Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/335—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Lactobacillus (G)

Definitions

• the present invention relates to a means for detecting lactic acid bacteria which affects the quality of a opaque bead by turbidity of the bead.
• the present invention also relates to a protein specific to lactic acid bacteria that makes beer turbid and a polynucleotide encoding the same.
• Beer has a limited carbon source, contains alcohol and carbon dioxide, is low in pH and is anaerobic, and contains hop-derived substances with antibacterial activity such as isohumulone.
• a beverage that is less susceptible to contamination and is less prone to microbial growth.
• certain lactic acid bacteria of the genus Lactobacillus or Pediococcus when mixed into beer under these conditions, proliferate and make the beer turbid, greatly affecting its quality. It is known to give Lactobacillus brevis (L. brevis), Pedococcus damnosus (P. damnosus), Lactobacillus lindneri (L. lindneri), and the like are typical, but other lactic acid bacteria have also been identified. ing.
• lactic acid bacteria belonging to the same species grow in beer and make the cloud turbid (hereinafter referred to as “beer turbid lactic acid bacteria”). ) And other lactate strains (hereinafter referred to as “beer non-turbid lactic acid bacteria”) are known to exist, and simply determining the genus alone does not necessarily mean that the beer is turbid lactic acid bacteria. Can not.
• the success or failure largely depends on the primer sequence and the primer sequence produced therefrom. That is, it is possible to easily detect a beer turbid lactic acid bacterium having a marker and a Braimer sequence produced therefrom, but cannot detect a beer turbid lactic acid bacterium which does not retain the sequence. Conversely, if there is a beer non-turbid lactic acid bacterium containing the sequence, this is erroneously detected as a beer turbid lactic acid bacterium.
• the conventional PCR-based determination method for beer turbid lactic acid bacteria has attempted to solve the above problem by making a primer based on the 16S liposomal RNA gene as a marker.
• the 16S ribosomal RNA gene is an essential gene for maintaining the life of bacteria and is highly conserved, but has a region, called the variable region, in which the DNA sequence differs depending on the species. This variable region is widely used for species classification and evolutionary phylogenetic analysis.Lactobacillus bacteria can also be used to detect Lactobacillus brevis, Pediococcus damnosus, Lactobacillus lindneri, etc. And can be determined. However, this method had two problems. One of them is related to a gene whose primer DNA sequence is not directly related to beer turbidity.Therefore, it may not be possible to detect beer turbid lactic acid bacteria with any mutation at this site. Is high.
• variable region of the 16S ribosomal RNA gene is considered to be a region where mutation is liable to occur.
• a beer turbid lactic acid bacterium having a mutation in the short region of the PCR primer is generated, it is considered that this cannot be detected.
• Another problem is that, as mentioned above, Lactobacillus previs and Pediococcus damnosus have lactic acid bacteria that are often of the same species, but are essentially lactobacilli that are both opaque to beer and opaque to beer.
• lactic acid bacteria containing beer and lactic acid not containing beer can be used for these lactic acid bacteria. Bacteria cannot be identified.
• a second preferable marker is a nucleotide sequence that is clearly found to be physically close to the causative gene that can impart the turbidity of the beads.
• the gene specific to beer turbid lactic acid bacteria was obtained as a hop resistance gene as an example of obtaining from Lactobacillus preforms; hor A and (journal of the American Society for brewing chemistry). 55 5 1 7—140, pp. 1997), considered to be a gene related to manganese uptake hit A (Federation of European Microbiological Societies and Netherlands Society for Microbiology 6th Lactic Acid Bacteria Symposium ⁇ ) Abstracts September 1999). These are considered to have prima facie effectiveness as a marker for beer turbid lactic acid bacteria in Lactobacillus previs.
• An object of the present invention is to provide a method for more accurately discriminating beer turbid lactic acid bacteria, and a probe, a primer, a primer pair, and an antibody for performing the method.
• the present invention also relates to a protein specific to lactic acid bacteria having beer turbidity, a method for producing the protein, a polynucleotide encoding the protein, a polynucleotide carrying the polynucleotide, and a vector comprising the polynucleotide. Its purpose is to provide a host transformed by one.
• the present inventors have now succeeded in obtaining a region (SEQ ID NO: 79) containing a specific gene widely found in beer turbid lactic acid bacteria (Example 1).
• the present inventors have also determined that beer turbid lactic acid bacteria can be distinguished at a very high frequency by using primers prepared based on this gene to determine beer turbid lactic acid bacteria by PCR. (Example 2).
• the polynucleotide probe for detection of beer turbid lactic acid bacteria comprises a nucleotide sequence consisting of at least 15 nucleotides which hybridizes to the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence.
• the method for detecting a beer turbid lactic acid bacterium according to the present invention is a method comprising a step of hybridizing a polynucleotide probe according to the present invention with a polynucleotide in a sample, and then detecting a hybridization complex.
• the polynucleotide primer used for detecting beer turbid lactic acid bacteria by the nucleic acid amplification reaction comprises a nucleotide sequence consisting of at least 15 nucleotides that hybridizes to the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence. is there.
• the primer pair according to the present invention comprises two types of primers according to the present invention, and is capable of amplifying a genomic sequence specific to beer turbid lactic acid bacteria by a nucleic acid amplification method.
• the method for detecting turbid lactic acid bacteria of beer according to the present invention is a method comprising the steps of: amplifying a polynucleotide in a sample by a nucleic acid amplification reaction using the primer pair of the present invention; and then detecting the amplified polynucleotide.
• the protein according to the present invention comprises the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence of SEQ ID NO: 7.
• the protein according to the present invention can also be obtained by culturing a host comprising a recombinant vector carrying a polynucleotide described below, and collecting a protein as an expression product of the polynucleotide from the culture.
• the polynucleotide according to the present invention encodes the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence of SEQ ID NO: 7.
• the recombinant vector according to the present invention carries the polynucleotide according to the present invention.
• a transformed host according to the present invention comprises a recombinant vector according to the present invention.
• the method for producing a protein according to the present invention comprises a step of culturing a host comprising a recombinant vector carrying a polynucleotide according to the present invention, and collecting a protein as an expression product of the polynucleotide from the culture. It becomes.
• the antibodies according to the invention are directed against the protein according to the invention.
• the method for detecting turbid lactic acid bacteria of beer according to the present invention comprises a step of reacting an antibody according to the present invention with a sample and detecting an antigen-antibody reaction.
• FIG. 1 is a diagram showing the positions of open reading frames and restriction sites in a gene region (SEQ ID NO: 79) specific to beer-turbid lactic acid bacteria. Detailed description of the invention
• Example 1 the present inventors succeeded in obtaining a gene region (SEQ ID NO: 79) specific to beer turbid lactic acid bacteria.
• ORFs open reading frames
• ORF 1 ⁇ ⁇ RF10 open reading frames
• ORF 1-3 genes form one operon, 0 RF 1 is considered to be a gene for glucosyltransferase or dolichol phosphate mannose synthase, and ORF 3 is a gene for ticolate galactosyltransferase. Can be Therefore, this operon is considered to be involved in the synthesis of sugar chains in the cell wall.
• this operon region is useful as a marker for determining turbidity, and the polynucleotide probe according to the present invention based on the nucleotide sequence in this region can be used for detecting beer turbid lactic acid bacteria.
• the present inventors further analyzed in detail up to which side of the operon both sides can be used as a marker for determining opacity, and the following results were obtained (see Example 2).
• lactic acid bacteria such as Lactobacillus plevis and Didococcus damnosus can be identified with extremely low frequency of lactic acid bacteria of beer Z and non-opaque lactic acid bacteria of beer.
• Lactobacillus brevis can identify beer-turbid lactic acid bacteria and beer-non-turbid lactic acid bacteria at a very high frequency, but it is not effective for the determination of Diococcus damnosus.
• ORF 5 cannot be used to identify beer turbid lactic acid bacteria Z-bille non-turbid lactic acid bacteria, at least within the range examined. (However, ORF 7 may be used to determine the species of lactic acid bacteria.) ).
• the prop according to the invention preferably has the nucleotide sequence of SEQ ID NO: 1
• the probe may be a probe consisting of at least 15 consecutive nucleotides of its complementary sequence.
• the polynucleotide probe according to the present invention preferably has a sequence in which at least 15 consecutive nucleotides are located at positions 2818 to 8056 of SEQ ID NO: 1 or a sequence complementary thereto (a sequence extending from 0RF 1 to 4, 8; see FIG. 1).
• SEQ ID NO: 2 (ORF 1), SEQ ID NO: 4 (ORF 2) or SEQ ID NO: 4 (ORF 2), most preferably at least 15 contiguous nucleotides 6 (ORF 3) or a probe consisting of at least 15 consecutive nucleotides of its complementary sequence.
• Probes consisting of at least 15 contiguous nucleotides of any of SEQ ID NO: 2 (ORF 1), SEQ ID NO: 4 (ORF2), and SEQ ID NO: 6 (ORF3) have the same 0 RF sequence but differ in arrangement. This is advantageous in that even if a beer-turbid lactic acid bacterium exists, the beer-turbid lactic acid bacterium can be detected without any problem.
• the probe according to the invention comprises at least 70%, preferably at least 80%, more preferably at least 80% of the nucleotide sequence consisting of 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence. Also included are those comprising a nucleotide sequence having an identity of 90%, most preferably at least 95%, and which hybridizes to a genomic sequence specific for beer turbid lactic acid bacteria.
• the “genomic sequence specific to beer turbid lactic acid bacteria” includes the nucleotide sequence of SEQ ID NO: 1 and its partial sequence, the nucleotide sequence of SEQ ID NO: 2 and its partial distribution sequence, the nucleotide sequence of SEQ ID NO: 4 and its partial sequence, and The nucleotide sequence of SEQ ID NO: 6 and a partial sequence thereof are exemplified.
• hybridize means that it hybridizes to a target nucleotide sequence under stringent conditions and does not hybridize to nucleotides other than the target nucleotide.
• Stringent conditions are determined by the Tm of the duplex between the probe sequence (or the primer sequence described below) and its complementary strand. (° C.) and the required salt concentration, etc., and it is a well-known technique to those skilled in the art to select stringent conditions after selecting a probe sequence (for example, J. Sambrook , EF Frisch, T. Maniatis; Molecular Cioning 2nd edition, Cold Spring Harbor Laboratory (1989)).
• the hybridization will specifically hybridize to sequences complementary to this nucleotide sequence and will not hybridize to sequences not complementary to this nucleotide.
• polynucleotide probe refers to a probe used for a means for detecting a nucleic acid such as Southern hybridization, Northern hybridization, colonization and the like.
• polynucleotide is used to include DNA, RNA, and PNA (peptide nucleic acid).
• the length of a polynucleotide probe according to the present invention consists of at least 15 nucleotides, more preferably at least 20 nucleotides.
• the polynucleotide probe according to the present invention is prepared by chemically synthesizing a nucleic acid according to an ordinary method such as the phosphite-triester method (Hunkapiller, M. et al., Nature, 310, 105, 1984).
• a total DNA of beer turbid lactic acid bacteria belonging to Lactobacillus brevis may be obtained according to the examples described later, and a DNA fragment containing the nucleotide sequence of interest based on the nucleotide sequence disclosed in the present specification may be obtained by PCR. Or the like may be appropriately obtained.
• the detection method using a probe can be carried out by hybridizing the polynucleotide probe of the present invention with a nucleic acid sample and detecting a hybridizing complex, ie, a nucleotide chain.
• a hybridizing complex ie, a nucleotide chain.
• the presence of the hybridizing complex indicates the presence of beer turbid lactic acid bacteria.
• hybridizing I This is as described in the section on nucleotide probes.
• a probe In a detection method using a probe, a probe can be labeled and used.
• the labeling example radioactivity (e.g., 3 2 P, 1 4 c , and 3 5 s), fluorescent
• FITC fluorescent chromase
• EuGase a label using an enzymatic reaction such as chemical coloring
• chemical coloring for example, peroxidase, alkaline phosphatase
• the detection of the hybridization complex can be carried out using well-known techniques such as Southern hybridization and colony hybridization (for example, J. Sambrook, EF Frisch, T. Maniatis: Molecular Cloning). 2nd edition, Cold Spring Harbor Laboratory (1989)).
• the test sample can be a sample suspected of containing beer turbid lactic acid bacteria, and specifically includes a bacterial colony detected by a microbe test of beer.
• the primer and primer pair according to the invention can each hybridize with a genomic sequence specific for beer turbid lactic acid bacteria. Therefore, the primer pair according to the present invention can be used for detection of beer turbid lactic acid bacteria by a nucleic acid amplification method such as the PCR method.
• the primer according to the present invention can be preferably a primer consisting of at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence.
• the “primer” refers to a nucleotide sequence used in a nucleic acid amplification method such as the PCR method.
• the “primer pair” refers to a pair of primers used in a nucleic acid amplification method such as the PCR method.
• the primer according to the invention consists of at least 15 nucleotides (preferably 15 to 30 nucleotides), preferably at least 20 nucleotides (more preferably 20 to 30 nucleotides). be able to.
• the primer pair according to the present invention can be selected so that a genomic sequence specific to beer turbid lactic acid bacteria can be amplified by a nucleic acid amplification method such as a PCR method.
• Nucleic acid amplification method are well known, and selection of a primer pair in a nucleic acid amplification method will be obvious to those skilled in the art.
• the PCR method one of the two primers is paired with one strand of a double-stranded DNA specific to beer turbid lactic acid bacteria, and the other primer is paired with the other strand of the double-stranded DNA.
• the primer can be selected such that it pairs with the other strand and the extended strand that has been extended by one primer.
• one primer consists of at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1 and the other primer has at least 15 contiguous sequences of the complement of the nucleotide sequence of SEQ ID NO: 1.
• Primers can be selected to consist of nucleotides.
• regions 0RF1, 2, 3, 4, and 8 are considered to be specific for beer turbid lactic acid bacteria, primers can be designed to amplify these regions (implementation See Examples 2 and 3).
• the primer pair according to the invention also preferably comprises one primer consisting of at least 15 contiguous nucleotides of the nucleotide sequence from position 2818 to position 8056 of SEQ ID NO: 1 (sequence spanning ORFs 1-4, 8), and the other primer One is a primer pair consisting of at least 15 consecutive nucleotides of the complementary sequence of the nucleotide sequence at positions 2818 to 8056 of SEQ ID NO: 1, more preferably one of the primers is a nucleotide sequence at positions 4202 to 7513 of SEQ ID NO: 1 (The sequence spanning ORF 1-3) consisting of at least 15 contiguous nucleotides, and the other primer consisting of at least 15 contiguous nucleotides of the complementary sequence of the nucleotide sequence from position 4202 to position 7513 of SEQ ID NO: 1.
• a pair of primers most preferably one primer is SEQ ID NO: 2, SEQ ID NO: 4, or The other primer consists of at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 6, SEQ ID NO: 4, or the complementary sequence of the nucleotide sequence of SEQ ID NO: 6
• a pair of brimmers can be
• the primer pairs of SEQ ID NO: 2 (ORF l), SEQ ID NO: 4 (ORF 2), and SEQ ID NO: 6 (ORF 3), which amplify one sequence or a partial sequence thereof, have the same 0RF sequence, There are beer turbid lactic acid bacteria strains whose arrangement is different Even so, it is advantageous in that lactic acid bacteria in beer turbidity can be detected without any problem.
• the primer according to the present invention comprises at least 70%, preferably at least 80%, more preferably at least 70% of the nucleotide sequence consisting of 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence. 90%, most preferably at least 95%, and also those comprising a nucleotide sequence that hybridizes to a genomic sequence specific to beer turbid lactic acid bacteria.
• the primer according to the present invention is based on the nucleotide sequence disclosed herein, for example, according to a conventional method such as the phosphite triester method (mmkapiller, M. et al., Nature, 310, 105, 1984). Can be chemically synthesized.
• the primer can be prepared, for example, according to “Bio-Experiment Illustrated 3 Really Increasing PCR” (by Hiroki Nakayama, Shujunsha).
• Hybridize in the detection method using a primer pair is as described in the section of the polynucleotide probe described above.
• the detection of beer turbid lactic acid bacteria using a primer pair is carried out in the usual manner using DNA obtained from a sample and using the primer according to the present invention as type I: DNA, as disclosed in the Examples below.
• PCR may be performed to detect the presence or absence of amplification of a DNA fragment specific for beer turbid lactic acid bacteria.
• the PCR technology itself is well known (for example, see “Bio Experimental Illustrated 3 Really Increasing PCR”), and those skilled in the art can appropriately carry out the method according to the present invention depending on the primer used.
• the presence of the amplification product indicates the presence of beer-turbid lactic acid bacteria.
• the test sample can be a sample suspected of containing beer turbid lactic acid bacteria, and specifically includes a bacterial colony detected by a microbe test of beer.
• the protein according to the present invention is specifically expressed in beer turbid lactic acid bacteria Therefore, it is an indicator of the presence of beer turbid lactic acid bacteria. Therefore, the protein according to the present invention is useful, for example, for producing an antibody according to the present invention described below.
• the protein according to the present invention comprises a protein having one or more modifications and having the amino acid sequence of SEQ ID NO: 3 having glucosyltransferase or dolicholic acid mannose synthase activity, and one or more And a protein comprising the amino acid sequence of SEQ ID NO: 7 having the following modification and having galactosyl ticolate transphorase activity.
• alteration means substitution, deletion, addition, and insertion.
• the number of modifications can be, for example, one to several, more specifically one to six. When there are a plurality of modifications, the types of the introduced modifications may be the same or different.
• the protein according to the present invention can be obtained by a step of culturing a host comprising a recombinant vector carrying the polynucleotide of the present invention and collecting a protein as an expression product of the polynucleotide, Can be identified.
• the polynucleotide according to the present invention encodes a protein specific to beer turbid lactic acid bacteria, and is useful for producing the protein according to the present invention described below by genetic recombination technology.
• Polynucleotides according to the present invention can be chemically synthesized DNA and naturally occurring DNA (derived from chromosomes, from plasmid).
• a method for obtaining a polynucleotide according to the present invention when obtaining a DNA having the nucleotide sequences of SEQ ID NOs: 2, 4, and 6, for example, a method is described in accordance with the Examples described below, which is used for the production of a beer cloudy lactic acid bacterium belonging to Lactobacillus brevis.
• Total DNA is obtained, a primer is appropriately prepared based on the nucleotide sequence disclosed herein so as to obtain a DNA fragment containing the target nucleotide sequence, and the DNA fragment is obtained by amplifying the DNA fragment by PCR.
• a DNA having a nucleotide sequence degenerately related to the nucleotide sequences of SEQ ID NOs: 2, 4, and 6 can be obtained by chemical synthesis of a nucleic acid.
• the sequence of the obtained DNA was determined by the Maxam Gilbert method (for example, Maxam, AM turbulence Gilbert, Natl. Acad. Sci. USA, 74, 560, 1977, etc.) and the Sanger method (for example, Sanger, F. & A. ILCoulson, J. Mol. BioL, 94, 441, 1975, Sanger,, Acad. Sci. USA, 74, 5463, 1977, and the like)).
• the recombinant vector according to the present invention can be constructed by incorporating the polynucleotide according to the present invention into a vector having a marker-gene that is replicable in a host and using a conventional genetic engineering technique. Can be.
• the recombinant vector according to the present invention can be produced according to the vector construction technique used. Specifically, when using Escherichia coli, which is a microorganism, as a host, for example, plasmid pUC119 (manufactured by Takara Shuzo Co., Ltd.) or phagemid pBluescriptll (manufactured by Straugene) is used. Can be. When yeast is used as a host, plasmid PYES2 (Invitrogen) can be used. PRC / RSV, pRC / CM when using mammalian cells as host
• a vector containing an autonomous replication origin derived from a virus such as pCEP4 (Invitrogen) can be used.
• a virus such as pCEP4 (Invitrogen)
• an insect virus such as baculovirus
• viruses such as baculovirus
• a transfer vector containing a nucleotide sequence homologous to the genome of the virus to be used can be used.
• transfer vectors include pBac PAK commercially available from Clonetech.
• Plasmids such as pAcUW31 can be mentioned.
• the polynucleotide according to the present invention is inserted into a transfer vector and the transfer vector and the viral genome are simultaneously introduced into a host, homologous recombination occurs between the transfer vector and the viral genome, and the polynucleotide according to the present invention is obtained.
• the virus integrated into the genome can be obtained.
• a vector capable of expressing a protein according to the present invention in a host comprises a polynucleotide according to the present invention and a control sequence operable in the host (for example, a promoter sequence and a one-minute-one-one sequence). And ligate them into a vector. It can be built by inserting.
• ⁇ operably linked '' means that a control sequence is linked to a polynucleotide according to the present invention so that the polynucleotide is expressed under the control of the control sequence in a host into which the polynucleotide according to the present invention is introduced.
• the motor can be connected upstream of the gene, and Yuichi Mine-Yuichi can be connected downstream of the gene.
• the promoter that can be used is not particularly limited as long as it exhibits a promoter activity in the host to be transformed.
• the host cell is an animal cell or fission yeast, for example, the early stage of adenovirus (Ad) Or late promoter, rous sarcoma virus (RSV) promoter overnight, cytomegalovirus (CMV) promoter overnight, simian virus (SV40) early or late promoter, mouse papillomavirus (MMTV) promoter overnight
• the promoter of the thymidine kinase (tk) gene of simple herpes virus (HSV) can be listed.
• the promoter of ADH1, GAL1 can be listed.
• the host is an insect cell, the baculovirus polyhedron promoter can be used overnight or the Drosophila metallothionein promoter. It can be.
• the promoter of the present invention and the polynucleotide of the present invention can be operably linked.
• plasmids pRCy / RSV, pRC / CMV, etc. have a cloning site downstream of a promoter operable in animal cells, insert the polynucleotide according to the present invention into the cloning site, and introduce it into animal cells. Then, the protein according to the present invention can be expressed.
• these plasmids contain the autonomous replication origin (ori) of SV40 in advance, when the plasmid is introduced into cultured cells transformed with the SV40 genome of ori (—), such as COS cells, Within this, the copy number of the plasmid is greatly increased, and as a result, the polynucleotide according to the present invention incorporated in the plasmid can be expressed in a large amount.
• the yeast plasmid pYES2 has a GAL1 promoter, and the plasmid or its derivative according to the present invention is downstream of the GAL1 promoter.
• the recombinant vector according to the present invention may be further linked with a marker gene for selecting a transformant.
• the recombinant vector according to the present invention further comprises a polynucleotide encoding the amino acid sequence of another protein or a part thereof directly or specific to a specific protease at the 5 ′ or 3 ′ side of the polynucleotide of the present invention. May be linked in frame via a polynucleotide encoding an amino acid sequence corresponding to a specific cleavage site.
• the amino acid sequence of another protein or a part thereof may have a signal peptide for secretion at its N-terminus, and in this case, ligation to the 5 'side is preferable.
• the method for introducing the recombinant vector according to the present invention into a host can be performed according to a method generally used depending on the host to be transformed.
• the calcium chloride method and the electroporation method described in Molecular Cloning, J. Sambrook et al., Cold Spring Harbor (1989) and the like can be used.
• the vector can be introduced using, for example, a Yeast transformation kit (manufactured by Invitrogen) based on the lithium method.
• a mammalian cell, an insect cell, or the like is used as a host, for example, the calcium phosphate method, the DAE dextran method, the electoral poration method, or the lipofection method can be used.
• the virus genome When a virus is used in a vector, the virus genome can be introduced into a host by the above-described general gene transfer method, or the virus genome can be transferred by infecting the host with a virus particle containing the virus genome. It can be introduced into a host.
• Transformants may be selected by a method according to the nature of each gene possessed by the introduced recombinant vector of the present invention.
• the marker gene is a resistance gene to a drug showing cell killing activity
• cells into which the recombinant vector of the present invention has been introduced may be cultured in a medium to which the drug is added.
• a drug resistance gene and a selected drug examples include the neomycin resistance gene and neomycin, the hygromycin resistance gene and hygromycin, the blasticidin S resistance gene and blasticidin S.
• the marker gene is a gene that complements the auxotrophy of the host, cells into which the recombinant vector of the present invention has been introduced may be cultured in a minimal medium that does not contain this nutrient.
• the recombinant vector of the present invention is linearized by digestion with a restriction enzyme or the like, followed by The cells may be introduced into a host by the above-described method and cultured usually for several weeks, and the transformed cells of interest may be selected using the introduced detection marker as an index.
• the recombinant vector according to the present invention having a resistance gene to the selective drug as a marker gene as described above is introduced into a host by the method described above, and subcultured for several weeks or more in a medium containing the selective drug, By picking up and purifying the selected drug-resistant clone that has survived in a colony, the transformant in which the gene of the present invention has been introduced into the chromosome of the host can be obtained.
• Such a transformant can be cryopreserved, and can be used after being awakened as needed, so that the effort for producing the transformant can be reduced as compared with a transient transgenic strain. This is advantageous in that the performance of the transformed cells can be kept constant.
• the protein according to the present invention can be produced by culturing the transformed host according to the present invention and collecting the protein according to the present invention from the culture.
• Proteins in which one or more of the N-terminus and zo or C-terminus are deleted due to processing in a host or the like can also be sufficiently used as an antigen for obtaining the antibody according to the present invention.
• the recombinant vector according to the present invention may have the amino acid sequence of another protein or a part thereof at the 5 ′ side or the 3 side of the gene of the present invention.
• the protein according to the present invention can It is expressed in the form of a fusion protein with the protein or a part thereof, which is also included in the protein according to the present invention.
• the carbon source used for normal culture of this microorganism nitrogen Culture can be performed using various media containing a source, organic salts, and inorganic salts as appropriate. Cultivation can be carried out in accordance with the usual method for culturing microorganisms. For example, solid culture, liquid culture (agitated culture (test tube shaking culture, reciprocating shaking culture, jaw fermenter) ) Cultivation) and static culture (including tank culture).
• the culture temperature can be appropriately changed within a range in which the microorganism grows.
• the culture may be performed in a culture temperature of about 15 ° C to about 40 ° C, pH of about 6 to;
• the culture time can be determined according to the culture conditions, but can usually be about 1 day to about 5 days.
• the host When the host is an animal cell, it can be cultured using a medium used for normal culture of the animal cell. For example, 10 v / v% and I'll become - cormorants using a medium of DMEM culture areas, such as the addition of FB S, at 37 ° 5 vZv% C0 2 presence, while replaced every few days the media culture can do.
• a medium of DMEM culture areas such as the addition of FB S, at 37 ° 5 vZv% C0 2 presence, while replaced every few days the media culture can do.
• the cells have grown to confluence, disperse them into individual cells using a 0.25 w / v% trypsin PBS solution, dilute them several times, inoculate them into new culture vessels, and continue culturing. Once the cells have grown to the required volume, collect the cells. By performing the subculture in this manner, the culture scale can be expanded to an arbitrary size.
• the host when it is an insect cell, for example, it can be obtained by subculturing at 25 ° C to 35 ° C using an insect cell medium such as Grace's medimn containing 10 vZv% FBS and 2 w / v% yeastlate. be able to.
• an insect cell medium such as Grace's medimn containing 10 vZv% FBS and 2 w / v% yeastlate.
• the culture is preferably terminated before the cell is killed by the cytoplasmic effect, for example, by 72 hours from the start of the culture.
• the protein of the present invention can be purified and isolated using known isolation and purification means.
• purifying the protein according to the present invention when the protein is produced intracellularly, for example, after the culture is completed, the cells are collected by centrifugation or the like, and the cells are collected in a normal buffer, for example, 20 mM HEPES pH 7, 1 mM. After suspending in a buffer consisting of EDTA, ImM DTT, 0.5 mM PMS F, etc., the cells are disrupted using a polytron, ultrasound, dounce homogenizer, etc.
• the fraction containing the protein according to the present invention can be obtained by ultracentrifuging at tens of thousands of xg for several tens of minutes to about 1 hour and collecting the supernatant fraction.
• a fraction containing the protein according to the present invention can be obtained as a supernatant fraction by centrifugation after completion of the culture.
• These supernatant fractions are subjected to salting out, solvent precipitation, dialysis, ultrafiltration, gel electrophoresis, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, affinity chromatography.
• the purified protein of the present invention can be recovered by appropriately combining purification means such as chromatography.
• the antibody according to the present invention is capable of recognizing protein specific to beer turbid lactic acid bacteria. Therefore, the antibody according to the present invention can be used for detecting beer turbid lactic acid bacteria by an antibody-antigen reaction.
• the antibodies according to the present invention include both polyclonal and monoclonal antibodies. Techniques for producing antibodies are well known, and therefore, whether using the protein according to the present invention as an immunogen, whether a polyclonal antibody or a monoclonal antibody according to the present invention, a person skilled in the art can easily prepare the protein according to each conventional method. Can be.
• the antibody according to the present invention comprises a protein according to the present invention as an antigen, a mammal generally used for producing an antibody such as a heron, a mouse, a rat, a goat, a sheep, a chicken, a hamster, a horse, a guinea pig, and the like.
• a polyclonal antibody can be obtained by administering it to birds or the like together with an adjuvant or the like, obtaining an antiserum from these animals, and performing fractionation and purification as it is or as necessary as described below.
• a monoclonal antibody for example, a mouse is used as the mammal, the spleen of the immunized mouse is removed, and lymphocytes prepared from the spleen and mouse myeloma (for example, p3X636.5.3 ATCCN o. CRL—1508) was fused with polyethylene glycol 150 (Peringer), and the positive cells were screened by limiting dilution from the fused cells. (See Milstein & G. Kohler 5 Nature, Vol. 256, p495 (1975)).
• hybrids expressing antibodies Cloning of the antibody gene or a part of the gene from the mammary cell can also be used to obtain a genetically engineered antibody molecule.
• a method for purifying an antibody from a material containing the antibody thus obtained that is, a culture supernatant such as an antiserum or a hybridoma
• a method generally used for protein purification protein A affinity chromatography, Affinity chromatography, such as protein G affinity chromatography, Avid gel chromatography, anti-immunoglobulin immobilized gel chromatography, cation exchange chromatography, anion exchange chromatography, lectin affinity chromatography, Dye adsorption chromatography, hydrophobic mutual chromatography, gel filtration chromatography, reverse-phase chromatography, hydroxy, hydroxypropylapatite chromatography, fluoroapatite chromatography Metal chelating chromatography, isoelectric point chromatography primary, there is a method of combining one or more of the preparative electrophoresis, and isoelectric focusing, etc.).
• a gel carrier or membrane in which the protein according to the present invention is chemically bound is prepared, and a material containing the antibody is added thereto, thereby adsorbing the antibody of interest.
• a method of eluting and recovering this under appropriate conditions, ie, antigen affinity purification, can also be used.
• Detection of the antibody-antigen reaction can be performed according to a known method, and can be performed, for example, as follows. After suspending the target lactic acid bacteria in a solution of 4 OmM Tris-HC1 (pH 7.5), 1 mM EDTA, and 15 OmM NaC1, several minutes with sufficient glass beads The cells are completely disrupted by vigorous stirring. Add SDS to a final concentration of 0.1%, stir again, and centrifuge to collect the supernatant fraction. The antibody or Fab ′ fragment derived from the antibody is prepared by binding a labeling enzyme such as horseradish peroxidase, a fluorescent substance, biotin, an isotope or the like.
• a labeling enzyme such as horseradish peroxidase, a fluorescent substance, biotin, an isotope or the like.
• the labeled antibody is mixed with the target lactic acid bacteria extract, and the antibody is sufficiently adsorbed to the antigen. Then, the excess labeled antibody is washed and removed, and the activity of the enzyme labeled with the antibody is measured. Turbid lactic acid bacteria can be detected. Many such detection methods are known, such as the Enzyme Linked Immunosorbent Assay (ELISA) method, the Western plot analysis method, and the Radioimunoassay method. The detailed procedure can be referred to Antibodies, A laboratory Manual, Ed Harlow and David Lane, Cold Spring Harbor Laboratory 1988, etc. Example
• Example 1 Acquisition of beer turbid lactic acid bacteria gene
• Lactobacillus brevis 3 turbid lactic acid bacteria and 2 non-turbid lactic acid bacteria were cultured in 100 ml of MRS medium (Oxoid) by stationary culture until stationary phase. After the cells were collected, the method of Douglas et al. (Applied and Environmental Microbiology, 46: 549- 552 (1983)), total DNA was obtained from each cell. The final DNA concentration was adjusted to about 10 to 20 mg / ml, and 2 ml of this DNA was used as type I DNA, and random polymorphic DNA PCR (RAPD PGR) (Nucleic Acid Research 22: 6531-6535 (1990)) was used. Done.
• MRS medium Oxoid
• a primer for gene mating which is a random 10-mer synthetic DNA
• PCR that uses 540 species from Kit AA to AZ as primers went.
• the primer used was 2 ml of 2 OmM, and the total volume of the reaction solution was adjusted to 50 ml.
• Takara Ex Taq kit of Takara Shuzo was used as a PCR reaction reagent, and Gene Amp PCR System 9600 of Perkin Elmer was used as a reaction device.
• the reaction program consisted of 45 cycles of 94 ° C for 1 minute, 36 ° C for 1 minute, and 72 ° C for 2 minutes.
• the reaction solution was electrophoresed on a 1% agarose gel, stained with ethidium amide solution, and the amplified band was analyzed.
• a gene specific to beer turbid lactic acid bacteria was recognized.
• Primers that is, primers that generate a band of a common size from the type III DNAs of the three types of beer-opaque lactic acid bacteria and that do not generate a band of the same size than the two types of non-beer-opaque lactic acid bacteria were selected.
• Primers thus selected were further subjected to PCR in the same manner using DNA extracted from 12 strains of turbid lactic acid bacteria in beer and 8 strains of turbid lactic acid bacterium as beer, and specific PCR was performed for lactic acid bacteria specific to beer. The primers that caused the band were further narrowed down.
• nucleotide sequences of the finally selected primers were as follows. OPAT 07 5 5 -ACTGCGACCA-3 5 ( SEQ ID NO: 8)
• OPAX 05 5 3 -AGTGCACACC-3 ' SEQ ID NO: 10.
• ORF4 8260th to 7766th
• ORF 7 9496th to 9867th
• DNA sequence data such as GENBANK etc. —Comparison with the evening base and function estimation. The results were as follows.
• ORF 1 Consists of 3 15 amino acids (SEQ ID NO: 3) and has 69% homology to an estimated 38.5 kDa protein similar to Bacillus subtilis glycosyl mannose synthase Therefore, it is considered that the protein encodes a protein having the function of glucosyltransferase or dolichol phosphate mannose synthase.
• ORF2 Consists of 644 amino acids (SEQ ID NO: 5). No homologous protein was found by comparison with the database.
• ORF3 Consists of 134 amino acids (SEQ ID NO: 7) and has 63% homology to Listeria monocytogenes (DgtcA, glycosylated ticoate protein); therefore, the function of ticolate galactosyltransferase It is thought to encode a protein with
• ORF4 Consists of 176 amino acids and has 86% homology to tract A of Lactococcus lactis, and is considered to encode a protein having the function of a nicking enzyme.
• ORF5 Consists of 69 amino acids and has 76% homology to the putative protein ORF0004 of the plasmid pMRCO1 of Lactococcus lactis (Molecular Micro Biology, 4: 1029-1038 (1998)).
• ORF6 Consists of 92 amino acids and has 77% homology to the putative protein ORF0003 of the plasmid pMRCO1 of Lactococcus lactis (Molecular Micro Biology, 4: 1029-1038 (1998)).
• ORF 7 Consists of 123 amino acids and has 62% homology to Orf A of IS 298 of Caulobacter crescentus. Therefore, it is considered that it encodes a protein having a transposase function.
• ORF 8 Consists of 305 amino acids and has 96% homology to the putative transposase of IS1070 of Leuconostoc lactis, and is considered to encode a protein having a transposase function.
• ORF9 Consists of 428 amino acids. No homologous protein was found by comparison with the database.
• ORF 10 Consists of 172 amino acids and has a homology of 49% with yxcB of Lactococcus lactis, and is considered to encode a protein having the function of a transcriptional regulator.
• ORFs 1, 2, and 3 are within one operon, these three ORFs are considered to be involved in the synthesis of some sugar chains.
• Example 2 Judgment of beer turbid lactic acid bacteria by PCR
• ORF 2- 1 5, -GCCCCGACTTGACCATTTGT-3 5 ( SEQ ID NO: 13).
• ORF4-2 5, -AGCCAAGCTTGATGCCGGCA-3 5 (SEQ ID NO: 18)
• ORF 8 5 '-TCTAATTCTTTGGCGCTAACCGTC-3 5 (SEQ ID NO: 26)
• the reaction program was 25 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute.
• the reaction solution after the reaction was electrophoresed on a 1.5% agarose gel, and the presence or absence of a band specific to beer turbid lactic acid bacteria was examined. Prior to the present invention, it was reported as a marker gene for judging beer turbidity lactic acid bacteria.]!
• Primers were synthesized from the it A and hor A genes, similar experiments were performed, and the results were compared. The results were as shown in Tables 1, 2 and 3.
• Lactobacillus previus was able to identify beer turbid lactic acid bacteria at exactly the same frequency as 0RF 1, 2, 3, 4, and 8. Bands were detected in all strains of D. coccus damnosus, and no bands were detected in single-type lactic acid bacteria of unknown taxonomic species.
• Example 3 Preparation of other primers and probe oligonucleotides for regions with high specificity for beer turbid lactic acid bacteria
• Primer pairs were designed based on the nucleotide sequences of ORFs 1, 2, 3, 4, and 8, which have particularly high specificity for beer turbid lactic acid bacteria. If necessary, primer design software (OLIGO primer analysis software ver.6.0: National al Biosciences, Inc.) may be used to design a primer pair. Oligonucleotides are prepared by chemical synthesis, and the resulting primer pairs are used in the PC III method. The PCR primer pair and hybridization conditions for each ORF region are as follows. In addition, these oligonucleotides can be used alone as a probe.
• primer design software OLIGO primer analysis software ver.6.0: National al Biosciences, Inc.
• the PCR reaction conditions are 30 cycles of 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 1 minute.
• the PCR reaction conditions are 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1/25 cycles.
• the PCR reaction conditions are 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1/25 cycles.
• the PCR reaction conditions are 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1/25 cycles.
• the PCR reaction conditions are 94 ° C for 30 seconds, 65 ° C for 30 seconds, and 72 ° C for 1/25 cycles.
• the PCR reaction conditions are 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 1/25 cycles.
• the PCR reaction conditions are 30 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1/25.
• the PCR reaction conditions are 94 ° C for 30 seconds, 65 ° C for 30 seconds, and 72 ° C for 1 minute and 25 cycles.
• the PCR reaction conditions are 30 cycles of 94 ° C for 30 seconds, 65 ° C for 30 seconds, and 72 ° C for 1/25.
• PCR reaction conditions are 30 seconds at 94 ° C, 30 seconds at 50 ° C, and 25 cycles of 72 ° C for 1 minute.
• the PCR reaction conditions are 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1/25 cycles.
• the PCR reaction conditions are 94 ° C for 30 seconds, 65 ° C for 30 seconds, and 72 ° C for 1/25 cycles.
• PCR reaction conditions are 94. C 30 seconds, 65 ° C 30 seconds, 72 ° C 1/25 cycle.
• the PCR reaction conditions are 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 1/25 cycles.
• the PCR reaction conditions are 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 1/25 cycles.
• the reaction conditions for P are 30 cycles of 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 1/25 cycles.
• the PCR reaction conditions are 30 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1/25.
• PCR reaction conditions were 94 ° C for 30 seconds and 65. C 30 sec, 72 ° C 25 min cycle.
• PCR reaction conditions are 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 1 minute 25 cycles.
• the PCR reaction conditions are 30 cycles of 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 1 minute.
• the PCR conditions are 25 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute.
• PCR reaction conditions are 30 cycles of 94 ° C for 30 seconds, 65 ° C for 30 seconds, and 72 ° C for 1 minute.
• the PCR conditions are 25 cycles of 94 ° C for 30 seconds, 65 ° C for 30 seconds, and 72 ° C for 1 minute.

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