CN103911337B - High-adhesiveness Clostridium butyricum and preparation method thereof - Google Patents
High-adhesiveness Clostridium butyricum and preparation method thereof Download PDFInfo
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Abstract
The present invention entitled high-adhesiveness Clostridium butyricum and preparation method thereof, belongs to gene recombinaton field.The technical problem to be solved is to provide a kind of Clostridium butyricum with high-adhesiveness and preparation method thereof, drip irrigation device is the attachment proteins gene that clone obtains lactobacillus, utilize gene recombinaton means that the recombinant plasmid transformed containing attachment proteins is entered Clostridium butyricum, it is thus achieved that Clostridium butyricum recombinant bacterial strain.By gene expression, make Clostridium butyricum autologous generation can have the albumen of adhesion function, substantially increase its adhesive capacity, and then improve the therapeutic efficiency of Clostridium butyricum.
Description
Technical field
The present invention relates to gene recombinaton field, Clostridium butyricum after a kind of gene recombinaton and preparation method thereof.
Background technology
Clostridium butyricum (Clostridiumbutyricum) is a kind of novel probiotic bacteria.Clostridium butyricum has another name called butyric acid bacteria, and it is a kind in Clostridium, is within 1933, to be entered closely to control doctor by Chiba, Japan medical university palace first find and report, is the most again Clostridium Butyricum.Nineteen thirty-five, doctor KingiMiyairi isolates Clostridium butyricum from the feces and soil of people, it is subsequently found in the filtrate of its Anaerobic culturel containing less fatty acid, there is extremely strong whole intestinal effect, it can suppress the pathogenic bacterium in intestinal, promotes the growth of probiotics such as bacillus bifidus and lactobacillus in intestinal.Clostridium butyricum has the effect that, (1) Clostridium butyricum can effectively suppress to cause the breeding of the staphylococcus of disease, candidiasis, klebsiella, Campylobacter, bacillus pyocyaneus, escherichia coli, dysentery bacterium and salmonella typhi and putrefaction bacteria, adjustment intestinal microecology restores balance, and decreases the generation (if these products can be damaged liver function by intestinal absorption) of the harmful substances such as Ammonia, amine, indoles and hydrogen sulfide simultaneously.(2) Clostridium butyricum can promote immunologic function with activating immune system, maintains the health status of body.(3) Clostridium butyricum can produce amylase, protease, glycosidase, cellulase in intestinal, and regeneration and reparation to gut epithelium tissue have very important significance.But, in the application process of Clostridium butyricum, its relatively low adhesion property has had a strong impact on its application effect, because major part Clostridium butyricum can not field planting in animal intestinal, need constantly to supplement from the external world, research at present is concentrated mainly on the yield improving Clostridium butyricum, and the such as patent documentation of Application No. 201110324228.2 discloses the method for the Clostridium butyricum of a kind of industrialized production superelevation cell concentration, considerably increases its application cost.
Summary of the invention
Present invention solves the technical problem that and be, a kind of Clostridium butyricum with high-adhesiveness and preparation method thereof is provided, the gene of the attachment proteins (adhensionprotein, AP) of lactobacillus is transferred to express in Clostridium butyricum by the method, thus improves the adhesive capacity of Clostridium butyricum.
Technical scheme is as follows:
The high-adhesiveness Clostridium butyricum recombinant bacterial strain that the present invention prepares is the Clostridium butyricum carrying recombiant plasmid, and described recombiant plasmid is the bacillus subtilis bacteria plasmid pHY300PLK containing lactobacillus attachment proteins gene.
Lactobacillus attachment proteins gene needed for the present invention can expand out from lactobacillus, and the primer is: 5 ＇-CGCGGATCCATGAATACTGTTGCTCCTC-3 ＇ and 5 ＇-CCGGAATTCATTTTTTTTACGTTTTTTTT-3 ＇ (primer synthesizes from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd).
The nucleotides sequence of described lactobacillus attachment proteins gene is classified as SEQIDNo:2.
The aminoacid sequence of the attachment proteins of described lactobacillus attachment proteins gene code is SEQIDNo:1.
A kind of method utilizing technique for gene engineering to prepare high-adhesiveness Clostridium butyricum, it is characterized by carrying the recombinant expression carrier of lactobacillus attachment proteins gene coding region, use any transgenic method to express lactobacillus attachment proteins in Clostridium butyricum, thus improve the adhesive capacity of Clostridium butyricum.Its step is as follows:
(1) method using gene clone obtains lactobacillus attachment proteins gene coding region；
(2) lactobacillus attachment proteins gene coding region is connected in expression regulation sequence, forms recombinant expression carrier；
(3) any transgenic method is used to be converted to Clostridium butyricum by recombinant expression carrier；
(4) screen under given conditions and identify Clostridium butyricum transformant；
(5) Clostridium butyricum positive colony is cultivated under the suitable conditions, it is thus achieved that the Clostridium butyricum after gene recombinaton.
The recombinant bacterial strain that the present invention relates to is the Clostridium butyricum carrying recombiant plasmid, and described recombiant plasmid is the bacillus subtilis bacteria plasmid pHY300PLK containing lactobacillus attachment proteins gene.Obtain restructuring Clostridium butyricum in aforementioned manners, it is characterized by: imported the attachment proteins gene order of lactobacillus, attachment proteins can have been expressed, thus improve adhesive capacity, be greatly improved its colonization ability in animal intestinal.
Described high-adhesiveness Clostridium butyricum, the chicken that antibiotic is caused, diarrhea of pigs, there is stronger therapeutical effect, optimum quantum of utilization is 106/kg body weight.
Described high-adhesiveness Clostridium butyricum, is used in mixed way with chitosan, oligosaccharide (such as oligomeric xylose, mannooligo saccharide etc.) etc., and better, optimum weight ratio is 1:1-100:1-100.
Described high-adhesiveness Clostridium butyricum, for aquaculture or improve water quality, concentration used is 104/milliliter.
In the present invention, can be selected for various carrier known in the art, such as commercially available carrier, including plasmid etc..
In the present invention, term " any transgenic method " includes 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 tumefaciems Ti-plasmids mediated gene, Agrobacterium rhizogenes mediated gene conversion etc..
In the present invention, term " screens and identifies Clostridium butyricum transformant " positive transformant selecting antibiotic resistance with culture medium flat plate under conditions of referring to cultivate with antibiotic (tetracycline, kanamycin, ampicillin etc.) under given conditions, and uses the modes such as PCR, Northern hybridization to identify the positive transformant of Clostridium butyricum.
In the present invention, term " is cultivated Clostridium butyricum positive colony under the suitable conditions " and is referred to the positive Clostridium butyricum transformant after identifying is carried out culture medium flat plate cultivation, and detect the expression of lactobacillus attachment proteins, filter out excellent transformant and cultivate.
In the present invention, we have cloned attachment proteins gene order from lactobacillus, gene clone technology is utilized to construct recombinant expression carrier, and genetic transformation Clostridium butyricum, make Clostridium butyricum autologous generation can have the albumen of adhesion function, improve the adhesive capacity of Clostridium butyricum, and carry out cultivating the Clostridium butyricum bacterial strain obtaining gene recombinaton by screening recon.
Protein electrophoresis analysis result finds, after induction, high efficient expression relative molecular weight is the albumen of 9.5KD.
Results of animal shows, the white mice diarrhoea that antibiotic is caused by the Clostridium butyricum after restructuring has stronger therapeutical effect, and usage amount is that cure rate during 106/kg body weight reaches 100%.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis figure of the PCR primer of the present invention, and swimming lane M is DNAmarker；Swimming lane 1 is for PCR primer.
Fig. 2 is the schematic diagram of carrier construction of the present invention.
Fig. 3 is that the double digestion of the recombiant plasmid pHY300PLK-AP of the present invention identifies collection of illustrative plates, and swimming lane M is DNAmarker；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 the restructuring Clostridium butyricum protein expression of the present invention, and swimming lane M is standard molecular weight albumen；Swimming lane 1 is the Clostridium butyricum expressing protein after restructuring；Swimming lane 2 is the Clostridium butyricum expressing protein of non-gene recombinaton；Visible swimming lane 1 is the protein band of a treaty 9.5kD than swimming lane more than 2, as shown by arrows in FIG..
Sequence 1 (SEQIDNO.1) is the aminoacid sequence of lactobacillus attachment proteins.
The nucleotide sequence of sequence 2 (SEQIDNO.2) lactobacillus attachment proteins gene
Detailed description of the invention
Below in conjunction with specific embodiment and accompanying drawing, further the present invention is illustrated.Should be understood that embodiment part is only used for understanding that the present invention need not limit the scope of the present invention.The test method of unreceipted actual conditions in the following example, generally presses the condition that chapter is conventional, such as " molecular cloning " (NewYork；ColdSpringHarborLaboratoryPress, 1989) condition described in or according to the condition proposed by manufacturer.
The clone of embodiment 1 lactobacillus attachment proteins gene order
Root it is documented (CompletegenomesequenceofLactobacilluskefiranofaciensZW3. JournalofBacteriology, 2011, 193 (16): 4280-4281) the lactobacillus attachment proteins gene order and described in NCBI (US National Biotechnology Information center) (GeneBank accession number: NC_015602), and according to the polyclone restriction enzyme site of bacillus subtilis bacteria plasmid pHY300PLK, design is carried the gene-specific primer of corresponding restriction enzyme site and is expanded genes of interest (lactobacillus attachment proteins gene), for building recombinant expression carrier.
The primer is: 5 ＇-CGCGGATCCATGAATACTGTTGCTCCTC-3 ＇ are (as forward primer; carry BamHI restriction enzyme site) and 5 ＇-CCGGAATTCATTTTTTTTACGTTTTTTTT-3 ＇ (as downstream primer; carrying EcoRI restriction enzyme site, protection base is respectively CGC and CCG).
From Kefir grains lactobacillus (Lactobacilluskefiranofaciens, be purchased from China General Microbiological 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), with this genomic DNA as template, (Shanghai Sheng Gong bio-engineering corporation i.e. uses PCR amplification kit to carry out PCR amplification, SK2081), PCR reaction system is: 20 microlitres；Course of reaction is as follows:
95℃30s
55℃30s
72℃1min
Carry out 30 circulations.
Agarose gel electrophoresis with 2% detects pcr amplification product, it is thus achieved that amplifying target genes (see Fig. 1).
Embodiment 2 construction recombination plasmid pHY300PLK-AP
nullAfter electrophoretic separation，The PCR primer (relative molecular weight is 271bp) of genes of interest is reclaimed (SK8131,SanPrep pillar DNA glue reclaims test kit,Shanghai Sheng Gong bio-engineering corporation)，Take the appropriate product that reclaims and carry out enzyme action with bacillus subtilis bacteria plasmid pHY300PLK (being purchased from China plasmid vector strain cell pnca gene preservation center) with restricted enzyme BamHI and EcoRI (restricted enzyme is purchased from Shanghai Sheng Gong bio-engineering corporation)，Then it is attached (vector construction process is shown in accompanying drawing 2) with T4DNA ligase，Convert DH5 α，On the LB flat board dual anti-containing ampicillin and tetracycline, screening obtains monoclonal，Shake bacterium amplification culture，Extracting plasmid，BamHI and EcoRI double digestion checking (see Fig. 3) also serves Hai Sheng work bio-engineering corporation order-checking checking further，Finally give the recombiant plasmid pHY300PLK-AP built.
The conversion of embodiment 3 Clostridium butyricum
Take the Clostridium butyricum bacterium solution cultivated to exponential phase of growth (about to cultivate 16 hours, Clostridium butyricum is separated by this laboratory, preserves) prepare competent cell, add plasmid pHY300PLK-AP, cell transformation is carried out with electric shocking method after mixing, cell after conversion is with 37 DEG C of cultivations, the coating LB culture medium flat plate containing tetracycline after 1.5 hours, 37 DEG C of Anaerobic culturel carried out bacterium colony PCR after 24 hours, electrophoresis detection obtains positive colony, getting rid of unconverted successful false positive clones, positive colony is the Clostridium butyricum after conversion.
Embodiment 4 lactobacillus attachment proteins expression (see Fig. 4) in Clostridium butyricum
Experimentation is as follows: the Clostridium butyricum after converting accesses to be cultivated in fluid medium to logarithmic (log) phase (about 16 hours), collects Clostridium butyricum bacterium solution, and 10000r/min is centrifuged 1min, and precipitation adds 10ml distilled water, carries out ultrasonic disruption.Solution 12000r/min after ultrasonic 3s/off, 4s/on, 50 cyclic ultrasonic breaking is centrifuged 10min.Taking 100 μ l supernatant in a centrifuge tube, this is Supernatant samples.Unconverted Clostridium butyricum (initial Clostridium butyricum) processes equally, compares.The two sample is carried out SDS-PAGE, carries out silver staining dyeing after electrophoresis, put into gel imaging system and take pictures.
Embodiment 5 zoopery
The Clostridium butyricum of this institute is through acute toxinology experiment (operating with reference to GB15193.3HornShi method) and Salmonella reversion test inspection, belong to safety non-toxic bacterial strain, and the white mice diarrhoea that antibiotic caused, there is stronger therapeutical effect, optimum quantum of utilization is 106/kg body weight.Specific experiment step is as follows:
Kunming kind healthy mice is provided by Zhejiang College Of Traditional Chinese Medicine animal center, body weight 20～22 grams, and receptacle temperature is 20～25 DEG C, and relative humidity is 40～60%.Animal productiong quality certification SCXK (Shanghai) 2002-0010, animal facility quality certification SYXK (Zhejiang) 2003-0003, the zoopery quality certification number (1996) the 22-00223rd.Mice is no-special pathogen (Specificpathogenfree, SPF).
Acute toxinology experiment operates with reference to GB15193.3HornShi method, if dosage is 1000mg/kg, 2150mg/kg, 4640mg/kg, 10000mg/kg, each 5 of every treated animal male and female, fasting 16 hours before experiment, gavage of per os, weighs and observe animal poisoning and death condition every day.Experiment is terminated after seven days.This experiment completes at Zhejiang College Of Traditional Chinese Medicine.
Salmonella reversion test uses flat board infiltration method.Carrying out prerun by the non-metabolism activation system of four strain bacterial strains, result does not occurs when dosage is 5000 μ g/ ware increasing bacterium or antibacterial phenomenon.Therefore during formal test, select 8,40,200,1000 and 5,000 five dosage groups of μ g/ ware, and weighing 0.8g and add 16 milliliters of distilled water, 121 DEG C of inactivations in 20 minutes are i.e. made into 5000 μ g/ wares.As maximum dose level, do 5 times of dilutions respectively, be made into 1000 μ g/ wares, 200 μ g/ wares, 40 μ g/ wares, the 8 μ each dosage of g/ ware, standby inspection.Setting blank group and positive controls, it is parallel that every kind of each test concentrations of bacterial strain sets three wares, tests under the conditions of adding S9 and being not added with S9 simultaneously.Retest is once.Observation index is directly to count returning of each bacterial strain in culture medium to become clump count.This experiment completes in Zhejiang Center For Disease Control and Prevention.
60 male Kun Ming mice are selected to be randomly divided into 6 groups, often group 10, wherein 1 group is Normal group, another 5 groups of lumbar injection penicillin sodium every days (0.9mg/g bw) are once, continuous 7 days, selecting 1 group at random is model group, inspection mice dysbacteriosis situation (normal group and model group are now surveyed)；Randomly select 1 group with sample diluting liquid gavage, as natural recovering group；Remaining 3 groups with Clostridium butyricum gavage every day once, and dosage is respectively 105/day (low dose group), 106/day (middle dosage group), 107/day (high dose group), gavage 5 days.
After each group of mice last lumbar injection penicillin sodium or gavage probiotic bacteria 24 hours, weigh, mice cervical dislocation is put to death, aseptic cecal content 0.2 gram is taked to be dissolved in 10mL diluent, 37 DEG C of water-baths are vibrated 15 minutes, 10 times of gradient dilutions the most successively, select suitable dilution factor, take 50 μ l diluents respectively and drip in each culture medium (every kind of each dilution factor of bacterium repeats three pieces of flat boards), measure enterobacteria in feces, enterococcus, bacillus bifidus, lactobacillus, bacillus perfringens and the situation of change of bacteroid, in conjunction with Gram’s staining, cell microscopic morphology and colonial morphology carry out strain and determine.
Note: chicken, pig experiment in, unused penicillin sodium makes dysbacteriosis model, directly tests with diarrhoeal diseases chicken, sick pig, takes feces and detect rapidly.
The experimental result (logCFU/g) of table 1 zoopery (object: chicken)
The experimental result of table 2 zoopery (object: pig)
Embodiment 6
During this strain Clostridium butyricum specifically used, it is used in mixed way with chitosan, oligosaccharide etc., better.The part by weight of Clostridium butyricum, chitosan and oligosaccharide (such as oligomeric xylose, mannooligo saccharide etc.) is 1:1～100:1～100.Specific experiment the results are shown in Table 3 and table 4.
Table 3 is recombinated Clostridium butyricum and chitosan, the mixed effect of oligosaccharide (zoopery object: chicken)
Table 4 is recombinated Clostridium butyricum and chitosan, the mixed effect of oligosaccharide (zoopery object: pig)
Embodiment 7 Clostridium butyricum of recombinating for aquaculture and improves water quality
Being cultivated by Clostridium butyricum after terminating, add in water quality, making Clostridium butyricum concentration in water body is 105/milliliter, and after 7 days, water sampling surveys light transmittance (spectrophotometric determination).Specific experiment the results are shown in Table 5.
Table 5 Clostridium butyricum of recombinating for aquaculture or improves water quality
SEQIDNo.1
MNTVAPHGEKIDKVHTVAPHGQRFKVNRNVTVPRAQSVNTVKSNSQHSELPQTGNDQQTNAAASILGGAAAAIGMIGLAGEKKRKKN
SEQIDNo.2
ATGAATACTGTTGCTCCTCATGGTGAAAAAATTGATAAAGTTCATACTGTTGCTCCTCATGGTCAACGTTTTAAAGTTAATCGTAATGTTACTGTTCCTCGTGCTCAAAGTGTTAATACTGTTAAAAGTAATAGTCAACATAGTGAATTACCTCAAACTGGTAATGATCAACAAACTAATGCTGCTGCTAGTATTTTAGGTGGTGCTGCTGCTGCTATTGGTATGATTGGTTTAGCTGGTGAAAAAAAACGTAAAAAAAAT
SEQUENCELISTING
<110>Chinese Marine University
<120>high-adhesiveness Clostridium butyricum and preparation method thereof
<130>2013
<160>2
<170>PatentInversion3.3
<210>1
<211>87
<212>PRT
<213>lactobacillus
<400>1
MetAsnThrValAlaProHisGlyGluLysIleAspLysValHisThr
151015
ValAlaProHisGlyGlnArgPheLysValAsnArgAsnValThrVal
202530
ProArgAlaGlnSerValAsnThrValLysSerAsnSerGlnHisSer
354045
GluLeuProGlnThrGlyAsnAspGlnGlnThrAsnAlaAlaAlaSer
505560
IleLeuGlyGlyAlaAlaAlaAlaIleGlyMetIleGlyLeuAlaGly
65707580
GluLysLysArgLysLysAsn
85
<210>2
<211>261
<212>DNA
<213>lactobacillus
<400>2
atgaatactgttgctcctcatggtgaaaaaattgataaagttcatactgttgctcctcat60
ggtcaacgttttaaagttaatcgtaatgttactgttcctcgtgctcaaagtgttaatact120
gttaaaagtaatagtcaacatagtgaattacctcaaactggtaatgatcaacaaactaat180
gctgctgctagtattttaggtggtgctgctgctgctattggtatgattggtttagctggt240
gaaaaaaaacgtaaaaaaaat261
Claims (2)
1. high-adhesiveness Clostridium butyricum, it is characterized in that, this high-adhesiveness Clostridium butyricum is the Clostridium butyricum carrying recombiant plasmid, described recombiant plasmid is the bacillus subtilis bacteria plasmid pHY300PLK containing lactobacillus attachment proteins gene, and the nucleotides sequence of described lactobacillus attachment proteins gene is classified as SEQIDNo:2.
2. according to the attachment proteins of the lactobacillus attachment proteins gene code described in claim 1, it is characterized in that: the aminoacid sequence of described albumen is SEQIDNo:1.
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| CN110484468A (en) * | 2019-08-20 | 2019-11-22 | 集美大学 | A kind of composite bacteria agent improving adhesiveness of the clostridium butyricum in common eel enteron aisle |
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN102329761A (en) * | 2011-10-24 | 2012-01-25 | 沈阳建筑大学 | Method for culturing Clostridium butyricum with ultra-high cell concentration |
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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| CN102329761A (en) * | 2011-10-24 | 2012-01-25 | 沈阳建筑大学 | Method for culturing Clostridium butyricum with ultra-high cell concentration |
Non-Patent Citations (1)
| Title |
|---|
| "丁酸梭菌体外粘附抗菌特性及对鮸鱼肠道生理的影响";潘晓东;《中国优秀硕士学位论文全文数据库-农业科技辑》;20080115;中文摘要部分最后1段，第19页最后1段-第20页第1段 * |
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