体外无细胞合成系统具有多种优点,如可表达对细胞有毒害作用或含有非天然氨基酸(如D-氨基酸)的特殊蛋白质,能够直接以PCR产物作为模板同时平行合成多种蛋白质,开展高通量药物筛选和蛋白质组学的研究。
其中，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，一种优选的蛋白质合成体系为体外蛋白质合成体系，在本发明中，体外蛋白质合成体系不受特别限制，一种优选的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，所述体外蛋白质合成体系包括：
(a)本发明第一方面所述的核酸构建物；和
(b)细胞提取物。
在另一优选例中，所述细胞提取物为可溶性的细胞提取物。
在另一优选例中，所述细胞提取物来源选自下组的一种或多种细胞：大肠杆菌、细菌、哺乳动物细胞(如HF9、Hela、CHO、HEK293)、植物细胞、酵母细胞、昆虫细胞、或其组合。
在另一优选例中，所述细胞提取物来源选自下组的一种或多种细胞：Hela、CHO、HF9、EμMyc、HEK293、BY-2、酵母、或其组合。
在另一优选例中，所述细胞提取物包括酵母细胞提取物。
在另一优选例中，所述酵母细胞选自下组的一种或多种来源的酵母：毕氏酵母、克鲁维酵母、或其组合；较佳地，所述的酵母细胞包括：克鲁维酵母，更佳地为马克斯克鲁维酵母、和/或乳酸克鲁维酵母。
在另一优选例中，所述的酵母细胞提取物为对酵母细胞的水性提取物。
在另一优选例中，所述酵母细胞提取物不含酵母内源性的长链核酸分子。
在另一优选例中，所述的酵母细胞提取物是用包括以下步骤的方法制备：
(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；和
(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。
在另一优选例中，所述的固液分离包括离心。
在另一优选例中，在液态下进行离心。
在另一优选例中，所述离心条件为5000-100000g，较佳地，8000-30000g。
在另一优选例中，所述离心时间为0.5min–2h，较佳地，20–50min。
在另一优选例中，所述离心在1-10℃下进行，较佳地，在2-6℃下进行。
在另一优选例中，所述的洗涤处理采用洗涤液在pH为7-8(较佳地，7.4)下进行处
在另一优选例中，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。
在另一优选例中，所述的破细胞处理包括高压破碎、冻融(如液氮低温)破碎。
在另一优选例中，所述外源蛋白合成体系中，组分(a)的浓度为5-30ng/μL，较佳地，8-20ng/μL，更佳地，10-16ng/μL，以所述外源蛋白合成体系的总体积计。
在另一优选例中，所述外源蛋白合成体系中，组分(b)的浓度(v/v)为20％-70％，较佳地，30-60％，更佳地，40％-50％，以所述外源蛋白合成体系的总体积计。
在另一优选例中，所述外源蛋白合成体系还包括选自下组的一种或多种组分：
(c1)镁离子；
(c2)钾离子；
(c3)缓冲剂；
(c4)能量再生系统；
(c5)聚乙二醇；
(c6)用于合成RNA的底物；
(c7)用于合成蛋白的底物；
(c8)RNA聚合酶；
(c9)任选的外源蔗糖；
(c10)任选的溶剂，所述溶剂为水或水性溶剂。
在另一优选例中，所述镁离子来源于镁离子源，所述镁离子源选自下组：醋酸镁、谷氨酸镁、或其组合。
在另一优选例中，所述钾离子来源于钾离子源，所述钾离子源选自下组：醋酸钾、谷氨酸钾、或其组合。
在另一优选例中，所述缓冲剂选自下组：4-羟乙基哌嗪乙磺酸、三羟甲基氨基甲烷、或其组合。
在另一优选例中，所述能量再生系统选自下组：磷酸肌酸/磷酸肌酸酶系统、糖酵解途径及其中间产物能量系统、或其组合。
在另一优选例中，所述的合成RNA的底物包括：核苷单磷酸、核苷三磷酸、或其组合。
在另一优选例中，所述的合成蛋白的底物包括：1-20种天然氨基酸、以及非天然氨基酸。
在另一优选例中，所述核苷三磷酸选自下组：腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸、尿嘧啶核苷三磷酸、或其组合。
在另一优选例中，所述氨基酸为选自下组：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、组氨酸、或其组合。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。
在另一优选例中，所述氨基酸包括D型氨基酸和/或L型氨基酸。
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)。
在另一优选例中，所述聚乙二醇选自下组：PEG3000、PEG8000、PEG6000、PEG3350、或其组合。
在另一优选例中，所述聚乙二醇包括分子量(Da)为200-10000的聚乙二醇，较佳地，分子量为3000-10000的聚乙二醇。
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T7RNA聚合酶。
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。
在另一优选例中，所述外源蛋白合成体系包括选自下组的成分：聚乙二醇、4-羟乙基哌嗪乙磺酸、醋酸钾、醋酸镁、核苷三磷酸、氨基酸、磷酸肌 酸、二硫苏糖醇(DTT)、磷酸肌酸激酶、蔗糖、本发明第一方面所述的核酸构建物(以萤火虫荧光素酶或绿色荧光蛋白为外源蛋白)、T7RNA聚合酶、酵母细胞提取物、或其组合。
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。
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
本发明第一方面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-Z3-Z4-Z5   (I)
式中，
Z1－Z5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为外源蛋白的编码序列；
Z2为无或连接序列；
Z3为终止密码子；
Z4为插入序列；
Z5为乳糖代谢基因的终止子序列；
并且，所述Z4的GC含量高于40％。
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.:1-20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，在本发明的体外蛋白质合成体系中应用本发明的核酸构建物，并在反应完成后使用biotin-binding技术，可以完成外源蛋白的表达和纯化，具体地，应用本发明的核酸构建物所合成的增强型绿色荧光蛋白量的相对光单位值相对较高。
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，如植物细胞、酵母细胞；或是高等真核细胞，如昆虫细胞、动物细胞等。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)在体外生物合成体系存在下，提供第一方面所述的核酸构建物；
(ii)在适合的条件下，孵育步骤(i)的体外生物合成体系一段时间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，将任选的外源蛋白的编码序列、或者任意的连接序列、终止密码子、GC含量高于40％的插入序列和乳糖代谢基因的终止子序列作为核酸构建物，应用于本发明的体外蛋白质合成体系中，可显著提高目标蛋白的产量，并可用于外源蛋白的表达与纯化。
(2)本发明的蛋白质表达纯化序列能够特异性识别并高效结合beads。这些序列的识别结合效率不仅超过传统的特异性氨基酸biotin序列，而且可以应用于蛋白质合成体系(尤其是体外蛋白合成体系)。
(3)本发明提供的蛋白质表达纯化序列表达稳定，只要在终止密码子(如TAA)和乳糖代谢基因的终止子序列(如LAC4)中插入一段GC的含量超过40％的任意核酸序列(如T2)，即使该插入序列存在其它的改变(如增加或减少部分序列、突变等)，均能有效提高目标蛋白质的含量。
(4)与其他细胞相比，乳酸克鲁维酵母因其安全性和高效性可以被应用于食品和药品领域蛋白质的生产，加上体外蛋白质合成体系的优点，如适应于高通量的蛋白质合成筛选，合成毒性蛋白质和时间短成本低等，所以乳酸 克鲁维酵母细胞来源的体外蛋白质合成体系在相关领域也能够得到广泛的应用。
(5)本发明提供的新的核酸构建物不仅能够提升目标外源蛋白的表达和纯化效果，更主要的是能够增加乳酸克鲁维酵母体外蛋白质合成体系的针对不同蛋白质合成的可能性。
(6)本发明首次公开了一种可提高目标蛋白表达以及纯化的新型核酸构建物，在本发明的蛋白质合成体系(尤其是体外蛋白合成体系)中应用本发明的核酸构建物，可以完成外源蛋白的表达和纯化，与不具备该结构的核酸相比，所合成的外源蛋白的荧光值有50％的提升。
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Sam brook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。
本发明实施例里的外源蛋白以eGFP为例。
实施例一
1.1序列设计：
如图1所示，以D2P1.08e为基础，在TAA后面添加如下碱基序列，标记为D2P1.08e-T2:CACTAATAAGTAAGTAAAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG，其中，AGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG为T7终止子；以D2P1.08e为基础，在TAA后面添加如下碱基序列，并标记为D2P1.08e-T3:aGCGGCCGCCACCAcCAcCAtCAtCAtCAcCACTAATAAGTAAGTAAAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG，其中，AGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG为T7终止子(如图2)。
这3种质粒模板序列如SEQ ID NO.:1-3所示。
比较D2P1.08e，D2P1.08e-T2,D2P1.08e-T3三种质粒IVTT表达后的EGFP荧光，荧光强弱可以直接反应EGFP蛋白表达量的高低。
1.2实验步骤：
1)分子克隆构建(所使用的分子克隆步骤为目前较为普遍的技术)
基于同源重组方案设计引物，进行PCR。PCR产物用DpnI消化后转化DH5α感受态细胞。转化后的单菌落经测序鉴定为正确序列后，得到插入目的片段的质粒，分别为D2P1.08e-T2，D2P1.08e-T3。
2)利用Ampi的方法扩增目的基因片段
将构建好的D2P质粒用Ampi体系进行扩增，D2P质粒模板浓度为1ng/uL，将混合物置于37度培养箱中，静置或以30rpm/min以下速度轻柔振摇，反应2小时后收集产物，即为基因扩增产物，反应产物可即可使用或保存于-20度。
3)按照使用说明，将Ampi后的DNA片段加入到自制的乳酸克鲁维酵母体外蛋白质合成体系中。并将上述反应体系置于30℃的环境中，静置孵育约3-6h。反应结束后，立即放置于Envision 2120多功能酶标仪(Perkin Elmer)，读数，检测eGFP信号强弱，相对荧光单位值(Relative Fluorescence Unit,RFU)作为活性单位，如图1所示。
1.3实验结果：
D2P1.08e-T2，D2P1.08e-T3的RFU荧光值均高于对照组D2P1.08e，其中D2P1.08e-T2的值较高，约为D2P1.08eRFU荧光值的1.5倍(如图3)。
本发明结果表明：在TAA终止密码子后面插入新的核酸序列可以提升目标蛋白的翻译效率，相应的提升目的蛋白质表达量。该序列设计能够应用于酵母体外蛋白质合成体系中,起始蛋白质合成的效率远远超过TAA和LAC4终止子直接相连的序列。增加了体外合成体系进行蛋白质表达纯化方式的选择性，极大增强了体外蛋白质合成体系的可用性。
筛选的包含T2序列的核苷酸序列在体外蛋白质合成体系中引起增强型绿色荧光蛋白发出的相对荧光单位值(Relative Fluorescence Units,RFU)均达到较高值，其中D2P1.08e-T2中EGFP的荧光值有50％的提升。
实施例二
2.1实验设计
由于D2P1.08e-T2活性高于D2P1.08e-T3，而前者插入的序列比后者短。为了进一步分析D2P1.08e-T2中较为重要的序列或区域，我们后续对D2P1.08e-T2 中插入的序列进行了逐步减少，构建了一系列截短的质粒，如图4所示。根据截短的序列由短到长，依次将这些质粒命名为D2P1.08e-T2-001至D2P1.08e-T2-015。
这15种质粒模板序列如SEQ ID NO.:4-18所示。
2.2实验步骤
同1.2所述步骤类似的方法进行分子克隆、扩增目的基因片段和测定相对荧光单位值。
2.3实验结果
结果发现，活性提升最为明显的一组仍是D2P1.08e-T2，如图5所示。
本发明结果表明：在D2P1.08e的基础上增加一段T2序列，可以明显提升质粒上目的基因的表达量。
实施例三
3.1实验设计
为了进一步方便克隆构建，我们在TAA终止密码子的5’端插入了XhoI内切酶酶切序列CTCGAG或NotIXhoI双酶切序列GCGGCCGCACTCGAG，将含有这两种序列的质粒分别标记为D2P1.08e-T2-BX和D2P1.08e-T2-BN。
这2种质粒模板序列如SEQ ID NO.:19-20所示。
3.2实验步骤
同1.2所述步骤类似的方法进行分子克隆、扩增目的基因片段和测定相对荧光单位值。
3.3实验结果
对D2P1.08e-T2-BX和D2P1.08e-T2-BN这两个质粒Ampi扩增后的IVTT活性，得到如下结果，如图6所示。
在D2P1.08e-T2的基础上增加酶切位点XhoI或NotIXhoI，对于D2P1.08e-T2的活性没有明显影响，且都比原始质粒D2P1.08e活性高。
本发明结果表明：本发明所述的核酸在目标蛋白质表达和纯化过程中具有极佳的稳定性。
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 (12)

  1. 一种核酸构建物，其特征在于，所述核酸构建物含有结构如式I所示的核酸序列：
    Z1-Z2-Z3-Z4-Z5    (I)
    式中，
    Z1－Z5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为外源蛋白的编码序列；
    Z2为无或连接序列；
    Z3为终止密码子；
    Z4为插入序列；
    Z5为乳糖代谢基因的终止子序列；
    并且，所述Z4的GC含量高于40％。
  2. 根据权利要求1所述的一种核酸构建物，所述Z4包含终止子元件；优选地，所述终止子元件选自下组：T7终止子、T3终止子、SP6终止子或其组合。
  3. 根据权利要求1或2所述的一种核酸构建物，所述Z4包含T7终止子；优选地，所述Z4包含T2序列，所述T2的核苷酸序列为：CACTAATAAGTAAGTAAAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG。
  4. 根据权利要求1-3任一项所述的一种核酸构建物，所述Z3的序列优选为TAA。
  5. 根据权利要求1-4任一项所述的一种核酸构建物，所述乳糖代谢基因优选为LAC4基因。
  6. 一种体外的外源蛋白合成体系，其特征在于，包括：
    (a)权利要求1-5中任一项所述的核酸构建物；和
    (b)细胞提取物。
  7. 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1-5中任一项所述的核酸构建物。
  8. 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1-5任一项所述的核酸构建物，或者所述基因工程细胞中含有权利要求7所述的载体或载体组合。
  9. 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1－5任一所述的构建物；
    (b)权利要求7所述的载体或载体组合；
    (c)权利要求8所述的基因工程细胞。
  10. 一种如权利要求1-5任一所述的构建物、权利要求7所述的载体或载体组合、权利要求8所述的基因工程细胞或权利要求9所述的试剂盒的用途，其特征在于，用于进行高通量的体外蛋白质合成。
  11. 一种体外高通量的外源蛋白的合成方法，其特征在于，包括步骤：
    (i)在体外生物合成体系存在下，提供权利要求1-5任一所述的核酸构建物；
    (ii)在适合的条件下，孵育步骤(i)的体外生物合成体系一段时间T1，从而合成所述外源蛋白。
  12. 如权利要求11所述的方法，其特征在于，还包括(iii)任选地从所述体外生物合成体系中，分离或检测所述外源蛋白。
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