WO2018062225A1 - Promoteur d'expression génique pour bactéries du genre bifidobacterium - Google Patents

Promoteur d'expression génique pour bactéries du genre bifidobacterium Download PDF

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WO2018062225A1
WO2018062225A1 PCT/JP2017/034854 JP2017034854W WO2018062225A1 WO 2018062225 A1 WO2018062225 A1 WO 2018062225A1 JP 2017034854 W JP2017034854 W JP 2017034854W WO 2018062225 A1 WO2018062225 A1 WO 2018062225A1
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promoter
motif
bifidobacterium
gene
nucleotide sequence
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PCT/JP2017/034854
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Japanese (ja)
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鈴木　徹
之郎 片岡
智也 小酒井
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株式会社アネロファーマ・サイエンス
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    • C12N15/09—Recombinant DNA-technology

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  • the present invention includes a promoter for gene expression of a bacterium belonging to the genus Bifidobacterium, which includes a TTGNNN motif and a TATANT motif arranged downstream of the TTGNNN motif, and is operable downstream of the promoter and the promoter.
  • the present invention relates to a method for producing a Bifidobacterium genus that expresses the gene, which comprises a step of transforming the Bifidobacterium genus using a vector containing the gene linked to.
  • Bifidobacterium is a kind of obligate anaerobic bacterium that inhabits tissues such as intestine, vagina, and oral cavity. Since Bifidobacterium is an anaerobic bacterium, it also survives and proliferates in a cancer tissue that is one of the hypoxic sites. Using this property, a vector expressing cytosine deaminase (CD) is transformed into a bacterium belonging to the genus Bifidobacterium, and the transformant and 5-fluorocytosine (5-FC) are used to treat cancer. It has been reported that it can be treated (Patent Document 1, Non-Patent Documents 1 and 2).
  • CD cytosine deaminase
  • 5-FC itself is not utilized in the body of mammals
  • CD is expressed from a transformed strain in cancer tissue, that is, a prodrug of 5-fluorouracil (5-FU) having antitumor activity, although it has low toxicity
  • 5-FC which is a (precursor)
  • 5-FU 5-fluorouracil
  • the target protein CD
  • One means for increasing the expression efficiency of the target protein is to modify the promoter to increase the promoter activity, that is, the transcription activity of mRNA encoding the target protein.
  • promoters have been improved in various bacterial species (Patent Documents 2 to 4). However, in Bifidobacterium, a promoter with improved promoter activity has not been reported so far.
  • An object of the present invention is to provide a promoter capable of efficiently expressing a gene in Bifidobacterium.
  • the present inventors paid attention to a promoter of a gene that is efficiently expressed in Bifidobacterium longum, and two consensus sequences (consensus sequence 1 “TTGTGC”) of such promoter. , And consensus sequence 2 “TACAAT”).
  • the second nucleotide residue (T), the third nucleotide residue (G), and the first nucleotide residue (T), second nucleotide residue (A) of the consensus sequence 2 (TACAAT)
  • the fourth nucleotide residue (A) and the sixth nucleotide residue (T) indicate that it is particularly necessary for improving promoter activity, and the third nucleotide residue (C) of the consensus sequence 2 As for, it was also found that the promoter activity was improved by substituting T.
  • the promoter activity was improved by modifying and optimizing a natural promoter (HU gene promoter derived from Bifidobacterium longum).
  • a natural promoter HU gene promoter derived from Bifidobacterium longum.
  • the “TTGNNN” motif, A promoter having a motif and a “TATANT” motif downstream has high promoter activity (transcription activity) in Bifidobacterium, and the base length between both motifs is about 11 base length or 17 base length In some cases, particularly when the length was about 17 bases, it was confirmed that the promoter activity was higher, and the present invention was completed.
  • the present invention is as follows.
  • a promoter for gene expression of a bacterium of the genus Bifidobacterium which comprises a TTGNNN motif and a TATANT motif arranged downstream of the TTGNNN motif.
  • [4] The promoter according to any one of [1] to [3] above, wherein the length between the TTGNNNN motif and the TATANT motif is 11 or 17 bases in length.
  • [5] 61-97th nucleotide residue of nucleotide sequence shown in SEQ ID NO: 14, 61-91st nucleotide residue of nucleotide sequence shown in SEQ ID NO: 20, or 61 of nucleotide sequence shown in SEQ ID NO: 21
  • a bacterium belonging to the genus Bifidobacterium is used.
  • a method for producing a Bifidobacterium genus that expresses the gene comprising a step of transforming.
  • the Bifidobacterium bacterium is used as a gene transport carrier for the treatment of anaerobic diseases such as solid tumors. Useful when performing missile therapy.
  • FIG. 1A is a diagram showing the results of analyzing the frequency of occurrence of nucleotide residues (A, C, G, and T) at the transcription start point (TSS; Transcriptional Start Sites).
  • 1B and 1C are diagrams showing the results of analyzing the length (base length) between TSS and the start codon.
  • FIG. 2A shows the putative Bifidobacterium promoter -35 and -10 regions.
  • FIG. 2B is a diagram showing the results of analyzing the frequency of appearance of 6-base long nucleotide residues (1 to 6 in the figure) in the ⁇ 35 region of the Bifidobacterium promoter.
  • FIG. 1A is a diagram showing the results of analyzing the frequency of occurrence of nucleotide residues (A, C, G, and T) at the transcription start point (TSS; Transcriptional Start Sites).
  • TSS transcription start point
  • 1B and 1C are diagrams showing the results of analyzing the length (base length) between TSS
  • FIG. 2C is a diagram showing the results of analyzing the frequency of appearance of 6-base nucleotide residues (7 to 12 in the figure) in the ⁇ 10 region of the Bifidobacterium promoter.
  • FIG. 2D is a diagram showing the results of comparison between promoter consensus sequences between Bifidobacterium (B. longum) and E. coli (E. coli).
  • FIG. 3A is a schematic diagram of the plasmid pKO403-TPCTcon.
  • FIG. 3B shows the result of measuring the promoter activity when a single base substitution was introduced into two consensus sequences (“TTGTGC” [consensus sequence 1] and “TACAAT” [consensus sequence 2]) in the promoter region of pKO403-TPCTcon.
  • substitution sites 1 to 6 indicate the sites corresponding to FIG. 2A, that is, the first to sixth nucleotide residues (T, T, G, T, G, and C) of the consensus sequence 1.
  • the results of “substitution sites 7 to 12” in the figure indicate the sites corresponding to FIG. 2A, that is, the first to sixth nucleotide residues (T, A, C, A, A, and T of consensus sequence 2).
  • FIG. 4A is a schematic diagram of two types of motifs (“TTGNNN” and “TANNNT”) used for spacer length analysis.
  • FIG. 4B is a diagram showing the results of analyzing the spacer length between the two motifs in the promoter of 130 genes derived from Bifidobacterium longum.
  • FIG. 4A is a schematic diagram of two types of motifs (“TTGNNN” and “TANNNT”) used for spacer length analysis.
  • FIG. 4B is a diagram showing the results of analyzing the spacer length between the two motifs in the promoter of 130 genes derived from Bifidobacterium longum.
  • FIG. 4C is a diagram showing the results (average value
  • FIG. 4D shows the results of analyzing the spacer length between the two motifs in the promoters derived from Bifidobacterium longum (each black bar graph) and E. coli K-12 strain (each gray bar graph).
  • FIG. Results of measuring promoter activity when the two motifs in the HU gene promoter (“TTCGCA” and “TAGTAT”) and the spacer length between both motifs were modified (mean value ⁇ 2 ⁇ standard deviation, n 3)
  • the “promoter activity level” on the vertical axis represents a relative value when the HU gene promoter (“hup-original”) activity before modification is 1.
  • a mouse interferon (mIFN) - ⁇ gene operably linked downstream of the DNA region containing the HU gene promoter from Bifidobacterium longum (DNA region [Hu1] consisting of the nucleotide sequence shown in SEQ ID NO: 18) It is a figure which shows typically the preparation method of the plasmid vector (pmIFNg33TL-Hu1) containing.
  • FIG. 10A results of measurement of viable cell count (FIG. 10A) and mIFN- ⁇ concentration (FIG. 10B) secreted into the culture medium when mIFNg33TL-Hu1 and mIFNg33TL-Hu2 transformants were cultured for 0 to 18 hours.
  • FIG. 10B results of measurement of viable cell count (FIG. 10A) and mIFN- ⁇ concentration (FIG. 10B) secreted into the culture medium when mIFNg33TL-Hu1 and mIFNg33TL-Hu2 transformants were cultured for 0 to 18 hours.
  • the promoter of the present invention is a TTGNN motif (motif consisting of the nucleotide sequence shown in SEQ ID NO: 1) (hereinafter referred to as “the present motif 1”), which has a limited use of “to express a gene in a genus Bifidobacterium”. And a TATANT motif (motif consisting of the nucleotide sequence shown in SEQ ID NO: 2) (hereinafter sometimes referred to as “this motif 2”) arranged downstream of the present motif 1 ( Hereinafter, it may be referred to as “the present promoter”.
  • expressing a gene means expression of mRNA of a gene and expression of a protein encoded by the mRNA of the gene.
  • the “promoter” means a region where RNA polymerase (preferably RNA polymerase and basic transcription factor) binds and initiates transcription of mRNA encoded by a gene located downstream thereof.
  • the present promoter usually includes a transcription start site (TSS).
  • downstream means a side closer to the gene when the gene is linked to the present promoter so that the gene can be operated
  • upstream means a side farther from the gene
  • A means an adenine residue
  • T means a thymine residue
  • G means a guanine residue
  • C means a cytosine residue
  • N means “ It means any nucleotide residue among “A”, “T”, “G”, and “C”.
  • the length of the promoter is not limited as long as RNA polymerase (preferably RNA polymerase and basic transcription factor) binds and initiates transcription by RNA polymerase, and is usually 20 bases or more in length. , Preferably 22 bases or more, more preferably 24 bases or more, even more preferably 26 bases or more, even more preferably 28 bases or more, particularly preferably 29 bases or more, most preferably 30 bases or more. .
  • the length of the present promoter is usually within 300 base length, preferably within 200 base length, more preferably within 100 base length, more preferably 90 base length. Is even more preferably within 80 base length, particularly preferably within 70 base length, particularly preferably within 60 base length, and most preferably within 50 base length.
  • the motif consisting of the nucleotide sequence shown in SEQ ID NO: 1, ie, T, T, G, N, N, and N (N is A, T, G, or C in order from the upstream of the promoter).
  • N is A, T, G, or C in order from the upstream of the promoter.
  • a TTGAGC motif, a TGTTCCC motif, a TTGTGA motif, a TGTGTGC motif, etc. a TTTGCC motif (consisting of the nucleotide sequence shown in SEQ ID NO: 3). ) Is preferred.
  • the position (region) of the motif 1 contained (arranged) in the promoter is not particularly limited as long as it is upstream of the motif 2, but the ⁇ 35 region is preferable.
  • the “-35 region” is usually in the range of 25 to 55 bases upstream from the transcription start point (preferably in the range of 25 to 50 bases, more preferably in the range of 25 to 45 bases, more preferably Means a site within a range of 25 to 40 bases in length, and most preferably within a range of 30 to 40 bases in length.
  • the motif consisting of the nucleotide sequence shown in SEQ ID NO: 2, ie, T, A, T, A, N, and T (N is A, T, G, or C in order from the upstream of the promoter).
  • N is A, T, G, or C in order from the upstream of the promoter.
  • TATAAT motif Is not particularly limited, for example, a TATAAT motif, a TATAATT motif, a TATAGT motif, or a TATAACT motif, but a TATAAT motif (consisting of a nucleotide sequence shown in SEQ ID NO: 4) Is preferred.
  • the position (region) of the motif 2 contained (arranged) in the promoter is not particularly limited as long as it is downstream of the motif 1, but the region ⁇ 10 is preferred.
  • the “ ⁇ 10 region” is usually within the range of 1 to 30 bases upstream from the transcription start point (preferably within the range of 3 to 24 bases, more preferably within the range of 4 to 20 bases, more preferably Means a site in the range of 6 to 16 bases in length, most preferably in the range of 8 to 13 bases in length.
  • the length between the motif 1 and the motif 2 may be as long as it does not adversely affect the binding ability of the RNA polymerase (preferably RNA polymerase and the basic transcription factor) to the promoter and transcription initiation.
  • the RNA polymerase preferably RNA polymerase and the basic transcription factor
  • the length between the motif 1 and the motif 2 means the sixth nucleotide residue (N) in the motif 1 and the first nucleotide residue in the motif 2 ( T).
  • the length between both motifs is 0 base length.
  • the present promoter includes nucleotide residues 61 to 97 of the nucleotide sequence shown in SEQ ID NO: 14, nucleotide residues 61 to 91 of the nucleotide sequence shown in SEQ ID NO: 20, or SEQ ID NO: 21.
  • a promoter comprising nucleotide residues 61 to 97 of the nucleotide sequence shown can be mentioned.
  • Bifidobacterium bacterium When a Bifidobacterium bacterium is transformed using a vector containing the promoter and a gene operably linked downstream of the promoter (hereinafter sometimes referred to as “the vector”), Bifidobacterium that expresses can be produced.
  • Bifidobacterium examples include, for example, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium infantis, Bifidobacterium infantis, Bifidobacterium thermophilum, Bifidobacterium pseudolongum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium Longum (Bifidobacterium longum) can be mentioned, and Bifidobacterium longum is preferable.
  • Bifidobacterium adolescentis Bifidobacterium animalis
  • Bifidobacterium infantis Bifidobacterium infantis
  • Bifidobacterium thermophilum Bifidobacterium pseudolongum
  • Bifidobacterium bifidum Bifidobacterium breve
  • Bifidobacterium Longum Bifidobacterium long
  • genes operably linked to the downstream of the present promoter refers to a gene derived from a species other than Bifidobacterium, even if it is a gene derived from Bifidobacterium. Also good.
  • genes derived from species other than Bifidobacterium include, for example, interferon (IFN) - ⁇ , ⁇ , ⁇ , granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin (IL) -1 ⁇ .
  • IFN interferon
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • IL interleukin
  • tumor Cytokines immunodeficiency virus
  • TNF necrosis factor
  • LT lymphotoxin
  • G-CSF granulocyte colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • MIF macrophage migration inhibitory factor
  • a gene encoding an angiogenesis inhibitor such as endostatin and angiostatin; 5-fluoro
  • the gene encoding the antibody; gene encoding cytosine deaminase is an enzyme that converts 5-fluorocytosine with a prodrug of uracil to 5-fluorouracil and the like.
  • genes contain a nucleotide sequence that encodes an affinity tag such as a histidine tag or the secretion of a protein encoded by a gene outside the Bifidobacterium genus for convenience in isolation of the protein encoded by the gene.
  • an affinity tag such as a histidine tag
  • secretion of a protein encoded by a gene outside the Bifidobacterium genus for convenience in isolation of the protein encoded by the gene.
  • a nucleotide sequence encoding a secretory signal peptide can be appropriately added.
  • the present vector further includes a nucleotide sequence of an enhancer region or a ribosome binding region (RBS) (for example, “AAGGAG” motif, “AAGAAGGATGCTTT” motif)
  • an enhancer region or a ribosome binding region (RBS)
  • RBS ribosome binding region
  • a drug resistance gene selection marker gene
  • the enhancer region is usually located upstream of the present promoter, and the RBS is usually located between the present promoter and the gene.
  • the method for transformation is not particularly limited, and examples thereof include a method using calcium ions, a general competent cell transformation method, a protoplast transformation method, and an electroporation method.
  • Bifidobacterium longum was cultured in an MRS (Lactobacilli MRS Broth, Difco) culture solution or on an MRS agar medium under anaerobic conditions at 37 ° C.
  • MRS Lacobacilli MRS Broth, Difco
  • RNA-seq analysis Preparation of total RNA and RNA-seq analysis. Preparation of total RNA derived from Bifidobacterium longum and RNA-seq using total RNA were performed according to the following procedures [1] to [8]. [1] 48 mL of a culture solution containing Bifidobacterium longum was centrifuged (6,500 rpm, 10 minutes, 4 ° C.) to remove the supernatant (culture solution), and then 500 ⁇ L TE buffer (10 mM Tris-HCl). [PH 8.0] and 1 mM EDTA) and 1 mL RNase inhibitor (RNA protect bacteria reagent, manufactured by Qiagen) was added.
  • RNA-seq analysis using total RNA was performed according to the instruction manual using MiSeq Sequencing System (manufactured by Illumina).
  • the nucleotide sequence in the table indicates the leading strand from upstream to downstream.
  • Two underlined parts in the nucleotide sequence indicate the -35 region and -10 region of the promoter, respectively.
  • the double line part in the nucleotide sequence indicates RBS.
  • the part surrounded by a square in the nucleotide sequence indicates a position where a single base substitution is introduced into the nucleotide sequence shown in SEQ ID NO: 5.
  • the part enclosed in double squares in the nucleotide sequence indicates TSS.
  • the transformant was selectively cultured on an MRS agar medium containing 75 ⁇ g / mL spectinomycin.
  • Colonies of the selected transformants were isolated and cultured in MRS medium until the absorbance at a wavelength of 412 nm reached 0.6.
  • TSS transcription start site
  • first nucleotide residue (T), second nucleotide residue (T), and third nucleotide residue (G) of consensus sequence 1 (TTGTGC) and consensus sequence 2 (TACAAT) The first nucleotide residue (T), the second nucleotide residue (A), the fourth nucleotide residue (A), and the sixth nucleotide residue (T) are particularly necessary for improving promoter activity.
  • the third nucleotide residue (C) of the consensus sequence 2 indicates that the promoter activity is improved by substituting T.
  • an optimized RBS (shown in SEQ ID NO: 19) in a DNA region containing the HU gene promoter derived from Bifidobacterium longum (DNA region consisting of the nucleotide sequence shown in SEQ ID NO: 18, Table 1)
  • a DNA region consisting of a nucleotide sequence (Table 1), a plasmid “hup-original” (Table 1) containing a DNA fragment having a CAT gene inserted downstream of the HU gene promoter, and a plasmid “hup- Based on “original”, the “TTCGCA” motif in the ⁇ 35 region of the HU gene promoter is replaced with consensus sequence 1 (“TTGTGC” motif), and the “TAGTAT” motif in the ⁇ 10 region is replaced with consensus sequence 2 ( Optimized TATAAT motif ("TATAAT” motif)
  • the plasmid “hup-optimal-11bp” (Table 1) containing a promoter (promoter consisting of nucleotide residues 61 to 91 of the nucle
  • the present promoter that is, a promoter having a “TTGNNN” motif and a “TATANT” motif downstream thereof, has high promoter activity (transcriptional activity) in Bifidobacterium. ). Furthermore, when the base length between both motifs is about 11 base length or 17 base length, especially when it is about 17 base length, it has also shown that promoter activity becomes higher.
  • pmIFNg33TL-Hu1 is an Escherichia coli-Bifidobacterium shuttle plasmid in which the mIFN- ⁇ gene is inserted downstream of Hu1 (DNA region including the HU gene promoter derived from Bifidobacterium longum [nucleotide sequence of SEQ ID NO: 18; Table 1]). It is a vector (FIG. 6).
  • pmIFNg33TL-Hu2 is an E. coli-Bifidobacterium shuttle plasmid vector in which the mIFN- ⁇ gene is inserted downstream of Hu2 (modified form of the above HU gene promoter [nucleotide sequence of SEQ ID NO: 14; Table 1]) (FIG. 7).
  • pBEshuttle-Hu2a Materials and methods [Preparation of pBEshuttle-Hu2a]
  • the pBEshuttle-Hu2a used for the preparation of pmIFNg33TL-Hu2 was obtained by using pBEshuttle (Reference Example 4 of International Publication No. 2011/093465), which is a bifidobacteria / E. coli shuttle plasmid vector containing a wild-type Hu promoter and an RBS region. Prepared in stages. An overview is shown in FIG. First, in the first stage, based on the plasmid vector pBEshuttle, the HU gene upstream fragment 361 base length promoter region and RBS region sequence contained in pBEshuttle, the HU gene upstream 218 base length and Hu2 DNA fragment.
  • PBEshuttle-Hu2 was prepared. Next, in the second stage, a DNA fragment having a length of 218 bases upstream of the HU gene was removed from pBEshuttle-Hu2 to prepare pBEshuttle-Hu2a. A more detailed operation procedure of the first stage and the second stage will be described below.
  • PCR products (PCR-A3 and PCR-B3; Table 2) were prepared using the template DNA and primer set (forward primer and reverse primer) shown in Tables 2 and 10. Specifically, to a 0.2 mL tube for PCR, 1 ng of template DNA, 1.6 ⁇ L each of 2.5 ⁇ M primers (forward primer and reverse primer), and PrimeSTAR HS (Premix) (manufactured by Takara Bio Inc.) 10 ⁇ L, The volume was adjusted to 20 ⁇ L with 0.1 ⁇ TE buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 7.5).
  • Each primer was designed such that the terminal 15 base length of the PCR product had the same sequence as the terminal 15 base length of the adjacent PCR product to be ligated in the next step In-Fusion reaction.
  • PCR conditions followed the product instructions of PrimeSTAR HS (Premix).
  • the extension reaction time was 1 minute per 1000 base length.
  • a portion of the PCR products (PCR-A3 and PCR-B3) were electrophoresed at 100 V on an agarose gel of an appropriate concentration to confirm that they were the expected sizes (88 and 3809 bases in length, respectively) Concentration was estimated.
  • PCR-A3 and PCR-B3 were combined by In-Fusion reaction to obtain In-Fusion reaction solution 1.
  • 2 ⁇ L of PCR product (PCR-A3 and PCR-B3), 5 ⁇ In-Fusion HD Emzyme Premix (Takara Bio Inc.), and 1 ⁇ L of Cloning Enhancer (Takara Bio Inc.) are added to a 0.2 mL tube.
  • E. coli HST16CR competent cell manufactured by Takara Bio Inc.
  • the transformation method followed the product instructions for E. coli HST16CR competent cells.
  • the transformed Escherichia coli suspension was applied to an LB agar medium containing 75 ⁇ g / mL spectinomycin and allowed to stand at 37 ° C. overnight to form colonies (transformant 1).
  • pmIFNg33TL-B The pmIFNg33TL-B used for the production of pmIFNg33TL-Hu1 and pmIFNg33TL-Hu2 was produced in four stages. The outline is shown in FIGS. 9-1 to 9-4. First, in the first stage, a nucleotide sequence encoding mIFN- ⁇ fused with a tag in which six histidines are linked (His ⁇ 6; hereinafter sometimes simply referred to as “histidine tag”) is inserted into pBEshuttle, and pmIFNg Was produced (FIG. 9-1).
  • a signal sequence (SP / Linker) for secretion in Bifidobacteria was inserted into pmIFNg to prepare pmIFNg33 (FIG. 9-2).
  • the pmIFNg33 Hu promoter was changed to the P30 promoter to prepare pmIFNg33-B (FIG. 9-3).
  • the nucleotide sequence encoding the histidine tag was removed from pmIFNg33-B to prepare pmIFNg33TL-B (FIG. 9-4). The detailed operation procedure of these first to fourth steps will be described below.
  • Plasmid vectors prepared according to the methods described in the above-mentioned [Preparation of pmIFNg33TL-Hu1] and [Preparation of pmIFNg33TL-Hu2] are respectively transformed into Bifidobacterium longum.
  • mIFNg33TL-Hu1 transformant and mIFNg33TL-Hu2 transformant were prepared. Specifically, 80 ⁇ L of Bifidobacterium longum competent cells were mixed with an appropriate amount of the above plasmid vector on ice, and then transferred to a BM cuvette (2 mm gap, manufactured by BM Instruments).
  • the mIFNg33TL-Hu1 transformed strain and the mIFNg33TL-Hu2 transformed strain were inoculated, respectively, and placed in a sealed container together with a deoxygenating / carbon dioxide generating agent (Aneropack Kenki, manufactured by Mitsubishi Gas Chemical Company), 37 ° C. For 24 hours.
  • the medium for main culture was a medium prepared by mixing 18 mL of DMEM medium (Ref: 12320-032, manufactured by Thermo Fisher Scientific), 2 mL of MRS medium, and 100 ⁇ L of ascorbic acid / cysteine solution overnight at 37 ° C. , And added 20 ⁇ L of 75 mg / mL spectinomycin solution.
  • mIFN- ⁇ concentration and viable cell count Using a part of the main culture after each time culture, the mIFN- ⁇ concentration in the culture supernatant and the viable count of Bifidobacterium longum were measured.
  • mIFN- ⁇ concentration a part of the main culture was transferred to a microtube (low protein adsorption, manufactured by Sumitomo Bakelite), centrifuged (15,000 rpm, 10 minutes, 4 ° C.), and the supernatant was recovered. Measurement was performed using Mouse IFN- ⁇ Quantikine ELISA Kit (manufactured by R & D Systems) (FIG. 10B).
  • the number of viable bacteria was measured by appropriately diluting the main culture after each time of culturing with an anaerobic diluent (Nippon Bifidobacteria Center, Tomokazu Mitsuoka, Research on Bifidobacteria, page 308), and then BL agar medium 3 100 ⁇ L each was applied to the sheet. These agar media were placed in a sealed container together with a deoxygenating / carbon dioxide generating agent and cultured at 37 ° C. for 3 days to form colonies.
  • the present invention contributes to missile therapy for cancer.

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Abstract

Le problème à la base de la présente invention concerne un promoteur capable d'exprimer efficacement des gènes dans des bactéries du genre Bifidobacterium. Dans la présente invention, un promoteur comprenant un motif TTGNNN et un motif TATANT situé en aval du motif TTGNNN, de préférence un promoteur comprenant un motif TTGTGC et un motif TATAAT situé à 11 ou 17 bases en aval du motif TTGTGC, est utilisé en tant que promoteur d'expression génique pour des bactéries du genre Bifidobacterium.
PCT/JP2017/034854 2016-09-28 2017-09-27 Promoteur d'expression génique pour bactéries du genre bifidobacterium WO2018062225A1 (fr)

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WO2021038975A1 (fr) 2019-08-28 2021-03-04 株式会社アネロファーマ・サイエンス Bifidobacterium spp. l'invention concerne l'expression et la sécrétion de bsab de type diabody.

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JPH08500739A (ja) * 1992-12-30 1996-01-30 バイオテクノロジスク インスティテュート 挿入プロモーター含有の組換え乳酸菌とその構築方法
WO2016117508A1 (fr) * 2015-01-19 2016-07-28 国立大学法人信州大学 Agent thérapeutique pour maladies ischémiques

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