CN103911337A - High adhesion clostridium butyricum and its preparation method - Google Patents
High adhesion clostridium butyricum and its preparation method Download PDFInfo
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- CN103911337A CN103911337A CN201310007380.7A CN201310007380A CN103911337A CN 103911337 A CN103911337 A CN 103911337A CN 201310007380 A CN201310007380 A CN 201310007380A CN 103911337 A CN103911337 A CN 103911337A
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- clostridium butylicum
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- attachment proteins
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Abstract
The invention discloses high adhesion clostridium butyricum and its preparation method, and belongs to the field of gene recombination. The invention provides the high adhesion clostridium butyricum and its preparation method to solve the technical problem, the main points of the technical proposal are that lactobacillus adhesion protein gene is obtained by cloning, and a lactobacillus adhesion protein gene-containing recombinant plasmid is converted into clostridium butyricum by use of a gene recombination method to obtain a clostridium butyricum recombinant strain. Through gene expression, the clostridium butyricum is capable of autologous production of a protein with the adhesion function, the adhesion ability is greatly improved, and the efficacy of the treatment of the clostridium butyricum is further improved.
Description
Technical field
The present invention relates to gene recombination field, is clostridium butylicum after a kind of gene recombination and preparation method thereof specifically.
Background technology
Clostridium butylicum (
clostridium butyricum) be a kind of novel probiotic bacterium.Clostridium butylicum has another name called Butylic acid bacteria, and it is a kind in Clostridium, is within 1933, to enter closely to control doctor by Chiba, Japan medical university palace first find and report, is therefore again Clostridium Butyricum.Nineteen thirty-five, Kingi doctor Miyairi isolates clostridium butylicum from people's ight soil and soil, find subsequently to contain less lipid acid in filtrate that its anaerobism cultivates, there is extremely strong whole intestines effect, it can suppress the pathogenic bacterium in enteron aisle, promotes in enteron aisle that probiotics is as the growth of bifidus bacillus and Bacterium lacticum.Clostridium butylicum has following effect, (1) clostridium butylicum can effectively suppress to cause staphylococcus, candidiasis, klebsiella, Campylobacter, Pseudomonas aeruginosa, intestinal bacteria, dysentery bacterium and the salmonella typhi of disease and the breeding of spoilage organism, adjustment intestinal microecology restores balance, and has also reduced the generation (if these products can be damaged liver function by intestinal absorption) of the objectionable impuritiess such as Ammonia, amine, indoles and hydrogen sulfide simultaneously.(2) clostridium butylicum can activating immune system, and Promote immunity function maintains the healthy state of body.(3) clostridium butylicum can produce amylase, proteolytic enzyme, Glycosylase, cellulase in enteron aisle, and regeneration and reparation to gut epithelium tissue have very important significance.But, in the application process of clostridium butylicum, its lower adhesion property has had a strong impact on its effect, because most of clostridium butylicum can not field planting in animal intestinal, need constantly to supplement from the external world, research at present mainly concentrates on the output that improves clostridium butylicum, and the patent documentation that for example application number is 201110324228.2 discloses a kind of method of clostridium butylicum of suitability for industrialized production superelevation cell concn, has greatly increased its application cost.
Summary of the invention
The technical problem that the present invention solves is, provide a kind of use to there is clostridium butylicum of high-adhesiveness and preparation method thereof, the method is expressed the transgenosis of the attachment proteins of Bacterium lacticum (adhension protein, AP) in clostridium butylicum, thereby improves the adhesive capacity of clostridium butylicum.
Technical scheme of the present invention is as follows:
The high-adhesiveness clostridium butylicum recombinant bacterial strain that the present invention makes is the clostridium butylicum that carries recombinant plasmid, and described recombinant plasmid is the bacillus subtilis bacteria plasmid pHY300PLK containing Bacterium lacticum attachment proteins gene.
Bacterium lacticum attachment proteins gene required for the present invention can increase out from Bacterium lacticum, and the primer is: 5 ＇-CGCGGATCCATGAATACTGTTGCTCCTC-3 ＇ and 5 ＇-CCGGAATTCATTTTTTTTACGTTTTTTTT-3 ＇ (primer is synthetic from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd).
The nucleotides sequence of described Bacterium lacticum attachment proteins gene is classified SEQ ID No:2 as.
The aminoacid sequence of the attachment proteins of described Bacterium lacticum attachment proteins genes encoding is SEQ ID No:1.
A kind of method of utilizing genetic engineering technique to prepare high-adhesiveness clostridium butylicum, be characterised in that and utilize the recombinant expression vector that carries Bacterium lacticum attachment proteins gene coding region, adopt any transgenic method in clostridium butylicum, to express Bacterium lacticum attachment proteins, thereby improve the adhesive capacity of clostridium butylicum.Its step is as follows:
(1) adopt the method for gene clone to obtain Bacterium lacticum attachment proteins gene coding region;
(2) Bacterium lacticum attachment proteins gene coding region is connected in to expression regulation sequence, forms recombinant expression vector;
(3) adopt any transgenic method that recombinant expression vector is converted in clostridium butylicum;
(4) screening and identification clostridium butylicum transformant under given conditions;
(5) under applicable condition, cultivate clostridium butylicum positive colony, obtain the clostridium butylicum after gene recombination.
The recombinant bacterial strain the present invention relates to is the clostridium butylicum that carries recombinant plasmid, and described recombinant plasmid is the bacillus subtilis bacteria plasmid pHY300PLK containing Bacterium lacticum attachment proteins gene.With aforesaid method acquisition restructuring clostridium butylicum, it is characterized by: imported the attachment proteins gene order of Bacterium lacticum, can express attachment proteins, thereby improved adhesive capacity, can greatly improve its colonization ability in animal intestinal.
Described high-adhesiveness clostridium butylicum, the chicken causing for microbiotic, diarrhea of pigs, have stronger therapeutic action, and optimum quantum of utilization is 10
6individual/kg body weight.
Described high-adhesiveness clostridium butylicum, mixes use with chitosan, oligosaccharide (as xylo-oligosaccharide, mannooligo saccharide etc.) etc., and effect is better, and optimum weight ratio is 1:1-100:1-100.
Described high-adhesiveness clostridium butylicum, for aquaculture or improve water quality, concentration used is 10
4individual/milliliter.
In the present invention, can select various carrier known in the art, as commercially available carrier, comprise plasmid etc.
In the present invention, term " any transgenic method " comprises electric shocking method gene transformation, ultrasonic-mediated gene transformation, the conversion of microinjection mediated gene, the conversion of laser microbeam mediated gene, the conversion of particle bombardment mediated gene, the conversion of agrobacterium tumefaciens Ti-plasmids mediated gene, the conversion of Agrobacterium rhizogenes mediated gene etc.
In the present invention, term " screening and identification clostridium butylicum transformant under given conditions " refers under the condition of cultivating with culture medium flat plate selects the positive transformant of antibiotics resistance with microbiotic (tsiklomitsin, kantlex, penbritin etc.), and identifies the positive transformant of clostridium butylicum by modes such as PCR, Northern hybridization.
In the present invention, term " is cultivated clostridium butylicum positive colony " and is referred to the positive clostridium butylicum transformant after qualification is carried out to culture medium flat plate cultivation under applicable condition, and detect the expression level of Bacterium lacticum attachment proteins, filter out good transformant and cultivate.
In the present invention, we have cloned attachment proteins gene order from Bacterium lacticum, utilize gene clone technology to build recombinant expression vector, and genetic transformation clostridium butylicum, make clostridium butylicum can autologous generation there is the albumen of adhesion function, improve the adhesive capacity of clostridium butylicum, and cultivated by screening recon the clostridium butylicum bacterial strain that has obtained gene recombination.
Protein electrophoresis analytical results is found, the albumen that high efficient expression relative molecular weight is 9.5 KD after induction.
Results of animal shows, the small white mouse diarrhoea that the clostridium butylicum after restructuring causes microbiotic has stronger therapeutic action, and usage quantity is 10
6individual/curative ratio when kg body weight reaches 100%.
Brief description of the drawings
Fig. 1 is the agarose gel electrophoresis figure of PCR product of the present invention, and swimming lane M is DNA marker; Swimming lane 1 is to be PCR product.
Fig. 2 is the schematic diagram of carrier construction of the present invention.
Fig. 3 is the double digestion qualification collection of illustrative plates of recombinant plasmid pHY300PLK-AP of the present invention, and swimming lane M is DNA marker; Swimming lane 1 is EcoR I and BamH I double digestion product; Swimming lane 2 is EcoR I single endonuclease digestion product; Swimming lane 3 is BamH I: single endonuclease digestion product.
Fig. 4 is the SDS-PAGE analysis chart of restructuring clostridium butylicum protein expression of the present invention, and swimming lane M is standard molecular weight albumen; Swimming lane 1 is the clostridium butylicum expressing protein after recombinating; Swimming lane 2 is the clostridium butylicum expressing protein of not gene recombination; Visible swimming lane 1 than swimming lane more than 2 protein band of a treaty 9.5kD, as shown by arrows in FIG..
Sequence 1(SEQ ID NO.1) be the aminoacid sequence of Bacterium lacticum attachment proteins.
Sequence 2(SEQ ID NO.2) nucleotide sequence of Bacterium lacticum attachment proteins gene
Embodiment
Below in conjunction with specific embodiment and accompanying drawing, further the present invention is set forth.Should be understood that embodiment part only need not limit the scope of the invention for understanding the present invention.The test method of unreceipted actual conditions in the following example, presses the condition of chapter routine, for example " molecular cloning " (New York conventionally; Cold Spring Harbor Laboratory Press, 1989) condition that the conditioned disjunction described in is advised according to manufacturer.
The clone of embodiment 1 Bacterium lacticum attachment proteins gene order
Root it is documented (Complete genome sequence of
lactobacillus kefiranofacienszW3. Journal of Bacteriology, 2011,193 (16): 4280-4281) and state-run biotechnology information center of the NCBI(U.S.) (GeneBank accession number: NC_015602) Bacterium lacticum attachment proteins gene order of recording, and according to the polyclone restriction enzyme site of bacillus subtilis bacteria plasmid pHY300PLK, the gene-specific primer that corresponding restriction enzyme site is carried in design increases to goal gene (Bacterium lacticum attachment proteins gene), for building recombinant expression vector.
The primer is: 5 ＇-CGC
gGATCCaTGAATACTGTTGCTCCTC-3 ＇ (as upstream primer, carries
bamHI restriction enzyme site) and 5 ＇-CCG
gAATTCaTTTTTTTTACGTTTTTTTT-3 ＇ (as downstream primer, carries
ecoRI restriction enzyme site, protection base is respectively CGC and CCG).
From Kai Feier Bacterium lacticum (
lactobacillus kefiranofaciensbe purchased from Chinese common micro-organisms culture presevation administrative center) in extracting genomic dna (the EZ-10 pillar bacterial genomes DNA extraction agent box of Shanghai Sheng Gong bio-engineering corporation), taking this genomic dna as template, (Shanghai Sheng Gong bio-engineering corporation uses pcr amplification test kit to carry out pcr amplification, SK2081), PCR reaction system is: 20 microlitres; Reaction process is as follows:
95℃ 30 s
55℃ 30 s
72℃ 1 min
Carry out 30 circulations.Agarose gel electrophoresis with 2% detects pcr amplification product, obtains amplifying target genes (see figure 1).
Embodiment 2 construction recombination plasmid pHY300PLK-AP
After electrophoretic separation, PCR product (relative molecular weight is 271bp) to goal gene reclaims (SK8131, SanPrep pillar DNA glue reclaims test kit, Shanghai Sheng Gong bio-engineering corporation), get appropriate recovery product and bacillus subtilis bacteria plasmid pHY300PLK (being purchased from Chinese plasmid vector strain cell pnca gene preservation center) restriction enzyme BamHI and EcoRI(restriction enzyme purchased from Shanghai Sheng Gong bio-engineering corporation) carry out enzyme and cut, then connect (vector construction process is shown in accompanying drawing 2) with T4 DNA ligase, transform DH5 α, screening obtains mono-clonal on containing penbritin and the dual anti-LB flat board of tsiklomitsin, shake bacterium enlarged culturing, extracting plasmid,
bamHI and
ecoRI double digestion checking (see figure 3) is also served further checking (see figure 5) of Hai Sheng work bio-engineering corporation order-checking, finally obtains the recombinant plasmid pHY300PLK-AP building.
The conversion of embodiment 3 clostridium butylicums
Get and be cultured to the clostridium butylicum bacterium liquid of exponential phase of growth and (approximately cultivate 16 hours, clostridium butylicum is separated, is preserved by this laboratory) prepare competent cell, add plasmid pHY300PLK-AP, after mixing, carry out cell transformation with electric shocking method, cell after conversion is with 37 DEG C of cultivations, the LB culture medium flat plate that after 1.5 hours, coating contains tsiklomitsin, 37 DEG C of anaerobism are cultivated after 24 hours and are carried out bacterium colony PCR, electrophoresis detection obtains positive colony, get rid of unconverted successful false positive clone, positive colony is the clostridium butylicum after conversion.
The expression (see figure 4) of embodiment 4 Bacterium lacticum attachment proteinses in clostridium butylicum
Experimentation is as follows: by being cultured to logarithmic phase (approximately 16 hours) in the clostridium butylicum access liquid nutrient medium after transforming, collect clostridium butylicum bacterium liquid, and centrifugal 1 min of 10000 r/min, precipitation adds 10 ml distilled water, carries out ultrasonic disruption.Ultrasonic 3 s/off, 4 s/on, centrifugal 10 min of solution 12000 r/min after 50 cyclic ultrasonic breakings.Get 100 μ l supernatant liquors in a centrifuge tube, this is supernatant sample.Unconverted clostridium butylicum (initial clostridium butylicum) is processed equally, compares.These two samples are carried out to SDS-PAGE, after electrophoresis, carry out silver dyeing, put into gel imaging system and take pictures.
Embodiment 5 experimentation on animalies
The clostridium butylicum that this institute uses, through acute toxinology experiment (with reference to the operation of GB15193.3 HornShi method) and Salmonella reversion test inspection, belongs to safety non-toxic bacterial strain, and the small white mouse that microbiotic is caused diarrhoea, has stronger therapeutic action, and optimum quantum of utilization is 10
6individual/kg body weight.Specific experiment step is as follows:
Kunming kind healthy mice is provided by Zhejiang College Of Traditional Chinese Medicine animal center, 20～22 grams of body weight, and receptacle temperature is 20～25 DEG C, relative humidity is 40～60%.Animal produces conformity certification SCXK (Shanghai) 2002-0010, animal facility conformity certification SYXK(Zhejiang) 2003-0003, experimentation on animals conformity certification numbers (1996) No. 22-00223.Mouse is no-special pathogen (Specific pathogen free, SPF).
Acute toxinology experiment operates with reference to GB15193.3 HornShi method, if dosage is 1000 mg/kg, 2150 mg/kg, 4640 mg/kg, 10000 mg/kg, each 5 of every treated animal male and female, fasting 16 hours before experiment, gavage of per os, weighs and observe the poisoning and death condition of animal every day.After seven days, finish experiment.This experiment completes at Zhejiang College Of Traditional Chinese Medicine.
Salmonella reversion test adopts dull and stereotyped infiltration method.Carry out prerun by the non-metabolism activation system of four strain bacterial strains, there is not increasing bacterium or antibacterial phenomenon in result in the time that dosage is 5000 μ g/ ware.Therefore select five dosage groups of 8,40,200,1000 and 5000 μ g/ wares when official test, take 0.8 g and add 16 ml distilled waters, 121 DEG C of deactivations in 20 minutes are made into 5000 μ g/ wares.As maximum dose level, do respectively 5 times of dilutions, be made into 1000 μ g/ wares, 200 μ g/ wares, 40 μ g/ wares, the each dosage of 8 μ g/ ware, standby inspection.Establish blank group and positive controls, it is parallel that every kind of each test concentrations of bacterial strain is established three wares simultaneously, tests adding S9 and do not add under S9 condition.Repeated test once.Observation index is that on direct census substratum, returning of each bacterial strain becomes colony number.This experiment completes in Zhejiang Center For Disease Control and Prevention.
Select 60 male Kunming kind small white mouses to be divided at random 6 groups, every group 10, wherein 1 group is Normal group, another 5 groups of abdominal injection benzylpenicillin sodiums every day (0.9 mg/gbw) once, continuous 7 days, select at random 1 group to be model group, inspection mouse flora imbalance situation (normal group and model group are now surveyed); Choose at random 1 group with sample diluting liquid gavage, as natural recovering group; All the other 3 groups with clostridium butylicum gavage every day once, and dosage is respectively 10
5individual/day (low dose group), 10
6individual/day (middle dosage group), 10
7individual/day (high dose group), gavage 5 days.
Give the each group of last abdominal injection benzylpenicillin sodium of mouse or gavage probiotic bacterium after 24 hours, weigh, the dislocation of mouse cervical vertebra is put to death, aseptic 0.2 gram of the cecal content of taking is dissolved in 10 mL diluents, 37 DEG C of water-baths are vibrated 15 minutes, 10 times of gradient dilutions successively again, select suitable extent of dilution, get respectively 50 μ l diluents and drip on each substratum (every kind of each extent of dilution of bacterium repeats three flat boards), measure enterobacteria in ight soil, faecalis, bifidus bacillus, Bacterium lacticum, the changing conditions of clostridium perfringens and bacterioide, in conjunction with gramstaining, cell microscopic morphology and colonial morphology carry out bacterial classification and determine.
Note: in the experiment of chicken, pig, do not make flora imbalance model with benzylpenicillin sodium, directly test with dysentery chicken, sick pig, get ight soil and detect rapidly.
The experimental result (logCFU/g) of table 1 experimentation on animals (object: chicken)
The experimental result of table 2 experimentation on animals (object: pig)
Embodiment 6
Use in this strain clostridium butylicum process concrete, mix use with chitosan, oligosaccharide etc., effect is better.The part by weight of clostridium butylicum, chitosan and oligosaccharide (as xylo-oligosaccharide, mannooligo saccharide etc.) is 1:1 ~ 100:1 ~ 100.Specific experiment the results are shown in Table 3 and table 4.
Table 3 recombinate clostridium butylicum and chitosan, the mixed effect of oligosaccharide (experimentation on animals object: chicken)
Table 4 recombinate clostridium butylicum and chitosan, the mixed effect of oligosaccharide (experimentation on animals object: pig)
Embodiment 7 recombinates clostridium butylicum for aquaculture and improves water quality
After clostridium butylicum cultivation is finished, add in water quality, making the concentration of clostridium butylicum in water body is 10
5individual/milliliter, after 7 days, water sampling is surveyed transmittance (spectrophotometric determination).Specific experiment the results are shown in Table 5.
Table 5 is recombinated clostridium butylicum for aquaculture or is improved water quality
SEQ ID No.1
MNTVAPHGEK IDKVHTVAPH GQRFKVNRNV TVPRAQSVNT VKSNSQHSEL PQTGNDQQTN AAASILGGAA AAIGMIGLAG EKKRKKN
SEQ ID No.2
ATGAATACTG TTGCTCCTCA TGGTGAAAAA ATTGATAAAG TTCATACTGT TGCTCCTCAT GGTCAACGTT TTAAAGTTAA TCGTAATGTT ACTGTTCCTC GTGCTCAAAG TGTTAATACT GTTAAAAGTA ATAGTCAACA TAGTGAATTA CCTCAAACTG GTAATGATCA ACAAACTAAT GCTGCTGCTA GTATTTTAGG TGGTGCTGCT GCTGCTATTG GTATGATTGG TTTAGCTGGT GAAAAAAAAC GTAAAAAAAA T
Claims (8)
1. high-adhesiveness clostridium butylicum, is characterized in that, this recombinant bacterial strain is the clostridium butylicum that carries recombinant plasmid, and described recombinant plasmid is the bacillus subtilis bacteria plasmid pHY300PLK containing Bacterium lacticum attachment proteins gene.
2. Bacterium lacticum attachment proteins gene according to claim 1, is characterized in that: the nucleotides sequence of this gene is classified SEQ ID No:2 as.
3. the attachment proteins of Bacterium lacticum attachment proteins genes encoding according to claim 1 and 2, is characterized in that: the aminoacid sequence of described albumen is SEQ ID No:1.
4. a method of utilizing genetic engineering technique to prepare high-adhesiveness clostridium butylicum, be characterised in that and utilize the recombinant expression vector that carries Bacterium lacticum attachment proteins gene coding region, adopt transgenic method in clostridium butylicum, to express Bacterium lacticum attachment proteins, thereby the adhesive capacity that improves clostridium butylicum, its step is as follows:
(1) adopt the method for gene clone to obtain Bacterium lacticum attachment proteins gene coding region;
(2) Bacterium lacticum attachment proteins gene coding region is connected in to expression regulation sequence, forms recombinant expression vector;
(3) recombinant expression vector is converted in clostridium butylicum;
(4) screening and identification clostridium butylicum transformant;
(5) cultivate clostridium butylicum positive colony, obtain the clostridium butylicum after gene recombination.
5. high-adhesiveness clostridium butylicum according to claim 1, is characterized in that it has imported the attachment proteins gene order of Bacterium lacticum, can express attachment proteins, thereby improve adhesive capacity, can greatly improve its colonization ability in animal intestinal.
6. high-adhesiveness clostridium butylicum according to claim 1, the chicken causing for microbiotic, diarrhea of pigs, have stronger therapeutic action, and optimum quantum of utilization is 10
6individual/kg body weight.
7. high-adhesiveness clostridium butylicum according to claim 1, mixes use with chitosan, oligosaccharide (as xylo-oligosaccharide, mannooligo saccharide etc.) etc., and effect is better, and optimum weight ratio is 1:1 ~ 100:1 ~ 100.
8. high-adhesiveness clostridium butylicum according to claim 1, for aquaculture or improve water quality, concentration used is 10
4individual/milliliter.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105641682A (en) * | 2016-02-26 | 2016-06-08 | 南昌大学 | Application of Bifidobacterium longum protein in improvement of antibiotics sensitivity of Salmonella typhimurium |
| CN110656039A (en) * | 2019-11-21 | 2020-01-07 | 浙江中跃医疗科技有限公司 | Laboratory is with antibiotic material efficiency detection device |
| WO2021031360A1 (en) * | 2019-08-20 | 2021-02-25 | 集美大学 | Complex microbial inoculant for improving adhesion of clostridium butyricum in intestinal tracts of eels |
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| CN101881751A (en) * | 2010-06-08 | 2010-11-10 | 南昌大学 | Method for screening and identifying adhesion proteins |
| CN101993887A (en) * | 2010-08-13 | 2011-03-30 | 江南大学 | Efficient bacillus secretory expression carrier and building method thereof |
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| CN101881751A (en) * | 2010-06-08 | 2010-11-10 | 南昌大学 | Method for screening and identifying adhesion proteins |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105641682A (en) * | 2016-02-26 | 2016-06-08 | 南昌大学 | Application of Bifidobacterium longum protein in improvement of antibiotics sensitivity of Salmonella typhimurium |
| CN105641682B (en) * | 2016-02-26 | 2019-02-01 | 南昌大学 | A kind of bifidobacterium longum protein is used to be promoted the application of salmonella typhimurium Antibiotic Sensitivity |
| WO2021031360A1 (en) * | 2019-08-20 | 2021-02-25 | 集美大学 | Complex microbial inoculant for improving adhesion of clostridium butyricum in intestinal tracts of eels |
| CN110656039A (en) * | 2019-11-21 | 2020-01-07 | 浙江中跃医疗科技有限公司 | Laboratory is with antibiotic material efficiency detection device |
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