WO2024032690A1 - 线性闭合dna的制备方法及该方法所用质粒 - Google Patents
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- plasmid
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- protelomerase
- exonuclease
- lcdna
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
Definitions
- the present invention relates to the technical field of genetic engineering, and specifically to a method for preparing linear closed DNA and a plasmid used in the method.
- Gene therapy is a method that uses nucleic acid drugs to repair, replace or regulate genes to prevent or treat diseases.
- Delivery vectors for gene therapy can be broadly divided into viral vectors and non-viral vectors. Thanks to their natural ability to infect cells and efficiently deliver genes, viral vectors are widely used in the fields of cell therapy and gene therapy. However, the use of viral vectors also carries varying degrees of risks. Lentiviral and retroviral vectors are mainly used to integrate target genes into the cell genome, which can also cause high-risk genetic mutations to cells. The risk of insertional mutations caused by adeno-associated viral vectors is low but still exists. Adenoviral vectors can exist free from the genome, but may cause toxicity and immunogenicity to cells.
- viral vectors such as AdV5 or AAV2
- AdV5 or AAV2 are widely distributed and many people have pre-existing immunity to them and cannot be used as gene delivery vectors.
- the cytotoxicity and immunogenicity of non-viral vectors are much lower than that of viral vectors, so the clinical research and commercial use of non-viral vectors in gene therapy and cell therapy are attracting more and more attention.
- Plasmid vectors are mainly composed of prokaryotic replicons, resistance genes and target genes.
- the prokaryotic replicon and resistance gene on the plasmid vector are and CpG sequences may cause cells to produce unnecessary immune responses and cell canceration. Therefore, scientists invented a method to produce linear closed DNA (LcDNA) using protelomerase TelN derived from E. coli phage N15.
- LcDNA only carries the target gene and TelN recognition sites at both ends, effectively removing sequences such as prokaryotic replicons and resistance genes on the plasmid that do not need to be delivered into cells.
- LcDNA has equivalent or even better expression levels in vitro or in vivo, is safer, and has higher transfection efficiency.
- LcDNA Small-scale production of LcDNA is not difficult, but large-scale industrial production under GMP conditions faces many difficulties, such as the circular plasmid backbone used to produce LcDNA is too large and unusable; the instability of large-scale enzyme digestion; and the purity cannot reach clinical standards. level, organic reagents such as Triton, isopropyl alcohol and ethanol may also be introduced, with high residual risks; the fidelity of in vitro amplification of polymerase Phi29 is lower than that of E.coli in vivo replication, and unnecessary mutations may occur.
- organic reagents such as Triton, isopropyl alcohol and ethanol may also be introduced, with high residual risks; the fidelity of in vitro amplification of polymerase Phi29 is lower than that of E.coli in vivo replication, and unnecessary mutations may occur.
- the present invention relates to a plasmid, which contains two editing regions of protelomerase target sequences connected in the same direction, and one or more restriction endonuclease enzyme cleavage sites are independently provided at both ends of the editing region.
- the length of the above-described plasmid is ⁇ 2500 bp, or ⁇ 2300 bp, or ⁇ 2100 bp. In some preferred embodiments, the length of the above plasmid is ⁇ 2100 bp.
- the plasmids described above further comprise a prokaryotic replicon and a selection gene.
- the restriction endonuclease cleavage site at each end is selected from any one or more of the following: AccI, AflII, AgeI, Apal, AscI, AvrII, BamHI, BglI, BglII, Bsal, BspQI, BstBI, BsteII, ClaI, EcoNI, EcoRI, EcoRV, FseI, HindIII, KpnI, MfeI, MluI, NcoI, NdeI, NheI, NotI, PacI, PstI, PvuI, PvuII, SacI, SacII, SalI, ScaI, SmaI,
- the restriction sites of SpeI, StuI, SwaI, XbaI, XhoI and XmaI are preferably selected from any one or more restriction sites of HindIII, SalI, SmaI, BamHI, EcoRV, XbaI, Kp
- HindIII, SalI, SmaI and BamHI restriction sites were inserted near one end of the editing region, and EcoRV, XbaI, KpnI and EcoRI restriction sites were inserted near the other end of the editing region.
- the protelomerase target sequence comprises the sequence shown in SEQ ID NO: 1, or is at least 80%, at least 85%, at least 90%, or at least 93% identical to the sequence shown in SEQ ID NO: 1 %, at least 95%, at least 97%, at least 99% identity and a nucleotide sequence capable of functioning as telomerase.
- the protelomerase target sequence is shown in SEQ ID NO: 1.
- the above-mentioned plasmid comprises a core having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 99% identity to the sequence shown in SEQ ID NO:2 nucleotide sequence. In some preferred embodiments, the sequence of the above plasmid is shown in SEQ ID NO: 2.
- the present invention also relates to a recombinant plasmid that inserts a gene of interest between the two protelomerase target sequences of the plasmid as described above.
- the invention also relates to a method for preparing linear closed DNA, which includes:
- the recombinant plasmid contains a protelomerase target sequence and a restriction endonuclease cleavage site, and a gene of interest is inserted between the protelomerase target sequences.
- the recombinant plasmid mentioned in the above preparation method can be a plasmid in which a gene of interest is inserted between two protelomerase target sequences of the plasmid as described above.
- the exonuclease in the above method includes Exonuclease III and/or T5 Exonuclease. In some embodiments, the exonuclease is added in an amount of 3000U/mg ⁇ 6000U/mg.
- the enzyme cutting temperature of the exonuclease is 34°C to 40°C, and the enzyme cutting time is 2 to 12 hours; preferably, the enzyme cutting time is 3 to 6 hours.
- EDTA at a final concentration of 1mM to 50mM is added and the temperature is raised to 70°C to 80°C for 5 to 30 minutes.
- the end of protelomerase digestion and/or after the end of restriction endonuclease digestion the temperature is raised to 70°C to 80°C for 5 to 30 minutes.
- the separation and purification method in the above preparation method includes one of gel filtration chromatography, anion chromatography, concentration and liquid replacement, and sterile filtration, or a combination thereof.
- the separation and purification method sequentially includes gel filtration chromatography and anion chromatography.
- the gel filtration chromatography filler is Bestarose 6FF, and the ⁇ 50mAu eluent of the first UV peak is collected.
- the filler of the anion chromatography is Fractogel EMD DEAE.
- the loading conditions for the anion chromatography include: using a loading solution containing 30% to 70% of chromatography solution B, and a loading capacity of 0.2 mg/mL to 1.0 mg/mL. The flow rate is ⁇ 120cm/h; the chromatography solution B contains 0.5M ⁇ 1.5M NaCl, 5mM ⁇ 15mM EDTA and buffer substances, with a pH of 7.2 ⁇ 7.8.
- the elution conditions of the anion chromatography include: 65% to 100% elution with chromatography solution B, an elution flow rate of 30cm/h to 120cm/h, and collecting ⁇ 100mAu eluate.
- the present invention also relates to a kit comprising protelomerase and the plasmid as described above;
- the kit further includes a restriction endonuclease and/or exonuclease capable of recognizing the restriction enzyme cleavage site.
- the invention also provides linear closed DNA prepared by the above preparation method.
- the present invention uses plasmids produced by bacterial fermentation as starting materials, which are not obtained through polymerase amplification methods such as Phi29, and have higher fidelity; and through specific design, the plasmids can maintain Small enough to increase the overall yield of LcDNA production from the source.
- FIG. 1 is a schematic diagram of the LcDNA production and purification process
- Figure 2A is the universal carrier skeleton CFL Universal map
- Figure 2B is a full sequence map of the vector of Figure 2A inserting the target sequence (GOI); among them, Kan promoter: kanamycin gene promoter, KanR: kanamycin resistance gene, pUC origin: pUC promoter, MCS: Multiple cloning site; GOI: target sequence; telR and telL: protelomerase target sequence;
- GOI target sequence
- telR and telL protelomerase target sequence
- Figure 3 shows the LcDNA agarose gel electrophoresis (AGE) detection.
- a and B are the AGE detection patterns of a batch of 60 mg circular plasmid digested and purified LcDNA containing the target sequence and the finished product.
- C is another batch of 60 mg containing the target sequence.
- lane M1 supercoiled DNA ladder
- lane M2 1kb DNA ladder
- Lane 1 TelN digestion product
- Lane 2 Exonuclease digestion product
- Lane 3 GF chromatography product
- Lane 4 AEX chromatography product
- Lane 1 LcDNA finished product
- Lane 2 1 ⁇ TAE buffer
- Lane 1 TelN digestion product
- Lane 2 Exonuclease digestion product
- Lane 3 GF chromatography product
- Lane 4 AEX chromatography product
- Lane 5 LcDNA finished product
- Figures 4A to 4E are LcDNA gel filtration chromatograms;
- Figures 4A and 4B are respectively the LcDNA gel filtration chromatograms of two batches of 60 mg circular plasmids containing the target sequence after digestion, and
- Figure 4C is a batch of 100 mg containing the target sequence.
- Figure 4D and 4E gel chromatogram of LcDNA after digestion of two batches of 150 mg circular plasmid containing the target sequence;
- Figures 5A to 5E are the anion chromatograms of LcDNA.
- Figures 5A and 5B are the anion chromatograms of two batches of 60 mg circular plasmid LcDNA containing the target sequence.
- Figure 5C is the anion chromatogram of a batch of 100 mg circular plasmid LcDNA containing the target sequence.
- Analysis chromatograms, Figures 5D and 5E are respectively the anion chromatography chromatograms of two batches of 150mg circular plasmid LcDNA containing the target sequence;
- Figures 6A to 6B are chromatograms of optimized loading conditions for LcDNA anion chromatography.
- Figure 6A is a loading solution containing 30% chromatography solution B
- Figure 6B is a loading solution containing 70% chromatography solution B;
- Figures 7A to 7B are chromatograms of optimized loading loading of LcDNA anion chromatography.
- the loading loading of Figure 7A is 0.25mg/mL, and the loading loading of Figure 7B is 1.0mg/mL;
- Figure 8 shows the linear elution of LcDNA anion chromatography (0-100% chromatography solution B gradient elution), in which the maximum ultraviolet peak appears when 67.5% chromatography solution B is eluted;
- curve a represents conductivity
- curve b represents ultraviolet peak
- the technical solution of "A, and/or, B, and/or, C, and/or, D” includes any one of A, B, C, and D (that is, they are all connected with "logical OR” technical solution), also includes any and all combinations of A, B, C, and D, that is, including combinations of any two or any three of A, B, C, and D, and also includes A, B, C , four combinations of D (that is, technical solutions that are all connected by "logical AND").
- the present invention refers to concentration values, and their meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2% can allow fluctuation within the range of ⁇ 0.1%. For values that are large or do not require too fine control, the meaning is also allowed to include larger fluctuations. For example, 100mM can allow fluctuations within the range of ⁇ 1%, ⁇ 2%, ⁇ 5%, etc. Referring to molecular weight, fluctuations of ⁇ 10% are allowed.
- the technical features described in open terms include closed technical solutions composed of the listed features, and also include open technical solutions including the listed features.
- the first aspect of the present invention relates to a plasmid, which contains two editing regions of protelomerase target sequences connected in the same direction, and one or more restriction endonuclease enzymes are independently provided at both ends of the editing region. cut point.
- the plasmid is ⁇ 2500bp in length, such as 2400bp, 2300bp, 2200bp, 2100bp, 2050bp, 2000bp, 1950bp, 1900bp, 1850bp, 1800bp or less.
- the plasmid further comprises a prokaryotic replicon and a selection gene.
- Typical screening genes include antibiotic resistance genes, such as kanamycin resistance gene (KanR), tetracycline resistance gene (tetR), neomycin phosphotransferase gene (npt), etc.
- the screening genes can also be certain compound detoxification enzyme genes, carbohydrate metabolism enzyme selection marker genes, etc.
- the number of restriction endonuclease cleavage sites at each end is independently selected from 1, 2, 3, 4, 5, 6 or more, and preferably the restriction endonuclease sites at the same end are The types of enzyme cleavage sites are different.
- restriction endonuclease cleavage sites located at the same end of the editing region overlap.
- all restriction enzyme cleavage sites are different.
- HindIII, SalI, SmaI and BamHI recognition sites are inserted near one end of the editing region, and EcoRV, XbaI, KpnI and EcoRI recognition sites are inserted near the other end of the editing region, thus providing 16 groups to choose from. enzyme digestion combination. So much more practical and broad spectrum.
- closed linear DNA molecules are generated by the action of protelomerase on DNA amplified from a DNA template comprising at least one protelomerase target sequence.
- a protelomerase target sequence is any DNA sequence present in the DNA template that enables its conversion into closed linear DNA by the enzymatic activity of protelomerase.
- the protelomerase target sequence is necessary for cleavage and religation of double-stranded DNA by protelomerase to form covalently closed linear DNA.
- protelomerase target sequences include any complete palindrome sequence, that is, any double-stranded DNA sequence with double rotational symmetry.
- the complete inverted repeat sequence In Borrelia burgdorferi, the complete inverted repeat sequence is 14 base pairs in length. In various mesophilic phages, the length of complete inverted repeats is 22 base pairs or more.
- the central complete inverted palindrome sequence is flanked by inverted repeats, forming part of a larger incomplete inverted palindrome sequence.
- the protelomerase target sequence is derived from E.coli N15 phage, Klebsiella Phi K02 phage, Yersinia Py54 phage, Halomonas Phi HAP phage, Vibrio VP882 phage or Borrelia burgdorferi lpB31.16, or a variant of any one of them.
- the protelomerase target sequence is derived from phage N15 TelN or a variant thereof.
- the protelomerase target sequence includes the sequence shown in SEQ ID NO: 1, or has at least 80% identity with the sequence shown in SEQ ID NO: 1 and can exert the function of telomerase.
- Nucleotide sequences such as nucleotide sequences that are at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical, such as one or several nucleotide truncations, insertions, Substitutions and/or deletions.
- the plasmid can be transformed from a conventional cloning vector, such as a prokaryotic vector, preferably an Escherichia coli plasmid vector, and more preferably a plasmid of less than 2000 bp. In some specific embodiments, it is modified from pUC57-Kan-mini (1824bp).
- the plasmid comprises a nucleotide sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 2, such as at least 85%, 90%, 95%, 96%, 97%, 98% , 99% identical nucleotide sequence, such as truncation, insertion, substitution and/or deletion of one or several nucleotides. In some specific embodiments, the plasmid comprises the nucleotide sequence set forth in SEQ ID NO:2.
- two protelomerase target sequences connected in the same direction are directly connected; in some embodiments, two protelomerase target sequences connected in the same direction also include a few A nucleotide sequence of less than 30 bp, or less than 20 bp, or less than 10 bp. These nucleotide sequences are preferably sites or related sequences that facilitate the insertion of the target gene.
- a second aspect of the invention relates to a recombinant plasmid inserting a gene of interest between two protelomerase target sequences of the plasmid as described above.
- the expression product of the target gene is interfering RNA or an aptamer.
- Interfering RNA can be selected from, for example, siRNA, shRNA or miRNA.
- the expression product of the gene of interest is mRNA, and such mRNA can preferably express proteins, for example, proteins that confer certain desired characteristics to target cells, such as fluorescent proteins that allow cell tracking, provide target cells with Active enzymes that are lost or altered, etc.
- the gene of interest may include, for example, a gene (nucleotide sequence) encoding a protein that is defective or missing in the recipient individual or target cell; encoding a protein with a desired biological or therapeutic effect (e.g., antibacterial, Genes for proteins with antiviral or antitumor/anticancer functions); nucleotide sequences encoding RNAs that inhibit or reduce the production of harmful or otherwise undesirable proteins (e.g., nucleosides encoding RNA interfering agents as defined above acid sequence); and/or a nucleotide sequence encoding an antigenic protein.
- a gene nucleotide sequence
- nucleotide sequences encoding RNAs that inhibit or reduce the production of harmful or otherwise undesirable proteins e.g
- Suitable genes of interest include, but are not limited to, nucleic acids encoding proteins useful in the treatment of endocrine, metabolic, hematological, cardiovascular, neurological, musculoskeletal, urinary, pulmonary and immune disorders, including, for example, cancer, inflammatory disorders, immune disorders , chronic and infectious conditions.
- Cancers such as bones, bone joints, muscles, lungs, trachea, heart, spleen, arteries, veins, blood, capillaries, lymph nodes, lymphatic vessels, lymph, mouth, pharynx, esophagus, stomach, duodenum, small intestine, colon , rectum, anus, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum, testicle, vas deferens, penis, eye, ear , nose, tongue, skin, brain, brainstem, medulla oblongata, spinal cord, cerebrospinal fluid, nerves, thyroid, parathyroid gland, adrenal gland, pituitary gland, pineal gland, pancreatic islet, thymus, gonads, sublingual gland and parot
- Suitable genes of interest include, but are not limited to, encoding at least one of the following multiple proteins: interferon (e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ ); insulin (e.g., nolin Helin, Humalog, Humalog, Humalog, Lantus, etc.); erythropoietin ("EPO"; for example, epoetin-alpha, darbepoetin-alpha, epoetin- ⁇ ; etc.); antibodies (e.g., monoclonal antibodies such as rituximab, infliximab, trastuzumab, HumiraTM (adalimumab monoclonal antibody), omasozumab, tositumomab, RaptivaTM (efalizumab), ErbituxTM (cetuximab), bevacizumab; etc.); including antigen-binding fragment
- the gene of interest can be inserted into the plasmid in the form of an expression cassette.
- the plasmid/recombinant plasmid may further comprise at least one of the following elements: a promoter, an enhancer, an internal ribosome entry site, a reporter gene, a coupling marker gene and a transcription termination signal. These elements are preferably located in the expression cassette of the gene of interest.
- a promoter is a nucleotide sequence that initiates and regulates the transcription of a polynucleotide. Promoters include inducible promoters (expression of a polynucleotide sequence operably linked to the promoter is induced by analytes, cofactors, regulatory proteins, etc.), repressor promoters (expression of a polynucleotide sequence operably linked to the promoter Expression of a ground-linked polynucleotide sequence is repressed by analytes, cofactors, regulatory proteins, etc.) and constitutive promoters.
- promoter is intended to include both full-length promoter regions and functional (eg, control of transcription or translation) fragments of these regions.
- Promoters preferably include promoters that initiate prokaryotic expression.
- a promoter used to control the expression of the target gene which can be a cell-free specific promoter, or can be "cell-specific” or “cell-specific” according to the desired location of administration of the product of the target gene.
- “Cell type specific”, “cell lineage specific” or “tissue specific” promoters which allow expression in restricted cell and tissue types respectively.
- Typical reporter genes include fluorescent protein genes, luciferase genes and LacZ.
- Fluorescent proteins and luciferase are both luminescent proteins, and fluorescent expression can be detected with a camera or similar device. Fluorescent proteins work by absorbing one color of light (excitation) and then emitting lower-energy light of a different color (emission). In contrast, luciferase (and other bioluminescent enzymes) emit light by catalyzing a chemical reaction with a substrate (i.e., luciferin). Unlike the two markers above, LacZ does not emit light. ⁇ -galactosidase, the product of the LacZ gene, can catalyze the conversion of X-gal into an opaque blue compound similar to indigo.
- fluorescent proteins such as green fluorescent protein, blue fluorescent protein, yellow fluorescent protein protein, orange fluorescent protein or red fluorescent protein.
- Green fluorescent protein can use common GFP, or modified GFP genes, such as enhanced GFP gene EGFP, etc.; blue fluorescent protein can be selected from EBFP, Azuritc, TagBFP, etc.; yellow fluorescent protein can be selected from EYFP, Ypct , PhiYFP, etc.; the orange fluorescent protein can be selected from mKO, mOrange, mBanana, etc.; the red fluorescent protein can be selected from TagRFP, mRuby, mCherry, mKatc.
- Typical conjugated marker genes are genes that can express biotin or its derivatives, where the biotin derivatives are D-biotin, activated biotin, biocytin, ethylenediamine biotin, cadaverine biotin, and desulfation Any of the biotins.
- the internal ribosome entry site is a genetically engineered element hundreds of base pairs (bp) long. IRES was first discovered in viruses, and later it was found that the mRNA of some eukaryotic cells also contains IRES elements. IRES can guide ribosomes into this sequence and translate the open reading frame in the 3' direction. Inserting an IRES sequence between two open reading frames (ORFs) under the same transcript can translate one transcript into two independent proteins.
- the transcription termination signal refers to the stem-loop structure formed by a sequence of RNA produced during the transcription process, which can be specifically recognized by the RNA polymerase transcription complex to terminate transcription.
- the target gene is a nucleic acid vaccine.
- the third aspect of the present invention relates to a method for preparing linear closed DNA, including:
- the recombinant plasmid contains a protelomerase target sequence and a restriction endonuclease cleavage site, and a gene of interest is inserted between the protelomerase target sequences.
- Miscellaneous nucleic acids refer to unnecessary or extra sequences other than the gene of interest. These unnecessary Or additional sequences typically include various elements in the plasmid as described above, such as replicons, selection genes, etc. However, it is easy to understand that the heteronucleic acid in the present invention refers to most but not all of the above-mentioned unnecessary or additional sequences. For example, at least the protelomerase target sequence (such as telL, telR) directly connected to the target gene is cannot be removed.
- the recombinant plasmid is the recombinant plasmid as defined above, and in this case, the content defined in the first and second aspects of the present invention also applies to the third aspect.
- the added amount of protelomerase is 8000U/mg to 15000U/mg, such as 9000U/mg, 10000U/mg, 11000U/mg, 12000U/mg, 13000U/mg, 14000U/mg.
- the enzyme cleavage temperature of the protelomerase is 25°C to 35°C, preferably 27°C to 33°C.
- the digestion time of the protelomerase is 1 to 8 hours, such as 2, 3, 4, 5, 6, or 7 hours.
- the added amount of each restriction enzyme is independently selected from 1000 U/mg to 6000 U/mg, such as 2000 U/mg, 3000 U/mg, 4000 U/mg, 5000 U/mg.
- the restriction enzyme has a cutting temperature of 32°C to 42°C, preferably 34°C to 40°C, such as 35°C, 36°C, 37°C, 38°C, and 39°C.
- the restriction endonuclease digestion time is 1 to 4 hours, such as 2 hours or 3 hours.
- the exonuclease includes Exonuclease III and/or T5 Exonuclease.
- the added amount of the exonuclease is 3000U/mg to 6000U/mg, such as 3500U/mg, 4000U/mg, 4500U/mg, 5000U/mg, 5500U/mg.
- the digestion temperature of the exonuclease is 34°C to 40°C, such as 35°C, 36°C, 37°C, 38°C, and 39°C.
- the exonuclease digestion time is 2 to 12 hours, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 hours, more preferably 3 to 6 hours .
- a final concentration of 1mM to 50mM EDTA is added, such as 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM.
- EDTA is added and the temperature is raised to 70°C to 80°C for 5 to 30 minutes, such as 10, 15, 20, or 25 minutes.
- EDTA and high temperature are used to inactivate the enzyme, effectively inhibiting the residual activity of endo- and exonucleases in the system and greatly improving the stability of large-scale production.
- the temperature is raised to 70°C to 80°C for 5 to 30 minutes, such as 10, 15, 20, 25 minutes. .
- the separation and purification method includes one of gel filtration chromatography, anion chromatography, concentration and liquid replacement, and sterile filtration, or a combination thereof.
- the separation and purification method sequentially includes gel filtration chromatography and anion chromatography. In other embodiments, the separation and purification method sequentially includes gel filtration chromatography, anion chromatography, and concentration and liquid replacement.
- the gel filtration chromatography filler is Bestarose 6FF, and the ⁇ 50 mAu eluent of the first UV peak is collected.
- the filler for the anion chromatography is Fractogel EMD DEAE.
- the loading conditions for anion chromatography include:
- a loading solution containing 30% to 70% chromatography solution B for example, 40%, 50%, 60%, as volume percentage
- a loading capacity of 0.20 mg/mL to 1.0 mg/mL for example, 0.25 mg/mL.
- 0.35mg/mL 0.35mg/mL
- 0.4mg/mL 0.45mg/mL
- 0.5mg/mL 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL
- sample flow rate ⁇ 120cm/h
- the chromatography solution B contains 0.5M ⁇ 1.5M NaCl, 5mM ⁇ 15mM EDTA and buffer substances, with a pH of 7.2 ⁇ 7.8.
- buffer substance refers to an aqueous solution or composition that resists changes in pH when an acid or base is added to the solution or composition. This resistance to pH changes is due to the buffering nature of such solutions. Therefore, solutions or compositions that exhibit buffering activity are called buffers or buffer solutions.
- the buffer that can be used in the method of the invention is preferably selected from phosphate buffer, phosphate buffered saline buffer (PBS), 2-amino-2hydroxymethyl-1,3-propanediol (TRIS) buffer, TRIS Buffered saline solution (TBS) and TRIS/EDTA (TE), etc.
- the loading conditions for anion chromatography include:
- a loading solution containing 50% chromatography solution B (volume percentage), with a loading capacity of 0.20 mg/mL to 1.0 mg/mL (such as 0.25 mg/mL, 0.3 mg/mL, 0.35 mg/mL, 0.4 mg/mL).
- the sample flow rate is ⁇ 120cm/h;
- the chromatography solution B contains 0.5M ⁇ 1.5M NaCl, 5mM ⁇ 15mM EDTA and 50mM ⁇ 150mM Tris-HCl, with a pH of 7.2 ⁇ 7.8.
- the chromatography solution B contains 1M NaCl, 10mM EDTA, 100mM Tris-HCl, and the pH is 7.5.
- the elution conditions of the anion chromatography include:
- chromatographic solution B elutes (for example, 70%, 80%, 90%, which is volume percentage), and the elution flow rate is 30cm/h to 120cm/h (for example, 40cm/h, 50cm/h, 60cm/h , 70cm/h, 80cm/h, 90cm/h, 100cm/h, 110cm/h), collect ⁇ 100mAu eluent.
- the elution conditions of the anion chromatography include:
- the method for concentrating liquid replacement is tangential flow filtration.
- a fourth aspect of the present invention relates to a kit comprising protelomerase and the plasmid as described above;
- the kit further includes a restriction endonuclease and/or exonuclease capable of recognizing the restriction endonuclease cleavage site.
- the kit can also include conventional components such as nucleic acid-free water and instructions for use.
- a fifth aspect of the present invention provides linear closed DNA obtained by the above preparation method.
- the linear closed DNA contains the gene of interest.
- the present invention also provides compositions comprising the linear closed DNA described above.
- the composition includes linear closed DNA as described above with a purity of at least 90%.
- the composition includes linear closed DNA as described above with a purity of at least 99%.
- the measurement parameters of raw material components are involved. Unless otherwise specified, there may be slight deviations within the range of weighing accuracy. Temperature and time parameters are involved, allowing for acceptable deviations due to instrument testing accuracy or operating accuracy.
- the specific design method is to first continuously insert two TelN enzyme recognition sequences TelRL (56bp) into pUC57-Kan-mini (1824bp), and then sequence them near the TelN recognition site of protelomerase at one end. Insert HindIII, SalI, SmaI and BamHI recognition sites, and insert EcoRV, XbaI, KpnI and EcoRI recognition sites near the TelN recognition site at the other end to provide 16 sets of optional enzymes for the second step of restriction endonuclease digestion.
- the modified CFL Universal length is 2017bp.
- the target sequence to be inserted only needs to be designed between the two recognition sites TelRL of TelN of CFL universal.
- the map is shown in Figure 2A (the sequence information is shown in SEQ ID NO:2), in which the GOI target gene is inserted as an example, with a length of 3169bp, as shown in Figure 2B.
- the designed circular plasmid sequence was entrusted to Nanjing GenScript Biotechnology Co., Ltd. for compliance synthesis, and then went through E. coli bacterial library construction, high-density fermentation, cell lysis, clarification and filtration, concentration and incubation, chromatography purification, concentration and exchange. liquid, sterile filtration and filling, and the high-purity and high-quality circular plasmid obtained is used as the starting material for LcDNA production.
- the three-step digestion of LcDNA is TelN digestion, exonuclease digestion and endonuclease digestion.
- the TelN enzyme digestion process parameters are: TelN enzyme addition amount 10000U/mg, enzyme digestion temperature 30°C, enzyme digestion time 3 hours, enzyme digestion termination temperature 75°C, Processing time 15 minutes.
- the process parameters for endonuclease digestion are: select an endonuclease that does not have a corresponding recognition site according to the inserted target sequence, such as HindIII, SalI, SmaI, BamHI, EcoRV, XbaI, KpnI and EcoRI, etc., and add two endonucleases.
- the addition amount of each endonuclease is 3000U/mg
- the enzyme cutting temperature is 37°C
- the enzyme cutting time is 2 hours
- the enzyme cutting stop temperature is 75°C
- the treatment time is 15 minutes.
- the exonuclease digestion process parameters are: exonuclease Exo III or T5 Exo, addition amount 4500U/mg, enzyme digestion temperature 37°C, enzyme digestion time 4 hours.
- the enzyme digestion reaction solution is centrifuged or filtered, and then subjected to gel filtration chromatography.
- the process parameters of gel filtration chromatography are: packing Bestarose 6FF, column height ⁇ 20cm, symmetry factor 0.8 ⁇ 1.8, number of plates >2000.
- the anion chromatography process parameters are: packing Fractogel EMD DEAE, chromatography column height ⁇ 20cm, symmetry factor 0.8 ⁇ 1.8, number of plates > 2000.
- the loading conditions include a loading solution containing 30-70% chromatographic solution B (volume ratio), a loading capacity of 0.25-1.0 mg/mL, a loading flow rate of ⁇ 120cm/h, and a retention time of ⁇ 6 minutes.
- the elution flow rate is 30-120cm/h, and the eluate of ⁇ 100mAu is collected.
- the component of chromatography solution A is 10mM EDTA, 100mM Tris-HCl, pH7.5
- the component of chromatography solution B is 1M NaCl
- 10mM EDTA 100mM Tris-HCl, pH7.5
- 100% chromatography solution A balance, 58% chromatography solution B washes impurities, and eluted with 65%-100% chromatography solution B.
- chromatography B elutes from 0-100% elution gradient, in which the maximum UV peak appears when 67.5% chromatography B elutes.
- the anion chromatography process optimization data is shown in Chart 1A and Table 1B, and the corresponding elution curves are shown in Figure 6A, Figure 6B, Figure 7A and Figure 7B.
- the collected eluate is subjected to sample concentration by tangential flow filtration. Shrink, concentrate to the dialysis point of 1.0mg/mL ⁇ 0.3mg/mL, and then use the final storage buffer such as Tris-EDTA, and replace the medium with 10 ⁇ 1 times the volume.
- the membrane cassette for tangential flow filtration has a pore size of 10kD and specifications of 50cm 2 , 0.01m 2 , 0.1m 2 , and 0.5m 2 . Just select the membrane cassette with appropriate specifications according to the production scale.
- the LcDNA sample is filled in a clean workbench or isolator according to the project packaging specifications. After filling, the finished LcDNA product is stored in an ultra-low temperature refrigerator at -70 ⁇ 10°C.
- the length of the example LcDNA is 3169 bp.
- the complete sequence map of the circular plasmid inserted into the LcDNA is shown in Figure 2B.
- Total yield LcDNA finished product amount/enzyme digested circular plasmid sample amount ⁇ 100%
- the circular plasmid is digested by TelN, separated by agarose gel electrophoresis (AGE), gel cut, purified with Axygen gel recovery kit (AXYGEN, Cat. No.: UE-GX-50), and LcDNA
- Axygen gel recovery kit AXYGEN, Cat. No.: UE-GX-50
- LcDNA The yield is low, less than 10%, and the quality does not meet clinical or commercial requirements.
- Using engineered bacterial fermentation that can produce LcDNA, followed by a one-step Q anion chromatography (NanoGel-50Q, Suzhou Nanotechnology Co., Ltd., Cat. No.: 04064-050200-2100) purification method the LcDNA yield is low, less than 10%, and the LcDNA The purity is not high and does not meet clinical or commercial requirements.
- the bacterial endotoxin in LcDNA was detected by gel method using Limulus reagent.
- the test results are summarized in Table 3.
- This LcDNA production process is very stable.
- the scale of the enzyme-digested circular plasmid was gradually expanded from 60 mg to 100 mg and 150 mg, and no abnormal deviation occurred in the production process. This process can also meet the enzyme digestion scale LcDNA production of greater than 150mg circular plasmid.
- This LcDNA production process has high overall yield.
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Abstract
提供了一种线性闭合DNA的制备方法及该方法所用质粒。该质粒包含两个同向串联的原核端粒酶靶序列的编辑区,所述编辑区的两端独立地设置有一个或多个限制性内切酶酶切位点。该方法能获得高纯度的LcDNA,能更好地应用于临床研究和商业化用途。
Description
相关申请的交叉引用
本申请要求2022年8月10日提交的申请号为202210955391.7的中国专利申请的优先权,其全部内容通过引入并入本文。
本发明涉及基因工程技术领域,具体而言,涉及一种线性闭合DNA的制备方法及该方法所用质粒。
基因治疗是利用核酸药物修复、替换或者调控基因来预防或治疗疾病的方法。基因治疗用的递送载体可以概括性的分为病毒载体和非病毒载体。得益于它们天然的侵染细胞的能力和高效递送基因的能力,病毒载体被广泛应用于细胞治疗和基因治疗领域。但是,病毒载体的使用也存在不同程度的风险。慢病毒和逆转录病毒载体主要用于将目的基因整合到细胞基因组上,这也会给细胞造成高风险的基因突变。腺相关病毒载体造成插入突变的风险低,但仍然存在。腺病毒载体可以游离于基因组存在,但是可能会给细胞造成毒性和免疫原性。某些病毒载体比如AdV5或AAV2,由于分布广泛,许多人已经预先存在对它们的免疫而不能被作为基因递送的载体。这些所有病毒载体家族,即使在人类中血清阳性率低,也不能被作为基因递送载体重复使用,因为人体会对其产生免疫力。而非病毒载体的细胞毒性和免疫原性远低于病毒载体,因此非病毒载体在基因治疗和细胞治疗的临床研究和商业化用途也越来越被关注。
常规的非病毒载体主要是质粒。质粒载体主要由原核的复制子,抗性基因和目的基因等元件组成。然而质粒载体上的原核复制子,抗性基因以
及CpG序列可能会导致细胞产生不必要的免疫应答和细胞癌变。因此,科学家利用来源于大肠杆菌噬菌体N15的原核端粒酶TelN发明了一种生产线性闭合DNA(Linear closed DNA,LcDNA)的方法。LcDNA只携带目的基因和两端的TelN识别位点,有效去除了质粒上的原核复制子和抗性基因等不需要递送进细胞的序列。相比于常规的质粒载体,LcDNA在体外或体内表达量相当,甚至更好,并且更安全,转染效率更高。
LcDNA小规模生产并不难,但是在GMP条件下大规模工业化生产面临了诸多难题,例如生产LcDNA的环状质粒骨架太大且不通用;大规模酶切的不稳定性;纯度达不到临床级别,还可能引入Triton,异丙醇和乙醇等有机试剂,残留风险高;聚合酶Phi29体外扩增的保真性相比于E.coli体内复制更低,可能产生不必要的突变等。
发明内容
本发明涉及质粒,其包含两个同向串联的原核端粒酶靶序列的编辑区,所述编辑区的两端独立地设置有一个或多个限制性内切酶酶切位点。
在一些实施方案中,上述质粒的长度≤2500bp,或≤2300bp,或≤2100bp。在一些优选实施方案中,上述质粒的长度≤2100bp。
在一些实施方案中,上述质粒还包含原核复制子和筛选基因。
在一些实施方案中,所述每一端的限制性内切酶酶切位点选自以下任意一种或多种:AccI、AflII、AgeI、ApaI、AscI、AvrII、BamHI、BglI、BglII、BsaI、BspQI、BstBI、BsteII、ClaI、EcoNI、EcoRI、EcoRV、FseI、HindIII、KpnI、MfeI、MluI、NcoI、NdeI、NheI、NotI、PacI、PstI、PvuI、PvuII、SacI、SacII、SalI、ScaI、SmaI、SpeI、StuI、SwaI、XbaI、XhoI和XmaI的酶切位点,优选地选自HindIII、SalI、SmaI、BamHI、EcoRV、XbaI、KpnI和EcoRI的酶切位点中的任意一种或多种。在一些优选实施方案中,
在所述编辑区一端附近插入HindIII、SalI、SmaI和BamHI酶切位点,所述编辑区另一端附近插入EcoRV、XbaI、KpnI和EcoRI酶切位点。
在一些实施方案中,所述原核端粒酶靶序列包含SEQ ID NO:1所示的序列,或与SEQ ID NO:1所示序列具有至少80%、至少85%、至少90%、至少93%、至少95%、至少97%、至少99%的一致性且能发挥端粒酶功能的核苷酸序列。在一些优选实施方案中,所述原核端粒酶靶序列如SEQ ID NO:1所示。
在一些实施方案中,上述质粒包含与SEQ ID NO:2所示序列具有至少80%、至少85%、至少90%、至少93%、至少95%、至少97%、至少99%一致性的核苷酸序列。在一些优选实施方案中,上述质粒的序列如SEQ ID NO:2所示。
本发明还涉及重组质粒,其在如上所述的质粒的两个原核端粒酶靶序列之间插入有目的基因。
本发明还涉及一种线性闭合DNA的制备方法,包括:
a)使用原核端粒酶酶切包含目的基因的重组质粒,以使所述目的基因和重组质粒中的杂核酸分离;
b)使用至少两种限制性内切酶酶切,以使得所述杂核酸的末端暴露,并采用核酸外切酶将所述杂核酸去除;
c)分离纯化含目的基因的线性闭合DNA;
所述重组质粒包含原核端粒酶靶序列和限制性内切酶酶切位点,所述原核端粒酶靶序列之间插入有目的基因。
在一些实施方案中,上述制备方法中提及的重组质粒可以是如上所述的质粒的两个原核端粒酶靶序列之间插入有目的基因的质粒。
在一些实施方案中,上述方法中所述核酸外切酶包括Exonuclease III和/或T5 Exonuclease。在一些实施方案中,所述核酸外切酶的添加量为
3000U/mg~6000U/mg。在一些具体实施方案中,所述核酸外切酶的酶切温度为34℃~40℃,酶切时间为2~12小时;优选地,所述酶切时间为3~6小时。在另一些具体实施方案中,在核酸外切酶酶切后,加入终浓度1mM~50mM EDTA并升温至70℃~80℃处理5~30分钟。在一些具体实施方案中,在原核端粒酶酶切结束后和/或限制性内切酶酶切结束后,升温至70℃~80℃处理5~30分钟。
在一些实施方案中,上述制备方法中所述分离纯化的方法包括凝胶过滤层析、阴离子层析、浓缩换液以及除菌过滤中的一种,或其组合。在一些优选的实施方案中,所述分离纯化的方法依次包括凝胶过滤层析和阴离子层析。
在一些具体实施方案中,所述凝胶过滤层析的填料为Bestarose 6FF,收集第一个紫外峰的≥50mAu洗脱液。
在另一些具体实施方案中,所述阴离子层析的填料为Fractogel EMD DEAE。在一些优选实施方案中,所述阴离子层析的上样条件包括:使用含30%~70%层析液B的上样液,上样载量0.2mg/mL~1.0mg/mL,上样流速≤120cm/h;所述层析液B包含0.5M~1.5M NaCl,5mM~15mM EDTA以及缓冲物质,pH为7.2~7.8。在一些优选实施方案中,所述阴离子层析的洗脱条件包括:65%~100%层析液B洗脱,洗脱流速30cm/h~120cm/h,收集≥100mAu洗脱液。
本发明还涉及试剂盒,其包含原核端粒酶以及如上所述的质粒;
或包含原核端粒酶以及如上所述的重组质粒。
在一些实施方案中,所述试剂盒还包含限制性内切酶和/或核酸外切酶,所述限制性内切酶能够识别所述限制性内切酶酶切位点。
本发明还提供了上述制备方法制备得到的线性闭合DNA。
本发明采用细菌发酵生产的质粒作为起始原料,不是通过Phi29等聚合酶扩增的方法获取,保真性更高;并且,通过特定设计,该质粒能够保持
足够小的大小,从而从源头上提升LcDNA生产的总收率。
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为LcDNA生产纯化工艺流程示意图;
图2A为通用载体骨架CFL Universal图谱;
图2B为图2A的载体插入目的序列(GOI)的全序列图谱;其中,Kan promoter:卡那霉素基因启动子,KanR:卡那霉素抗性基因,pUC origin:pUC启动子,MCS:多克隆位点;GOI:目标序列;telR和telL:原核端粒酶靶序列;
图3为LcDNA琼脂糖凝胶电泳(AGE)检测,A和B为一批60mg含目的序列的环状质粒酶切纯化的LcDNA中间样品及成品AGE检测图谱,C为另一批60mg含目的序列的环状质粒酶切纯化的LcDNA中间样品及成品AGE检测图谱;
在A~C中,泳道M1:超螺旋DNA Ladder;泳道M2:1kb DNA Ladder;
A:泳道1:TelN酶切产物;泳道2:外切酶酶切产物;泳道3:GF层析产物;泳道4:AEX层析产物;
B:泳道1:LcDNA成品;泳道2:1×TAE缓冲液;
C:泳道1:TelN酶切产物;泳道2:外切酶酶切产物;泳道3:GF层析产物;泳道4:AEX层析产物;泳道5:LcDNA成品;
图4A~图4E为LcDNA凝胶过滤层析图谱;其中图4A和4B分别为两批60mg含目的序列的环状质粒酶切后LcDNA凝胶过滤层析图谱,图4C为一批100mg含目的序列的环状质粒酶切LcDNA凝胶过滤层析图谱,图4D和4E两批150mg含目的序列的环状质粒酶切后LcDNA凝胶层析图谱;
图5A~图5E为LcDNA阴离子层析图谱,图5A和5B分别为两批60mg含目的序列的环状质粒LcDNA阴离子层析图谱,图5C为一批100mg含目的序列的环状质粒LcDNA阴离子层析图谱,图5D和5E分别为两批150mg含目的序列的环状质粒LcDNA阴离子层析图谱;
图6A~图6B为LcDNA阴离子层析上样条件优化的图谱,图6A为含30%层析液B的上样液,图6B为含70%层析液B的上样液;
图7A~图7B为LcDNA阴离子层析上样载量优化的图谱,图7A的上样载量为0.25mg/mL,图7B的上样载量为1.0mg/mL;
图8为LcDNA阴离子层析线性洗脱(0-100%层析液B梯度洗脱),其中67.5%层析液B洗脱时出现最大紫外峰的图谱;
在图4~图8中,曲线a均代表电导率,曲线b均代表紫外峰。
现将详细地提供本发明实施方式的参考,其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上,对本领域技术人员而言,显而易见的是,可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如,作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中,来产生更进一步的实施方式。
除非另有说明,用于披露本发明的所有术语(包括技术和科学术语)的意义与本发明所属领域普通技术人员所通常理解的相同。通过进一步的指导,随后的定义用于更好地理解本发明的教导。本文中在本发明的说明书中所
使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。
本文所使用的术语“和/或”、“或/和”、“及/或”的选择范围包括两个或两个以上相关所列项目中任一个项目,也包括相关所列项目的任意的和所有的组合,所述任意的和所有的组合包括任意的两个相关所列项目、任意的更多个相关所列项目、或者全部相关所列项目的组合。需要说明的是,当用至少两个选自“和/或”、“或/和”、“及/或”的连词组合连接至少三个项目时,应当理解,在本申请中,该技术方案毫无疑问地包括均用“逻辑与”连接的技术方案,还毫无疑问地包括均用“逻辑或”连接的技术方案。比如,“A及/或B”包括A、B和A+B三种并列方案。又比如,“A,及/或,B,及/或,C,及/或,D”的技术方案,包括A、B、C、D中任一项(也即均用“逻辑或”连接的技术方案),也包括A、B、C、D的任意的和所有的组合,也即包括A、B、C、D中任两项或任三项的组合,还包括A、B、C、D的四项组合(也即均用“逻辑与”连接的技术方案)。
本发明中所使用的术语“含有”、“包含”和“包括”是同义词,其是包容性或开放式的,不排除额外的、未被引述的成员、元素或方法步骤。
本发明中用端点表示的数值范围包括该范围内所包含的所有数值及分数,以及所引述的端点。
本发明中涉及浓度数值,其含义包括在一定范围内的波动。比如,可以在相应的精度范围内波动。比如2%,可以允许±0.1%范围内波动。对于数值较大或无需过于精细控制的数值,还允许其含义包括更大波动。比如100mM,可以允许±1%、±2%、±5%等范围内的波动。涉及分子量,允许其含义包括±10%的波动。
本发明中,涉及“多个”、“多种”等描述,如无特别限定,指在数量上指大于等于2。
本发明中,以开放式描述的技术特征中,包括所列举特征组成的封闭式技术方案,也包括包含所列举特征的开放式技术方案。
本发明中,“优选”、“更好”、“更佳”、“为宜”仅为描述效果更好的实施方式或实施例,应当理解,并不构成对本发明保护范围的限制。本发明中,“可选地”、“可选的”、“可选”,指可有可无,也即指选自“有”或“无”两种并列方案中的任一种。如果一个技术方案中出现多处“可选”,如无特别说明,且无矛盾之处或相互制约关系,则每项“可选”各自独立。
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。除非和本申请的发明目的和/或技术方案相冲突,否则,本发明涉及的引用文献以全部内容、全部目的被引用。本发明中涉及引用文献时,相关技术特征、术语、名词、短语等在引用文献中的定义也一并被引用。本发明中涉及引用文献时,被引用的相关技术特征的举例、优选方式也可作为参考纳入本申请中,但以能够实施本发明为限。应当理解,当引用内容与本申请中的描述相冲突时,以本申请为准或者适应性地根据本申请的描述进行修正。
本发明的第一方面涉及一种质粒,其包含两个同向串联的原核端粒酶靶序列的编辑区,所述编辑区的两端独立地设置有一个或多个限制性内切酶酶切位点。
在一些实施方式中,所述质粒的长度≤2500bp,例如2400bp、2300bp、2200bp、2100bp、2050bp、2000bp、1950bp、1900bp、1850bp、1800bp或更小。
在一些实施方式中,所述质粒还包含原核复制子和筛选基因。
筛选基因典型的例如抗生素抗性基因,例如卡那霉素抗性基因(KanR)、四环素抗性基因(tetR)、新霉素磷酸转移酶基因(npt)等。筛选基因也可以是某些化合物解毒酶基因、糖类代谢酶选择标记基因等。
在一些实施方式中,每一端的限制性内切酶酶切位点的数量独立地选自1、2、3、4、5、6个或更多个,且优选位于同一端的限制性内切酶酶切位点种类均不同。
在更为优选的实施方式中,位于编辑区同一端的限制性内切酶酶切位点之间发生重叠。
在更为优选的实施方式中,所有的限制性内切酶酶切位点均不同。
所述每一端的限制性内切酶酶切位点可以选自以下任意一种或多种:AccI、AflII、AgeI、ApaI、AscI、AvrII、BamHI、BglI、BglII、BsaI、BspQI、BstBI、BsteII、ClaI、EcoNI、EcoRI、EcoRV、FseI、HindIII、KpnI、MfeI、MluI、NcoI、NdeI、NheI、NotI、PacI、PstI、PvuI、PvuII、SacI、SacII、SalI、ScaI、SmaI、SpeI、StuI、SwaI、XbaI、XhoI和XmaI的酶切位点,优选地选自HindIII、SalI、SmaI、BamHI、EcoRV、XbaI、KpnI和EcoRI的酶切位点中的任意一种或多种。在一个具体的实施方式中,在编辑区一端附近插入HindIII、SalI、SmaI和BamHI识别位点,编辑区另一端附近插入EcoRV、XbaI、KpnI和EcoRI识别位点,从而可提供16组可供选择的酶切组合。如此实用性和广谱性更好。
根据本发明,通过原核端粒酶对从包含至少一个原核端粒酶靶序列的DNA模板扩增的DNA的作用生成闭合线状DNA分子。原核端粒酶靶序列是存在于DNA模板中使得能够通过原核端粒酶的酶活性将其转化成闭合线状DNA的任何DNA序列。换言之,原核端粒酶靶序列对于通过原核端粒酶裂解并再连接双链DNA以形成共价闭合线状DNA是必需的。
原核端粒酶及其靶序列为本领域技术人员所公知,其描述例如2011-12-28所公开的CN102301010A中所描述的原核端粒酶及其靶序列。典型地,原核端粒酶靶序列包括任何完整回文序列,即任何具有双重旋转对称的双链DNA序列。在伯氏疏螺旋体中,完整反转重复序列的长度为14个碱基对。在各种常温噬菌体中,完整反转重复序列的长度为22个碱基对或更长。此外,在一些情况下,例如大肠杆菌N15,中心完整反转回文序列侧接反转重复顺序,即形成了更大的不完整反转回文序列的一部分。在一些具体实施方式中,所述原核端粒酶靶序列来源于大肠杆菌(E.coli)N15噬菌体、克雷伯氏菌(Klebsiella)Phi K02噬菌体,耶尔森式菌(Yersinia)
Py54噬菌体,盐单胞菌(Halomonas)Phi HAP噬菌体,弧菌(Vibrio)VP882噬菌体或伯氏疏螺旋体(Borrelia burgdorferi)lpB31.16,或任其一的变异体。优选所述原核端粒酶靶序列来源于噬菌体N15 TelN或其变异体。在一些具体实施方式中,所述原核端粒酶靶序列包含SEQ ID NO:1所示的序列,或与SEQ ID NO:1所示序列具有至少80%一致性且能发挥端粒酶功能的核苷酸序列,例如至少85%、90%、95%、96%、97%、98%、99%一致性的核苷酸序列,例如发生一个或若干个核苷酸的截短、插入、替换和/或缺失。
所述质粒可由常规的克隆载体改造得到,如原核载体,优选为大肠杆菌质粒载体,更优选为小于2000bp的质粒。在一些具体的实施方式中,其为pUC57-Kan-mini(1824bp)改造得到。在一些实施方式中,所述质粒包含与SEQ ID NO:2所示序列具有至少80%一致性的核苷酸序列,例如至少85%、90%、95%、96%、97%、98%、99%一致性的核苷酸序列,例如发生一个或若干个核苷酸的截短、插入、替换和/或缺失。在一些具体实施方案中,所述质粒包含SEQ ID NO:2所示的核苷酸序列。
所述编辑区中,在一些实施方式中,两个同向串联的原核端粒酶靶序列直接相连;在一些实施方式中,两个同向串联的原核端粒酶靶序列之间还包含少于30bp,或少于20bp,少于10bp的核苷酸序列,这些核苷酸序列优选为利于目的基因的插入的位点或相关序列。
本发明的第二方面涉及重组质粒,其在如上所述的质粒的两个原核端粒酶靶序列之间插入有目的基因。
在一些实施方式中,所述目的基因的表达产物为干扰RNA或适配体。
干扰RNA可以选自例如siRNA、shRNA或miRNA。
在一些实施方式中,所述目的基因的表达产物为mRNA,此类mRNA优选能表达蛋白质,例如,向靶细胞赋予某些所需特征的蛋白质,如允许细胞示踪的荧光蛋白、提供靶细胞中丢失或改变的活性酶,等等。
所述目的基因可包括例如编码受体个体或靶细胞中有缺陷或缺少的蛋白质的基因(核苷酸序列);编码具有所需生物或治疗效果(例如,抗细菌、
抗病毒或抗肿瘤/抗癌功能)的蛋白质的基因;编码抑制或减少有害或另外不希望有的蛋白质的产生的RNA的核苷酸序列(例如,编码如上所定义的RNA干扰剂的核苷酸序列);和/或编码抗原蛋白的核苷酸序列。
适合的目的基因包括但不限于编码用于治疗以下疾病的蛋白质的核酸:内分泌、代谢、血液、心血管、神经、肌肉骨骼、泌尿、肺和免疫病症,包括如癌症、炎性病症、免疫病症、慢性和感染性病症。癌症例如骨、骨连接、肌肉、肺、气管、心脏、脾脏、动脉、静脉、血液、毛细血管、淋巴结、淋巴管、淋巴液、口腔、咽、食管、胃、十二指肠、小肠、结肠、直肠、肛门、阑尾、肝、胆、胰腺、腮腺、舌下腺、泌尿肾、输尿管、膀胱、尿道、卵巢、输卵管、子宫、阴道、外阴部、阴囊、睾丸、输精管、阴茎、眼、耳、鼻、舌、皮肤、脑、脑干、延髓、脊髓、脑脊液、神经、甲状腺、甲状旁腺、肾上腺、垂体、松果体、胰岛、胸腺、性腺、舌下腺以及腮腺中任一处病变生成的肿瘤;免疫病症例如系统红斑狼疮、多发性硬化症、I型糖尿病、银屑病、溃疡性结肠炎、Sjogren综合征、硬皮病、多肌炎、类风湿关节炎、混合性结缔组织病、原发性胆汁性肝硬变、自身免疫性溶血性贫血、桥本甲状腺炎、Addisons病、白斑、Graves病、自身免疫性重症肌无力、强直性脊柱炎、变应性骨关节炎、变应性血管炎、自身免疫性噬中性白细胞减少症、特发性血小板减少性紫癜、狼疮性肾炎、慢性萎縮性胃炎、自身免疫性不育、子宫内膜异位症、Pasture病、天疱疮、盘状狼疮或致密沉积物疾病;感染性病症可以为病毒、细菌、真菌和寄生虫中的任一种感染,或其联合感染。
适合的目的基因包括但不限于编码以下多种蛋白质中的至少一种:干扰素(例如,IFN-γ、IFN-α、IFN-β、IFN-ω、IFN-τ);胰岛素(例如,诺和灵、优泌林、优泌乐、来得时(Humalog)、优乐停(Lantus)等);促红细胞生成素(“EPO”;例如,依泊汀-α、达贝泊汀-α、依泊汀-β;等等);抗体(例如,单克隆抗体,如,利妥昔单抗、英夫利昔单抗、曲妥单抗、HumiraTM(阿达木
单抗)、奥马佐单抗、托西莫单抗、RaptivaTM(依法珠单抗)、ErbituxTM(西妥昔单抗)、贝伐单抗;等等);包括单克隆抗体的抗原结合片段(例如,雷珠单抗);血液因子(例如,阿替普酶、组织纤溶酶原活化蛋白、重组人因子VIIa、因子VIIa、因子VIII、因子IX、β-球蛋白、血红蛋白;等等);集落刺激因子(例如,(非格司亭;G-CSF)、Neulasta(聚乙二醇化非格司亭)、粒细胞集落刺激因子(G-CSF)、粒细胞单核细胞集落刺激因子、巨噬细胞集落刺激因子、巨核细胞集落刺激因子;等等);生长激素(例如,生长激素(somatotropin)、生长激素;等等);白介素(例如,IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9;等);生长因子(例如,(beclapermin;PDGF)、(曲弗明;bFGF)、(安西司亭;干细胞因子)、角质细胞生长因子、酸性成纤维细胞生长因子、干细胞因子、碱性成纤维细胞生长因子、肝细胞生长因子;等等);可溶性受体(例如,TNF-α结合可溶性受体,如(依那西普)、可溶性VEGF受体、可溶性白介素受体、可溶性γ/δT细胞受体;等等);酶(例如,α-葡糖苷酶、β-葡糖脑苷脂酶、阿糖苷酶)、酶激活剂(例如,组织型纤溶酶原激活剂);趋化因子(例如,IP-10、Mig、Groα/IL-8、RANTES、MIP-1α、MIP-1β、MCP-1、PF-4;等等);血管生成剂(例如,血管内皮生长因子(VEGF);抗血管生成剂(例如,可溶性VEGF受体);蛋白疫苗;神经活性肽,如血管舒缓激肽、缩胆囊肽、胃泌素、分泌素、催产素、促性腺激素释放激素、β-内啡肽、脑啡肽、P物质、生长激素释放抑制因子、催乳素、甘丙肽、生长激素释放激素、韩蛙皮素、强啡肽、神经降压素、胃动素、促甲状腺激素、神经肽Y、黄体生成素、降血钙素、胰岛素、胰高血糖素、加压素、血管紧张素II、促甲状腺激素释放激素、血管活性肠肽、睡眠肽等;其他蛋白,如溶解血栓剂、心钠肽、骨形态发生蛋白、促血小板生成素、松弛素、胶质细胞原纤维酸性蛋白、促卵泡激素、人α-1抗胰蛋白酶、白血病抑制因子、转化生长因子、胰岛素样生长因子、黄体生成素、巨噬细胞活化因子、肿瘤坏死因子、嗜中性白细胞趋化因子、神经生长因子、金属蛋白酶的组织
抑制剂;血管活性肠肽、血管生成素、促血管素、纤维蛋白;水蛭素;白血病抑制因子;IL-1受体拮抗剂(例如,(阿那白滞素));离子通道,例如,囊性纤维化跨膜传导调节蛋白(CFTR);肌营养不良蛋白(dystrophin);utrophin(一种肿瘤抑制剂);溶酶体酶酸α-葡糖苷酶(GAA);等等。适合的目的基因还包括编码任何上述蛋白的功能性片段的那些;以及编码任何上述蛋白的功能性变体的核酸。
在一些实施方式中,目的基因可以表达盒的形式插入所述质粒。
容易理解,所述质粒/重组质粒还可以包含如下元件中的至少一种:启动子、增强子、内部核糖体进入位点、报告基因、偶联标记基因和转录终止信号。这些元件优选位于所述的目的基因的表达盒中。
启动子为起始并调节多聚核苷酸转录的核苷酸序列。启动子包括诱导型启动子(与该启动子可操作地连接的多聚核苷酸序列的表达受到分析物、辅因子、调节蛋白等的诱导)、阻抑启动子(与该启动子可操作地连接的多聚核苷酸序列的表达受到分析物、辅因子、调节蛋白等的阻抑)和组成型启动子。术语“启动子”意图包括全长启动子区和这些区的功能性(例如控制转录或翻译)片段。启动子优选包括始于原核表达的启动子。但当存在目的基因时,亦可包含与用于控制目的基因表达的启动子,可以为无细胞特异性启动子,或者可根据目的基因的产物期望的施用位置,可为“细胞特异性”、“细胞类型特异性”、“细胞谱系特异性”或“组织特异性”启动子,其允许分别在种类受限的细胞和组织类型中表达。
报告基因典型的例如荧光蛋白基因、荧光素酶基因以及LacZ。荧光蛋白和荧光素酶都属于发光蛋白,可用照相机或类似的装置来检测荧光表达。荧光蛋白通过吸收一种颜色的光(激发),然后发出不同颜色(发射)的较低能量光来起作用。相反,荧光素酶(和其他生物发光酶)通过催化底物(即荧光素)发生化学反应而发光。与上述两种标记不同,LacZ不发光。LacZ基因的产物β-半乳糖苷酶可以催化X-gal转化成类似于靛蓝的不透明蓝色化合物。其中荧光蛋白例如绿色荧光蛋白、蓝色荧光蛋白、黄色荧光
蛋白、橙色荧光蛋白或红色荧光蛋白。绿色荧光蛋白可以采用常见的GFP,也可以采用经过改造后的GFP基因,例如增强型GFP基因EGFP等;蓝色荧光蛋白可以选自EBFP、Azuritc、TagBFP等;黄色荧光蛋白可以选自EYFP、Ypct、PhiYFP等;橙色荧光蛋白可以选自mKO、mOrange、mBanana等;红色荧光蛋白可以选自TagRFP、mRuby、mCherry、mKatc。
偶联标记基因典型的例如能够表达生物素或其衍生物的基因,其中生物素的衍生物为D-生物素、活化生物素、生物胞素、乙二胺生物素、尸胺生物素以及脱硫生物素中的任一种。
内部核糖体进入位点(IRES)是一个长数百碱基对(bp)的基因工程元件。IRES最初发现于病毒中,后来发现一部分真核细胞的mRNA上也带有IRES元件。IRES能引导核糖体进入该序列,转译3'方向的开放阅读框。将IRES序列插入同一转录本下的两个开放阅读框(ORF)中间,可以使一个转录本转译产生两个独立的蛋白质。
转录终止信号指转录过程产生RNA的一段序列所形成的茎-环结构,可特异性地被RNA聚合酶转录复合体识别而使转录终止。
在一些实施方式中,所述目的基因为核酸疫苗。
本发明的第三方面涉及线性闭合DNA的制备方法,包括:
a)使用原核端粒酶酶切包含目的基因的重组质粒,以使所述目的基因和重组质粒中的杂核酸分离;
b)使用至少两种限制性内切酶酶切,以使得所述杂核酸的末端暴露,并采用核酸外切酶将所述杂核酸去除;
c)分离纯化含目的基因的线性闭合DNA;
所述重组质粒包含原核端粒酶靶序列和限制性内切酶酶切位点,所述原核端粒酶靶序列之间插入有目的基因。
杂核酸是指除目的基因之外的不必要的或额外的序列。这些不必要的
或额外的序列典型地包括如上所述是质粒中的各种元件,例如复制子、筛选基因等。但容易理解,本发明中的杂核酸是指上述不必要的或额外的序列的绝大部分但并非全部,例如,至少与目的基因直接相连的原核端粒酶靶序列(如telL、telR)是无法被除去的。
在一些实施方式中,所述重组质粒为如上所定义的重组质粒,此时本发明第一方面和第二方面所定义的内容也适用于与第三方面。
在一些实施方式中,所述原核端粒酶添加量为8000U/mg~15000U/mg,例如9000U/mg、10000U/mg、11000U/mg、12000U/mg、13000U/mg、14000U/mg。
在一些实施方式中,所述原核端粒酶的酶切温度为25℃~35℃,优选27℃~33℃。
在一些实施方式中,所述原核端粒酶的酶切时间为1~8小时,例如2、3、4、5、6、7小时。
在一些实施方式中,每种限制性内切酶的添加量独立地选自1000U/mg~6000U/mg,例如2000U/mg、3000U/mg、4000U/mg、5000U/mg。
在一些实施方式中,所述限制性内切酶的酶切温度为32℃~42℃,优选34℃~40℃,如35℃、36℃、37℃、38℃、39℃。
在一些实施方式中,所述限制性内切酶的酶切时间为1~4小时,例如2小时、3小时。
在一些实施方式中,所述核酸外切酶包括Exonuclease III和/或T5 Exonuclease。
在一些实施方式中,所述核酸外切酶的添加量为3000U/mg~6000U/mg,例如3500U/mg、4000U/mg、4500U/mg、5000U/mg、5500U/mg。
在一些实施方式中,所述核酸外切酶的酶切温度为34℃~40℃,例如35℃、36℃、37℃、38℃、39℃。
在一些实施方式中,所述核酸外切酶的酶切时间为2~12小时,例如3、4、5、6、7、8、9、10、11小时,较为优选的为3~6小时。
在一些实施方式中,在核酸外切酶酶切后,加入终浓度1mM~50mM EDTA,例如5mM、10mM、15mM、20mM、25mM、30mM、35mM、40mM、45mM。
在一些实施方式中,在核酸外切酶酶切后,加入EDTA并升温至70℃~80℃处理5~30分钟,例如10、15、20、25分钟。
在核酸外切酶酶切后同时采用EDTA和高温两种方式灭酶,有效抑制体系中内切酶和外切酶残留的活性,大大提高大规模生产的稳定性。
在一些实施方式中,在原核端粒酶酶切结束后和/或限制性内切酶酶切结束后,升温至70℃~80℃处理5~30分钟,例如10、15、20、25分钟。
在一些实施方式中,所述分离纯化的方法包括凝胶过滤层析、阴离子层析、浓缩换液以及除菌过滤中的一种,或其组合。
在一些实施方式中,所述分离纯化的方法依次包括凝胶过滤层析和阴离子层析。在另一些实施方式中,所述分离纯化的方法依次包括凝胶过滤层析、阴离子层析和浓缩换液。
采用凝胶过滤层析和阴离子层析相结合的工艺纯化LcDNA,既提高了LcDNA产品的纯度和质量,也更合规;此外,该方法能够在保证纯度的前提下还显著提高产品收率,因而更加适用于商业化用途。
在一些实施方式中,所述凝胶过滤层析的填料为Bestarose 6FF,收集第一个紫外峰的≥50mAu洗脱液。
在一些实施方式中,所述阴离子层析的填料为Fractogel EMD DEAE。
在一些实施方式中,所述阴离子层析的上样条件包括:
使用含30%~70%层析液B的上样液(例如40%、50%、60%,为体积百分数),上样载量0.20mg/mL~1.0mg/mL(例如0.25mg/mL、0.3mg/mL、
0.35mg/mL、0.4mg/mL、0.45mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL),上样流速≤120cm/h;
所述层析液B包含0.5M~1.5M NaCl,5mM~15mM EDTA以及缓冲物质,pH为7.2~7.8。
如本文使用的术语“缓冲物质”指水溶液或组合物,当酸或碱加入该溶液或组合物中时,所述水溶液或组合物抵抗pH中的变化。这种对pH变化的抗性是由于此类溶液的缓冲性质。因此,显示出缓冲活性的溶液或组合物被称为缓冲液或缓冲溶液。可以在本发明的方法中使用的缓冲液优选选自磷酸盐缓冲液、磷酸盐缓冲盐水缓冲液(PBS)、2-氨基-2羟甲基-1,3-丙二醇(TRIS)缓冲液、TRIS缓冲盐水溶液(TBS)和TRIS/EDTA(TE)等。
在一些优选实施方式中,所述阴离子层析的上样条件包括:
使用含50%层析液B的上样液(体积百分数),上样载量0.20mg/mL~1.0mg/mL(例如0.25mg/mL、0.3mg/mL、0.35mg/mL、0.4mg/mL、0.45mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL),上样流速≤120cm/h;
所述层析液B包含0.5M~1.5M NaCl,5mM~15mM EDTA以及50mM~150mM Tris-HCl,pH为7.2~7.8。
更优选地,所述层析液B包含1M NaCl,10mM EDTA,100mM Tris-HCl,pH为7.5。
在一些实施方式中,所述阴离子层析的洗脱条件包括:
65%~100%层析液B洗脱(例如70%、80%、90%,为体积百分数),洗脱流速30cm/h~120cm/h(例如40cm/h、50cm/h、60cm/h、70cm/h、80cm/h、90cm/h、100cm/h、110cm/h),收集≥100mAu洗脱液。
在一些优选实施方式中,所述阴离子层析的洗脱条件包括:
80%层析液B洗脱(体积百分数),洗脱流速30cm/h~120cm/h(例如
40cm/h、50cm/h、60cm/h、70cm/h、80cm/h、90cm/h、100cm/h、110cm/h),收集≥100mAu洗脱液。
在一些实施方式中,所述浓缩换液的方法为切向流过滤。
本发明的第四方面涉及试剂盒,其包含原核端粒酶以及如上所述的质粒;
或包含原核端粒酶以及如上所述的重组质粒。
在一些实施方式中,试剂盒还包含限制性内切酶和/或核酸外切酶,所述限制性内切酶能够识别所述限制性内切酶酶切位点。
试剂盒中还可包含无核酸水、使用说明书等常规组分。
本发明第五方面提供上述制备方法得到的线性闭合DNA。所述线性闭合DNA包含目的基因。
本发明还提供组合物,所述组合物包含上述线性闭合DNA。在一些实施方案中,所述组合物中包含纯度至少90%的上述线性闭合DNA。在一些优选实施方案中,所述组合物中包含纯度至少99%的上述线性闭合DNA。
本发明第一、第二方面的描述也适用于第四方面、第五方面。
下面将结合实施例对本发明的实施方案进行详细描述。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,优先参考本发明中给出的指引,还可以按照本领域的实验手册或常规条件,还可以参考本领域已知的其它实验方法,或者按照制造厂商所建议的条件。
下述的具体实施例中,涉及原料组分的量度参数,如无特别说明,可能存在称量精度范围内的细微偏差。涉及温度和时间参数,允许仪器测试精度或操作精度导致的可接受的偏差。
实施例1 LcDNA生产的环状质粒通用型载体CFL universal的设计
通过设计通用型LcDNA模板质粒骨架CFL universal,使模板质粒骨架的大小最小化,从源头上提升LcDNA的理论回收率。具体设计方法是先在pUC57-Kan-mini(1824bp)上连续插入两个TelN酶识别序列TelRL(56bp,序列信息见SEQ ID NO:1),然后在一端原核端粒酶TelN识别位点附近依次插入HindIII、SalI、SmaI和BamHI识别位点,另一端TelN识别位点附近插入EcoRV、XbaI、KpnI和EcoRI识别位点,为第二步限制性内切酶酶切提供16组可供选择的酶切组合,大大提高CFL Universal的实用性和广谱性。改造后的CFL Universal长度为2017bp。待插入的目的序列只需要设计在CFL universal的TelN两个识别位点TelRL之间即可。图谱见图2A(序列信息如SEQ ID NO:2所示),其中插入GOI目的基因作为示例,长度3169bp,见图2B。设计好的环状质粒序列委托南京金斯瑞生物科技有限公司进行合规性合成,然后依次经过大肠杆菌菌库构建、高密度发酵、细胞裂解、澄清过滤、浓缩孵育、层析纯化、浓缩换液、除菌过滤和灌装,获得的高纯度高质量的环状质粒作为LcDNA生产的起始材料。
实施例2 LcDNA酶切和纯化
(1)LcDNA酶切
LcDNA的三步酶切依次为TelN酶切,外切酶酶切和内切酶酶切。当选择CFL universal载体作为起始材料环状质粒的骨架时,TelN酶切工艺参数为:TelN酶添加量10000U/mg,酶切温度30℃,酶切时间3小时,酶切终止温度75℃,处理时间15分钟。内切酶酶切工艺参数为:根据插入的目的序列选择没有相应识别位点的内切酶,如HindIII、SalI、SmaI、BamHI、EcoRV、XbaI、KpnI和EcoRI等,加入两种内切酶,每种内切酶添加量3000U/mg,酶切温度37℃,酶切时间2小时,酶切终止温度75℃,处理时间15分钟。外切酶酶切工艺参数为:外切酶Exo III或T5 Exo,添加量4500U/mg,酶切温度37℃,酶切时间4小时。酶切终止时,先加入终浓度15mM EDTA,在75℃,处理时间10-20分钟。
(2)LcDNA凝胶过滤层析(gel filtration chromatography,GF)
LcDNA三步酶切之后,离心或过滤处理酶切反应液,然后进行凝胶过滤层析。凝胶过滤层析工艺参数为:填料Bestarose 6FF,层析柱高≥20cm,对称因子0.8~1.8,塔板数>2000。上样体积≤0.3柱体积,上样流速60cm/h,洗脱流速≤120cm/h,平衡和洗脱缓冲液GF Buffer A(成分0.5M NaCl,5mM EDTA,50mM Tris-HCl)收集第一个紫外峰的≥50mAu洗脱液。
(3)LcDNA阴离子层析(anion exchange chromatography,AEX)
凝胶过滤层析之后,收集的洗脱液,进行阴离子层析。阴离子层析工艺参数为:填料Fractogel EMD DEAE,层析柱高≤20cm,对称因子0.8~1.8,塔板数>2000。上样条件含30-70%层析液B(体积比)的上样液,上样载量0.25-1.0mg/mL,上样流速≤120cm/h,保留时间≥6分钟。洗脱流速30-120cm/h,收集≥100mAu洗脱液。层析液A成分10mM EDTA,100mM Tris-HCl,pH7.5,层析液B成分1M NaCl,10mM EDTA,100mM Tris-HCl,pH7.5。100%层析液A平衡,58%层析液B洗杂,65%-100%层析液B洗脱。对于洗脱条件的优化,如图8所示,层析液B洗脱从0-100%洗脱梯度洗脱,其中67.5%层析液B洗脱时出现最大紫外峰的图谱。80%层析液B洗脱时,阴离子层析工艺优化数据见图表1A和表1B,相应的洗脱曲线见图6A、图6B、图7A和图7B。
表1 LcDNA阴离子层析优化数据汇总表
表1A 上样条件优化
表1B 上样载量优化
(4)LcDNA浓缩换液
阴离子层析结束后,用切向流过滤的方法对收集的洗脱液进行样品浓
缩,浓缩到1.0mg/mL±0.3mg/mL的透析点,然后用最终保存缓冲液比如Tris-EDTA,换液10±1倍体积。切向流过滤的膜包孔径10kD,规格50cm2,0.01m2,0.1m2,0.5m2,根据生产规模选择合适规格的膜包即可。
(5)LcDNA除菌过滤
浓缩换液之后的LcDNA中间样品,进行除菌过滤操作。除菌滤器孔径0.22μm,规格针头滤器,1L滤杯,囊式滤器。根据浓缩换液之后样品的体积选择合适规格的滤器即可。
(6)LcDNA灌装和保存
除菌过滤之后,对LcDNA样品按项目分装规格在超净工作台或隔离器中进行灌装。灌装完成后的LcDNA成品保存在-70±10℃超低温冰箱中。
实施例3 LcDNA质量检测
示例LcDNA长度为3169bp,插入LcDNA的环状质粒全序列图谱见图2B。对LcDNA中间样品及成品进行质量检测,检测方法和对应的结果如下:
3.1琼脂糖凝胶电泳(AGE)检测LcDNA中间样品及成品
使用0.7%琼脂糖凝胶对LcDNA中间样品及成品进行检测,1×TAE作为电泳缓冲液。电泳电压150V,电泳时间30分钟,电泳结束后,用Image Lab软件拍胶成像。AGE检测结果见图3和表2。
表2 LcDNA生产数据汇总表
备注:总收率=LcDNA成品量/酶切环状质粒样品量×100%
相比于小规模的生产方法,环状质粒经过TelN酶切,琼脂糖凝胶电泳(AGE)分离、切胶,用Axygen胶回收试剂盒(AXYGEN,货号:UE-GX-50)纯化,LcDNA收率低,不足10%,且质量不满足临床或商业化要求。利用能生产LcDNA的工程菌发酵,后续一步Q阴离子层析(NanoGel-50Q,苏州纳微科技股份有限公司,货号:04064-050200-2100)纯化方法,LcDNA收率低,不足10%,且LcDNA纯度不高,不满足临床或商业化要求。
3.2高效液相色谱(IEC-HPLC)检测LcDNA纯度
使用Agilent 1260高效液相色谱仪对LcDNA成品进行纯度检测,检测结果汇总见表3。
3.3 Sanger测序验证LcDNA
根据插入的目的序列设计合适的测序引物,对LcDNA样品进行Sanger全质粒测序验证,测序结果汇总见表3。
3.4 Nanorange荧光检测LcDNA中总蛋白残留
使用NanoOrange试剂盒(ThermoFisher,货号:N10271)检测LcDNA成品中总蛋白残留,用酶标仪读取数值。检测结果汇总见表3。
3.5荧光定量PCR法(qPCR)检测LcDNA中宿主DNA(E.coli源)残留
使用实时荧光定量PCR仪(ThermoFisher,型号:7500)和E.coli残留DNA检测试剂盒(申科生物,货号:SK030202E100)检测LcDNA中宿主DNA(E.coli源)残留,检测结果汇总见表3。
3.6凝胶法(LAL)检测LcDNA中细菌内毒素
使用鲎试剂对LcDNA中细菌内毒素进行凝胶法检测,检测结果汇总见表3。
3.7直接接种法检测LcDNA是否无菌
用一次性无菌注射器或经过灭菌的移液器吸取LcDNA样品,接种至
100mL硫乙醇酸盐FTM培养基中,30-35℃培养14天,检测LcDNA样品是否无菌。检测结果汇总见表3。
表3 LcDNA成品检测结果汇总表
结论
1)此LcDNA生产工艺很稳定。酶切环状质粒的规模从60mg逐步放大到100mg,150mg规模,生产过程未发生异常偏差。此工艺也可以满足大于150mg环状质粒的酶切规模的LcDNA生产。
2)此LcDNA生产工艺总收率高。三种环状质粒酶切规模,共5批次的生产,总收率高达25%-33%。
3)此工艺生产的LcDNA成品纯度和质量都能满足药典的要求,可以用做基因治疗等领域的临床研究和商业化用途。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准,说明书及附图可以用于解释权利要求的内容。
Claims (24)
- 质粒,其包含两个同向串联的原核端粒酶靶序列的编辑区,所述编辑区的两端独立地设置有一个或多个限制性内切酶酶切位点。
- 根据权利要求1所述的质粒,其长度≤2500bp,或≤2300bp,或≤2100bp。
- 根据权利要求1所述的质粒,其还包含原核复制子和筛选基因。
- 根据权利要求3所述的质粒,所述每一端的限制性内切酶酶切位点选自以下任意一种或多种:AccI、AflII、AgeI、ApaI、AscI、AvrII、BamHI、BglI、BglII、BsaI、BspQI、BstBI、BsteII、ClaI、EcoNI、EcoRI、EcoRV、FseI、HindIII、KpnI、MfeI、MluI、NcoI、NdeI、NheI、NotI、PacI、PstI、PvuI、PvuII、SacI、SacII、SalI、ScaI、SmaI、SpeI、StuI、SwaI、XbaI、XhoI和XmaI的酶切位点,优选地选自HindIII、SalI、SmaI、BamHI、EcoRV、XbaI、KpnI和EcoRI的酶切位点中的任意一种或多种。
- 根据权利要求4所述的质粒,在所述编辑区一端附近插入HindIII、SalI、SmaI和BamHI酶切位点,所述编辑区另一端附近插入EcoRV、XbaI、KpnI和EcoRI酶切位点。
- 根据权利要求1~5中任一项所述的质粒,所述原核端粒酶靶序列包含SEQ ID NO:1所示的序列,或与SEQ ID NO:1所示序列具有至少80%一致性且能发挥端粒酶功能的核苷酸序列。
- 根据权利要求1~6中任一项所述的质粒,其包含与SEQ ID NO:2所示序列具有至少80%一致性的核苷酸序列。
- 重组质粒,其在权利要求1~7中任一项所述的质粒的两个原核端粒酶靶序列之间插入有目的基因。
- 线性闭合DNA的制备方法,包括:a)使用原核端粒酶酶切包含目的基因的重组质粒,以使所述目的基因和重组质粒中的杂核酸分离;b)使用至少两种限制性内切酶酶切,以使得所述杂核酸的末端暴露,并采用核酸外切酶将所述杂核酸去除;c)分离纯化含目的基因的线性闭合DNA;所述重组质粒包含原核端粒酶靶序列和限制性内切酶酶切位点,所述原核端粒酶靶序列之间插入有目的基因。
- 根据权利要求9所述的方法,所述重组质粒为权利要求8所述的重组质粒。
- 根据权利要求9或10所述的方法,所述核酸外切酶包括Exonuclease III和/或T5 Exonuclease。
- 根据权利要求11所述的方法,所述核酸外切酶的添加量为3000U/mg~6000U/mg。
- 根据权利要求12所述的方法,所述核酸外切酶的酶切温度为34℃~40℃,酶切时间为2~12小时;优选地,所述酶切时间为3~6小时。
- 根据权利要求9或10所述的方法,在核酸外切酶酶切后,加入终浓度1mM~50mM EDTA并升温至70℃~80℃处理5~30分钟。
- 根据权利要求9~14中任一项所述的方法,在原核端粒酶酶切结束后和/或限制性内切酶酶切结束后,升温至70℃~80℃处理5~30分钟。
- 根据权利要求9~15中任一项所述的方法,所述分离纯化的方法包括凝胶过滤层析、阴离子层析、浓缩换液以及除菌过滤中的一种,或其组合。
- 根据权利要求16所述的方法,所述分离纯化的方法依次包括凝胶过滤层析和阴离子层析。
- 根据权利要求17所述的方法,所述凝胶过滤层析的填料为Bestarose 6FF,收集第一个紫外峰的≥50mAu洗脱液。
- 根据权利要求18所述的方法,所述阴离子层析的填料为Fractogel EMD DEAE。
- 根据权利要求19所述的方法,所述阴离子层析的上样条件包括:使用含30%~70%层析液B的上样液,上样载量0.2mg/mL~1.0mg/mL,上样流速≤120cm/h;所述层析液B包含0.5M~1.5M NaCl,5mM~15mM EDTA以及缓冲物质,pH为7.2~7.8。
- 根据权利要求20所述的方法,所述阴离子层析的洗脱条件包括:65%~100%层析液B洗脱,洗脱流速30cm/h~120cm/h,收集≥100mAu洗脱液。
- 试剂盒,其包含原核端粒酶以及权利要求1~7中任一项所述的质粒;或包含原核端粒酶以及权利要求8所述的重组质粒。
- 根据权利要求22所述的试剂盒,其还包含限制性内切酶和/或核酸外切酶,所述限制性内切酶能够识别所述限制性内切酶酶切位点。
- 根据权利要求9~21中任一项所述的方法制备得到的线性闭合DNA。
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