CN101705236B - Protein sequence of FbpA in lactobacillus casei Zhang - Google Patents
Protein sequence of FbpA in lactobacillus casei Zhang Download PDFInfo
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Abstract
The invention relates to a protein sequence of FbpA in lactobacillus casei Zhang and provides a new nucleotide sequence for coding a lactobacillus casei heat shock protein FbpA. The sequence is a gene correlated with the stress of lactobacillus casei Zhang and has 99 percent of similarity with the known nucleotide sequence FM177140 of the lactobacillus casei BL23 and the nucleotide sequence CP000423 of the lactobacillus casei ATCC334. The invention also provides a nucleotide sequence of FbpA gene in the lactobacillus casei Zhang, which is SEQ ID NO: 1. The invention also provides an aminoacid sequence of the separated lactobacillus casei heat shock protein FbpA, which is SEQ ID NO:2.
Description
[technical field]
The invention belongs to the genetically engineered field, particularly, relate to a kind of albumen coded sequence, particularly the protein sequence of FbpA in a kind of Lactobacillus casei Zhang.
[background technology]
About more than 1000 kinds of symbiotic microorganisms are arranged in the human intestinal, and the summation of its genetic information is " microorganism group ", and encoding gene has more than 1,000,000.If want to keep the state of health of human body self, must make to keep mutually coordination, perfect harmony between intestinal microflora and the people.Bacterium lacticum is a class zymohexose, produces the general name of the bacterium take lactic acid as main metabolites.They stick the Growth and reproduction that can effectively suppress pathogenic bacteria with field planting as one of dominant microflora in the enteron aisle at host's gi tract epithelium, have played very important effect for keeping the intestinal microecology balance.In recent years, stick for Bacterium lacticum in the research of characteristic, except adopting conventional Physiology and biochemistry means qualitative detection Bacterium lacticum to stick activeconstituents and the biological characteristics thereof, many investigators also utilize the analytical procedure of information biology also to carry out intensive analysis to sticking relevant albumen.At present, although a part of member's function has obtained checking in these genes, the adherence mechanism of Bacterium lacticum it be unclear that.Along with development and the maturation of modern molecular biology investigative technique, the database of correlation function gene certainly will become better and approaching perfection day by day, and this also helps the identification of this mechanism.
FbpA albumen is dimer glycoprotein, and molecular weight is about 450kD.Two kinds of existence forms are arranged usually: a kind of free being present in the blood plasma; A kind of with the fibrous extracellular matrix that exists.About the existing nearly 60 years history of the research of fbpA albumen, particularly in pathogenic bacterium, comprise streptococcus pneumoniae and micrococcus scarlatinae, it is proved to be at Growth of Cells, break up and stick very important effect is arranged in the process, therefore also is considered to one of main virulence factor in the pathogenic bacteria.In Bacterium lacticum, the investigator has also carried out fruitful exploration for fbpA albumen latent effect in sticking process.For example: in short lactobacillus, the investigator finds that S-layer proteins has the zone that connects with fibronectin at the N end, is comprised of 96-245 amino acid; In Lactobacterium acidophilum, the transgenation result has also proved fbpA indispensable effect in sticking process, and the ability that the strain cell that lacks the fbpA gene sticks Caco-2 significantly descends.Although lactobacterium casei also is one of focus of studying at present, up to now, seldom there is the scholar to inquire into for the fbpA albumen of encoding in the lactobacterium casei.
The present invention is with reference to other bacterial strain fbpA gene order, cloning and sequencing obtains the fbpA gene order in the Lactobacillus casei Zhang (Lactobacillus casei Zhang), and different plant species fbpA gene nucleic acid and aminoacid sequence carried out similarity relatively, for carry out from now on lactobacterium casei in fbpA albumen and host stick ability correlative study work basic data be provided.
" Wang Junguo etc., Lactobacillus casei Zhang Journal of Nutrition the 1st phase of the 31st volume in 2009, disclose the Lactobacillus casei Zhang bacterial strain in open 2009 2 months day to the impact of Antioxidation Ability of Rats at document.
At Chinese patent application CN200810175856.7, publication number CN101418270A in open day on April 29th, 2009, discloses the Lactobacillus casei Zhang bacterial strain in the title page, page 2, the 6th page, and has illustrated that preserving number is CGMCC1697.
At number of patent application 2006101273622, publication number CN 101144062A, open day is on March 19th, 2008, and it is lactobacterium casei that Classification And Nomenclature is provided, and strain name is that the biomaterial preservation of the bacterial strain of zhang proves, and preserving number is CGMCC 1697.
Above-mentioned literature content is incorporated herein by reference.
[summary of the invention]
The invention provides heat shock protein(HSP) FbpA encoding sequence in a kind of Lactobacillus casei Zhang, this sequence is that Lactobacillus casei Zhang stress closely-related gene, and its nucleotide sequence has 99% similarity with the nucleotide sequence FM177140 of the lactobacterium casei BL23 that has announced and the nucleotide sequence CP000423 of lactobacterium casei ATCC334.
Particularly, the invention provides the encoding sequence of the heat shock protein(HSP) FbpA in a kind of Lactobacillus casei Zhang, the nucleotides sequence of this sequence is classified SEQ ID NO:1 as,
The present invention also provides the FbpA albumen in a kind of isolated Lactobacillus casei Zhang, and the aminoacid sequence of this albumen is SEQ ID NO:2.
The present invention also provides polypeptide or its conservative property variation polypeptide or its active fragments of the aminoacid sequence shown in the SEQ ID NO:2, or its reactive derivative.
The present invention also provides heat shock protein(HSP) FbpA gene cloning and sequence measurement in a kind of Lactobacillus casei Zhang.The method comprising the steps of: (1) PCR design of primers is with synthetic; (2) extraction of lactobacterium casei DNA; (3) pcr amplification of purpose segment; (4) agarose gel electrophoresis detects; (5) the goal gene segment reclaims and identifies; (6) cloning and identification of goal gene segment; (7) order-checking, it is characterized in that the PCR primer is SEQ ID NO:3(forward) and SEQ ID NO:4(reverse), its sequence information is as follows:
SEQ?ID?NO:3：5’-GCTTTTTGCTATGCCACCC-3’
SEQ?ID?NO:4：5’-TGACAGCGACCGTCTTTTG-3’
Particularly, the detailed content of each step is as follows:
(1) the PCR design of primers is with synthetic
The forward primer that the PCR primer is right and reverse primer information are as follows:
SEQ ID NO:3(forward): 5 '-GCTTTTTGCTATGCCACCC-3 ';
SEQ ID NO:4(is reverse): 5 '-TGACAGCGACCGTCTTTTG-3 ';
(2) extraction of lactobacterium casei DNA
The extraction of total DNA is extracted the test kit specification sheets with reference to the bacterial genomes of QIAGEN and is carried out.Concrete operation step is as follows:
Inoculum 5ml, centrifugal 1 minute of 10,000rpm abandons supernatant.Add an amount of lysozyme soln, making its ultimate density is 20mg/ml, thoroughly suspends, and processes 30-60 minute for 37 ℃.Add 20 μ l Proteinase K (20mg/ml) solution, mixing.Add 220 μ l buffered soln GB, vibrated 15 seconds.Placed 30 minutes for 70 ℃.Add 220 μ l dehydrated alcohols, fully vibrated 15 seconds.Solution is added in the adsorption column centrifugal 30 seconds of 12,000rpm.Add 500 μ l protein liquid removal GD, centrifugal 30 seconds of 12,000rpm abandons waste liquid.Add 700 μ l rinsing liquid GW, centrifugal 30 seconds of 12,000rpm abandons waste liquid.Add 500 μ l rinsing liquid GW, centrifugal 30 seconds of 12,000rpm discards waste liquid.Again 12, centrifugal 2 minutes of 000rpm places room temperature number minute with adsorption column.Adsorption column is changed in the clean centrifuge tube, add 50-200 μ l elution buffer TE, room temperature was put 2-5 minute, centrifugal 30 seconds of 12,000rpm.The centrifugal solution that obtains adds in the adsorption column again, and room temperature was placed 2 minutes, and centrifugal 2 minutes of 12,000rpm namely obtains the Lactobacillus casei Zhang genomic dna.
(3) pcr amplification of purpose segment
Pcr amplification system: 0.2 μ l Taq polysaccharase (5U/ μ l, Takara Tokyo, Japan), 2.5 μ l, 10 * PCR damping fluid (without Mg
2+), 2 μ l dNTP (2.5mM each), 2 μ l MgCl
2(25mM), 0.2 μ l forward primer (50pM), 0.2 μ l reverse primer (50pM), 1 μ l genomic dna and 17.4 μ l ddH
2O.
Pcr amplification condition: 97 ℃ of sex change 5min; 95 ℃ of 30s, 54.5 ℃ of 30s, 72 ℃ of 30s so carry out 35 circulations; 72 ℃ are extended 10min, 4 ℃ of 10min.
(4) agarose gel electrophoresis detects
Get PCR product 5 μ l, mix with 1 μ l sample-loading buffer, point sample adds 5 μ l DL2000 Marker, electrophoresis 30min under the 120V/cm voltage in another hole in the sepharose hole.Electrophoresis is left and taken photo at gel imaging system GDS-8000 System (UVP Biomaging Systems) after finishing.
(5) the goal gene segment reclaims and identifies
The recovery of fragment is carried out according to the explanation that the QIAGEN sepharose reclaims test kit.Concrete operation step is as follows:
The Agarose plug that will contain rapidly purpose fragment band under ultraviolet lamp is cut off, and puts into the centrifuge tube of 1.5ml.Add 3 times of volume sol solutions PN in the blob of viscose, put 50 ℃ of water-baths and placed 10 minutes, Agarose plug is dissolved fully.Agarose plug after will dissolving moves among adsorption column CA1 or the CA2, and centrifugal 30 seconds of 13,000rpm outwells the liquid in the collection tube.Adsorption column is put into same collection tube, in adsorption column, add 700 μ l rinsing liquid PW, 13,000rpm, centrifugal 30 seconds, outwell the liquid in the collection tube, adsorption column is put into same collection tube.Add 500 μ l rinsing liquid PW in adsorption column, centrifugal 30 seconds of 13,000rpm outwells waste liquid.Centrifugal adsorbing column CA1 or CA2 are put back in the collection tube, and centrifugal 2 minutes of 13,000rpm removes rinsing liquid as far as possible.Adsorption column is placed room temperature or 50 ℃ of incubator numbers minute, dry.Adsorption column is put into a clean centrifuge tube, and to the elution buffer EB of an amount of 65-70 ℃ of water-bath preheating of the unsettled dropping in adsorption film mid-way, room temperature was placed 2 minutes.13,000rpm collected dna solution in centrifugal 1 minute.
(6) cloning and identification of goal gene segment
(6.1) preparation of competent cell
Picking JM109 intestinal bacteria original seed, LB agar lining out.The fresh single bacterium colony of picking after the incubated overnight is inoculated in the 100ml LB substratum, and 37 ℃ are shaken 2-3 hour (160 rev/mins) of bacterium cultivation, to OD
600When reaching 0.4-0.5 bacterium liquid is moved in the 100ml centrifuge tube of sterilization precooling ice bath 10-15 minute.4,000rpm, 4 ℃ centrifugal 10 minutes, reclaim bacterial precipitation.Add 20ml through filtration sterilization and through the 0.1MCaCl of precooling
2The suspension bacterial precipitation.After ice bath 10-15 minute in 4,000rpm, 4 ℃ centrifugal 10 minutes, reclaim bacterial precipitation.Add 4ml 0.1M CaCl
2The suspension bacterial precipitation.This cell can be directly used in transformation experiment.Glycerol adding to final concentration is 15%-20%, mixing, and every part is distributed into 100 μ l in 1.5ml EP pipe, frozen in-70 ℃ of refrigerators.
(6.2) reclaim fragment with the connection of T carrier
Connect the test kit with TaKaRa pMD 18-T Vector.
Specific operation process is as follows:
At first in the EP pipe, prepare following ligation liquid:
Then above-mentioned reaction solution was reacted 30 minutes at 16 ℃, product is used for the transformed competence colibacillus cell.
(6.3) conversion of plasmid
From-70 ℃ of refrigerators, take out the competent cell of preserving, ice bath hydrotropy.100 μ l competent cells are added 10 μ l connect product, ice bath 42 ℃ of 90 seconds of water-bath heat stress, was inserted in the ice bath 2 minutes after 30 minutes immediately.Add 400 μ l liquid LB substratum, 37 ℃ bring back to life and to get 100 μ l after 50 minutes and be laid on the LB flat board, and flat board contains 0.1(V/V) ammonia benzyl mould Amp(100mg/ml) X-gal(100 μ g/ml) and IPTG(1mM).At last in 37 ℃ of constant temperature culture 10-15 hours.The white bacterial plaque should contain recombinant plasmid.
(6.4) PCR that transforms bacterium colony identifies and cultivation
Transform single bacterium colony of cultivating with the marking pen mark, dip white single bacterium colony with the toothpick of sterilizing.Dentiscalprum head is placed in to be added with in the PCR reaction solution EP pipe of (not containing template) rocks, carry out pcr amplification.The PCR product is carried out agarose gel electrophoresis together with Marker, differentiate on the T carrier whether contain the purpose fragment.After obtaining the purpose fragment, be taken at it corresponding single bacterium colony on the flat board with sterilization rifle choicest, put into 40ml and contain 0.1%(V/V) benzylpenicillin sodium (100mg/ml) LB liquid nutrient medium, 37 ℃ are spent the night and shake bacterium and cultivate, and are used for that plasmid reclaims and order-checking.
(7) order-checking
Obtain the nucleotide sequence of the coding heat shock protein(HSP) FbpA shown in the SEQ ID NO:1 through order-checking.
FbpA gene in the Lactobacillus casei Zhang of the present invention sticks at Bacterium lacticum very important effect in the process.This gene is carried out Cloning and sequencing, and this provides the basic research material for milk-acid bacteria probiotic strain genetic modification and application.
[embodiment]
Following embodiment is used for further specifying of the present invention, but is not used for limiting protection scope of the present invention.
Embodiment 1
The Cloning and sequencing of heat shock protein(HSP) FbpA encoding gene in the Lactobacillus casei Zhang.
The segment that the present invention's order-checking obtains relatively is the complete sequence of heat shock protein(HSP) FbpA encoding gene through similarity.
1, experimental technique
1.1 the PCR design of primers is with synthetic
The correlated series that the PCR primer provides according to GenBank (Accession No:FM177140; CP000423) adopt Primer 5.0 designed, designeds, synthetic by Shanghai Sani's bio tech ltd, the right nucleotide sequence of this primer is respectively SEQ ID NO:3(forward) and SEQ ID NO:4(reverse), sequence information is as follows:
SEQ ID NO:3(forward): 5 '-GCTTTTTGCTATGCCACCC-3 ';
SEQ ID NO:4(is reverse): 5 '-TGACAGCGACCGTCTTTTG-3 ';
1.2 the extraction of lactobacterium casei DNA.
The extraction of total DNA is extracted the test kit specification sheets with reference to the bacterial genomes of QIAGEN and is carried out.Concrete operation step is as follows:
Inoculum 5ml, centrifugal 1 minute of 10,000rpm abandons supernatant, adds an amount of lysozyme soln, and making its ultimate density is 20mg/ml, thoroughly suspends, and processes 50 minutes, and adds 20 μ l Proteinase K solution (20mg/ml), mixing for 37 ℃.Add 220 μ l buffered soln GB, vibrated 15 seconds.Placed 30 minutes for 70 ℃.Add 220 μ l dehydrated alcohols, fully vibrated 15 seconds.Solution is added in the adsorption column centrifugal 30 seconds of 12,000rpm.Add 500 μ l protein liquid removal GD, centrifugal 30 seconds of 12,000rpm abandons waste liquid.Add 700 μ l rinsing liquid GW, centrifugal 30 seconds of 12,000rpm abandons waste liquid.Add 500 μ l rinsing liquid GW, centrifugal 30 seconds of 12,000rpm discards waste liquid.Again 12, centrifugal 2 minutes of 000rpm places room temperature number minute with adsorption column.Adsorption column is changed in the clean centrifuge tube, add 200 μ l elution buffer TE, room temperature was put 4 minutes, centrifugal 30 seconds of 12,000rpm.The centrifugal solution that obtains adds in the adsorption column again, and room temperature was placed 2 minutes, and centrifugal 2 minutes of 12,000rpm namely obtains the Lactobacillus casei Zhang genomic dna.
1.3 the pcr amplification of purpose segment
Pcr amplification system: 0.2 Μ l Taq polysaccharase (5U/ μ l, Takara Tokyo, Japan), 2.5 μ l10 * PCR damping fluid (without Mg
2+), 2 μ l dNTP (2.5mM each), 2 μ l MgCl
2(25mM), 0.2 μ l forward primer (SEQ ID NO:3) (50pM), 0.2 μ l reverse primer (SEQID NO:4) (50pM), 1 μ l genomic dna and 17.4 μ l ddH
2O.
Pcr amplification condition: 97 ℃ of sex change 5min; 95 ℃ of 30s, 54.5 ℃ of 30s, 72 ℃ of 30s so carry out 35 circulations; 72 ℃ are extended 10min, 4 ℃ of 10min.
1.4 agarose gel electrophoresis detects
Get PCR product 5 μ l, mix with 1 μ l sample-loading buffer, point sample adds 5 μ l DL2000 Marker, electrophoresis 30min under the 120V/cm voltage in another hole in the sepharose hole.Electrophoresis is left and taken photo at gel imaging system GDS-8000 System (UVP Biomaging Systems) after finishing.
1.5 the goal gene segment reclaims and identifies
The recovery of fragment is carried out according to the explanation that the QIAGEN sepharose reclaims test kit.
The Agarose plug that will contain rapidly purpose fragment band under ultraviolet lamp is cut off, and puts into the centrifuge tube of 1.5ml.Add 3 times of volume sol solutions PN in the blob of viscose, put 50 ℃ of water-baths and placed 10 minutes, Agarose plug is dissolved fully.Agarose plug after will dissolving moves among the adsorption column CA1, and centrifugal 30 seconds of 13,000rpm outwells the liquid in the collection tube.Adsorption column is put into same collection tube, in adsorption column, add 700 μ l rinsing liquid PW, 13,000rpm, centrifugal 30 seconds, outwell the liquid in the collection tube, adsorption column is put into same collection tube.Add 500 μ l rinsing liquid PW in adsorption column, centrifugal 30 seconds of 13,000rpm outwells waste liquid.Centrifugal adsorbing column CA1 or CA2 are put back in the collection tube, and centrifugal 2 minutes of 13,000rpm removes rinsing liquid as far as possible.Adsorption column is placed room temperature or 50 ℃ of incubator numbers minute, dry.Adsorption column is put into a clean centrifuge tube, and to the elution buffer EB of an amount of 65-70 ℃ of water-bath preheating of the unsettled dropping in adsorption film mid-way, room temperature was placed 2 minutes.13,000rpm collected dna solution in centrifugal 1 minute.
1.6 the cloning and identification of goal gene segment
1.6.1 the preparation of competent cell
Picking JM109 intestinal bacteria original seed, LB agar lining out.The fresh single bacterium colony of picking after the incubated overnight is inoculated in the 100ml LB substratum, and 37 ℃ are shaken 3 hours (160 rev/mins) of bacterium cultivation, to OD
600When reaching 0.4-0.5 bacterium liquid is moved in the 100ml centrifuge tube of sterilization precooling ice bath 15 minutes.4,000rpm, 4 ℃ centrifugal 10 minutes, reclaim bacterial precipitation.Add 20ml through filtration sterilization and through the 0.1MCaCl of precooling
2The suspension bacterial precipitation.Ice bath after 15 minutes in 4,000rpm, 4 ℃ centrifugal 10 minutes, reclaim bacterial precipitation.Add 4ml 0.1M CaCl
2The suspension bacterial precipitation.This cell can be directly used in transformation experiment.Glycerol adding to final concentration is 15%-20%, mixing, and every part is distributed into 100 μ l in 1.5ml EP pipe, frozen in-70 ℃ of refrigerators.
1.6.2 reclaim fragment with the connection of T carrier
Connect the test kit with TaKaRa pMD 18-T Vector.Operating process is as follows:
At first in the EP pipe, prepare following ligation liquid:
16 ℃ of reactions 30 minutes, product was used for the transformed competence colibacillus cell with above-mentioned reaction solution.
1.6.3 the conversion of plasmid
From-70 ℃ of refrigerators, take out the competent cell of preserving, ice bath hydrotropy.100 μ l competent cells are added 10 μ l connect product, ice bath 42 ℃ of 90 seconds of water-bath heat stress, was inserted in the ice bath 2 minutes after 30 minutes immediately.Add 400 μ l liquid LB substratum, 37 ℃ bring back to life and to get 100 μ l after 50 minutes and be laid on the LB flat board, and flat board contains 0.1(V/V) ammonia benzyl mould Amp(100mg/ml) X-gal(100 μ g/ml) and IPTG(1mM).At last in 37 ℃ of constant temperature culture 15 hours.The white bacterial plaque should contain recombinant plasmid.
1.6.4 transforming the PCR of bacterium colony identifies and cultivation
Transform single bacterium colony of cultivating with the marking pen mark, dip white single bacterium colony with the toothpick of sterilizing.Dentiscalprum head is placed in to be added with in the PCR reaction solution EP pipe of (not containing template) rocks, carry out pcr amplification.The PCR product is carried out agarose gel electrophoresis together with Marker, differentiate on the T carrier whether contain the purpose fragment.After obtaining the purpose fragment, be taken at it corresponding single bacterium colony on the flat board with sterilization rifle choicest, put into 40ml and contain 0.1%(V/V) benzylpenicillin sodium (100mg/ml) LB liquid nutrient medium, 37 ℃ are spent the night and shake bacterium and cultivate, and are used for that plasmid reclaims and order-checking.
2, experimental result
Obtain the nucleotide sequence of the FbpA of coding shown in SEQ ID NO:1 gene through order-checking.
3, conclusion
The full length nucleotide sequence of the FbpA gene that cloning and sequencing obtains.
Embodiment 2: the sequence information of FbpA gene and bioinformatic analysis in the Lactobacillus casei Zhang
The length of the lactobacterium casei FbpA full length coding region sequence that the present invention is new is 1704bp, and detailed sequence is seen SEQ ID NO:1.Derive the aminoacid sequence of FbpA in the Lactobacillus casei Zhang according to total length CDS, totally 365 amino-acid residues, see SEQ ID NO:2 for details, on-line prediction (http://www.expasy.ch/tools/pi_tool.html) result shows, its molecular weight is 64301 dalton, and iso-electric point (pI) is 9.21.
Full length nucleotide sequence and coded protein thereof that lactobacterium casei FbpA is relevant carry out Nucleotide and protein similarity retrieval with blast program in the Non-redundant data storehouse of GenBank, found that it and the lactobacterium casei FbpA that announces sequence have 99% similarity on nucleotide level and amino acid levels.With other Bacterium lacticum as: plant lactobacillus WCSF1(Lactobacillus plantarum WCSF1), lactobacillus salivarius UCC118(Lactobacillus salivarius UCC118) and short lactobacillus ATCC367(Lactobacillus brevis ATCC 367) etc. the FbpA gene of coding higher similarity (concrete similarity is referring to table 1) is also arranged.This shows that the FbpA gene has higher conservative property between the Bacterium lacticum different plant species.By the Interpro database of retrieval by European bioinformation institute (EMI) exploitation, we find that it has the typical structural domain feature of FbpA albumen, a N end ATP function calmodulin binding domain CaM (ATP binding domain) and a C end binding domain polypeptide territory (Polypeptide-binding domain).FbpA has been proved to be at Bacterium lacticum in other Bacterium lacticum stress have very important effect in the process, can think FbpA lactobacterium casei stress process in also have similar effect.
3, conclusion
By aforesaid operations, show that the present invention has obtained gene order and the aminoacid sequence thereof of the FbpA albumen in the coding Lactobacillus casei Zhang.
Table 1: the FbpA gene order that the present invention obtains in the coding Lactobacillus casei Zhang with other Bacterium lacticum has very high similarity
Claims (1)
1. heat shock protein(HSP) fbpA gene cloning and sequence measurement in the Lactobacillus casei Zhang, the method comprising the steps of: (1) PCR design of primers is with synthetic; (2) extraction of lactobacterium casei DNA; (3) pcr amplification of purpose segment; (4) agarose gel electrophoresis detects; (5) the goal gene segment reclaims and identifies; (6) cloning and identification of goal gene segment; (7) order-checking, the forward primer that it is characterized in that the PCR primer is that SEQ ID NO:3 and reverse primer are SEQ ID NO:4, its sequence information is as follows:
SEQ?ID?NO:3：5’-GCTTTTTGCTATGCCACCC-3’
SEQ?ID?NO:4：5’-TGACAGCGACCGTCTTTTG-3’。
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| CN1552845A (en) * | 2003-05-30 | 2004-12-08 | ������ҵ�ɷ�����˾ | Cheese lactobacillus Bd-II strain and use for decreasing blood fat |
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| CN1552845A (en) * | 2003-05-30 | 2004-12-08 | ������ҵ�ɷ�����˾ | Cheese lactobacillus Bd-II strain and use for decreasing blood fat |
Non-Patent Citations (2)
| Title |
|---|
| Antonsson M et al.fibronectin-binding protein [Lactobacillus paracasei subsp. paracasei 8700:2]，EEQ64941，567 aa linear.《NCBI》.2009,1. * |
| Antonsson M et al.fibronectin-binding protein [Lactobacillus paracasei subsp. paracasei 8700:2],ZP_04674506,567 aa linear.《NCBI》.2009,1. * |
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