WO2021107072A1 - Molécule de liaison à l'antigène, composition d'acide nucléique, composition de vecteur et procédé de purification de cellules et bifidobacterium longum - Google Patents

Molécule de liaison à l'antigène, composition d'acide nucléique, composition de vecteur et procédé de purification de cellules et bifidobacterium longum Download PDF

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WO2021107072A1
WO2021107072A1 PCT/JP2020/044146 JP2020044146W WO2021107072A1 WO 2021107072 A1 WO2021107072 A1 WO 2021107072A1 JP 2020044146 W JP2020044146 W JP 2020044146W WO 2021107072 A1 WO2021107072 A1 WO 2021107072A1
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acid sequence
amino acid
seq
antigen
binding molecule
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Japanese (ja)
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学 中藤
浄 井上
ひかる 井上
真嗣 福田
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地方独立行政法人神奈川県立産業技術総合研究所
学校法人慶應義塾
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a method for purifying an antigen-binding molecule, a nucleic acid composition, a vector composition, cells and Bifidobacterium longum.
  • intestinal flora saliva
  • intestinal flora mainly the large intestine
  • human intestinal bacteria are closely related to each other and are intricately balanced, and are extremely diverse and different depending on the individual, and also differ depending on factors such as diet and country of residence.
  • Bifidobacterium longum (hereinafter referred to as "B. longum”) is known as a bacterium that has a positive effect on human health. This, B. There is an example of producing an antibody against longum (for example, Non-Patent Document 1).
  • Non-Patent Document 1 has been shown to be cross-reactive with other types of bacteria of the genus Bifidobacterium, and there is room for improvement in terms of specificity.
  • the present invention has been made in view of such a background, and B.I.
  • An object of the present invention is to provide an antigen-binding molecule having high specificity for longum.
  • the main invention of the present invention for solving the above-mentioned problems is an antigen-binding molecule that binds to Bifidobacterium longum.
  • an antigen-binding molecule having high specificity for Bifidobacterium longum can be obtained.
  • the present invention has the following configurations.
  • [Item 2] Do not bind to other Bifidobacterium genera, The antigen-binding molecule according to Item 1.
  • [Item 3] The other genus Bifidobacterium is Bifidobacterium pseudobacterium, Bifidobacterium influenza, Bifidobacterium breve, Bifidobacterium pseudobacterium, Bifidobacterium, Bifidobacterium. 2.
  • the antigen-binding molecule is an antibody, antibody fragment or peptide.
  • the amino acid sequence the amino acid sequence set forth in any of SEQ ID NOs: 3 to 8 or the amino acid sequence having 90% or more homology with the amino acid sequence set forth in any of SEQ ID NOs: 3 to 8.
  • the antigen-binding molecule according to any one of items 1 to 3, wherein the antigen-binding molecule comprises.
  • the antigen-binding molecule is an antibody or antibody fragment. An amino acid sequence having 90% or more homology with the amino acid set forth in any of SEQ ID NOs: 3 to 5 or the amino acid sequence set forth in any of SEQ ID NOs: 3 to 5 as a heavy chain amino acid sequence.
  • the antigen-binding molecule according to item 4 wherein the antigen-binding molecule comprises.
  • [Item 7] A nucleic acid composition comprising a nucleic acid sequence encoding the antigen-binding molecule according to any one of items 1 to 6, or a plurality of nucleic acid sequences.
  • [Item 8] The nucleic acid sequence according to item 7, or a vector composition containing a plurality of nucleic acid sequences, or a vector composition comprising a plurality of vectors.
  • a cell comprising the vector composition according to item 8.
  • the antigen-binding molecule according to the present embodiment is, for example, a monoclonal antibody that strongly and specifically binds to a specific antigen, a polyclonal antibody, or a multispecific antibody formed from at least two different epitope-binding fragments (for example, bispecificity).
  • Antibody human antibody, humanized antibody, camel family antibody, chimeric antibody, short chain Fv, single chain antibody, single domain antibody, domain antibody, Fab fragment, F (ab') 2 fragment, antibody fragment disulfide-bound Fv , And anti-idiotype antibodies, intracellular antibodies, antibody fragments, and any of the above epitope-binding fragments, including polypeptides, peptides, nucleic acids, synthetic small molecules, synthetic polymers, and the like.
  • the monoclonal antibody (hereinafter referred to as the present antibody) as the antigen-binding molecule according to the present embodiment is described in B.I. It is highly specific for longum. Therefore, by using the antigen-binding molecule according to the present embodiment, B.I. From the sample containing longum, B. longum. It is possible to isolate or concentrate longum.
  • the antibody does not bind to other Bifidobacterium spp.
  • B. From samples such as feces containing bacteria other than longum, B. longum. It is possible to isolate or concentrate longum.
  • B examples of the genus Bifidobacterium other than longum include Bifidobacterium pseudobacterium, Bifidobacterium infantis, Bifidobacterium breve, and Bifidobacterium bacteria.
  • the antibody has no crossing property with Gram-positive and Gram-negative bacteria.
  • this antibody it is possible to obtain B.I. It is possible to isolate and concentrate longum.
  • the antibody may be of any isotype (eg, IgG, IgA, IgM, IgD, IgA, and IgY), or a subisotype thereof, or an allotype.
  • the antibody is used in any mammal, such as, but not limited to, humans, monkeys, rabbits, pigs, horses, rats, dogs, cats, mice, sheep, camels, etc.
  • it may be derived from another animal, such as a bird (eg, a chicken).
  • a part of the amino acid sequence of this antibody is disclosed below.
  • Equation 1 is SEQ ID NO: 3
  • Equation 2 is SEQ ID NO: 4
  • Equation 3 is SEQ ID NO: 5
  • Equation 4 is SEQ ID NO: 6
  • Equation 5 is SEQ ID NO: 7
  • Equation 6 is SEQ ID NO: It is 8.
  • the heavy chain amino acid sequence of the present antibody is shown in SEQ ID NO: 1 and the light chain amino acid sequence of the present antibody is shown in SEQ ID NO: 2, which are separately described in the sequence listing.
  • the present antibody has 90% or more homology as an amino acid sequence with respect to the amino acid sequence set forth in any of SEQ ID NOs: 3 to 8 or the amino acid sequence set forth in any of SEQ ID NOs: 3 to 8. It preferably contains an amino acid sequence having sex.
  • the present antibody preferably contains, as an amino acid sequence, an amino acid sequence having 90% or more homology with the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2.
  • the present antibody is 90% or more homologous to the amino acid set forth in any of SEQ ID NOs: 3 to 5 or the amino acid sequence set forth in any of SEQ ID NOs: 3 to 5 as a heavy chain amino acid sequence. 90% or more homology between the amino acid sequence having sex and the amino acid sequence set forth in any of SEQ ID NOs: 6 to 8 or the amino acid sequence set forth in any of SEQ ID NOs: 6 to 8 as the amino acid sequence of the light chain. It is preferable to include an amino acid sequence having.
  • the present antibody preferably contains, as an amino acid sequence, an amino acid sequence having 90% or more homology with the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2.
  • the antibody is A VHCDR1 region containing an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having 90% or more homology with respect to the amino acid sequence set forth in SEQ ID NO: 3.
  • a VHCDR2 region containing an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having 90% or more homology with respect to the amino acid sequence of SEQ ID NO: 4.
  • a VHCDR3 region containing an amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having 90% or more homology with respect to the amino acid sequence set forth in SEQ ID NO: 5.
  • a VLCDR1 region containing an amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 6.
  • the present antibody preferably contains, as an amino acid sequence, an amino acid sequence having 90% or more homology with the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2.
  • This antibody is based on B.I. It can bind to the surface antigen of longum, but preferably does not specifically bind to other polypeptides. As a result, B. From samples such as feces containing bacteria other than longum, more accurately B. longum. It is possible to isolate or concentrate longum.
  • the base sequence encoding a part of the amino acid sequence of the present antibody is disclosed below.
  • Equation 7 is SEQ ID NO: 11
  • Equation 8 is SEQ ID NO: 12
  • Equation 9 is SEQ ID NO: 13
  • Equation 10 is SEQ ID NO: 14
  • Equation 11 is SEQ ID NO: 15,
  • Equation 12 is SEQ ID NO: 16.
  • the nucleotide sequence shown in SEQ ID NO: 9 is the nucleotide sequence encoding the full length of the heavy chain amino acid sequence of this antibody
  • the nucleotide sequence shown in SEQ ID NO: 10 is the nucleotide sequence encoding the full length of the light chain amino acid sequence. It is an array.
  • the base sequence encoding the amino acid sequence of the present antibody with respect to the amino acid sequence set forth in any of SEQ ID NOs: 11 to 16 or the base sequence set forth in any of SEQ ID NOs: 11 to 16. It preferably contains a base sequence having 90% or more homology.
  • the nucleic acid composition also includes a nucleic acid sequence encoding an antigen-binding molecule according to the present embodiment, or a nucleic acid composition containing a plurality of nucleic acid sequences.
  • the present embodiment also includes the nucleic acid sequence, a vector composition containing a plurality of nucleic acid sequences, or a plurality of vector compositions. Further, in this embodiment, the vector is also included.
  • the vector means a nucleic acid molecule that carries another linked nucleic acid.
  • a vector is a plasmid.
  • the plasmid is a circular double-stranded DNA formed by ligating another DNA fragment therein.
  • a virus vector there is a virus vector.
  • the viral vector can ligate another DNA fragment into the viral genome.
  • Certain vectors can replicate autonomously in the host cell into which they are introduced.
  • the cell means a cell into which an expression vector is introduced.
  • Host cells include bacterial cells, microbial cells, plant cells, or animal cells.
  • the antibody for example, a method of producing a hybridoma, secondary lymphoid tissue transplantation; can be obtained by (S econdary L ymphoid O rgan T ransplantation SLOT) immunization containing treatment.
  • SLOT procedure can be performed by the method described in Japanese Patent No. 5996437, paragraphs 0031 to 0058. An example of the method for producing this antibody will be described below.
  • BACTRON 300 manufactured by SHEL LAB
  • Immunity Alum Adjuvant manufactured by Invitrogen
  • an antigen for intraperitoneal administration is added to the antigen in an equal amount (hereinafter referred to as an antigen for intraperitoneal administration), and then the mouse is intraperitoneally (ip) administered.
  • an antigen for intraperitoneal administration is added to the antigen in an equal amount (hereinafter referred to as an antigen for intraperitoneal administration), and then the mouse is intraperitoneally (ip) administered.
  • iv caudal venule administration
  • the antigen for intraperitoneal administration was administered to 5 BALB mice twice a week. p. Administer.
  • mice 3 mice in secondary lymphoid tissue transplantation; and (S econdary L ymphoid O rgan T ransplantation SLOT) usually immunized group without the treatment (Conventional immunize group), the spleen was excised from 2 mice rest, Used for SLOT treatment.
  • the final immunity was given to the mice that had undergone the SLOT treatment. v. After carrying out by the route, the spleen is removed and fused with myeloma cells to prepare a hybridoma. From the obtained hybridomas, those that show a specific reaction to the antigen and have the ability to produce an antibody having low cross-reactivity to other bacteria are selected by flow cytometry.
  • the antigen B In preparing the monoclonal antibody, which is an example of this embodiment, first, the antigen B. The longum was adjusted.
  • the supernatant was removed by centrifugation at 4 ° C. for 15 minutes. Sterilized PBS was added to the remaining pellets for washing. The cells were washed 3 times in total. A part of the recovered cells was dried for 24 hours using a VD-800R freeze-dryer (manufactured by TAITEC), and the ratio of the dry weight to the cell weight was determined. The cells / PBS were diluted with sterilized PBS so as to be 100 ⁇ g / 100 ⁇ l, and used as an antigen below.
  • mice immunization was performed using the prepared antigen.
  • the tail venule (iv) was administered, only the antigen was additionally administered to the mice.
  • the adjusted antigen was applied to 5 BALB mice twice a week i. p. It was administered. Of the five mice, three were in the conventional immune group without SLOT treatment, and the spleens were removed from the remaining two mice and used for SLOT treatment.
  • mice that had undergone the SLOT treatment were undergone the SLOT treatment. v. After carrying out by the route, the spleen was removed and fused with myeloma cells to prepare a hybridoma.
  • test culture condition 1 The culture conditions for the first test bacterial group (hereinafter referred to as test culture condition 1) are as follows. To 5 ml of GAM liquid medium (manufactured by Nissui Pharmaceutical Co., Ltd.), 20 ⁇ l was added from the glycerol stock of each bacterium, and the cells were cultured in an anaerobic chamber BACTRON 300 (manufactured by SHEL LAB) for 24 hours.
  • the first test bacterial group was confirmed to be a single bacterium after culturing using the Gram stain method shown below.
  • 5 ⁇ l of the above-mentioned bacterial culture solution was applied to a slide glass and fixed by flame.
  • 100 ⁇ l of crystal violet was allowed to act on each spot, stained for 1 minute, and then washed with water.
  • 100 ⁇ l of iodine was allowed to act, the mixture was stained for 30 seconds, and then washed with water. Iodine solution staining was repeated once more.
  • An ethanol / acetone mixed decolorizing solution was allowed to act in an amount of 100 ⁇ l per spot, washed with water and dried.
  • 100 ⁇ l of the Paifel solution was allowed to act, stained for 1 minute, and then washed with water. After drying, it was observed under an optical microscope and an image was acquired using ToupView (manufactured by ToupTek Photonics).
  • an antibody-containing supernatant a cell culture supernatant containing an antibody (hereinafter referred to as an antibody-containing supernatant) was prepared. Adding the hybridoma cells 2X10 a flask cell culture medium was included in 50 ml 6, and one week cultured under 37 °C 5% CO 2 environment. The solution in the flask was transferred to a 50 ml tube and centrifuged at 1000 rpm, 5 min, 4 ° C. The supernatant was filtered through a 0.22 um filter to remove contaminants and collected in 50 ml tubes. NaN 3 was added to a final concentration of 0.05%, and the mixture was stored at 4 ° C. until use.
  • a flow cytometer Accuri C6 plus (manufactured by BD) was used to measure the specificity and cross-reactivity of the antibody in the antibody-containing supernatant.
  • the test culture condition 1 the target bacterial species was cultured, and the culture solution was centrifuged at 8000 ⁇ g, 5 min, 4 ° C., and the culture supernatant was removed. Then, after washing twice with PBS, the cell pellet was resuspended with PBS.
  • IgG1 Isotype Control manufactured by SIGMA
  • SIGMA which has no reactivity with the first test bacterial group
  • 1 ml of sterilized PBS was added and washed, and the mixture was centrifuged at 17,800 ⁇ g, 5 min, 4 ° C., and the supernatant was removed. After repeating this process twice, 100 ⁇ l of the secondary antibody was added, and the mixture was allowed to stand at 4 ° C. for 30 minutes and used for the study.
  • the secondary antibody was obtained by diluting Goat anti-mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor (registered trademark) 488 (manufactured by Thermo Fisher Scientific) with 2% BSA / PBS. Further, immediately before the FACS measurement, 1 ⁇ l of Propidium iodide (PI: manufactured by Invitrogen) was added to distinguish it from dead bacteria. The acquired FCS version 3.0 file was analyzed by FlowJo (FlowJo LLC).
  • FIG. 2 shows the results of the FACS using a hybridoma culture supernatant named Jih1 (hereinafter referred to as Jih1 culture supernatant) as an antibody-containing supernatant.
  • Jih1 culture supernatant a hybridoma culture supernatant named Jih1 (hereinafter referred to as Jih1 culture supernatant) as an antibody-containing supernatant.
  • the results show that the antibody contained in the Jih1 culture supernatant (hereinafter referred to as Jih1 antibody) is described in B.I. It was shown that it binds to longum but not to other Bifidobacterium genus shown in FIG.
  • the bacterial species purchased from JCM shown in FIG. 3, hereinafter referred to as the second test bacterial group
  • the Jih1 culture supernatant The reactivity of was examined.
  • the culture conditions of each bacterium were the same as those described in the test culture condition 1, the culture supernatant was the same as that described in paragraph 0044, and the FACS method used was that described in paragraph 0045.
  • FIG. 4 shows the results of FACS in which the Jih1 culture supernatant was reacted with the second test bacterial group. The results showed that the Jih1 culture supernatant did not react with the second test bacterial group. As a result, the Jih1 antibody becomes B.I. It proved to be an antibody with high specificity for longum and low cross-reactivity to other bacteria.
  • a flow cytometer Accuri C6 plus (manufactured by BD) was used to measure the specificity and cross-reactivity of the antibody in the antibody-containing supernatant.
  • the test culture condition 1 the target strain of the intestinal bacterium was cultured, and the culture solution was centrifuged at 8000 ⁇ g, 5 min, 4 ° C., and the culture supernatant was removed. Then, after washing twice with PBS, the cell pellet was resuspended with PBS.
  • IgG1 Isotype Control manufactured by SIGMA
  • SIGMA which has no reactivity with the gut microbiota reference strain
  • 1 ml of sterilized PBS was added and washed, and the mixture was centrifuged at 17,800 ⁇ g, 5 minutes at 4 ° C., and the supernatant was removed. After repeating this process three times, 100 ⁇ l of the secondary antibody was added, and the mixture was allowed to stand at 4 ° C. for 30 minutes and used for the study.
  • an Anti-mouse IgG PE-labeled antibody (manufactured by abcam) diluted 2000-fold with 2% BSA / PBS was used. After washing in the same manner as in the primary antibody washing, 400 ⁇ l of 2% BSA / PBS was added, and the mixture was stored at 4 ° C. until measurement. The acquired FCS file was analyzed with the analysis software FlowJo version 10.5.3.
  • Anti-PE labeled MACS registered trademark
  • BSA / PBS Anti-PE labeled MACS (registered trademark) beads (manufactured by Miltenyi Biotec) diluted 20-fold with 1% BSA / PBS was added to the culture solution in an amount of 200 ⁇ l each and reacted at 15 min and 4 ° C. 1 ml of sterilized PBS was added and suspended, and 14000 r. p. m. The supernatant was removed by centrifugation at 5 minutes and 4 ° C. After performing this washing step a total of 3 times, the mixture was suspended in 500 ⁇ l of 0.5% BSA / PBS (containing 2 mM EDTA) solution.
  • This suspension was separated using MACS (registered trademark) midi emulsion (manufactured by Miltenyi Biotec), and separated into a target fraction (Elution) to which magnetic beads were bound and another fraction (Flow Through). These separated solutions were centrifuged at 8,000 xg, 15 min, 4 ° C. and suspended in 400 ⁇ l of 2% BSA / PBS. Using Accuri C6 plus (manufactured by BD), the reactivity of various bacterial suspensions, Flow Through and Elution, with the Jih1 culture supernatant before and after separation by MACS (registered trademark) was analyzed. The acquired FCS file was analyzed with the analysis software FlowJo version 10.5.3. In addition, a part of Flow Through and Elution is 14000r. p. m. Bacterial pellets were collected at 10 min and 4 ° C and stored at ⁇ 80 ° C until use.
  • FIG. 6 shows the examination results of MACS (registered trademark). It was found that there was a difference between the Flow Through that flowed out without reacting with the Jih1 antibody and the Elution that flowed out from the antibody after reacting with the Jih1 antibody.
  • the DNA concentration was measured using Nanodrop, the sample concentration was diluted to 100 ng using Pure Water (manufactured by Wako), and stored at ⁇ 30 ° C. Based on the measured DNA concentration, the DNA diluted to 100 ng was diluted 10-fold with Pure Water.
  • RT-PCR was performed using diluted DNA as a template and SYBR (registered trademark) Premix Ex Taq2 (manufactured by TakaRa). The PCR conditions were initial denaturation 95 ° C., 30 sec ⁇ (96 ° C., 30 sec, 55 ° C., 45 sec, 72 ° C., 1 min) ⁇ 40 cycles. The primers used are shown in FIG.
  • the analysis method was based on the gene expression level amplified by 16S rRNA sequence-specific primers (Fig. 7) targeting Eubacterium.
  • the gene expression levels amplified by the longum-specific primer (Fig. 7) and the Bacteroides genus-specific primer (Fig. 7) were analyzed by the comparative Ct method.
  • FIG. 8 shows the results of RT-PCR.
  • B. Results using longum-specific primers show that B. longum-specific primers were used during Elution. It proved to contain longum.
  • Jih1 antibody B.I. It was found that longum can be specifically isolated and concentrated.
  • the culture solution before MACS separation and each solution of Flow Through and Elution after separation were centrifuged at 17,800 ⁇ g, 5 min, and 4 ° C., and suspended in 400 ⁇ l with 2% BSA / PBS. 50 ⁇ l of the suspended solution was sprinkled on an agar medium using a sterilized spreader and cultured. After culturing for 24 hours, the colonies formed on the plate were photographed.
  • hybridomas producing Jih1 antibody was performed as follows. Frozen vial tubes containing hybridomas were rapidly thawed at 37 ° C. Add 10 ml of cell culture medium (RPMI / 10% FCS / 1% sodium pyruvate solution / 1X penicillin-streptomycin-glutamine solution (manufactured by GIBCO)) to 1 ml of hybridoma cell solution, and centrifuge at 1000 rpm, 5 min, 4 ° C. separated. The supernatant was removed, resuspended in 1 ml of medium, transferred to a culture flask containing 14 ml of medium, and cultured in a CO 2 environment at 37 ° C. and 5%. Immediately before reaching saturation, passage was continued to multiply the extracellular fluid by a factor of 10.
  • cell culture medium RPMI / 10% FCS / 1% sodium pyruvate solution / 1X penicillin-streptomycin-glutamine solution (manufactured
  • Jih1 antibody-producing hybridoma cells were prepared as follows. The cultured hybridoma was collected and centrifuged at 1000 rpm, 5 min, and 4 ° C. The supernatant was removed and the cells were resuspended in 10 ml sterile PBS. Centrifugation was performed at 1000 rpm, 5 min, and 4 ° C., sterile PBS was added, and the number of cells was counted using a hemocytometer. The cells were adjusted to 2 x 10 7 / ml, 100 ⁇ l of cell solution was dispensed into a new tube, 900 ⁇ l of RNA later was added and suspended, and the sample was incubated overnight at 4 ° C. and the sample was used for DNA sequence analysis. There was.
  • the obtained antibody is B.I. It has high specificity for longum, and B. longum. Longum can be specifically purified. Therefore, this antibody is based on B.I. From samples such as feces containing bacteria other than longum, more accurately B. longum. Since longum can be isolated and further concentrated, it can contribute to the fields of medicine, health care and the like.
  • B. cerevisiae which is one of the intestinal bacteria constituting the intestinal flora.
  • B. longum is used. From a bacterial population containing longum, target B. longum. Only longum can be efficiently isolated and concentrated.

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Abstract

Le problème décrit par la présente invention concerne la fourniture d'une molécule de liaison à l'antigène qui a une spécificité élevée vis-à-vis de Bifidobacterium longum. La solution selon l'invention porte sur une molécule de liaison à l'antigène qui se lie à B. longum.
PCT/JP2020/044146 2019-11-29 2020-11-27 Molécule de liaison à l'antigène, composition d'acide nucléique, composition de vecteur et procédé de purification de cellules et bifidobacterium longum WO2021107072A1 (fr)

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JP2019-217338 2019-11-29
JP2019217338A JP2021083433A (ja) 2019-11-29 2019-11-29 抗原結合分子、核酸組成物、ベクター組成物、細胞およびＢｉｆｉｄｏｂａｃｔｅｒｉｕｍ ｌｏｎｇｕｍの精製方法

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004531245A (ja) * 2001-01-30 2004-10-14 ソシエテ デ プロデユイ ネツスル ソシエテ アノニム Ｎｃｃ２７０５−ビフィドバクテリウムのゲノム

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
JP2004531245A (ja) * 2001-01-30 2004-10-14 ソシエテ デ プロデユイ ネツスル ソシエテ アノニム Ｎｃｃ２７０５−ビフィドバクテリウムのゲノム
JP2004531229A (ja) * 2001-01-30 2004-10-14 ソシエテ デ プロデユイ ネツスル ソシエテ アノニム ビフィズス菌中のセルピン

Non-Patent Citations (4)

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Title
AMROUCHE, T. ET AL.: "Production and characterization of anti-bifidobacteria monoclonal antibodies and their application in the development of an immune-culture detection method", JOURNAL OF MICROBIOLOGICAL METHODS, vol. 65, 2006, pages 159 - 170, XP027926820 *
CHIRON, C. ET AL.: "Flow cytometry: a versatile technology for specific quantification and viability assessment of micro-organisms in multistrain probiotic products", J OF APPLIED MICROBIOLOGY, vol. 124, 2017, pages 572 - 584, XP055654957, DOI: 10.1111/jam.13666 *
SUZUKI, K. ET AL.: "Adhesion properties of a putative polvmorphic fimbrial subunit protein from Bifidobacterium longum subsp. longum'', Bioscience of Microbiota", FOOD AND HEALTH, vol. 35, no. 1, 2016, pages 19 - 27, XP055832766 *
XU, F. ET AL.: "Preparation of High Specific Polyclonal Antibody against Bifidobacterium longum", CHINESE JOURNAL OF BIOLOGICALS, vol. 23, no. 2, 2010, pages 182 - 184 *

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