CN106834162A - Lactobacillus brevis DM9218, genetic fragment and the recombinant protein of blood uric acid can be reduced - Google Patents

Lactobacillus brevis DM9218, genetic fragment and the recombinant protein of blood uric acid can be reduced Download PDF

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Publication number
CN106834162A
CN106834162A CN201611128934.9A CN201611128934A CN106834162A CN 106834162 A CN106834162 A CN 106834162A CN 201611128934 A CN201611128934 A CN 201611128934A CN 106834162 A CN106834162 A CN 106834162A
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lactobacillus brevis
uric acid
recombinant protein
seq
dna
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袁杰力
郑鹏远
李明
杨殿斌
王凤娇
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Dalian Medical University
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Dalian Medical University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/86—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/02—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amides (3.5.2)
    • C12Y305/0201—Creatininase (3.5.2.10)

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Abstract

The present invention discloses a kind of Lactobacillus brevis DM9218 fast to purine nucleosides decomposition rate, the reduced blood uric acid that resolution ratio is high, genetic fragment and recombinant protein, Lactobacillus brevis DM9218（Lactobacillus brevis）It has been deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number：CGMCC No.12797；The gene segment DNA sequence dna of Lactobacillus brevis DM9218 such as SEQ ID NO：Shown in 2；The recombinant protein of the gene segment of Lactobacillus brevis DM9218, it is to enter performing PCR reaction by template of the DNA of Lactobacillus brevis DM9218, the DNA sequence dna of the recombinant protein that gained PCR primer is obtained using gene clone method, the PCR reaction sense primers and anti-sense primer is respectively such as SEQ ID NO：3、SEQ ID NO：Shown in 4.

Description

Lactobacillus brevis DM9218, genetic fragment and the recombinant protein of blood uric acid can be reduced
Technical field
The invention belongs to biological field, especially a kind of, reduced blood that resolution ratio high fast to purine nucleosides decomposition rate The Lactobacillus brevis DM9218 of uric acid, genetic fragment and recombinant protein.
Background technology
Blood uric acid is not only as the index for judging goat, while being also blood pressure, blood sugar and blood fat severe illness One important Judging index, blood uric acid is exceeded close with various metabolic diseases such as disorders of lipid metabolism, diabetes, hypertension, obesity Cut is closed.Outside existing hyperuricemia is divided by drug therapy, will typically be aided with strict diet control, i.e., reduce as far as possible Exogenous purine substance intake.However, due in various foods purine substance content do not have accurately final conclusion, particularly Improve the tasty agents, its Main Ingredients and Appearance 5 '-the sapidity nucleotide disodium such as chickens' extract, oyster sauce, the fish sauce of food flavor（Including 5 '-guanosine Acid disodium and IMP）Purine substance is, therefore strict diet control is extremely difficult.
Enteron aisle lactic acid bacteria is the profitable strain in body gastrointestinal tract, although have confirmed that it has to purine, purine metabolism The absorption function of intermediate product inosine, guanosine and uric acid, but existing bacterial strain is equal to the decomposition rate and resolution ratio of purine nucleosides It is relatively low, it is impossible to enough to meet the clinical requirement for reducing blood uric acid.
The content of the invention
The present invention is to solve the above-mentioned technical problem existing for prior art, there is provided a kind of to decompose speed to purine nucleosides The Lactobacillus brevis DM9218 of the reduced blood uric acid that rate is fast, resolution ratio is high, genetic fragment and recombinant protein.
Technical solution of the invention is：
A kind of Lactobacillus brevis DM9218 for reducing blood uric acid（Lactobacillus brevis）, it has been deposited in China Microbiological Culture presevation administration committee common micro-organisms center, preserving number：CGMCC No. 12797.
The DNA sequence dna such as SEQ ID NO of the Lactobacillus brevis DM9218 of blood uric acid can be reduced：Shown in 1.
A kind of gene segment of the above-mentioned Lactobacillus brevis DM9218 for reducing blood uric acid, its DNA sequence dna such as SEQ ID NO： Shown in 2.
A kind of recombinant protein of the gene segment of the above-mentioned Lactobacillus brevis DM9218 for reducing blood uric acid, is with Lactobacillus brevis The DNA of DM9218 enters performing PCR reaction for template, by gained PCR primer using the recombinant protein obtained by gene clone method, institute The DNA sequence dna of PCR reaction sense primers and anti-sense primer is stated respectively such as SEQ ID NO：3、SEQ ID NO：Shown in 4.
Lactobacillus brevis DM9218 of the invention（Lactobacillus brevis）The purine such as rapid decomposable inosine, guanosine Nucleosides, its decomposition rate and resolution ratio are above prior art, belong to country allow to be applied to it is prebiotic in health food category Bacterium, can be applied to prepare reduces high lithemia medicine, safe and free of toxic and side effects.
Brief description of the drawings
Fig. 1 is the electrophoretogram after embodiment of the present invention genetic fragment is connected double digestion with carrier.
Fig. 2 is the SDS-PAGE of embodiment of the present invention bacterium solution after induced expression.
Fig. 3 is the recombinant protein SDS-PAGE of embodiment of the present invention expression.
Fig. 4 is embodiment of the present invention target protein SDS-PAGE after purification.
Fig. 5 is the e. coli bl21 thalline and inosine, the chromatogram of guanosine reaction solution that the embodiment of the present invention is transferred to empty carrier Figure.
Fig. 6 is the recombinant bacterial strain and inosine, the chromatogram of guanosine reaction solution that the embodiment of the present invention is not induced.
Fig. 7 is embodiment of the present invention target protein after purification and inosine, the chromatogram of guanosine reaction solution.
Fig. 8 is that embodiment of the present invention target protein after purification is contrasted with other materials to the resolution ratio of inosine and guanosine Figure.
Lactobacillus brevis DM9218 preservation dates：On July 20th, 2016；
Classification And Nomenclature：Lactobacillus brevis（Lactobacillus brevis）；
Depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center（CGMCC）；
Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preserving number：CGMCC No. 12797.
Specific embodiment
Embodiment 1：
With lactic acid bacteria Selective agar medium to salt marsh Chinese cabbage（Sauerkraut）Zymotic fluid is cultivated, afterwards 37 DEG C of Anaerobic culturels again, according to The isolated Lactobacillus brevis DM9218 of conventional method, has been deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms Center, preserving number：CGMCC No. 12797.
Experiment：
1. checking Lactobacillus brevis DM9218 decomposes the ability of purine nucleosides
Lactobacillus brevis DM9218 is inoculated in MRS culture medium Anaerobic culturel 48h, thalline is collected by centrifugation, contained to addition in thalline The neutral phosphate buffer liquid of inosine, guanosine.37 DEG C under anaerobic, 120r/min shaken cultivations 60min, subsequent centrifuging and taking Supernatant presses 9：1 ratio adds 0.1mol/l HClO4Solution, for RP-HPLC analyses.Chromatographic condition：High performance liquid chromatography Instrument：LC-20A；Reversed-phase liquid chromatography post：Cosmosil-5C18-AR-；Mobile phase：0.1μmol/l NaClO4–0.187mol/ l H3PO4；Flow velocity：1ml/min；Column temperature：40℃；Retention time：40min；Determine wavelength：254nm.
Based on HPLC chromatogram and standard curve, residual nucleoside amount and decomposition rate in supernatant are calculated, as a result such as the institute of table 1 Show.
Table 1
As it can be seen from table 1 Lactobacillus brevis DM9218 is decomposed, purine nucleosides speed is fast, and resolution ratio is high.
2. Lactobacillus brevis DM9218 cholate tolerances are verified
DM9218 bacterial strains are seeded to containing in 0.1% cholate MRS culture mediums with 20% inoculum concentration, are passed on after culture 24h, and passage is twice Normal liquid MRS culture mediums are connected to 5% inoculum concentration afterwards, 24 h are cultivated；0.2%MRS containing cholate is seeded to 20% inoculum concentration again to train Support in base, repeat aforesaid operations and tamed；Then it is seeded in the culture mediums of 0.3%MRS containing cholate with 20% inoculum concentration, passage two Take appropriate nutrient solution after secondary to be coated on MRS solid mediums, 37 DEG C of culture 48h.The single bacterium colony that picking grows, is seeded to normal Liquid MRS culture mediums.
Purine nucleosides capacity of decomposition measure, DM9218 bacterium are carried out to the bacterial strain obtained after domestication using same HPLC methods Purine nucleosides ability measurement result such as table 2 is decomposed before and after strain domestication.
Table 2
Result shows that nucleotide cleavage ability does not have and weakens because of domestication.
3. Lactobacillus brevis DM9218 bacterial strains decompose the experiment of nucleosides capability analysis
Using same HPLC methods analysis Lactobacillus brevis DM9218 strain culturings lean liquor and cellular content to inosine and guanosine Capacity of decomposition, result is that the culture lean liquor of the bacterial strain is respectively 16% and 11.6% to the resolution ratio of inosine and guanosine, and bacterial strain Cellular content is respectively 83.1% and 84.5% to the resolution ratio of inosine and guanosine, thus proves that the strains for degrading nucleosides can advocate If being played a role by the hydrolase of Lactobacillus brevis DM9218 bacterial strains intracellular generation.
4. effect of Lactobacillus brevis DM9218 bacterial strain blood uric acids is verified in zoopery
32 6 week old male Wistar rats, after adaptability raises 3d, are randomly divided into hyperuricemia model group, Lactobacillus brevis DM9218 bacterium intervention group, allopurinol intervention group, 4 groups of Normal group.First three groups feed purine mouse grain high, and Normal group is given Common mouse grain is given, each group equal free water starts experiment after feeding 7d.
1 ~ 7 day：First three groups press 350mg/100g body weight intraperitoneal injection Oxonic acid potassium salt daily CMC-Na suspensions, while being aided with purine mouse grain high, free water；
8 ~ 14 days：Lactobacillus brevis DM9218 bacterium intervention groups：MRS Anaerobic culturel Lactobacillus brevis DM9218 bacterium, centrifugation thalline, with Physiological saline is adjusted to No. 4 Maxwell turbidity（1.2×109cfu/ml）, every rat oral gavage 1.0ml bacteria suspension, lasting 7d；Not Purine alcohol intervention group：Daily every rat 4.2mg/100g gavage allopurinol CMC-Na suspension, continues 7d；Hyperuricemia Model group：Gavage 1.0ml CMC-Na solution, continues 7d.
0d, the 14d for starting in zoopery respectively, collect blood, and 3000r/min centrifugal separation plasmas determine blood Uric acid（UA）Content, four groups of rat Uric Acid Contents（Mean ± SD, n=8）It is shown in Table 3.
Table 3
Note：* represent that there is significant difference, P ＜ 0.05 compared with model group；# is represented compared with allopurinol group to be had significantly Sex differernce, P ＜ 0.05
It can be seen from Table 3 that Lactobacillus brevis DM9218 has obvious reduction hyperuricemia model rat serum uric acid level Effect.
Embodiment 2：
The genome sequencing of Lactobacillus brevis DM9218 bacterial strains.
The Lactobacillus brevis DM9218 that will have been activated, through culture, be collected by centrifugation thalline, washing thalline, lysozyme broken wall, point Bacterial genomes DNA is obtained from steps such as, purifying.Shanghai's style Sen Nuo bio tech ltd is to DM9218 full genomes in commission Group is determined, DNA sequence dna such as SEQ ID NO：Shown in 1.
Embodiment 3：
By the way that to Lactobacillus brevis DM9218 complete genome sequencings, the coding inosine hydrolase of Gene A 0008 may participate in decomposition flesh Glycosides, the DNA sequence dna such as SEQ ID NO of Gene A 0008：Shown in 2.
Embodiment 4：
The recombinant protein of the gene segment of the Lactobacillus brevis DM9218 of blood uric acid can be reduced, is prepared in accordance with the following steps successively：
A. using the genomic DNA of DNA extraction kit rapid extraction Lactobacillus brevis DM9218, then with Lactobacillus brevis DM9218 genomic DNAs are template, are utilized as PCR and expand the gene segment of the Lactobacillus brevis DM9218 that can reduce blood uric acid and set The primer of meter（SEQ ID NO：3 、SEQ ID NO：Primer shown in 4）Enter performing PCR amplification, will expand the PCR primer that obtains with Carrier pET28a uses restriction enzyme respectivelyXho IWithNco IDigestion, digestion products are attached after purification, construction expression Carrier.By expression vector convert toE.coli In BL21 Host Strains, the LB fine jades containing 50 μ g/mL kanamycins are then applied to On fat flat board, 37 DEG C of incubated overnights.The picking single bacterium colony from flat board, is inoculated in the LB fluid nutrient mediums of 50 μ g/mL kanamycins, 37 DEG C, 220rpm/min incubated overnights.Plasmid is extracted, is usedXho IWithNco IDouble digestion electroresis appraisal.Result as shown in figure 1, Show that genes of interest has succeeded and connected with carrier pET28a and be transferred to Host StrainsE.coli In BL21.
B. after recombinant plasmid being transferred into e. coli bl21, under the conditions of 37 DEG C cultivate, to OD be 0.6 ~ 0.8 when, use IPTG（Final concentration of 0.1mM）Induced expression, the bacterium solution for collecting different time carries out SDS-PAGE electrophoretic analysis, and electrophoresis pattern is such as Shown in Fig. 2.
Figure it is seen that after being induced through IPTG, the Escherichia coli containing recombinant plasmid have molecular weight about 35kD's Protein band, further proves that A0008 gene recombinant vectors are successfully constructed.
C. the bacterium solution after the completion of induction is taken, bacterial sediment is collected by centrifugation, the resuspended thalline of PBS are added, 4 after ultrasonication bacterium DEG C centrifugation；SDS-PAGE analyses are carried out to its supernatant precipitation respectively, electrophoresis pattern is as shown in Figure 3.
From figure 3, it can be seen that the recombinant protein majority of expression exists in the form of inclusion body, but also there is solubility in supernatant The expression of fusion protein.
D. supernatant is through Ni post affinitive layer purification albumen.
Imidazole gradient using 10-100mM during Ni post affinity chromatographys is eluted, and collects sample after post, in case SDS-PAGE electrophoretic analysis.Electrophoresis pattern as shown in figure 4, obtain clearly protein band, i.e. A0008 gene recombinant proteins.
Experiment：
The external Enzyme activity assay of one .A0008 gene recombinant proteins：According to the method that Yang Dianbin etc. has been delivered, after taking expression and purification Albumen carry out the analysis of external enzyme activity.Based on RP-HPLC methods, by detection containing inosine, the reaction solution of guanosine and induction After recombinant protein culture 60min after purification, inosine and guanosine changes of contents in reaction solution, PD purine after detection induction The ability of nucleosides, using the recombinant bacterial strain that is transferred to the e. coli bl21 thalline of empty carrier and does not induce as control, as a result distinguishes As shown in Fig. 5, Fig. 6, Fig. 7, as a result show that the recombinant bacterial strain for being transferred to the e. coli bl21 thalline of empty carrier and not inducing does not have The ability of degraded nucleosides, and the recombinant protein through IPTG inductions after purification has the ability of degraded inosine higher.
Two, are equally based on RP-HPLC methods, and detection is containing inosine, the reaction solution of guanosine and MRS nutrient solutions, Lactobacillus brevis DM9218 thalline, do not induce recombinant protein supernatant and culture 60min after, inosine and guanosine changes of contents, each material in reaction solution To the resolution ratio of inosine and guanosine with shown in Fig. 8.Show Lactobacillus brevis DM9218 thalline recombinant protein tool with induction after purification There is stronger purine nucleosides resolution ratio.
Sequence table
<110>Dalian Medical Univ
<120>Lactobacillus brevis DM9218, genetic fragment and the recombinant protein of blood uric acid can be reduced
<160> 4
<170> PatentIn version 3.2
<210> 1
<211>
<212> DNA
<213> Lactobacillus brevis
<400> 1
CACTTGTCACCGGCCCATGACTAACTTAGCTGCTTTGAAGAAGGCCCCAGAAATTGCCACAGAAATGGGCAAC ATGACCTTCATGGGTGGCGCCTTAACCAGGTAACGTAACCCGTTTCGCTGAAGCAAACATTAACCAAGATGCCGAAG CCGCTAACGCCGTCCTCACTAGCCCACTCCACTCAACGATGGTTGGCCTAGATGTTACGCTACGGACTTTGTTGACT AAGAAAGAAACCCAACAAGCCTACAAGCAATTTAACGACAACTTGGGTGGCTGTGCACTGCACGACCCATTAGCCGT TGGGGTAGCGTTAGACCCTAGCTTTGTGACAACGATTGATCTCAATATGGTCAACACCAATAAGGAAACTTATGGCC GAACTATCGGTGATGACAACCGTTTGAACGACCCAACAA
<210> 2
<211>
<212> DNA
<213>Lactobacillus brevisA0008 genes
<400> 2
MILDLDTGIDDSLAIAYALGAPDVDLIGIIGSYGNVVIEEGGKNALKILELLGHTDIPVYLGESHSSTSDHFD RMEVSANIHGQNGIGEVDLPEPTRAIESEGVDFLIDAVHQYGKDLTLVPTGPMTNLAAALKKAPEIATEMGNMTFMG GALTMPGNVTRFAEANINQDAEAANAVLTSPLHSTMVGLDVTRTLLTKKETQQWRDLGTTSGEKFADIVDYYIEAYK QFNDNLGGCALHDPLAVGVALDPSFVTTIDLNMVVNTNKETYGRTIGDDNRLNDPTNVKVAMVDKDRYLKVFMDYLT NLFKEN
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
CCATGGCAAAACGCAAAATG 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
CTCGAGGTTTTCTTTGAATA 20

Claims (4)

1. a kind of Lactobacillus brevis DM9218 for reducing blood uric acid（Lactobacillus brevis）, it has been deposited in Chinese micro- life Thing culture presevation administration committee common micro-organisms center, preserving number：CGMCC No.12797.
2. the Lactobacillus brevis DM9218 for reducing blood uric acid according to claim 1, it is characterised in that DNA sequence dna such as SEQ ID NO：Shown in 1.
3. a kind of gene segment of the Lactobacillus brevis DM9218 that can reduce blood uric acid as claimed in claim 1 or 2, it is characterised in that DNA sequence dna such as SEQ ID NO：Shown in 2.
4. a kind of recombinant protein of the gene segment of the Lactobacillus brevis DM9218 that can reduce blood uric acid as claimed in claim 3, its It is characterised as entering performing PCR reaction by template of the DNA of Lactobacillus brevis DM9218, gained PCR primer is utilized into gene cloning side The DNA sequence dna of the recombinant protein that method is obtained, the PCR reaction sense primers and anti-sense primer is respectively such as SEQ ID NO：3、SEQ ID NO：Shown in 4.
CN201611128934.9A 2016-12-09 2016-12-09 Lactobacillus brevis DM9218, genetic fragment and the recombinant protein of blood uric acid can be reduced Pending CN106834162A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108048368A (en) * 2018-01-25 2018-05-18 吉林省命之元生物科技有限公司 One UA-416 plants of lactobacillus plantarum and its application
CN110184209A (en) * 2019-04-24 2019-08-30 杭州娃哈哈科技有限公司 One plant of Lactobacillus rhamnosus that can reduce blood uric acid
CN111944711A (en) * 2020-07-07 2020-11-17 浙大宁波理工学院 Lactobacillus brevis capable of degrading purine nucleosides and application thereof
CN112458002A (en) * 2020-10-22 2021-03-09 宁波大学 Lactobacillus acidophilus strain for reducing urine blood, screening method thereof and application of lactobacillus acidophilus strain in preparing functional yoghourt

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015182155A1 (en) * 2014-05-29 2015-12-03 日東薬品工業株式会社 Novel lactic acid bacterium and composition including said lactic acid bacterium

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Publication number Priority date Publication date Assignee Title
WO2015182155A1 (en) * 2014-05-29 2015-12-03 日東薬品工業株式会社 Novel lactic acid bacterium and composition including said lactic acid bacterium

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MING LI等: ""Screening and Characterization of Purine Nucleoside Degrading Lactic Acid Bacteria Isolated from Chinese Sauerkraut and Evaluation of the Serum Uric Acid Lowering Effect in Hyperuricemic Rats"", 《PLOS ONE》 *
NCBI: ""Pyrimidine ribonucleoside hydrolase rihA"", 《GENBANK DATABASE》 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108048368A (en) * 2018-01-25 2018-05-18 吉林省命之元生物科技有限公司 One UA-416 plants of lactobacillus plantarum and its application
CN108048368B (en) * 2018-01-25 2019-01-01 吉林省命之元生物科技有限公司 One UA-416 plants of lactobacillus plantarum and its application
CN110184209A (en) * 2019-04-24 2019-08-30 杭州娃哈哈科技有限公司 One plant of Lactobacillus rhamnosus that can reduce blood uric acid
CN111944711A (en) * 2020-07-07 2020-11-17 浙大宁波理工学院 Lactobacillus brevis capable of degrading purine nucleosides and application thereof
CN112458002A (en) * 2020-10-22 2021-03-09 宁波大学 Lactobacillus acidophilus strain for reducing urine blood, screening method thereof and application of lactobacillus acidophilus strain in preparing functional yoghourt
CN112458002B (en) * 2020-10-22 2023-05-12 宁波大学 Lactic acid bacteria strain for reducing haematuria acid, screening method thereof and application for preparing functional yoghurt

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