CN110452860A - A kind of streptococcus salivarius and its application in treatment inflammatory bowel medicine - Google Patents
A kind of streptococcus salivarius and its application in treatment inflammatory bowel medicine Download PDFInfo
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- CN110452860A CN110452860A CN201910903985.1A CN201910903985A CN110452860A CN 110452860 A CN110452860 A CN 110452860A CN 201910903985 A CN201910903985 A CN 201910903985A CN 110452860 A CN110452860 A CN 110452860A
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- streptococcus salivarius
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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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
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
Abstract
A kind of streptococcus salivarius and its application in treatment inflammatory bowel medicine, are related to field of biotechnology.The streptococcus salivarius (Streptococcus salivarius) 1802, collection deposit number are CCTCC NO:M 2019251.The screening technique of streptococcus salivarius: by healthy human faecal mass, after being resuspended using sterile water, it is diluted to 10^6 times, is separately cultured with plate streak in brain heart oxoid culture medium, obtains the streptococcus salivarius (Streptococcus salivarius) 1802.The streptococcus salivarius (Streptococcus salivarius) 1802 can be in the application in preparation treatment inflammatory bowel medicine.It can be applied in the drug of inflammatory reaction for alleviating inflammatory bowel disease.It can be applied in the drug for alleviating inflammatory bowel disease Injured colonic mucosa.
Description
Technical field
The present invention relates to field of biotechnology, more particularly, to a kind of streptococcus salivarius and its in treatment inflammatory bowel disease medicine
Application in object.
Background technique
Inflammatory bowel disease (InflammationBowelDisease, IBD) is the enteron aisle chronic disease of one group of idiopathic
It is referred to as, according to position difference is involved, is divided into ulcerative colitis (Ulcerativecolitis, UC) and Crohn's disease
(Crohn ' s disease, CD).Ulcerative colitis usually it is starting cash be involve rectum mucous layer and submucosa it is continuous
Property inflammation, gradually spread to total colectomy, and Crohn disease often involves the noncontinuity holostrome inflammation of ileocecus enteron aisle.Inflammatory bowel
The cardinal symptom of disease shows as abdominal pain, vomits, tenesmus, the abdominal symptoms such as bloody stool and anaemia, fever, the whole bodies such as malnutrition
Symptom, with protracted course, the colorectal cancer risk of patient is significantly raised.The cause and onset of disease mechanism of inflammatory bowel disease at present
It is still not clear, mainly by heredity, being immunized influences with factors such as intestinal floras.Currently, China's inflammatory bowel disease disease incidence be in by
Year increases trend, has important clinical meaning for the treatment new method of inflammatory bowel disease[1]。
Currently, clinical be directed to inflammatory bowel disease mainly based on drug symptomatic treatment, common such as salicylazosulfapyridine is U.S. husky
Aminosalicylic acids preparation, the patients heavier for the state of an illness such as piperazine is drawn to be often used glucocorticoid, such as prednisone to alleviate disease
Shape.These conventional medication side effects are big, and in recent years, clinic is carried out inflammatory using drug therapies such as biological agent such as classes gram
Enteropathy obtains certain effect, but expensive, and serious adverse events can occur.It is ineffective for Morbidity control, it is poisoned
Property Hirschsprung's disease, the patient of the severe complications such as enterobrosis, it usually needs emergency operation[2]。
It is nearest the study found that intestinal flora is in the pathogenic process of inflammatory bowel disease, the vital effect of performance.
Patients with inflammatory bowel disease generally entails serious intestinal bacilli illness, after correcting flora imbalance to patients with inflammatory bowel disease,
Patient can maintain the disease-free state of long period.It is compared with the traditional method, the transplanting of intestines bacterium has cure rate high, holds time
Long, the advantages such as adverse reaction is few, and expense is low.But intestines bacterium transplants the problems such as short there is healthy donors at present[3]。
So far basic research field for intestinal flora Mechanism Study still not deeply, can for over the course for the treatment of
The specific bacterial strain for playing beneficial effect understands few.If can be separated in intestines bacterium transplantation therapy, has for inflammatory bowel disease and control
The bacterial strain for the treatment of effect, carries out specific aim transplanting, and the transplanting of intestines bacterium can be made more efficient, reliable and safe.
Bibliography:
[1] Wang Yufang, et al., inflammatory bowel disease Advance of Epidemiological Research gastroenterology, 2013.18 (01): 48-
51.
[2] in National Consensus (Guangzhou in 2012) of Hu Pinjin, et al., China's diagnosis status of inflammatory bowel disease and treatment
Section's theory and practice, 2013.8 (01): 61-75.
[3] Guo Yuecheng, et al., caprophyl transplantation treatment status and its application world the disease for digest in inflammatory bowel disease
Magazine, 2018.38 (05): 294-296+305.
Summary of the invention
The purpose of the present invention is to provide a kind of streptococcus salivarius (Streptococcus salivarius) 1802.
Another object of the present invention is to provide answering for streptococcus salivarius (Streptococcus salivarius) 1802
With.
The streptococcus salivarius (Streptococcus salivarius) 1802 was preserved on 04 11st, 2019
China typical culture collection center, address: Wuhan, China Wuhan University, postcode: 430072, collection deposit number is
CCTCC NO:M 2019251.
The screening technique of the streptococcus salivarius is as follows:
By healthy human faecal mass, after being resuspended using sterile water, it is diluted to 10^6 times, is trained with plate streak in brain heart oxoid
Feeding base is separately cultured, and the streptococcus salivarius (Streptococcus salivarius) 1802 is obtained.
The streptococcus salivarius (Streptococcus salivarius) 1802 can treat inflammatory bowel disease medicine in preparation
Application in object.
The streptococcus salivarius (Streptococcus salivarius) 1802 can be in the inflammation for alleviating inflammatory bowel disease
It is applied in the drug of reaction.
The streptococcus salivarius Streptococcus salivarius 1802 can alleviate inflammatory bowel disease colonic mucosa
It is applied in the drug of damage.
The present invention passes through the animal model for establishing inflammatory bowel disease, and comprehensive analysis is horizontal multinomial with molecular water equality in tissue
Physical signs, it is demonstrated experimentally that the streptococcus salivarius bacterial strain can be obviously improved the intestinal inflammatory of inflammatory bowel disease model mice,
Mitigate intestinal mucosal injury.Streptococcus salivarius of the present invention can be applied in preparation treatment inflammatory bowel medicine, can be clinic
Patients with inflammatory bowel disease provides new method.
Detailed description of the invention
Fig. 1 is the murine colonic inflammation contracture result statistical chart that streptococcus salivarius alleviates DSS induction.
Fig. 2 is the index result figure for the murine colonic inflammation that streptococcus salivarius mitigates DSS induction.
Fig. 3 is the mouse Colon injury tissue appraisal result figure that streptococcus salivarius mitigates DSS induction.
Specific embodiment
Following embodiment will be further elaborated technical solution of the present invention in conjunction with attached drawing.But these embodiments are only
Be it is exemplary, it is not intended to limit the scope of the present invention in any way.
Embodiment 1: the separation and identification of streptococcus salivarius
Healthy People fecal sample is taken, the gradient dilution of 10^-1 to 10^-10 is made of aqua sterilisa, with sterile glass rod in BHI
Coated plate on solid medium, 37 ° are cultivated 2~3 days.
With aseptic inoculation ring, picking single colonie, after purifying is passed on 3 times, picking single colonie comes into 5mlBHI fluid nutrient medium,
37 ° of overnight incubations take 1mL bacterium solution, and 12000g/min is centrifuged 10min, after abandoning supernatant, use full formula gold EasyPure Genomic
DNA Kit (EE101) extracts DNA of bacteria.
PCR amplification: amplification reaction system:
Amplification system (50 μ L):
| 2×EasyTaq PCR SuperMix | 25μL |
| DNA profiling | 2μL |
| Preceding primer | 1μL |
| Primer afterwards | 1μL |
| Ultrapure water | 21μL |
Primer sequence:
Preceding primer, 27F:5'-AGAGTTTGATCCTGGCTCAG-3'
Primer afterwards, 1492R:5'-TACCTTGTTACGACTT-3'
Amplification condition: 95 ° of 15min；(95 ° of 30S, 60 ° of 60S, 72 ° of 90S) X30 circulation；72°10min；4 ° of holdings；
Purified pcr product: with 1.5% agarose gel electrophoresis (200V, 20min), gel imaging cuts the position 1500bp
Band, use the band after full formula gold EasyPure Quick Gel Extraction Kit (EG101) extraction purification.
To the PCR product of extraction, be sequenced and be compared in NCBI.
Embodiment 2: the foundation and grouping of animal model
By the inoculum concentration of 1 ︰ 10, the streptococcus salivarius that logarithmic phase is grown is inoculated in the BHI culture medium of sterilizing, is trained in 37 °
It supports overnight, after, 3000G/min is centrifuged 10min, after abandoning supernatant, is resuspended, is adjusted to 10^7CFU/mL with PBS.
22~27g of weight, 8 week old, male C57/B6 mouse are randomly divided into Control group, and S.S group, is raised by every group 10
It supports in Mr. Yu university Experimental Animal Center SPF environment.Animal adapts to start to be administered after a week.Two groups give 2.5% sulfuric acid Portugal
Glycan (mpbio, 9011-18-1) free water 6 days.Control group gives 0.2mL/ only daily, and successive administration 3 days；S.S group
Give the streptococcus salivarius 0.2mL/ of PBS resuspension only, successive administration 3 days.After overnight starvation, cervical dislocation puts to death mouse, measurement
Enteron aisle length collects intestinal tissue, and a part of 4% paraformaldehyde is fixed, and is used for paraffin section, and -80 ° of another part preservations are used
Inflammatory factor expression is detected in qPCR.As shown in Figure 1, control group mice colon, because of inflammation contracture, length is obviously shortened, by saliva
The mouse of liquid streptococcus stomach-filling processing, colon contracture mitigate, and colon shortens to obtain part recovery.T examines discovery, and colon lengths change
Becoming has statistical significance.
Embodiment 3: the qPCR detection of the intestinal inflammatory factor
The enteron aisle sample for taking -80 ° of preservations after grinding in liquid nitrogen, after extracting total serum IgE using TRIzol method, uses
TransScript Two-Step RT-PCR SuperMix (AT401) reverse transcription cDNA.
Quantitative pcr amplification:
Amplification reaction system: amplification system (20 μ L):
| TransStart Green qPCR SuperMix | 10μL |
| DNA profiling | 2μL |
| Preceding primer | 1μL |
| Primer afterwards | 1μL |
| Ultrapure water | 6μL |
Primer sequence:
| mouse-TNF-a-F | CCCTCACACTCAGATCATCTTCT |
| mouse-TNF-a-R | GCTACGACGTGGGCTACAG |
| mouse-IFN-g-F | ATGAACGCTACACACTGCATC |
| mouse-IFN-g-R | CCATCCTTTTGCCAGTTCCTC |
| mouse-GAPDH-F | AGGTCGGTGTGAACGGATTTG |
| mouse-GAPDH-R | TGTAGACCATGTAGTTGAGGTCA |
Amplification program: 95 ° of 15min；(95 ° of 30S, 60 ° of 30S) X40 circulation.
It data analysis and calculates: according to parameters such as amplification cycles numbers, calculating mRNA differential expression using GAPDH as control group,
The relative expression levels of each gene mRNA are calculated according to formula 2^ (- Δ Δ CT).
As shown in Fig. 2, compared with blank control, by the mouse that streptococcus salivarius stomach-filling is handled, inflammation index TNF-a,
IFN-g shows to be decreased obviously, and t has statistical significance after examining.
Embodiment 4: the HE dyeing of intestinal tissue
The intestinal tissue that 4% paraformaldehyde fixes 24 hours is taken, carry out dehydration embedding treatment: the program of dehydration is 50%
The ethyl alcohol of ethyl alcohol 25min → 70% ethyl alcohol of 25min → 80% ethyl alcohol of 25min → 95% ethyl alcohol 30min → 100% of 25min → 100%
Ethyl alcohol 30min → ethyl alcohol/dimethylbenzene 30min → I 20min of dimethylbenzene → II 20min of dimethylbenzene → dimethylbenzene/paraffin 30min →
I 1.5h of paraffin → paraffin, II 1.5h.
Embedding treatment: tissue is wrapped in paraffin using suitable grinding tool.The thin slice with a thickness of 4 μm is cut out with slicer,
Then 40 DEG C of exhibition pieces, 60 DEG C of baking pieces.
Dyeing processing: 100% dimethylbenzene dewax 15min, totally 3 times；Ethanol concentration gradient dehydration, slide cross 100% respectively
Ethyl alcohol, 90% ethyl alcohol, 80% ethyl alcohol and 70% ethyl alcohol, every cylinder infiltrate 5min；After distilled water flushing, haematoxylin dyeing 5~
The time of 10min, different tissues is not identical；Distilled water flushing for several times, enters 0.1% hydrochloride alcohol differentiation 2s, and distilled water is of short duration clear
It washes；The anti-indigo plant 2s of ammonium hydroxide, the of short duration cleaning of distilled water；0.5% eosin stain contaminates 1min, is then dehydrated, and successively crosses 80% ethyl alcohol, 90%
Ethyl alcohol and 100% ethyl alcohol, respectively infiltrate 5min；Dimethylbenzene penetrating 2 times are finally crossed, each 1min；Neutral gum mounting.
It takes pictures under light microscopic, carries out histopathology scoring.As shown in figure 3, histological score result is shown, with blank control phase
Than by the mouse that streptococcus salivarius stomach-filling is handled, intestinal inflammatory and inflammatory bowel disease Injured colonic mucosa are relieved, t inspection
It issues after examination and approval now, histological scores have statistical significance.
Sequence table
<110>Xiamen University
<120>a kind of streptococcus salivarius and its application in treatment inflammatory bowel medicine
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1424
<212> DNA
<213> Streptococcus salivarius
<400> 1
ctatacatgc agtagaacgc tgaagagagg agcttgctct tcttggatga gttgcgaacg 60
ggtgagtaac gcgtaggtaa cctgccttgt agcgggggat aactattgga aacgatagct 120
aataccgcat aacaatggat gacacatgtc atttatttga aaggggcaat tgctccacta 180
caagatggac ctgcgttgta ttagctagta ggtgaggtaa cggctcacct aggcgacgat 240
acatagccga cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct 300
acgggaggca gcagtaggga atcttcggca atgggggcaa ccctgaccga gcaacgccgc 360
gtgagtgaag aaggttttcg gatcgtaaag ctctgttgta agtcaagaac gagtgtgaga 420
gtggaaagtt cacactgtga cggtagctta ccagaaaggg acggctaact acgtgccagc 480
agccgcggta atacgtaggt cccgagcgtt gtccggattt attgggcgta aagcgagcgc 540
aggcggtttg ataagtctga agttaaaggc tgtggctcaa ccatagttcg ctttggaaac 600
tgtcaaactt gagtgcagaa ggggagagtg gaattccatg tgtagcggtg aaatgcgtag 660
atatatggag gaacaccggt ggcgaaagcg gctctctggt ctgtaactga cgctgaggct 720
cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt aaacgatgag 780
tgctaggtgt tggatccttt ccgggattca gtgccgcagc taacgcatta agcactccgc 840
ctggggagta cgaccgcaag gttgaaactc aaaggaattg acgggggccc gcacaagcgg 900
tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatcccga 960
tgctatttct agagatagaa agttacttcg gtacatcggt gacaggtggt gcatggttgt 1020
cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccctattgtt 1080
agttgccatc attcagttgg gcactctagc gagactgccg gtaataaacc ggaggaaggt 1140
ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatggt 1200
tggtacaacg agttgcgagt cggtgacggc aagctaatct cttaaagcca atctcagttc 1260
ggattgtagg ctgcaactcg cctacatgaa gtcggaatcg ctagtaatcg cggatcagca 1320
cgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg 1380
taacacccga agtcggtgag gtaacctttt ggagccagcc gcct 1424
Claims (5)
1. a kind of streptococcus salivarius, the streptococcus salivarius (Streptococcus salivarius) 1802 is in 2019 04
It is preserved within 11st China typical culture collection center the moon, collection deposit number is CCTCC NO:M 2019251.
2. the screening technique of the streptococcus salivarius (Streptococcus salivarius) 1802 as described in claim 1,
It is characterized in that the specific method is as follows:
By healthy human faecal mass, after being resuspended using sterile water, it is diluted to 10^6 times, with plate streak in brain heart oxoid culture medium
It is separately cultured, obtains the streptococcus salivarius (Streptococcus salivarius) 1802.
3. streptococcus salivarius (Streptococcus salivarius) 1802 as described in claim 1 is inflammatory in preparation treatment
Application in enteropathy drug.
4. the streptococcus salivarius (Streptococcus salivarius) 1802 as described in claim 1 is inflammatory in alleviation
It is applied in the drug of the inflammatory reaction of enteropathy.
5. the streptococcus salivarius (Streptococcus salivarius) 1802 as described in claim 1 is inflammatory in alleviation
It is applied in the drug of enteropathy Injured colonic mucosa.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111117935A (en) * | 2020-02-10 | 2020-05-08 | 农业农村部食物与营养发展研究所 | Microbial agent for inhibiting muscle synthesis and application thereof |
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| CN111117935A (en) * | 2020-02-10 | 2020-05-08 | 农业农村部食物与营养发展研究所 | Microbial agent for inhibiting muscle synthesis and application thereof |
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Application publication date: 20191115 |