WO2011105429A1 - Site-specific recombination method and kit - Google Patents

Site-specific recombination method and kit Download PDF

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WO2011105429A1
WO2011105429A1 PCT/JP2011/053990 JP2011053990W WO2011105429A1 WO 2011105429 A1 WO2011105429 A1 WO 2011105429A1 JP 2011053990 W JP2011053990 W JP 2011053990W WO 2011105429 A1 WO2011105429 A1 WO 2011105429A1
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attp
attb
integrase
dna
gene
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高橋　秀夫
森田　健太郎
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学校法人日本大学
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10331—Uses of virus other than therapeutic or vaccine, e.g. disinfectant

Definitions

  • the present invention relates to a site-specific recombination method and kit using integrase.
  • the so-called genome engineering technology that modifies the genome structure of eukaryotes and manipulates their gene expression control is an important method not only for gene therapy but also for breeding various eukaryotes.
  • targeted integration of foreign genes into the eukaryotic genome is particularly important.
  • the integration site is difficult to control, and there is a problem that abnormal gene expression due to random foreign gene incorporation and disruption of important genes are by-produced.
  • Cre recombinase of Escherichia coli phage P1, FLP recombinase of yeast, etc. have been confirmed to cause site-specific DNA recombination and function in eukaryotic cells, but also catalyze excision of the incorporated gene. Therefore, the gene transfer frequency is low as a result, and it is more widely used for creating deletion mutations than gene transfer.
  • integrase of E. coli phage lambda catalyzes site-specific gene integration, but cannot be directly applied to eukaryotic genome modification such as requiring host E. coli factors. Therefore, a recombinase effective for the integration of a foreign gene into the genome is preferably one that catalyzes the integration of a site-specific gene with an enzyme alone, and that cannot excise the incorporated gene with an enzyme alone.
  • a recombinase effective for the gene cloning method is preferably a recombinase that catalyzes site-specific gene incorporation with the enzyme alone, and that the enzyme alone cannot catalyze the excision of the incorporated gene.
  • Serine-type integrase was first discovered by Actinophage R4 by Matsuura et al. And was found to cause specific recombination reactions in Escherichia coli, yeast cells, animal cells, etc. (Matsuura M, Noguchi T, Yamaguchi D, Aida T, Asayama M, Takahashi H, Shirai M. J Bacteriol., 1996 Jun; 178 (11): 3374-6.).
  • Non-Patent Document 11 the integrase produced by actinophage ⁇ C31 is also serine type. Serine-type integrase, unlike tyrosine type, does not require the presence of host factors, and both attB and attP cause site-specific recombination between relatively short sequences of about 40 bp. Use as a tool is expected (Non-Patent Document 11).
  • Patent Document 2 An integrase gene derived from actinophage TG1 (For, F., et al., Gene, 39, 11-16 (1985)) (Patent Document 2).
  • the gene cloning method is generally as follows. 1. Amplify DNA with the gene of interest using PCR. 2. Treat the amplified DNA fragment and the plasmid vector to be inserted with restriction enzyme treatment and modification enzyme treatment, respectively. 3. Mix each DNA fragment and perform a ligase binding reaction (Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • the method using lambda phage integrase has the advantage that the above-mentioned enzyme treatment can be performed at once by site-specific recombination reaction. (Invitrogen http://www.invitrogen.co.jp/gateway/index.shtml).
  • the present inventor has constructed a site-specific recombination system in vitro for several types of actinophage integrase. As a result, it was found that the actinophage TG1 integrase exhibits strong and efficient recombination activity in vitro and in vitro against the corresponding attB / attP sequences.
  • the present inventor provides the following site-specific recombination method and kit for site-specific recombination.
  • Item 1 Recombination between the first DNA sandwiched between two attBs and the second DNA sandwiched between two attPs using integrase, or between the first DNA sandwiched between two attPs and the second DNA sandwiched between two attBs A site-specific recombination method, wherein the integrase is a TG1 phage integrase.
  • Item 2. Item 2. The item 1, wherein the attB and attP are sites including a minimum base sequence that functions as attB (TT), attP (TT), attB (TA), and attP (TA) shown below.
  • AttB 5'-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3 ' attP
  • TT 5'-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3 ' attB
  • TA 5'-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3 ' attP
  • TA 5'-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3 '.
  • Item 4. 1) a polynucleotide having a recognition sequence (attP), 2) a polynucleotide having a recognition sequence (attB), and 3) A kit for site-specific recombination containing a polypeptide of TG1 phage integrase or a gene encoding TG1 phage integrase.
  • the present invention has made it possible to carry out site-specific gene recombination and cloning with very high efficiency.
  • the method of the present invention has a recombination efficiency much higher than that of the conventional method, and was able to develop a highly efficient and simple cloning technique.
  • FIG. 1 Schematic diagram of cloning method using TG1 integrase
  • a Construction of a plasmid vector (Acceptor vector) with a lethal gene (ccdB gene, etc.) sandwiched between attB sites of two TG1 phages or by cutting between attB and modifying the ends so that self-ligation does not occur To do.
  • b A DNA fragment (Donor DNA) having the target gene sandwiched between the attP sites of the TG1 phage is prepared using GSP (Gene-Specific Primer) -attP primer and AdaptorAdPCR using attP primer.
  • GSP Gene-Specific Primer
  • the target gene is inserted into AcceptorAcvector by site-specific recombination reaction.
  • b Schematic diagram of Donor DNA construction.
  • EGFP was amplified using an EGFP primer to which 48 bp attP was added, and the EGFP gene sandwiched between 48 bp attP was designated as Donor DNA.
  • c Schematic diagram of site-specific recombination reaction using pAVOTT and Donr DNA.
  • TG1 integrase causes a site-specific recombination reaction between attB and attP, and the EGFP gene in Donor DNA is incorporated into pAVOTT. Since Escherichia coli having pAVOTT before recombination has a LacZa gene, it becomes a blue colony on a medium containing X-gal.
  • the site-specific recombination efficiency by TG1 integrase was calculated as the number of white colonies / total number of colonies * 100.
  • Results of recombination efficiency of TG1 integrase and GATEWAY system The results of recombination efficiency of TG1 integrase and GATEWAY system are shown.
  • the vertical axis represents the number of colonies into which the target gene has been incorporated / the total number of colonies * 100 as the recombination efficiency.
  • the horizontal axis represents the amount of insert DNA.
  • Electrophoresis photograph of plasmid extracted from recombinant The plasmid extracted from the recombinant was cleaved with EcoRI and HindIII and confirmed by agarose gel electrophoresis. M. 1 kb plus ladder (Invitrogen), 1. pAVOTT, 2. Plasmid extracted from blue colony, 3-5. Plasmid extracted from white colony. The left represents the length (bp) indicated by the marker. A DNA fragment containing LacZa can be confirmed in 1 and 2, and a DNA fragment containing EGFP can be confirmed in 3 to 5. Electrophoresis photograph.
  • the upper figure is an electrophoresis photograph of 1.2% SeakemGTG, each lane from the left, markers, pUCattP (TT) and 50bp attB (TT), pUCattP (TT) and 50bp attB (TA), pUCattB (TT) and 50bp attP (TT), pUCattB (TT) and 50bp attP (TA), pUCattP (TA) and 50bp attB (TT), pUCattP (TA) and 50bp attB (TA), pUCattP (TA) and 50bp attB (TA), pUCattB (TA) and 50bp attP (TT), pUCattB ( TA) and 50bp attP (TT), pUCattB ( TA) and 50bp ⁇ ⁇ attP (TA), respectively, are recombination reactions and markers.
  • Each of-and + indicates the absence or addition of TG1 Integr
  • the integrase of the present invention is a TG1 integrase and is registered in the database under Accession No. AB251920.
  • each of the first DNA and the second DNA may be any DNA, for example, one may be a marker gene and the other may be any gene intended for introduction.
  • the marker gene include a drug resistance gene, a fluorescent protein (eg, GFP, YFP, etc.) gene, a luminescent enzyme (eg, luciferase) gene, a colored protein (phycocyanin, phycoerythrin, etc.), and target genes include enzymes, antibodies, Examples include genes that encode proteins that constitute intracellular organs such as receptors, hormones, cytoskeletons, and organelles.
  • the integrase of the present invention is obtained by cloning a gene encoding an enzyme having the amino acid sequence shown in SEQ ID NO: 1 from Actinophage TG1, specifically an integrase gene consisting of the base sequence shown in SEQ ID NO: 2, and using this. Prepare.
  • AttB and attP are sites containing the minimum base sequences that function as attB (TT), attP (TT), attB (TA), and attP (TA) shown below: attB (TT): 5'-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3 ' attP (TT): 5'-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3 ' attB (TA): 5'-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3 ' attP (TA): 5'-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3 '. Furthermore, it is possible to use a gene further comprising an SD sequence (Shine-Dalgarno Sequence) upstream of the gene encoding integrase, and by using such a gene, the amount of enzyme produced is
  • AttB (TT) and attP (TT) have the core sequence TT
  • attB (TA) and attP (TA) have the core sequence replaced with TA.
  • the core sequences correspond to the two attB or attP sandwiching the first DNA and the two attP or attB sandwiching the second DNA. That is, for example, when two attB sandwiching the first DNA is the attB (TT), it is preferable that the two attP sandwiching the second DNA is the attP (TT), When two attB sandwiching the first DNA is the attB (TA), the two attP sandwiching the second DNA is preferably the attP (TA), When two attB sandwiching the first DNA is one of the attB (TT) and attB (TA), the two attP sandwiching the second DNA is one of the attP (TT) and one of the attP (TA).
  • the two attB sandwiching the second DNA are preferably the attB (TT)
  • the two attB sandwiching the second DNA are preferably the attB (TA)
  • two attP sandwiching the first DNA is one of the attP (TT) and attP (TA)
  • two attB sandwiching the second DNA is one of the attB (TT) and one of the attB (TA).
  • One of the two attB or attP sandwiching the first DNA attB (TT): 5'-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3 'or attP (TT): 5'-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3 '
  • the other is attB (TA): 5'-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3 'or attP (TA): 5'-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3 'and one of the two attP or attB sandwiching the second DNA is attP (TT): 5'-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3 'or attB (TT): 5'-TCGATCAGCTCCGC
  • Gene cloning, preparation of an expression vector using a cloned DNA fragment, preparation of an integrase using an expression vector, etc. are well-known techniques for engineers belonging to the field of molecular biology. For example, it can be performed according to the method described in “Molecular Cloning” (edited by Maniatis et al., Cold Spring Harbor Laboratories, Cold Spring Harbor, New York (1982)).
  • the TG1 integrase gene was inserted into an appropriate vector and injected into a eukaryotic cell, or mRNA of the integrase gene was injected into a eukaryotic cell and sandwiched between two attB and two attP. Site-specific recombination can be induced between DNA.
  • references Thiagarajan, B., et al., Mol. Cell Biol., 21, 3926-3934 (2001), Gtoth, A. C., et al., Genetics, 166, 1775-1782 ( 2004)).
  • a vector for producing integrase in eukaryotic cells is prepared.
  • an expression control signal used for producing integrase in a eukaryotic cell it is desirable to use a transcription initiation signal and a translation initiation signal that can be artificially controlled and can control the productivity of integrase.
  • vectors that cannot replicate in eukaryotic cells such as various plasmids can be used.
  • a non-replicating plasmid containing a first DNA sequence between two attB (or attP) is prepared, introduced into a target eukaryotic cell, and integrated into a chromosome.
  • Many methods have already been reported for transforming eukaryotic cells, and may be appropriately selected according to the organism used as a host.
  • plasmid that contains a gene (second DNA) intended to be introduced between two attP (or attB) and cannot replicate in eukaryotes is prepared.
  • this vector it is desirable for this vector to have a selection marker different from the previous attB (or attP) -containing vector.
  • the attP (or attB) -containing plasmid and the integrase gene-containing vector are simultaneously injected into the prepared attB (or attP) -containing cells.
  • the integrase is temporarily expressed by the introduced integrase gene-containing vector, and site-specific recombination occurs between attB (or attP) on the chromosome and the introduced attP (or attB) DNA sequence, and attP
  • the (or attB) containing plasmid can be integrated into the attB (or attP) site on the chromosome.
  • integrase mRNA is synthesized in vitro using the integrase gene as a template according to a conventional method, and the integrase is transiently synthesized by injecting the mRNA into the cell instead of the integrase gene-containing vector.
  • the attP (or attB) -containing plasmid injected at the same time can be integrated into the chromosome.
  • the DNA concentration at the time of transformation can be limited, and the integration of a non-site-specific attP (or attB) -containing plasmid can be suppressed. .
  • a plasmid vector that has a lethal gene (for example, ccdB gene) sandwiched between attB sites of two TG1 phages or that has been cleaved between attB and modified so that self-ligation does not occur
  • a DNA fragment having a target gene sandwiched between attP sites of TG1 phage is prepared using PCR.
  • PCR reaction is performed using a primer (GSP-attP primer) of a target gene to which a full length attP sequence is added.
  • Donor DNA is created (FIG. 1b).
  • a site-specific recombination reaction occurs between the attB site on Acceptor vector and the attP site on Donor DNA, and the target gene on Donor DNA is Acceptor vector
  • the above lethal gene is replaced, and the target gene is inserted on the Acceptor vector (Fig. 1c).
  • transformation into E. coli results in replication of the acceptor vector into which the target gene has been inserted.
  • Escherichia coli having an unreacted Acceptor vector has a lethal gene and cannot grow, and only positive clones can be selected.
  • Example 1 Subcloning method using TG1 integrase First, a vector DNA into which a target gene was inserted was constructed. A LacZa gene sandwiched between 48 bp attB (TT) sequences was synthesized downstream of the lac promoter and inserted into the NdeI-HindIII site of pUC57 to construct pAVOTT (Genscript). Next, Donor DNA containing the target gene was prepared.
  • TT bp attB
  • Primers were designed to amplify the EGFP gene sandwiched between 48 bp attP (TT) sequences, and PCR reaction was performed using KOD plus ver.2 (TOYOBO) (the primers used were attP-pLac- full_5: 5'-TTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATCGCAACGCAATTAATGTGAG-3 'and attP-EGFP-full_3: 5'-ATCCCGCCCAACTGGGTAAGAGCAAAGACTAACACTGTTGGGCTGGAATTACTTGTACAGCTCGTCCATGC-3'). The conditions were 95 degrees 2 minutes, (98 degrees 10 seconds, 68 degrees 1 minute) ⁇ 30 cycles.
  • PCR products were purified using the Wizard (R) SV Gel and PCR Clean-Up System (Promega). Cloning reaction was performed using the prepared Donor DNA and vector DNA. 15 pmol purified TG1 integral and 300 ng ( ⁇ 0.15 pmol) pAVOTT, 0 pmol, 0.15 pmol, 0.45 pmol Donor DNA and Reaction buffer (20 mM Tris-HCl pH 7.5, 10 mM EDTA, 25 mM NaCl, 10 mM Spermidine, 1 mM DTT, 0.1 mg / ml BSA) was mixed to a total volume of 20 microliters and reacted at 37 ° C. for 1 hour.
  • Primers were designed so that the EGFP gene sandwiched between attB sequences recognized by BP Clonase of the GATEWAY system was amplified, and PCR reaction was performed using KOD plus ver.2 (TOYOBO) (the primer used was attB1 -pLacEGFP-5: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCGCAACGCAATTAATGTGAG-3 'and attB2-EGFP-3: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTACTTGTACAGCTCGTCCA-3'). The conditions were 95 degrees 2 minutes (98 degrees 10 seconds, 68 degrees 1 minute) ⁇ 30 cycles.
  • PCR products were purified using the Wizard (R) SV Gel and PCR Clean-Up System (Promega). Cloning reaction was performed using the prepared Donor DNA and vector DNA. Mix 4 microliters of BP Clonase Mix (Invitrogen) with 300 ng of pDONR (-), 0 pmol, 0.15 pmol, 0.45 pmol of Donor DNA and 5x Reaction buffer to a total volume of 20 microliters, and react at 37 ° C for 1 hour. went. After the reaction, 2 microliters of 2 mg / ml proteinase K was added and reacted at 37 ° C. for 10 minutes to stop the recombination reaction.
  • pDONR pDONR
  • reaction solution was added to 50 microliters of Competent Quick DH5a (TOYOBO), and the reaction was performed at 42 ° C. for 30 seconds. Thereafter, 450 microliters of LB medium was added and reacted at 37 ° C. for 1 hour. 20 microliters in the reaction solution was smeared on an LB selective medium containing 0.1 mM IPTG, 0.04 mg / ml X-gal, 30 microgram / ml Kanamycin, and cultured at 37 ° C. overnight. Thereafter, the number of colonies was counted, and the number of fluorescent colonies / total number of colonies * 100 was calculated as the recombination efficiency.
  • TG1 integration using an attachment site with a substituted core sequence In order to verify whether the site-specific recombination reaction by TG1 integrase occurs between sequences different from the original attachment site, a plasmid was prepared by substituting the core sequences of attB and attP with TA. The presence or absence of integration activity was measured by the combination of attP (TT) and attB (TT), attP (TA) and attB (TA), attP (TT) and attB (TA), and attP (TA) and attB (TA).
  • the composition of the reaction solution used for the recombination reaction was sterile ultrapure water, 1 ⁇ reaction buffer, 0.06 pmol plasmid, 0.6 pmol 50 bp attsite fragment, 12 pmol TG1 integrase.For dilution of TG1 integrase, 0.5 ⁇ PreScission buffer Using 50% Glycerol, the final concentration in the reaction solution was adjusted to 10%, and the total amount of the reaction solution was adjusted to 20 microliters. This reaction solution was reacted at 30 ° C. for 2 hours, and then subjected to heat shock at 75 ° C. for 10 minutes to stop the enzyme reaction.
  • a plasmid was extracted from the obtained colonies, subjected to restriction enzyme treatment and confirmed, and as a result, a band of the desired size could be confirmed.
  • FIG. 5 as a result of substituting the core sequence of att TG site recognized by TG1 integrase, recombination reactions were observed with pUCattP (TT) and attB (TT), pUCattB (TT) And attP (TT), pUCattP (TA) and attB (TA), pUCattB (TA) and attP (TA), and the core sequence is TT or TA. Confirmed and suggested that unidirectional cloning is possible.
  • TG1 integrase gene cloning can be performed more simply and efficiently than the conventional cloning method.

Abstract

Disclosed is a novel integrase which can catalyse site-specific recombination rapidly at low substrate concentrations. The site-specific recombination method comprises the use of an integrase to recombine a first DNA flanked by two attB and a second DNA flanked by two attP, or a first DNA flanked by two attP and a second DNA flanked by two attB, where the integrase is a TG1 phage integrase.

Description

部位特異的組換え方法及びキットSite-specific recombination method and kit
　本発明は、インテグラーゼを用いた部位特異的組換え方法及びキットに関するものである。 The present invention relates to a site-specific recombination method and kit using integrase.
　真核生物のゲノム構造を改変し、その遺伝子発現制御を操作する、いわゆるゲノム工学技術は、遺伝子治療のみならず、様々な真核生物の育種において重要な方法である。中でも外来遺伝子の真核生物ゲノム中の標的組み込みが特に重要である。従来の真核生物への遺伝子の導入では、組み込み部位の制御が難しく、ランダムな外来遺伝子の組み込みによる他の遺伝子発現の異常や重要な遺伝子の破砕などが副生する問題がある。  The so-called genome engineering technology that modifies the genome structure of eukaryotes and manipulates their gene expression control is an important method not only for gene therapy but also for breeding various eukaryotes. In particular, targeted integration of foreign genes into the eukaryotic genome is particularly important. In conventional gene introduction into eukaryotes, the integration site is difficult to control, and there is a problem that abnormal gene expression due to random foreign gene incorporation and disruption of important genes are by-produced.
　ＤＮＡの相同性を利用した標的遺伝子組み込みは有効であるが、組換え頻度が極めて低いという問題がある。この問題解決手段として、近年は部位特異的な組換えをするレコンビナーゼが注目を浴びている。 Although target gene integration using DNA homology is effective, there is a problem that recombination frequency is extremely low. In recent years, recombinases that perform site-specific recombination have attracted attention as means for solving this problem.
　大腸菌ファージＰ１のＣｒｅレコンビネナーゼや酵母のＦＬＰレコンビナーゼなどは、部位特異的なＤＮＡ組換えを起こし、真核細胞内で機能できることが確認されているが、同時に組み込まれた遺伝子の切出しも触媒するため、結果的に遺伝子導入頻度は低く、遺伝子導入より欠失変異の作製に広く用いられている。 Cre recombinase of Escherichia coli phage P1, FLP recombinase of yeast, etc. have been confirmed to cause site-specific DNA recombination and function in eukaryotic cells, but also catalyze excision of the incorporated gene. Therefore, the gene transfer frequency is low as a result, and it is more widely used for creating deletion mutations than gene transfer.
　また、大腸菌ファージラムダのインテグラーゼは、部位特異的な遺伝子組み込みを触媒するが、宿主大腸菌の因子を必要とするなど真核生物のゲノム改変には直接適用できない。そのため、外来遺伝子のゲノムへの組み込みに有効なレコンビナーゼとしては、酵素単独で部位特異的な遺伝子の組み込みを触媒し、さらに酵素単独では組み込んだ遺伝子の切出しを触媒できないものが望ましい。 In addition, integrase of E. coli phage lambda catalyzes site-specific gene integration, but cannot be directly applied to eukaryotic genome modification such as requiring host E. coli factors. Therefore, a recombinase effective for the integration of a foreign gene into the genome is preferably one that catalyzes the integration of a site-specific gene with an enzyme alone, and that cannot excise the incorporated gene with an enzyme alone.
　Ｃａｌｏｓらは、放線菌ファージ（アクチノファージ）φＣ３１のインテグラーゼに着目し、その真核細胞における標的遺伝子組み込みへの利用を検討している。φＣ３１のインテグラーゼは、ファージと宿主のゲノム上のａｔｔａｃｈｍｅｎｔｓｉｔｅ（ａｔｔＰとａｔｔＢ）の間で組換えを起こし、また組み込み遺伝子の切り出しにはＸｉｓというファージ蛋白質を要求するので、切出しを伴わない遺伝子の部位特異的な導入に有効である（Thorp, H. M., and Smith, M. C. M., Proc. Natl. Acad. Sci. USA, 95, 5505-5510 (1998), Kuhstoss, S. and Rao, R. N., J. Mol. Biol., 222, 897-908 (1991), Rausch, H., and Lehmann, M., Nucleic Acids Res, 19, 5187-5189 (1991)）。 Calos et al. Are paying attention to the integrase of actinomyces phage (actinophage) φC31 and are examining its use for target gene integration in eukaryotic cells. The φC31 integrase undergoes recombination between the phage and the attachment sites (attP and attB) on the host genome, and requires a phage protein called Xis for excision of the integrated gene. Effective for specific introduction (Thorp, H. M., and Smith, M. C. M., Proc. Natl. Acad. Sci. USA, 95, 5505-5510 (1998), Kuhstoss, S. and Rao, R. N., J. Mol. Biol., 222, 897-908 (1991), Rausch, H., and Lehmann, M., Nucleic Acids Res, 19, 5187-5189 (1991)).
　また、Ｃａｌｏｓらは、φＣ３１のインテグラーゼとａｔｔＢ／ａｔｔＰの組み合わせは、動物細胞内でも機能し、部位特異的組換えを生じることを確認し、さらにトランスジェニックショウジョウバエの作製にも成功している（非特許文献８～１０）。 Calos et al. Also confirmed that the combination of φC31 integrase and attB / attP also functions in animal cells to cause site-specific recombination, and succeeded in the production of transgenic Drosophila ( Non-Patent Documents 8 to 10).
　遺伝子のクローニング法に有効なリコンビナーゼとしては、酵素単独で部位特異的な遺伝子の組み込みを触媒し、さらに酵素単独では組み込んだ遺伝子の切出しを触媒できないものが望ましいと考えられる。セリン型インテグラーゼは、最初松浦らによってアクチノファージR4より発見され、大腸菌、酵母細胞、動物細胞などで特異的な組換え反応を起すことが明らかにされた (Matsuura M, Noguchi T, Yamaguchi D, Aida T, Asayama M, Takahashi H, Shirai M. J Bacteriol., 1996 Jun; 178(11):3374-6.)。次いで、アクチノファージφC31の産生するインテグラーゼもセリン型であることが明らかにされた。セリン型インテグラーゼは、チロシン型と異なり、宿主因子の存在を必要とせず、attB、attPともに40 bpほどの比較的短い配列の間で部位特異的な組換えを起すことから、遺伝子ターゲティングなどのツールとしての利用が期待されている(非特許文献１１)。 It is considered that a recombinase effective for the gene cloning method is preferably a recombinase that catalyzes site-specific gene incorporation with the enzyme alone, and that the enzyme alone cannot catalyze the excision of the incorporated gene. Serine-type integrase was first discovered by Actinophage R4 by Matsuura et al. And was found to cause specific recombination reactions in Escherichia coli, yeast cells, animal cells, etc. (Matsuura M, Noguchi T, Yamaguchi D, Aida T, Asayama M, Takahashi H, Shirai M. J Bacteriol., 1996 Jun; 178 (11): 3374-6.). Next, it was revealed that the integrase produced by actinophage φC31 is also serine type. Serine-type integrase, unlike tyrosine type, does not require the presence of host factors, and both attB and attP cause site-specific recombination between relatively short sequences of about 40 bp. Use as a tool is expected (Non-Patent Document 11).
　本発明者は、先にアクチノファージＴＧ１（Foor, F., et al., Gene, 39, 11-16 (1985)）由来のインテグラ－ゼ遺伝子を明らかにした(特許文献2)。 The present inventor previously revealed an integrase gene derived from actinophage TG1 (For, F., et al., Gene, 39, 11-16 (1985)) (Patent Document 2).
ＷＯ２００５－１０７７９０号公報WO2005-107790 gazette 特開2007-228920号公報JP 2007-228920 A
　遺伝子のクローニング方法は一般的には以下のようなものである。
1、目的の遺伝子を持つDNAをPCR法を用いて増幅させる。
2、増幅させたDNA断片と挿入させたいプラスミドベクターをそれぞれ制限酵素処理、修飾酵素処理を行う。
3、それぞれのDNA断片を混ぜ、リガーゼによる結合反応を行う(Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, NY.)。 The gene cloning method is generally as follows.
1. Amplify DNA with the gene of interest using PCR.
2. Treat the amplified DNA fragment and the plasmid vector to be inserted with restriction enzyme treatment and modification enzyme treatment, respectively.
3. Mix each DNA fragment and perform a ligase binding reaction (Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
　しかし、上記のような従来の方法では実験操作が複雑なことから操作に伴う失敗も多く、また所要時間も多くかかる。また、ラムダファージのインテグラーゼを用いた方法では部位特異的組換え反応により、上記酵素処理を一度に行える利点があるが、インテグラーゼの他に大腸菌宿主の因子を必要とすることや、特異的に認識する部位の長さが長いなどの問題がある (Invitrogen http://www.invitrogen.co.jp/gateway/index.shtml)。 However, in the conventional method as described above, since the experimental operation is complicated, there are many failures due to the operation and much time is required. In addition, the method using lambda phage integrase has the advantage that the above-mentioned enzyme treatment can be performed at once by site-specific recombination reaction. (Invitrogen http://www.invitrogen.co.jp/gateway/index.shtml).
　本発明者は、数種のアクチノファージのインテグラーゼについてin vitro における部位特異的組換え系の構築を行なった。その結果、アクチノファージTG1のインテグラーゼが対応するattB/attP配列に対してin vivo、in vitroで強く効率の良い組換え活性を示すことを見出した。 The present inventor has constructed a site-specific recombination system in vitro for several types of actinophage integrase. As a result, it was found that the actinophage TG1 integrase exhibits strong and efficient recombination activity in vitro and in vitro against the corresponding attB / attP sequences.
　本発明者は、以下の部位特異的組換え方法及び部位特異的組換え用キットを提供するものである。
項１．　インテグラーゼを用いて２つのattBに挟まれた第１DNAと２つのattPに挟まれた第２DNA間、または２つのattPに挟まれた第１DNAと２つのattBに挟まれた第２DNA間の組換えを行うことを特徴とする部位特異的組換え方法であって、前記インテグラーゼがＴＧ１ファージインテグラーゼである、部位特異的組換え方法。
項２．　前記attB及びattPが、下記で示されたattB(TT)、attP(TT)、attB(TA)、attP(TA)として機能する最小塩基配列を含む部位であることを特徴とする項１に記載の部位特異的組換え方法：
attB(TT)：5’-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3’
attP(TT)：5’-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3’
attB(TA) :5’-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3’
attP(TA) :5’-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3’。
項３．　第１DNAを挟む２つのattBまたはattPの一方が
attB(TT)：5’-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3もしくは
attP(TT)：5’-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3’であり、
他方が
attB(TA)：5’-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3’もしくは
attP(TA)：5’-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3’であり、かつ
第２DNAを挟む２つのattPまたはattBの一方が
attP(TT)：5’-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3’もしくはattB(TT)：5’-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3であり、
他方が
attP(TA)：5’-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3’もしくは
attB(TA)：5’-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3’である、請求項２に記載の部位特異的組換え方法。
項４．　1) 認識配列（attP）を有するポリヌクレオチド、
2) 認識配列（attB）を有するポリヌクレオチド、及び
3) ＴＧ１ファージインテグラーゼのポリペプチド若しくはＴＧ１ファージインテグラーゼをコードする遺伝子を含有する部位特異的組換え用キット。 The present inventor provides the following site-specific recombination method and kit for site-specific recombination.
Item 1. Recombination between the first DNA sandwiched between two attBs and the second DNA sandwiched between two attPs using integrase, or between the first DNA sandwiched between two attPs and the second DNA sandwiched between two attBs A site-specific recombination method, wherein the integrase is a TG1 phage integrase.
Item 2. Item 2. The item 1, wherein the attB and attP are sites including a minimum base sequence that functions as attB (TT), attP (TT), attB (TA), and attP (TA) shown below. Site-specific recombination methods:
attB (TT): 5'-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3 '
attP (TT): 5'-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3 '
attB (TA): 5'-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3 '
attP (TA): 5'-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3 '.
Item 3. One of the two attB or attP sandwiching the first DNA
attB (TT): 5'-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3 or
attP (TT): 5'-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3 '
The other is
attB (TA): 5'-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3 'or
attP (TA): 5'-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3 'and one of the two attP or attB sandwiching the second DNA is
attP (TT): 5'-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3 'or attB (TT): 5'-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3
The other is
attP (TA): 5'-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3 'or
The site-specific recombination method according to claim 2, which is attB (TA): 5'-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3 '.
Item 4. 1) a polynucleotide having a recognition sequence (attP),
2) a polynucleotide having a recognition sequence (attB), and
3) A kit for site-specific recombination containing a polypeptide of TG1 phage integrase or a gene encoding TG1 phage integrase.
　本発明により、非常に高い効率で部位特異的遺伝子組換え及びクローニングを実施できるようになった。本発明の方法は、従来の方法と比較して組換え効率が格段に高く、高効率で簡便なクローニング技術を開発することができた。 The present invention has made it possible to carry out site-specific gene recombination and cloning with very high efficiency. The method of the present invention has a recombination efficiency much higher than that of the conventional method, and was able to develop a highly efficient and simple cloning technique.
TG1 インテグラーゼを用いたクローニング法の模式図TG1インテグラーゼを用いたクローニング法の模式図を示す。a. 2つのTG1ファージのattBサイトに挟まれた致死遺伝子(ccdB遺伝子など)を持った、もしくはattB間で切断し、セルフライゲーションが起こらないように末端を修飾したプラスミドベクター(Acceptor vector)を構築する。b. GSP (Gene-Specific Primer)-attP primerとattP primerを用いたAdaptor PCR を用いてTG1ファージのattPサイトに挟まれた目的遺伝子を持つDNA断片(Donor DNA)を作成する。c. Acceptor vectorとDonor DNAと精製したTG1インテグラーゼを反応させることで、部位特異的組換え反応によって、Acceptor vector上に目的遺伝子が挿入される。Schematic diagram of cloning method using TG1 integrase A schematic diagram of cloning method using TG1 integrase is shown. a. Construction of a plasmid vector (Acceptor vector) with a lethal gene (ccdB gene, etc.) sandwiched between attB sites of two TG1 phages or by cutting between attB and modifying the ends so that self-ligation does not occur To do. b. A DNA fragment (Donor DNA) having the target gene sandwiched between the attP sites of the TG1 phage is prepared using GSP (Gene-Specific Primer) -attP primer and AdaptorAdPCR using attP primer. c. By reacting Acceptor vector, Donor DNA and purified TG1 integrase, the target gene is inserted into AcceptorAcvector by site-specific recombination reaction. TG1インテグラーゼによる部位特異的組換えシステムを用いたサブクローニング法の確立のための実験の模式図。a. pAVOTTの模式図。Lacプロモーター制御下に48bp attB-LacZa-48bp attBカセットを挿入したプラスミドである。b. Donor DNA構築の模式図。48bp attPを付加したEGFPプライマーを用いて、EGFPを増幅させ、48bp attPに挟まれたEGFP遺伝子をDonor DNAとした。c. pAVOTTとDonr DNAを用いた部位特異的組換え反応の模式図。TG1インテグラーゼにより、attB-attP間で部位特異的組換え反応が生じ、Donor DNA中のEGFP遺伝子がpAVOTTに組込まれる。組換え前のpAVOTTを持つ大腸菌には、LacZa遺伝子が存在するため、X-gal含有培地上で青色のコロニーとなる。TG1インテグラーゼによる部位特異的組換え後のプラスミドはLacZa遺伝子を持たないため、白色のコロニーとなる。TG1インテグラーゼによる部位特異的組換え効率は白色コロニー数/全コロニー数*100として算出した。Schematic diagram of experiment for establishing subcloning method using site-specific recombination system with TG1 integrase. a. Schematic diagram of pAVOTT. This is a plasmid in which a 48 bp attB-LacZa-48bp attB cassette is inserted under the control of the Lac promoter. b. Schematic diagram of Donor DNA construction. EGFP was amplified using an EGFP primer to which 48 bp attP was added, and the EGFP gene sandwiched between 48 bp attP was designated as Donor DNA. c. Schematic diagram of site-specific recombination reaction using pAVOTT and Donr DNA. TG1 integrase causes a site-specific recombination reaction between attB and attP, and the EGFP gene in Donor DNA is incorporated into pAVOTT. Since Escherichia coli having pAVOTT before recombination has a LacZa gene, it becomes a blue colony on a medium containing X-gal. Since the plasmid after site-specific recombination with TG1 integrase does not have the LacZa gene, it becomes a white colony. The site-specific recombination efficiency by TG1 integrase was calculated as the number of white colonies / total number of colonies * 100. TG1インテグラーゼとGATEWAYシステムの組換え効率の結果TG1インテグラーゼとGATEWAYシステムの組換え効率の結果を示す。縦軸は目的の遺伝子が組み込まれたコロニー数/全コロニー数*100を組換え効率として算出したものである。横軸はinsert DNAの量を表す。Results of recombination efficiency of TG1 integrase and GATEWAY system The results of recombination efficiency of TG1 integrase and GATEWAY system are shown. The vertical axis represents the number of colonies into which the target gene has been incorporated / the total number of colonies * 100 as the recombination efficiency. The horizontal axis represents the amount of insert DNA. 組換え体から抽出したプラスミドの電気泳動写真。組換え体から抽出したプラスミドをEcoRI、HindIIIで切断し、アガロースゲル電気泳動で確認した。M. 1kb plus ladder (Invitrogen)、1. pAVOTT、2. 青色コロニーから抽出したプラスミド、3～5. 白色コロニーから抽出したプラスミド。左はマーカーの示す長さ(bp)を表す。1、2ではLacZaを含むDNA断片、3～5では、EGFPを含むDNA断片が確認できる。Electrophoresis photograph of plasmid extracted from recombinant. The plasmid extracted from the recombinant was cleaved with EcoRI and HindIII and confirmed by agarose gel electrophoresis. M. 1 kb plus ladder (Invitrogen), 1. pAVOTT, 2. Plasmid extracted from blue colony, 3-5. Plasmid extracted from white colony. The left represents the length (bp) indicated by the marker. A DNA fragment containing LacZa can be confirmed in 1 and 2, and a DNA fragment containing EGFP can be confirmed in 3 to 5. 電気泳動写真。上の図は1.2% SeakemGTGの電気泳動写真で、各レーンは左から、マーカー、pUCattP(TT)と50bp attB(TT)、pUCattP(TT)と50bp attB(TA)、pUCattB(TT)と50bp attP(TT)、pUCattB(TT) と50bp attP(TA)、pUCattP(TA)と50bp attB(TT)、pUCattP(TA)と50bp attB(TA)、pUCattB(TA)と50bp attP(TT)、pUCattB(TA)と50bp attP(TA)のそれぞれの組換え反応、マーカーとなっている。それぞれの-と+はTG1 Integraseの未添加、添加を示している。下の図は、上記のTG1 Integraseを添加した反応物をXmnIのsingle digestionにより処理したもので、uncutは未処理、cutは酵素を付加した事を示す。Electrophoresis photograph. The upper figure is an electrophoresis photograph of 1.2% SeakemGTG, each lane from the left, markers, pUCattP (TT) and 50bp attB (TT), pUCattP (TT) and 50bp attB (TA), pUCattB (TT) and 50bp attP (TT), pUCattB (TT) and 50bp attP (TA), pUCattP (TA) and 50bp attB (TT), pUCattP (TA) and 50bp attB (TA), pUCattB (TA) and 50bp attP (TT), pUCattB ( TA) and 50bp 反 応 attP (TA), respectively, are recombination reactions and markers. Each of-and + indicates the absence or addition of TG1 Integrase. The lower figure shows that the reaction product to which TG1 Integrase was added was treated with XmnI single digestion, uncut was untreated, and cut was enzyme added.
　本発明のインテグラーゼはTG1インテグラーゼであり、Accession No. AB251920でデータベースに登録されている。 The integrase of the present invention is a TG1 integrase and is registered in the database under Accession No. AB251920.
　本明細書において、第１ＤＮＡと第２ＤＮＡは、各々任意のＤＮＡでよく、例えば一方はマーカー遺伝子であり、他方は導入を目的とする任意の遺伝子であってもよい。マーカー遺伝子としては、薬剤耐性遺伝子、蛍光タンパク質(例えばＧＦＰ，ＹＦＰなど)遺伝子、発光酵素(例えばルシフェラーゼ)遺伝子、着色蛋白質（フィコシアニン、フィコエリトリン等）などが挙げられ、目的遺伝子としては、酵素、抗体、受容体、ホルモン、細胞骨格・オルガネラなどの細胞内器官を構成するタンパク質をコードする遺伝子が挙げられる。 In the present specification, each of the first DNA and the second DNA may be any DNA, for example, one may be a marker gene and the other may be any gene intended for introduction. Examples of the marker gene include a drug resistance gene, a fluorescent protein (eg, GFP, YFP, etc.) gene, a luminescent enzyme (eg, luciferase) gene, a colored protein (phycocyanin, phycoerythrin, etc.), and target genes include enzymes, antibodies, Examples include genes that encode proteins that constitute intracellular organs such as receptors, hormones, cytoskeletons, and organelles.
　本発明のインテグラーゼは、アクチノファージＴＧ１から配列番号１に示すアミノ酸配列を有する酵素をコードする遺伝子、具体的には配列番号２に示す塩基配列からなるインテグラーゼ遺伝子をクローニングし、これを使用して調製する。
attB及びattPについては、下記で示されたattB(TT)、attP(TT)、attB(TA)、attP(TA)として機能する最小塩基配列を含む部位である：
attB(TT)：5’-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3’
attP(TT)：5’-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3’
attB(TA)：5’-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3’
attP(TA)：5’-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3’。
さらに、インテグラーゼをコードする遺伝子の上流にさらにＳＤ配列（Ｓｈｉｎｅ－Ｄａｌｇａｒｎｏ Ｓｅｑｕｅｎｃｅ）を含んでなる遺伝子を利用することも可能で、このような遺伝子を利用することで、酵素の生産量を著しく増加させることができる。 The integrase of the present invention is obtained by cloning a gene encoding an enzyme having the amino acid sequence shown in SEQ ID NO: 1 from Actinophage TG1, specifically an integrase gene consisting of the base sequence shown in SEQ ID NO: 2, and using this. Prepare.
AttB and attP are sites containing the minimum base sequences that function as attB (TT), attP (TT), attB (TA), and attP (TA) shown below:
attB (TT): 5'-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3 '
attP (TT): 5'-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3 '
attB (TA): 5'-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3 '
attP (TA): 5'-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3 '.
Furthermore, it is possible to use a gene further comprising an SD sequence (Shine-Dalgarno Sequence) upstream of the gene encoding integrase, and by using such a gene, the amount of enzyme produced is significantly increased. Can be made.
　なお、attB(TT)及びattP(TT)はコア配列がTTであり、attB(TA)及びattP(TA)はコア配列がTAに置換されている。 Note that attB (TT) and attP (TT) have the core sequence TT, and attB (TA) and attP (TA) have the core sequence replaced with TA.
　第１DNAを挟む２つのattBまたはattPと、第２DNAを挟む２つのattPまたはattBとはコア配列が対応することが好ましい。すなわち、例えば、第１DNAを挟む２つのattBが前記attB(TT)である場合は、第２DNAを挟む２つのattPは前記attP(TT)であることが好ましく、
第１DNAを挟む２つのattBが前記attB(TA)である場合は、第２DNAを挟む２つのattPは前記attP(TA)であることが好ましく、
第１DNAを挟む２つのattBが前記attB(TT)及びattB(TA)の１ずつである場合は、第２DNAを挟む２つのattPは前記attP(TT)及びattP(TA)の１ずつであることが好ましく、
第１DNAを挟む２つのattPが前記attP(TT)である場合は、第２DNAを挟む２つのattBは前記attB(TT)であることが好ましく、
第１DNAを挟む２つのattPが前記attP(TA)である場合は、第２DNAを挟む２つのattBは前記attB(TA)であることが好ましく、
第１DNAを挟む２つのattPが前記attP(TT)及びattP(TA)の１ずつである場合は、第２DNAを挟む２つのattBは前記attB(TT)及びattB(TA)の１ずつであることが好ましい。 It is preferable that the core sequences correspond to the two attB or attP sandwiching the first DNA and the two attP or attB sandwiching the second DNA. That is, for example, when two attB sandwiching the first DNA is the attB (TT), it is preferable that the two attP sandwiching the second DNA is the attP (TT),
When two attB sandwiching the first DNA is the attB (TA), the two attP sandwiching the second DNA is preferably the attP (TA),
When two attB sandwiching the first DNA is one of the attB (TT) and attB (TA), the two attP sandwiching the second DNA is one of the attP (TT) and one of the attP (TA). Is preferred,
When two attP sandwiching the first DNA is the attP (TT), the two attB sandwiching the second DNA are preferably the attB (TT),
When two attP sandwiching the first DNA is the attP (TA), the two attB sandwiching the second DNA are preferably the attB (TA),
When two attP sandwiching the first DNA is one of the attP (TT) and attP (TA), two attB sandwiching the second DNA is one of the attB (TT) and one of the attB (TA). Is preferred.
　本発明のより好ましい態様において、
第１DNAを挟む２つのattBまたはattPの一方が
attB(TT)：5’-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3’もしくは
attP(TT)：5’-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3’であり、
他方が
attB(TA)：5’-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3’もしくは
attP(TA)：5’-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3’であり、かつ
第２DNAを挟む２つのattPまたはattBの一方が
attP(TT)：5’-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3’もしくはattB(TT)：5’-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3’であり、
他方が
attP(TA)：5’-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3’もしくは
attB(TA)：5’-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3’
とすることができる。 In a more preferred embodiment of the present invention,
One of the two attB or attP sandwiching the first DNA
attB (TT): 5'-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3 'or
attP (TT): 5'-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3 '
The other is
attB (TA): 5'-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3 'or
attP (TA): 5'-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3 'and one of the two attP or attB sandwiching the second DNA is
attP (TT): 5'-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3 'or attB (TT): 5'-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3'
The other is
attP (TA): 5'-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3 'or
attB (TA): 5'-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3 '
It can be.
　斯かる態様においては、効率よく一方向性のクローニングが実現される。これは、後述の実施例で示すように、attPとattBとの間では同じコア配列同士の場合に効率よく組換えを生じるためと考えられる。第一DNAもしくは第二DNAを挟む２つのattBもしくはattPが同じコア配列を有する場合、例えば図１ｃにおいて第一DNAの左側と第二DNAの右側や、第一DNAの右側と第二DNAの左側のattPとattB間において意図しないアタッチメントサイト間で組換え反応が生じてしまうと考えられる。このような場合、組換え産物は直鎖状となり、大腸菌内で複製できなくなってしまうため、結果としてクローニングの効率が低下する。本願発明のより好ましい態様においては、このような意図しない組換え反応が抑制されることにより、効率よい一方向性のクローニングが実現されると推測される。 In such an embodiment, efficient unidirectional cloning is realized. This is presumably because recombination occurs efficiently between attP and attB when the same core sequence is present, as shown in the Examples described later. When two attB or attP sandwiching the first DNA or the second DNA have the same core sequence, for example, in FIG. 1c, the left side of the first DNA and the right side of the second DNA, the right side of the first DNA and the left side of the second DNA It is considered that a recombination reaction occurs between unintended attachment sites between attP and attB. In such a case, the recombination product becomes linear and cannot be replicated in E. coli, resulting in a reduction in cloning efficiency. In a more preferred embodiment of the present invention, it is presumed that efficient unidirectional cloning is realized by suppressing such unintended recombination reaction.
　遺伝子のクローニング、クローン化したＤＮＡ断片を用いた発現ベクターの調製、発現ベクターを用いたインテグラーゼの調製などは、分子生物学の分野に属する技術者にとっては周知の技術であり、具体的には、例えば「Molecular Cloning」（Maniatisら編、Cold Spring Harbor Laboratories, Cold Spring Harbor、New York(1982)）に記載の方法に従って行うことができる。 Gene cloning, preparation of an expression vector using a cloned DNA fragment, preparation of an integrase using an expression vector, etc. are well-known techniques for engineers belonging to the field of molecular biology. For example, it can be performed according to the method described in “Molecular Cloning” (edited by Maniatis et al., Cold Spring Harbor Laboratories, Cold Spring Harbor, New York (1982)).
　ＴＧ１インテグラーゼ遺伝子を適当なベクターに挿入し、真核生物細胞に注入すること、あるいはインテグラーゼ遺伝子のｍＲＮＡを真核生物細胞に注入することで、２つのａｔｔＢと２つのａｔｔＰで各々挟まれたＤＮＡ間で部位特異的組換えを誘導することができる。具体的には、文献（Thyagarajan, B., et al., Mol. Cell Biol., 21, 3926-3934 (2001)、Gtoth, A. C., et al., Genetics, 166, 1775-1782 (2004)）などの方法に準じて実施できる。 The TG1 integrase gene was inserted into an appropriate vector and injected into a eukaryotic cell, or mRNA of the integrase gene was injected into a eukaryotic cell and sandwiched between two attB and two attP. Site-specific recombination can be induced between DNA. Specifically, references (Thyagarajan, B., et al., Mol. Cell Biol., 21, 3926-3934 (2001), Gtoth, A. C., et al., Genetics, 166, 1775-1782 ( 2004)).
　まず、インテグラーゼを真核細胞内で生産させるためのベクターを作製する。インテグラーゼを真核生物細胞内で産生させるために使用する発現制御シグナルとしては、人為的制御が可能で、インテグラーゼの産性を制御できるような転写開始並びに翻訳開始シグナルを用いることが望ましい。ベクターとしては、種々のプラスミドなど、真核生物細胞内で複製できないベクターが使用可能である。 First, a vector for producing integrase in eukaryotic cells is prepared. As an expression control signal used for producing integrase in a eukaryotic cell, it is desirable to use a transcription initiation signal and a translation initiation signal that can be artificially controlled and can control the productivity of integrase. As the vector, vectors that cannot replicate in eukaryotic cells such as various plasmids can be used.
　次に、２つのａｔｔＢ（もしくはａｔｔＰ）間に第１ＤＮＡ配列を含有する、複製機能を持たないプラスミドを作製し、対象とする真核生物細胞に導入し、染色体にインテグレートさせる。真核生物細胞を形質転換する方法はすでに多くの方法が報告されており、宿主として使用する生物に応じて適宜選択すればよい。 Next, a non-replicating plasmid containing a first DNA sequence between two attB (or attP) is prepared, introduced into a target eukaryotic cell, and integrated into a chromosome. Many methods have already been reported for transforming eukaryotic cells, and may be appropriately selected according to the organism used as a host.
　また、２つのａｔｔＰ（もしくはａｔｔＢ）間に導入を目的とする遺伝子(第２ＤＮＡ)を含有し、真核生物内で複製できないプラスミドを作製する。なお、このベクターは先のａｔｔＢ（もしくはａｔｔＰ）含有ベクターとは、異なる選択マーカーを有する事が望ましい。 Also, a plasmid that contains a gene (second DNA) intended to be introduced between two attP (or attB) and cannot replicate in eukaryotes is prepared. In addition, it is desirable for this vector to have a selection marker different from the previous attB (or attP) -containing vector.
　次に作製したａｔｔＢ（もしくはａｔｔＰ）含有細胞にａｔｔＰ（もしくはａｔｔＢ）含有プラスミドとインテグラーゼ遺伝子含有ベクターを同時に注入する。導入されたインテグラーゼ遺伝子含有ベクターにより一時的にインテグラーゼを発現させ、染色体上のａｔｔＢ（もしくはａｔｔＰ）と導入されたａｔｔＰ（もしくはａｔｔＢ）ＤＮＡ配列間で部位特異的な組換えを起こさせ、ａｔｔＰ（もしくはａｔｔＢ）含有プラスミドを染色体上のａｔｔＢ（もしくはａｔｔＰ）部位に組み込むことができる。 Next, the attP (or attB) -containing plasmid and the integrase gene-containing vector are simultaneously injected into the prepared attB (or attP) -containing cells. The integrase is temporarily expressed by the introduced integrase gene-containing vector, and site-specific recombination occurs between attB (or attP) on the chromosome and the introduced attP (or attB) DNA sequence, and attP The (or attB) containing plasmid can be integrated into the attB (or attP) site on the chromosome.
　また、インテグラーゼ遺伝子を鋳型としてインテグラーゼのｍＲＮＡを常法に従い試験管内で合成し、インテグラーゼ遺伝子含有ベクターの代わりに該ｍＲＮＡを細胞内に注入することで、インテグラーゼを一過的に合成させて、同時に注入されたａｔｔＰ（もしくはａｔｔＢ）含有プラスミドを染色体内に組み込むこともできる。なお、この際に本発明のインテグラーゼ遺伝子を利用すれば、形質転換時のＤＮＡ濃度を制限することができ、もって部位非特異的なａｔｔＰ（もしくはａｔｔＢ）含有プラスミドの組み込みを抑制することができる。 In addition, integrase mRNA is synthesized in vitro using the integrase gene as a template according to a conventional method, and the integrase is transiently synthesized by injecting the mRNA into the cell instead of the integrase gene-containing vector. Thus, the attP (or attB) -containing plasmid injected at the same time can be integrated into the chromosome. In this case, if the integrase gene of the present invention is used, the DNA concentration at the time of transformation can be limited, and the integration of a non-site-specific attP (or attB) -containing plasmid can be suppressed. .
　以下、TG1インテグラーゼを用いたクローニング法についての一例を述べる。まず、2つのTG1ファージのattBサイトに挟まれた致死遺伝子(例えばccdB遺伝子など)を持つ、もしくは、attB間で切断し、セルフライゲーションが起こらないように末端を修飾したプラスミドベクター(Acceptor vector)を構築する(図 1a)。次に、PCRを用いてTG1ファージのattPサイトに挟まれた目的遺伝子を持つDNA断片(Donor DNA)を作成する。方法は、全長のattP配列を付加した目的遺伝子のプライマー(GSP-attP primer)を用いてPCR反応を行う。この結果、Donor DNAが作成される(図1b)。Acceptor vectorとDonor DNAと精製したTG1インテグラーゼを反応させることで、Acceptor vector上のattBサイトとDonor DNA上のattPサイト間で部位特異的組換え反応が生じ、Donor DNA上の目的遺伝子がAcceptor vector上の致死遺伝子と入れ替わり、Acceptor vector上に目的遺伝子が挿入される(図 1c)。その後、反応液をそのまま用いて、大腸菌にトランスフォーメーションすることにより、目的遺伝子を挿入された Acceptor vectorが複製される。未反応のAcceptor vectorを持つ大腸菌は、致死遺伝子を持つため、増殖できず、ポジティブクローンのみ選抜できる。 The following describes an example of a cloning method using TG1 integrase. First, a plasmid vector (Acceptor vector) that has a lethal gene (for example, ccdB gene) sandwiched between attB sites of two TG1 phages or that has been cleaved between attB and modified so that self-ligation does not occur Build (Figure 1a). Next, a DNA fragment (Donor DNA) having a target gene sandwiched between attP sites of TG1 phage is prepared using PCR. In the method, PCR reaction is performed using a primer (GSP-attP primer) of a target gene to which a full length attP sequence is added. As a result, Donor DNA is created (FIG. 1b). By reacting Acceptor vector and Donor DNA with purified TG1 integrase, a site-specific recombination reaction occurs between the attB site on Acceptor vector and the attP site on Donor DNA, and the target gene on Donor DNA is Acceptor vector The above lethal gene is replaced, and the target gene is inserted on the Acceptor vector (Fig. 1c). Thereafter, using the reaction solution as it is, transformation into E. coli results in replication of the acceptor vector into which the target gene has been inserted. Escherichia coli having an unreacted Acceptor vector has a lethal gene and cannot grow, and only positive clones can be selected.
　以下、TG1インテグラーゼによる部位特異的組換えシステムを用いたサブクローニング法の確立のための実験の具体的な実験方法を示すが、本発明はこれら実施例に限定されない。 Hereinafter, specific experimental methods for experiments for establishing a subcloning method using a site-specific recombination system using TG1 integrase are shown, but the present invention is not limited to these examples.
実施例１
(1)TG1インテグラーゼを用いたサブクローニング法
　まず、目的遺伝子を挿入させるvector DNAの構築を行った。lacプロモーターの下流に48bpのattB(TT)配列に挟まれたLacZa遺伝子を合成し、pUC57のNdeI-HindIIIサイトに挿入し、pAVOTTを構築した（Genscript）。次に、目的遺伝子を含むDonor DNAを調製した。48bpのattP(TT)配列に挟まれたEGFP遺伝子が増幅されるようにプライマーを設計し、KOD plus ver.2(TOYOBO)を用いてPCR反応を行った(用いたプライマーは、attP-pLac-full_5：5'-TTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATCGCAACGCAATTAATGTGAG-3'及びattP-EGFP-full_3：5'-ATCCCGCCCAACTGGGTAAGAGCAAAGACTAACACTGTTGGGCTGGAATTACTTGTACAGCTCGTCCATGC-3'。)。条件は95度2分、(98度10秒、68度1分)×30サイクルで行った。PCR産物はWizard(R)SV Gel and PCR Clean-Up System（Promega）を用いて精製した。調製したDonor DNAと vector DNAを用いてcloning反応を行った。15pmolの精製したTG1 integraseと300ng(≒0.15pmol)のpAVOTT、0pmol、0.15pmol、0.45pmolのDonor DNAとReaction buffer(20mM Tris-HCl pH 7.5、10mM EDTA、25mM NaCl、10mM Spermidine、1mM DTT、0.1mg/ml BSA)を全量20マイクロリットルになるように混合し、37℃で1時間反応を行った。反応後、2マイクロリットルの2mg/ml Proteinase Kを添加し、37℃で10分間反応を行い、組換え反応を停止させた。1マイクロリットルの反応液を50マイクロリットルのCompetent Quick DH5a(TOYOBO)に加え、42℃で30秒反応を行った。その後、450マイクロリットルのLB培地を加え、37℃で1時間反応を行った。反応液中の20マイクロリットルを0.1mM IPTG、0.04mg/ml X-gal、100マイクログラム/ml Carbenicillinを含むLB選択培地に塗抹し、37℃で一晩培養を行った。その後、コロニー数を計測し白コロニー数/全コロニー数*100を組換え効率として算出した(図2)。 Example 1
(1) Subcloning method using TG1 integrase First, a vector DNA into which a target gene was inserted was constructed. A LacZa gene sandwiched between 48 bp attB (TT) sequences was synthesized downstream of the lac promoter and inserted into the NdeI-HindIII site of pUC57 to construct pAVOTT (Genscript). Next, Donor DNA containing the target gene was prepared. Primers were designed to amplify the EGFP gene sandwiched between 48 bp attP (TT) sequences, and PCR reaction was performed using KOD plus ver.2 (TOYOBO) (the primers used were attP-pLac- full_5: 5'-TTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATCGCAACGCAATTAATGTGAG-3 'and attP-EGFP-full_3: 5'-ATCCCGCCCAACTGGGTAAGAGCAAAGACTAACACTGTTGGGCTGGAATTACTTGTACAGCTCGTCCATGC-3'). The conditions were 95 degrees 2 minutes, (98 degrees 10 seconds, 68 degrees 1 minute) × 30 cycles. PCR products were purified using the Wizard (R) SV Gel and PCR Clean-Up System (Promega). Cloning reaction was performed using the prepared Donor DNA and vector DNA. 15 pmol purified TG1 integral and 300 ng (≈0.15 pmol) pAVOTT, 0 pmol, 0.15 pmol, 0.45 pmol Donor DNA and Reaction buffer (20 mM Tris-HCl pH 7.5, 10 mM EDTA, 25 mM NaCl, 10 mM Spermidine, 1 mM DTT, 0.1 mg / ml BSA) was mixed to a total volume of 20 microliters and reacted at 37 ° C. for 1 hour. After the reaction, 2 microliters of 2 mg / ml proteinase K was added and reacted at 37 ° C. for 10 minutes to stop the recombination reaction. One microliter of the reaction solution was added to 50 microliters of Competent Quick DH5a (TOYOBO), and the reaction was performed at 42 ° C. for 30 seconds. Thereafter, 450 microliters of LB medium was added and reacted at 37 ° C. for 1 hour. 20 microliters in the reaction solution was smeared on an LB selective medium containing 0.1 mM IPTG, 0.04 mg / ml X-gal, 100 microgram / ml Carbenicillin, and cultured overnight at 37 ° C. Thereafter, the number of colonies was counted, and the number of white colonies / total number of colonies * 100 was calculated as the recombination efficiency (FIG. 2).
(2)GATEWAYシステムを用いたサブクローニング法
　GATEWAYシステムの組換え効率を比較するために、pDONR221からccdB遺伝子を除いたプラスミドpDONR(-)を構築した。pDONR221をScaIとXmnIで切断し、セルフライゲーションさせpDONR(-)を構築した。次に、目的遺伝子を含むDonor DNAを調製した。GATEWAYシステムのBP Clonaseが認識するattB配列に挟まれたEGFP遺伝子が増幅されるようにプライマーを設計し、KOD plus ver.2(TOYOBO)を用いてPCR反応を行った（用いたプライマーは、attB1-pLacEGFP-5：5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCGCAACGCAATTAATGTGAG-3'及びattB2-EGFP-3：5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTACTTGTACAGCTCGTCCA-3'。）。条件は95度 2分、(98度10秒、68度1分)x30 サイクルで行った。PCR産物はWizard(R)SV Gel and PCR Clean-Up System（Promega）を用いて精製した。調製したDonor DNAとvector DNAを用いてcloning反応を行った。4マイクロリットルのBP Clonase Mix(Invitrogen)と300ngのpDONR(-)、0pmol、0.15pmol、0.45pmolのDonor DNA と5xReaction bufferを全量20マイクロリットルになるように混合し、37℃で1時間反応を行った。反応後、2マイクロリットルの2mg/ml Proteinase Kを添加し、37℃で10分間反応を行い、組換え反応を停止させた。1マイクロリットルの反応液を50マイクロリットルのCompetent Quick DH5a(TOYOBO)に加え、42℃で30秒反応を行った。その後、450マイクロリットルのLB培地を加え、37℃で1時間反応を行った。反応液中の20マイクロリットルを0.1mM IPTG、0.04mg/ml X-gal、30マイクログラム/ml Kanamycinを含むLB選択培地に塗抹し、37℃で一晩培養を行った。その後、コロニー数を計測し、蛍光コロニー数/全コロニー数*100を組換え効率として算出した。 (2) Subcloning method using GATEWAY system In order to compare the recombination efficiency of the GATEWAY system, a plasmid pDONR (-) was constructed by removing the ccdB gene from pDONR221. pDONR221 was cut with ScaI and XmnI and self-ligated to construct pDONR (-). Next, Donor DNA containing the target gene was prepared. Primers were designed so that the EGFP gene sandwiched between attB sequences recognized by BP Clonase of the GATEWAY system was amplified, and PCR reaction was performed using KOD plus ver.2 (TOYOBO) (the primer used was attB1 -pLacEGFP-5: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCGCAACGCAATTAATGTGAG-3 'and attB2-EGFP-3: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTACTTGTACAGCTCGTCCA-3'). The conditions were 95 degrees 2 minutes (98 degrees 10 seconds, 68 degrees 1 minute) × 30 cycles. PCR products were purified using the Wizard (R) SV Gel and PCR Clean-Up System (Promega). Cloning reaction was performed using the prepared Donor DNA and vector DNA. Mix 4 microliters of BP Clonase Mix (Invitrogen) with 300 ng of pDONR (-), 0 pmol, 0.15 pmol, 0.45 pmol of Donor DNA and 5x Reaction buffer to a total volume of 20 microliters, and react at 37 ° C for 1 hour. went. After the reaction, 2 microliters of 2 mg / ml proteinase K was added and reacted at 37 ° C. for 10 minutes to stop the recombination reaction. One microliter of the reaction solution was added to 50 microliters of Competent Quick DH5a (TOYOBO), and the reaction was performed at 42 ° C. for 30 seconds. Thereafter, 450 microliters of LB medium was added and reacted at 37 ° C. for 1 hour. 20 microliters in the reaction solution was smeared on an LB selective medium containing 0.1 mM IPTG, 0.04 mg / ml X-gal, 30 microgram / ml Kanamycin, and cultured at 37 ° C. overnight. Thereafter, the number of colonies was counted, and the number of fluorescent colonies / total number of colonies * 100 was calculated as the recombination efficiency.
(3)コア配列を置換したアタッチメントサイトを使用したTG1 integration
　TG1インテグラーゼによる部位特異的組換え反応が、元々のアタッチメントサイトとは異なった配列間で起こるかどうかを検証するために、attB、attPそれぞれのコア配列をTAに置換したプラスミドを作成した。attP(TT)とattB(TT)、attP(TA)とattB(TA)、attP(TT)とattB(TA)、attP(TA)とattB(TA)の組み合わせでintegration 活性の有無を測定した。組換え反応に用いた反応液の組成は、滅菌超純水、1× reaction buffer、0.06 pmol plasmid、0.6pmol 50bp attsite fragment、12pmol TG1 integraseであり、TG1 integraseの希釈には、0.5× PreScission buffer・50% Glycerolを使用し、反応液における最終濃度を 10% にし、反応液の全量は 20 マイクロリットルとなるように調整した。この反応液を 30℃で2hr反応させた後、75℃で10min Heat Shockを施し酵素反応を停止させた。反応後、XmnIで切断し、アガロースゲル電気泳動を行った。組換えが起こっていない場合、plasmidは制限酵素処理によりlinearとなり、長さが2780bp の band が出る。組換えが起こっているものは元々linearのため、制限酵素処理によって、1915bpと865bpの二つのbandが確認された。 (3) TG1 integration using an attachment site with a substituted core sequence
In order to verify whether the site-specific recombination reaction by TG1 integrase occurs between sequences different from the original attachment site, a plasmid was prepared by substituting the core sequences of attB and attP with TA. The presence or absence of integration activity was measured by the combination of attP (TT) and attB (TT), attP (TA) and attB (TA), attP (TT) and attB (TA), and attP (TA) and attB (TA). The composition of the reaction solution used for the recombination reaction was sterile ultrapure water, 1 × reaction buffer, 0.06 pmol plasmid, 0.6 pmol 50 bp attsite fragment, 12 pmol TG1 integrase.For dilution of TG1 integrase, 0.5 × PreScission buffer Using 50% Glycerol, the final concentration in the reaction solution was adjusted to 10%, and the total amount of the reaction solution was adjusted to 20 microliters. This reaction solution was reacted at 30 ° C. for 2 hours, and then subjected to heat shock at 75 ° C. for 10 minutes to stop the enzyme reaction. After the reaction, it was cleaved with XmnI and subjected to agarose gel electrophoresis. When recombination has not occurred, plasmid becomes linear by restriction enzyme treatment and a band of 2780 bp in length appears. Since recombination was originally linear, two bands of 1915 bp and 865 bp were confirmed by restriction enzyme treatment.
　図３で示されるように、TG1インテグラーゼを用いたサブクローニング法とGATEWAYシステムとの比較した結果、TG1インテグラーゼを用いたサブクローニング法の場合、組換え効率は、Donor DNAが0.15pmolで15.7±3.6%、0.45pmolで54.7±3.6%であった。GATEWAYシステムの場合、組換え効率は、Donor DNAが0.15pmolで3.2±0.2%、0.45pmolで4.1±1.4%であった。この結果より、TG1インテグラーゼを用いたサブクローニング法は従来の方法であるGATEWAYシステムよりも高効率であることがわかった。図４で示されるように、得られたコロニーからプラスミドを抽出し、制限酵素処理を行い確認した結果、目的のサイズのバンドが確認できた。また、図5で示されるように、TG1インテグラーゼが認識するatt siteのコア配列を置換させた結果、組換え反応が見られたのはpUCattP(TT)とattB(TT)、pUCattB(TT)とattP(TT)、pUCattP(TA)とattB(TA)、pUCattB(TA)とattP(TA)でありコア配列がTTまたはTA同士の場合であり、同じコア配列同士のみ組換えを生じることが確認され、一方向性のクローニングが可能であることが示唆された。 As shown in FIG. 3, as a result of comparison between the subcloning method using TG1 integrase and the GATEWAY system, in the case of the subcloning method using TG1 integrase, the recombination efficiency was 15.7 ± 3.6 at 0.15 pmol of Donor DNA. %, 0.45 pmol was 54.7 ± 3.6%. In the case of the GATEWAY system, the recombination efficiency was 3.2 ± 0.2% at 0.15 pmol and 4.1 ± 1.4% at 0.45 pmol. From these results, it was found that the subcloning method using TG1 integrase is more efficient than the conventional GATEWAY system. As shown in FIG. 4, a plasmid was extracted from the obtained colonies, subjected to restriction enzyme treatment and confirmed, and as a result, a band of the desired size could be confirmed. In addition, as shown in FIG. 5, as a result of substituting the core sequence of att TG site recognized by TG1 integrase, recombination reactions were observed with pUCattP (TT) and attB (TT), pUCattB (TT) And attP (TT), pUCattP (TA) and attB (TA), pUCattB (TA) and attP (TA), and the core sequence is TT or TA. Confirmed and suggested that unidirectional cloning is possible.
　本発明により、TG1インテグラーゼを利用することで、従来のクローニング法よりも簡便で高効率に遺伝子のクローニングが可能となる。 According to the present invention, by using TG1 integrase, gene cloning can be performed more simply and efficiently than the conventional cloning method.

Claims (4)

  1. インテグラーゼを用いて２つのattBに挟まれた第１DNAと２つのattPに挟まれた第２DNA間、または２つのattPに挟まれた第１DNAと２つのattBに挟まれた第２DNA間の組換えを行うことを特徴とする部位特異的組換え方法であって、前記インテグラーゼがＴＧ１ファージインテグラーゼである、部位特異的組換え方法。 Recombination between the first DNA sandwiched between two attB and the second DNA sandwiched between two attP using integrase, or the first DNA sandwiched between two attP and the second DNA sandwiched between two attB A site-specific recombination method, wherein the integrase is a TG1 phage integrase.
  2. 前記attB及びattPが、下記で示されたattB(TT)、attP(TT)、attB(TA)、attP(TA)として機能する最小塩基配列を含む部位であることを特徴とする請求項１に記載の部位特異的組換え方法：
    attB(TT) ：5’-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3’
    attP(TT) ：5’-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3’
    attB(TA) ：5’-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3’
    attP(TA) ：5’-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3’。     The attB and attP are sites including the minimum base sequence functioning as attB (TT), attP (TT), attB (TA), and attP (TA) shown below. The site-specific recombination method described:
    attB (TT): 5'-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3 '
    attP (TT): 5'-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3 '
    attB (TA): 5'-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3 '
    attP (TA): 5'-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3 '.
  3. 第１DNAを挟む２つのattBまたはattPの一方が
    attB(TT)：5’-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3もしくは
    attP(TT)：5’-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3’であり、
    他方が
    attB(TA)：5’-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3’もしくは
    attP(TA)：5’-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3’であり、かつ
    第２DNAを挟む２つのattPまたはattBの一方が
    attP(TT)：5’-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3’もしくはattB(TT)：5’-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3であり、
    他方が
    attP(TA)：5’-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3’もしくは
    attB(TA)：5’-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3’である、請求項２に記載の部位特異的組換え方法。     One of the two attB or attP sandwiching the first DNA
    attB (TT): 5'-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3 or
    attP (TT): 5'-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3 '
    The other is
    attB (TA): 5'-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3 'or
    attP (TA): 5'-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3 'and one of the two attP or attB sandwiching the second DNA is
    attP (TT): 5'-GTTCCAGCCCAACAGTGTTAGTCTTTGCTCTTACCCAGTTGGGCGGGATA-3 'or attB (TT): 5'-TCGATCAGCTCCGCGGGCAAGACCTTCTCCTTCACGGGGTGGAAGGTCGG-3
    The other is
    attP (TA): 5'-GTTCCAGCCCAACAGTGTTAGTCTTAGCTCTTACCCAGTTGGGCGGGATA-3 'or
    The site-specific recombination method according to claim 2, which is attB (TA): 5'-TCGATCAGCTCCGCGGGCAAGACCTACTCCTTCACGGGGTGGAAGGTCGG-3 '.
  4. 1) 認識配列（attP）を有するポリヌクレオチド、
    2) 認識配列（attB）を有するポリヌクレオチド、及び
    3) ＴＧ１ファージインテグラーゼのポリペプチド若しくはＴＧ１ファージインテグラーゼをコードする遺伝子を含有する部位特異的組換え用キット。     1) a polynucleotide having a recognition sequence (attP),
    2) a polynucleotide having a recognition sequence (attB), and
    3) A kit for site-specific recombination containing a polypeptide of TG1 phage integrase or a gene encoding TG1 phage integrase.
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JP2007512838A (en) * 2003-12-01 2007-05-24 インヴィトロジェン コーポレーション Nucleic acid molecules containing recombination sites and methods of use thereof
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