CN110305824A - Show the Ko subtilis and preparation method thereof of organophosphor hydrolytic enzyme - Google Patents
Show the Ko subtilis and preparation method thereof of organophosphor hydrolytic enzyme Download PDFInfo
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Abstract
The present invention relates to the Ko subtilis and preparation method thereof for showing organophosphor hydrolytic enzyme, belong to field of biotechnology, for the biological prosthetic of organophosphorus pesticide environmental pollution.There is the nucleotide sequence of coding organophosphor hydrolytic enzyme OPH, capsid protein CotG and own promoter after Ko subtilis host of the invention is engineered.After carrying out raw born of the same parents' culture by nutrition failure method, the fusion protein of capsid protein CotG and organophosphor hydrolytic enzyme OPH are expressed in host and show on the surface of Ko subtilis.The Ko subtilis has the ability of hydrolysis organophosphorus pesticide, also has good resistance to complex environment, can be used as environmentally protective pesticide cleanser for the biological prosthetic of contaminated environment.
Description
Technical field
The present invention relates to the Ko subtilis and preparation method thereof for showing organophosphor hydrolytic enzyme OPH, belong to field of biotechnology,
For the biological prosthetic of organophosphorus pesticide environmental pollution.
Background technique
It is the part organic phosphorus compound of representative by the activity of cholinesterase in inhibition body using organophosphorus ester, to machine
Body damages.Never poison and most of civilian insecticide in chemical warfare agent are namely based on the design and manufacture of this principle.
For various countries, the protection and pollution control of organic phosphorus compound are concerning country and public safety.Tradition, which is administered, mainly passes through object
Reason and chemical degradation also bring some problems although obtaining good result, such as cannot be directly used to human body or accurate instrument
Device, decontaminant cause secondary pollution etc. often with toxic or corrosivity, to environment.Organophosphor hydrolytic enzyme can be catalyzed organic phosphatization
Hydrate hydrolysis has the advantages that effect fast, mild condition, nontoxic, non-corrosive, has been applied to organic phosphorous insecticide and has polluted
The fields such as improvement, the protection of chemical poison and decontamination, the detection of organic phosphorus compound.
The application of organophosphor hydrolytic enzyme can reduce the use of chemical substance, mitigate logistic implications and environmental pollution, but
There is also some technical problems in actual production and application.Such as: isolating and purifying for enzyme is cumbersome, and to the activity of enzyme
It can have an impact；Organophosphor hydrolytic enzyme is more sensitive to temperature and pH variation, and storage stability is not high, is unfavorable in natural ring
It is used under the even mal-condition of border.Also, pure enzyme needs and the combinations such as salting liquid, foam matrix when in use.Accordingly, there exist
The demand of more stable organophosphor hydrolytic enzyme is developed, so that the use of organophosphor hydrolytic enzyme is more convenient, application range is wider.
Ko subtilis display technique, which refers to, shows foreign protein in brood cell's table conducive to genetic engineering means or suction-operated
Face, and the space conformation for keeping its relatively independent and original bioactivity.External source egg is promoted by the excellent resistance of brood cell
White storage stability is suitble to apply in complex conditions；Brood cell's display technique avoids isolating and purifying for enzyme, reduces life
Produce cost.Therefore Ko subtilis display technique is quickly grown in recent years, is showed in fields such as vaccine preparation, biocatalysis
Good application prospect out.However, in the prior art and be not present blanket surface display system, because of external source egg
White whether can effectively show is influenced by various factors, and the size and characteristic of target protein are to determine Bacillus subtillis
Brood cell show validity principal element (refer to Wang He etc., Appl Microbiol Biotechnol (2017),
101:933-949).But brood cell there is no to show the report of organophosphor hydrolytic enzyme.
Summary of the invention
Stability is poor when applying in the actual environment present invention aim to address organophosphor hydrolytic enzyme, isolates and purifies program
Cumbersome technical problem provides the Ko subtilis and preparation method thereof for showing organophosphor hydrolytic enzyme.
Bacillus subtillis (Bacillus subtilis) of the present invention, is preserved in China on March 8th, 2018
Microbiological Culture Collection administration committee common micro-organisms center, deposit number are CGMCC No.15427, preservation address Beijing
The institute 3 of city, North Star West Road, Chaoyang District 1, Institute of Microorganism, Academia Sinica.
The present invention solves the above problems the technical solution of use: showing the Ko subtilis of organophosphor hydrolytic enzyme, the Ko subtilis table
Face shows the fusion protein of organophosphor hydrolytic enzyme OPH and capsid protein CotG, the biology with OPH degrading organic phosphor compound
Activity has excellent resistance to complex environment；Wherein, the amino acid sequence of organophosphor hydrolytic enzyme OPH are as follows:
MSAQAMRSIRARPIT
ISEAGFTLTHEDISAARQDSCVLGQSSSVAQSSSGKGCERIARQSGWRANDCRCVDFRYRS RRQFIGRGFA
GCRRSYLAATGLWFDPPLSMRLRYVEELTLVLPAVRFNMASKYTGIRAGII KVATTGKATPFQELVLKAAARASL
ATGVPVTTHTAASQRDGERGRPPFLSPKLEPSRVCIG HSDDTDDLSYLTALLRGYLIGLDHIPHSAIGLEDNASA
SPLLGIRSWQTRALLIKALIDQGY MKQILVSNDWLFGFSSYVTNIMDVMDRVNPDGMAFIH, capsid protein CotG's
Amino acid sequence are as follows: MGHYSHSDIEEAVKSAKKEGLKDYLYQEPHGKKRSHKKSHRTHKKSRSHKKSYCSH KKS RS
HKKSFCSHKKSRSHKKSYCSHKKSRSHKKSYRSHKKSRSYKKSYRSYKKSRSYKKSCRS YKKSRSYKKSYCSHKK
KSRSYKKSCRTHKKSYRSHKKYYKKPHHHCDDYKRHDDYDSK KEYWKDGNCWVVKKKYK.For the first time in Ko subtilis
Surface display organophosphor hydrolytic enzyme, using the excellent resistance promotion organophosphor hydrolytic enzyme of brood cell in the steady of complex condition
It is qualitative, promote its popularization and application in real life.
Steps are as follows for the Ko subtilis preparation method of displaying organophosphor hydrolytic enzyme of the invention:
(1) Bacillus subtillis genome is extracted as template, passes through PCR amplification capsid protein CotG and its own starting
The nucleotide sequence of son；Use the restriction enzyme nucleotide sequence to above-mentioned amplification and the shuttle of large intestine-withered grass respectively
Plasmid vector carries out double digestion；Two kinds of digestion products are mixed with 1: 3~1: 5 ratio, are connected using nucleic acid ligase, structure
Build the recombinant plasmid comprising capsid protein CotG and its own promoter nucleotide sequence；Above-mentioned recombinant plasmid is passed through into chemistry
Conversion method imports escherichia coli cloning bacterial strain host, by selecting monoclonal after antibiotic-screening culture；To above-mentioned monoclonal into
Row culture amplification, and extract recombinant plasmid and carry out restriction enzyme digestion and electrophoresis verifying and gene sequencing identification；It has chosen in this step from withered
The capsid protein CotG of careless bacillus itself is as the anchorin with organophosphor hydrolytic enzyme OPH amalgamation and expression, in maturation
Abundance in brood cell is higher, and will not influence brood cell's structure after modifying it transformation；In addition, in order to reduce foreign protein to place
Advocate peace the influence of sporulation, the promoter of capsid protein CotG itself has been used in this step, so as to the expression of albumen, adjust
It controls closer to nature；
(2) it is compiled according to gene of the codon preference of Escherichia coli and Bacillus subtillis to organophosphor hydrolytic enzyme OPH
Code sequence optimizes, and the nucleotide sequence of organophosphor hydrolytic enzyme OPH: atgagcgcgc is synthesized by artificial synthesized mode
aggcgatgcg tagcattcgt gcgcgtccga ttaccattag cgaagcgggc tttaccctga cccatgaaga
tattagcgcg gcgcgtcagg atagctgcgt gctgggccag agcagcagcg tggcgcagag cagcagcggc
aaaggctgcg aacgtattgc gcgtcagagc ggctggcgtg cgaacgattg ccgttgcgtg gattttcgtt
atcgtagccg tcgtcagttt attggccgtg gctttgcggg ctgccgtcgt agctatctgg cggcgaccgg
cctgtggttt gatccgccgc tgagcatgcg tctgcgttat gtggaagaac tgaccctggt gctgccggcg
gtgcgtttta acatggcgag caaatatacc ggcattcgtg cgggcattat taaagtggcg accaccggca
aagcgacccc gtttcaggaa ctggtgctga aagcggcggc gcgtgcgagc ctggcgaccg gcgtgccggt
gaccacccat accgcggcga gccagcgtga tggcgaacgt ggccgtccgc cgtttctgag cccgaaactg
gaaccgagcc gtgtgtgcat tggccatagc gatgataccg atgatctgag ctatctgacc gcgctgctgc
gtggctatct gattggcctg gatcatattc cgcatagcgc gattggcctg gaagataacg cgagcgcgag
cccgctgctg ggcattcgta gctggcagac ccgtgcgctg ctgattaaag cgctgattga tcagggctat
atgaaacaga ttctggtgag caacgattgg ctgtttggct ttagcagcta tgtgaccaac attatggatg
Tgatggatcg tgtgaacccg gatggcatgg cgtttattca ttaa, and pass through PCR amplification；Use restriction enzyme
The nucleotide sequence to above-mentioned amplification and the middle recombinant plasmid constructed of step (1) carry out double digestion to enzyme respectively；Two kinds of digestions are produced
Object is mixed with 1: 3 to 1: 5 ratio, and organophosphor hydrolytic enzyme gene is connected to capsid protein CotG base using nucleic acid ligase
The C-terminal of cause, weight of the building comprising capsid protein CotG, CotG own promoter and organophosphor hydrolytic enzyme OPH nucleotide sequence
Group plasmid；Above-mentioned recombinant plasmid is imported into escherichia coli cloning bacterial strain host by chemical transformation, is trained by antibiotic-screening
Monoclonal is selected after supporting；Culture amplification is carried out to above-mentioned monoclonal, and extracts recombinant plasmid and carries out restriction enzyme digestion and electrophoresis verifying and gene
Sequencing identification；For organophosphor hydrolytic enzyme OPH, the technology of inclusion body easy to form is difficult during recombinant expression in this step
Topic, has carried out codon preference sex modification to its encoding gene, this gene order has originality；
(3) recombinant plasmid constructed in step (2) is imported into Bacillus subtillis expressive host by electrotransformation, passed through
Monoclonal is selected after antibiotic-screening culture；Culture amplification is carried out to above-mentioned monoclonal, and extracts recombinant plasmid and carries out digestion electricity
Swimming verifying；This step constructs the withered grass engineered strain for showing organophosphor hydrolytic enzyme OPH, and source is U.S.'s food medicine
The grade-safe bacterial strain Bacillus subtilis that object management board and the Ministry of Agriculture ratify；
(4) the Bacillus subtillis expressive host constructed in step (3) is subjected to raw born of the same parents' culture by nutrition failure method,
Culture solution, DSM culture medium, that is, 8g nutrient broth medium, 1g potassium chloride, 0.25g are collected after cultivating 1~2 day in DSM culture medium
Bitter salt, tetra- chloride hydrate manganese of 2mg, sodium hydroxide tune pH to 7.0, distilled water are settled to 1L, 121 DEG C of high pressure sterilizations
The 1ml 1M calcium nitrate solution and 1ml 1mM ferric sulfate of filter membrane degerming is added in 20min；
(5) using aqua sterilisa to above-mentioned culture solution dilution 106Afterwards, 100~200 microlitres are taken to be coated on screening flat board, overnight
Total bacteria count is counted after culture, separately takes above-mentioned culture solution in 80 DEG C of incubation 15min or more, dilution 106After take 100~200 microlitres of paintings
It is distributed in screening flat board, brood cell's number is counted after being incubated overnight, the results showed that brood cell's production rate of engineered strain is up to 60~70%；This
Raw born of the same parents lead to be led close with the life born of the same parents of Bacillus subtillis under natural conditions, and the expression and displaying for showing organophosphor hydrolytic enzyme OPH are not
It will affect the sporulation of host；
(6) brood cell in step (4) culture solution is purified, 4000~20000g centrifugation collection bacterium, abandon supernatant after with
2mg/ml lysozyme soln is resuspended, and brood cell is collected by centrifugation in 37 DEG C of incubations 30min~1h, 4000~20000g, uses 1M respectively
NaCl and 1M KCl washing, uses pure water 3~5 times, brood cell is collected by centrifugation in 4000~20000g, is resuspended in 10ml later
PBS buffer solution；
(7) brood cell in step (6) after purification is resuspended in the strip buffer of 1/10th volumes, 0.1M DTT,
0.5% SDS, 0.1M NaCl；Brood cell's capsid protein of 70 DEG C or more high temperature incubation 30min or more, acquisition pass through dialysis desalination
The affine column purification of histidine is used afterwards, and purifying protein is analyzed by protein immunoblot and point Blot experiment, with control group
There is significant chemiluminescence compared to the extraction albumen of Recombinant spores, the results showed that the fusion protein molecule of each brood cell's expression reaches
105The order of magnitude；The experiment of this step show organophosphor hydrolytic enzyme OPH can successful expression into Recombinant spores；
(8) using the brood cell in source of mouse polyhistidine antibody incubation step (6) after purification, with sheep anti mouse-FITC after PBS washing
Secondary antibody is incubated for, and after PBS is washed, is added dropwise to glass slide, is placed in observation analysis under laser confocal microscope, with control group phase
There is significant fluorescence than Recombinant spores, show fusion protein by successful presentation on the surface of Ko subtilis；The experiment table of this step
It is bright, organophosphor hydrolytic enzyme OPH can successful presentation on the surface of Recombinant spores；
(9) using paraoxon as reaction substrate, in 75mM Tris-HCl, 50 μM of CoCl2, pH=8.0 reaction environment in
The spore suspension of 20% step (6) preparation is added, uses UV spectrophotometer measuring reaction solution after 37 DEG C of reaction 2min
A410, the results showed that, it shows that the Ko subtilis of organophosphor hydrolytic enzyme has significant hydrolysing activity, reaches 15.81U/mg；
Ko subtilis detects work after organic reagent, high temperature, freeze thawing, acidity, alkalinity, Protease Treatment brood cell is respectively adopted
Property, the results showed that, show the Ko subtilis hydrolysis vigor of organophosphor hydrolytic enzyme in organic reagent methanol or ether, 40~60 DEG C
High temperature, -15~-20 DEG C of freeze thawing, pH3~5, pH 9~11, the tolerance in trypsase or Proteinase K environment significantly increase
By force；The experiment of this step shows that the Recombinant spores of displaying organophosphor hydrolytic enzyme OPH have the activity of hydrolysis organophosphorus pesticide, and
It is significantly improved for the tolerance of a variety of poor environments, P is less than 0.001.
Beneficial effects of the present invention:
1. successfully organophosphor hydrolytic enzyme OPH can be expressed by the Bacillus subtillis engineered strain that genetic recombination improves
And show that on the surface of brood cell, the Ko subtilis of acquisition has the catalytic activity of hydrolysis organophosphorus pesticide, can be used as mild, ring
The pesticide cleanser of guarantor is biological prosthetic for contaminated environment；
2. showing that stability of the Ko subtilis of organophosphor hydrolytic enzyme OPH under a variety of adverse circumstances increases compared with pure enzyme
By force, and Ko subtilis prepares simple, eliminates separation, the purification step of pure enzyme complexity, reduces production cost, favorably
In the popularization and application in real life；
3. hypopus of the brood cell as engineered strain sprouts autonomous regeneration under optimum conditions, sustainable, circulation can be reached
It utilizes.
Detailed description of the invention
Fig. 1 recombinant plasmid pHY300PLK-cotG-opdcb schematic diagram
1% agarose electrophoresis figure is identified in the digestion of Fig. 2 recombinant vector
In figure: swimming lane 1 is HindIII the and BamHI double digestion electrophoresis knot of recombinant plasmid pHy300PLK-cotG-opdcb
Fruit；Swimming lane 2 is the BamHI single endonuclease digestion electrophoresis result of recombinant plasmid pHY300PLK-cotG-opdcb；Swimming lane 3 is recombinant plasmid
HindIII the and BamHI double digestion electrophoresis result of pHY300PLK-cotG；Swimming lane 4 is recombinant plasmid pHY300PLK-cotG's
BamHI single endonuclease digestion electrophoresis result；Swimming lane 5 is 1kb DNA Marker.
The recombinant bacterial strain of Fig. 3 surface display OPH counts lithograph
In figure: the 1st is classified as the raw total bacterium of born of the same parents' culture solution of bacterial strain pHY300PLK-cotG/DB104；2nd is classified as bacterial strain
PHY300PLK-cotG/DB104 gives birth to born of the same parents' culture solution brood cell；3rd is classified as the raw born of the same parents of bacterial strain pHY300PLK-cotG-opdcb/DB104
The total bacterium of culture solution；4th is classified as the raw born of the same parents' culture solution brood cell of bacterial strain pHY300PLK-cotG-opdcb/DB104.
The capsid protein western blot figure of Fig. 4 Recombinant spores
In figure: swimming lane 1 is albumen Marker；Swimming lane 2 is recombinant bacterial strain pHY300PLK-cotG-opdcb/DB104 brood cell
Capsid protein；Swimming lane 3 is the capsid protein of recombinant bacterial strain pHY300PLK-cotG/DB104 brood cell.
The capsid protein point trace figure of Fig. 5 Recombinant spores
In figure: group 1 is the capsid protein of recombinant bacterial strain pHY300PLK-cotG/DB104 brood cell；Group 2 is carrying histidine
The standard protein of label.
The immunofluorescence microscopy figure of Fig. 6 Recombinant spores
In figure: I is light field microscopy figure；II is fluorescence microscopy figure；III is integration map；A is Recombinant spores sample；B is wild
Brood cell's sample.
The FCM analysis figure of Fig. 7 Recombinant spores
In figure: a is the flow cytometer detection result of recombinant bacterial strain pHY300PLK-cotG/DB104 brood cell；B is recombinant bacterial strain
The flow cytometer detection result of pHY300PLK-cotG-opdcb/DB104 brood cell；Ordinate is brood cell's quantity, and abscissa is fluorescent value.
The Activity determination figure of Fig. 8 Recombinant spores
In figure: group 1 is wild brood cell's sample；Group 2 is Recombinant spores sample；Ordinate is the enzyme activity of every milligram of brood cell's dry weight
Property.
The tolerance of Fig. 9 Recombinant spores detects figure
In figure: group 1 is untreated；Group 2 is methyl alcohol process；Group 3 is handled for ether；Group 4 is freeze thawing treatment；Group 5 is high temperature
Processing；Group 6 is basic treatment；Group 7 is acidic treatment；Group 8 is trypsin treatment；Group 9 is handled for Proteinase K；Ordinate is
Account for the percentage of original enzymatic activity；Recombinant spores group is represented,Represent pure enzyme group.
Specific embodiment
Technical solution of the present invention is clearly and completely illustrated below in conjunction with drawings and examples.
Method in following embodiments is unless otherwise instructed conventional method；Percentage composition in following embodiments,
It unless otherwise instructed, is mass percentage.
Main experimental material
High-fidelity DNA polymerase (Thermo Fisher Scientific), dNTP Mix (Thermo Fisher
Scientific), PCR Master Mix 2 × (Thermo Fisher Scientific), plasmid extraction reagent (Thermo
Fisher Scientific), DNA gel recovery purifying kit (Thermo Fisher Scientific) is restricted interior
Enzyme cutting (Thermo Fisher Scientific), T4 DNA ligase (Thermo Fisher Scientific), DTT
(Thermo Fisher Scientific), protein molecular weight standard (Thermo Fisher Scientific), DNA
Ladder (Fermentas), peptone (Britain OXOID), yeast extract (OXOID), Tris alkali (Amresco), glycine
(Amresco), skimmed milk power (BD), HRP substrate chemiluminescence detection agent (Tiangen), agarose (Biowest), agar
(Biowest), dimethylthiazole diphenyltetrazoliumbromide bromide MTT (Sigma), (BeiJing, China's couple stars is raw for coomassie brilliant blue R_250
Object company), dodecyl sodium sulfate SDS (Amresco), vector plasmid pHY300PLK (TaKaRa), clone strain
Escherichia coli DH5 α (TIANGEN), Bacillus subtillis Bacillus subtilis DB104 (Chinese people's solution
The tenth research institute, Fang Jun Military Medical Science Institute), anti-His mouse monoclonal antibody (Tiangen), horseradish peroxidase mark
The goat anti-mouse IgG antibodies (Sino Biological) of note, the anti-mouse goat antibody (ZSGB-BIO) of FITC label, algae red
The anti-His mouse monoclonal antibody (abcam) of protein labeling.
Embodiment 1
The acquisition of the display carrier of organophosphor hydrolytic enzyme
1, the building of the recombinant vector containing capsid protein gene
Bacillus subtillis DB104 genome is extracted, is reacted as template by PCR using genome and obtains capsid protein cotG
(SEQ ID NO:4) and its promoter gene, primer are cotG-F (5-gccttt gaattc agtgtccctagctccgag-
3 ' (SEQ ID NO:6)) and cotG-R (5-ctattg ggatcc tgaacccccacctcc
Tttgtatttctttttgacta-3 ' (SEQ ID NO:7)).By PCR, flexible peptide chain (Gly-Gly-Gly-Gly-Ser)
It is introduced into the end capsid protein CotG.PCR amplification condition is 95 DEG C of initial denaturation 5min, then expands 35 circulations: 95 DEG C of 30s,
55 DEG C of 30s, 72 DEG C of 2min, 10 min of last 72 DEG C of extensions.PCR product is carried out by purification kit after purification, to use limit
Property restriction endonuclease EcoRI and BamHI processed carries out digestion processing to it and purifies.
The escherichia coli cloning bacterial strain DH5 α containing shuttle plasmid pHY300PLK is cultivated, extracts plasmid vector after amplification.Make
Digestion processing is carried out to carrier with restriction enzyme EcoRI and BamHI and is purified.Capsid protein cotG gene will be contained
DNA enzymatic slice section is attached with the carrier segments after digestion reacts (recombinant plasmid schematic diagram is shown in Fig. 1).Use Escherichia coli
Clone strain DH5 α competence converts connection product.Screening and culturing is carried out by the solid medium containing tetracycline,
Digestion identification and sequencing analysis are carried out after the transformant culture amplification of acquisition, the electrophoresis result of digestion identification is shown in Fig. 2 swimming lane 3,4.
The result shows that the structure and sequence of recombinant vector are correct, the Insert Fragment containing 1060bp, and through sequencing identification there is no
Gene mutation.
2, the building of the recombinant vector containing organic phosphorus hydrolase gene
Organophosphor hydrolytic enzyme gene opdcb (SEQ ID NO:3) is obtained by artificial synthesized mode, and in gene end
Histidine tag His × 6 are added convenient for subsequent purification identification (the prosperous Biotechnology Co., Ltd of Beijing AudioCodes).Expanded by PCR
The protein gene of gaining, primer are opdcb-F (5-actg ggatcc agcgcgcaggcgatgcgtag-3 ' (SEQ ID
NO:8)) expand with opdcb-R (5-agag aagctt tta gtggtggtggtggtggtg-3 ' (SEQ ID NO:9)), PCR
Increasing condition is 95 DEG C of initial denaturation 5min, and then expand 35 circulations: 95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min, last 72 DEG C are prolonged
Stretch 10min.PCR product is carried out after purification, using restriction enzyme HindIII and BamHI to it by purification kit
It carries out digestion processing and purifies.
The escherichia coli cloning bacterial strain DH5 α containing recombinant plasmid pHY300PLK-cotG is cultivated, recombination matter is extracted after amplification
Grain.Digestion processing is carried out to carrier using restriction enzyme HindIII and BamHI and is purified.Organophosphor hydrolytic enzyme will be contained
The DNA enzymatic slice section of opdcb gene is attached with the carrier segments after digestion reacts.
Secondary connection product is converted using escherichia coli cloning bacterial strain DH5 α competence.By containing Fourth Ring
The solid medium of element carries out screening and culturing, carries out digestion identification and sequencing analysis, digestion after the transformant culture amplification of acquisition
The electrophoresis result of identification is shown in Fig. 2 swimming lane 1,2.The result shows that the structure and sequence of recombinant vector are correct, the insertion containing 915bp
Segment, and there is no gene mutations through sequencing identification.
Embodiment 2
It can show the acquisition of the recombinant bacterial strain of organophosphor hydrolytic enzyme
DB104 is extracellular neutral proteinase (nprE) and serine protease (aprA) deficiency Bacillus subtillis,
According to FUJIO KAWAMURA and ROY H.DOI, Construction of a Bacillus subtilis Double
Mutant Deficient in Extracellular Alkaline and Neutral Proteases, Journal of
It is prepared by Bacteriology, Vol.160, No.1, p.442-444, the method recorded in 10 months 1984.
It will show that host Bacillus subtillis DB104 was cultivated to plateau, transfer in competence growth medium (10g/L
Peptone, 5g/L yeast extract, 10g/L sodium chloride, 0.5M sorbierite) it cultivates to OD600Between 0.85~0.95.Ice bath
Collect bacterium after 10min, uses electroporation buffer (0.5M sorbierite, 0.5M mannitol, 10% glycerol) lotion thallus of pre-cooling, weight
It is 4 times multiple.Be resuspended in electroporation buffer at 1: 40, and with 60 μ l packing, -80 DEG C are saved for use.
The escherichia coli cloning bacterial strain DH5 α containing recombinant plasmid pHY300PLK-cotG-opdcb is cultivated, is extracted after amplification
By the plasmid of genetic recombination twice.It takes Bacillus subtillis DB104 competent cell ice bath to dissolve, 50ng recombination is added and carries
Body ice bath 10min is transferred to the 2mm electricity revolving cup of pre-cooling.Recombinant vector is imported into Bacillus subtillis by the method for electrotransformation
DB104 competent cell, it is 2.5kv that electricity, which turns parameter, is shocked by electricity 1 time.The renewal cultivation of 1ml preheating is added after electric shock immediately
Base (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 0.5M sorbierite, 0.38M mannitol), 37 DEG C,
150rpm oscillation recovery culture 3h.With the solid medium screening and culturing containing tetracycline after collection bacterium, to transformant
PHY300PLK-cotG-opdcb/DB104 carries out PCR identification.Condition is 95 DEG C of initial denaturation 5min, then expands 25 circulations:
95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, last 72 DEG C of extensions 10min.
Embodiment 3
The culture and purifying of Recombinant spores
By recombinant bacterial strain pHY300PLK-cotG-opdcb/DB104 with 1: 100 be inoculated in brood cell generate culture medium (DSM:
8% nutrient broth, 0.1% potassium chloride, 0.025% bitter salt, 0.01mM manganese chloride, 1mM calcium nitrate, 0.01mM sulphur
Sour iron, pH to 7.0) culture 48h.The bacterial strain sum in culture medium is calculated by colony counting method, 80 DEG C of culture solution are incubated for
Brood cell's number is calculated after 15min.Count results (see Fig. 3) show in raw born of the same parents' culture solution brood cell's concentration be (9.35 ± 0.83) ×
106Cfu/ml, raw born of the same parents lead as (61.61 ± 16.09) %.
High speed centrifugation collects brood cell, and 37 DEG C of incubation 30min after being resuspended with 2mg/ml lysozyme soln use 1M respectively
NaCl and 1M KCl washing, uses pure water 3 times.It is stand-by that brood cell's sample is resuspended in pure water.
Embodiment 4
Expression and displaying identification of the organophosphor hydrolytic enzyme in Recombinant spores
Capsid protein strip buffer (0.1M DTT, 0.5%SDS, 0.1M NaCl) is added to spore suspension, 70 DEG C incubate
30min is educated, supernatant dialysis desalination 4h is repeated 3 times.Using histidine column purification dialyzate, refined solution is super with 10K super filter tube
It is lyophilized after filtering out salt, the dry powder albumen of acquisition is resuspended with ultrapure water.Above-mentioned protein sample is subjected to SDS-PAGE electrophoresis, to obtaining
The running gel obtained carries out electric transfer, and 110V transfers 90min, albumen is transferred to 0.2 μm of pvdf membrane.Later with 5% cow's serum
Albumin solution closes 1h, and source of mouse polyhistidine antibody is incubated for 1h, and TBS-T washs 5min, is repeated 4 times, sheep anti mouse-HRP secondary antibody is incubated
1h is educated, TBS-T washs 5min, is repeated 4 times, and ECL developing solution is added dropwise on film and develops the color and photographs to record, western blotting qualification
As a result see Fig. 4.The result shows that recombinant bacterial strain pHY300PLK-cotG-opdcb/DB104 can express organophosphor hydrolytic enzyme, and
It is assembled in Recombinant spores.
A blotting experiments are carried out using above-mentioned protein purification sample.By the standard egg of protein sample and carrying histidine tag
White GFP-6 × His doubling dilution takes 2 μ l protein samples and its dilution, standard protein and its dilution to be added dropwise to 0.2 respectively
μm pvdf membrane, with 5%BSA solution close 1h, source of mouse polyhistidine antibody be incubated for 1h, TBS-T wash 5min, be repeated 4 times, sheep
Anti- mouse-HRP secondary antibody is incubated for 1h, and TBS-T washs 5min, is repeated 4 times, and ECL developing solution is added dropwise on film and develops the color and photographs to record,
The result of point trace identification is shown in Fig. 5.The result shows that each brood cell of recombinant bacterial strain pHY300PLK-cotG-opdcb/DB104
About 8.74 × 10 can averagely be expressed4A OPH molecule.
It takes spore suspension to wash 3 times with PBS, is incubated at room temperature 30min, 10000g centrifugation using 3% bovine serum albumin solution
5min discards supernatant liquid.It is resuspended using source of mouse polyhistidine antibody, 4 DEG C of overnight incubations are washed 3 times with PBS.Use sheep anti mouse-
FITC secondary antibody is incubated for 2h, is washed 3 times with PBS.By about 20 μ l, treated that sample is added dropwise to glass slide, and it is aobvious to be placed in laser co-focusing
Micro- microscopic observation, and photograph to record, immunofluorescence microscopic examination result is shown in Fig. 6.The result shows that organic phosphorus hydrolysis of the expression to brood cell
Enzyme can be located in the surface of Recombinant spores.It takes spore suspension to wash 3 times with PBS, uses 3% bovine serum albumin solution room temperature
It is incubated for 30min, 10000g is centrifuged 5min, discards supernatant liquid.It is resuspended using source of mouse histidine monoclonal antibody, is incubated for 2h, with
PBS is washed 3 times.Taking treated, sample carries out FCM analysis and records, and FCM analysis result is shown in Fig. 7.As a result table
It is bright, show that the Recombinant spores average fluorescent strength of OPH is (55.93 ± 3.54), positive rate 5.65% is all remarkably higher than pair
According to group (P < 0.001).
Embodiment 5
The organophosphor hydrolytic enzyme activity identification of Recombinant spores
Using paraoxon as reaction substrate, it is outstanding to sequentially add 200 μ l Recombinant spores pHY300PLK-cotG-opdcb/DB104
Liquid, 700 μ l survey buffer (75mM Tris-HCl, 50 μM of CoCl living2, pH=8.0) and 100 μ l 20mM paraoxon methanol
Solution is mixed evenly, 37 DEG C of reaction 2min.Supernatant is taken after centrifugation, detects A to survey after liquid living dilutes 3 times410, in triplicate.
Using Recombinant spores pHY300PLK-cotG/DB104 suspension as negative control and the influence of nature hydrolysis is deducted, according to nitro
Phenol standard curve calculates the Recombinant spores enzyme activity for showing organophosphor hydrolytic enzyme.Separately take 1ml Recombinant spores pHY300PLK-
The freeze-drying weighing of cotG-opdcb/DB104 suspension.The enzyme activity unit (U) of Recombinant spores is defined as: at 37 DEG C, discharge per minute
Recombinant spores amount needed for 1nmol p-nitrophenol.The specific activity of enzyme of Recombinant spores is defined as: the institute of every milligram of Recombinant spores
The enzyme activity unit (U/mg) contained.Activity determination result is shown in Fig. 8, the results showed that Recombinant spores hydrolysis paraoxon activity be
15.81U/mg brood cell's dry weight has be obviously improved (p < 0.001) compared with the control group.That is recombinant bacterial strain pHY300PLK-
The Recombinant spores that cotG-opdcb/DB104 is generated have the ability of degrading organic phosphor substrate, and organophosphor hydrolytic enzyme can be with tool
There is the form of bioactivity to show on the surface of Recombinant spores.
100 μ l Recombinant spores pHY300PLK-cotG-opdcb/DB104 suspensions and the pure enzyme sample of OPH are taken, is separately added into
Methanol or ether, 37 DEG C of incubation 1h are measured, its activity is in the above way detected, three groups is repeated and records.Take 100 μ l Recombinant spores
PHY300PLK-cotG-opdcb/DB104 suspension and the pure enzyme sample of OPH, 40 DEG C of incubation 1h in the above way detect its activity,
It repeats three groups and records.100 μ l Recombinant spores pHY300PLK-cotG-opdcb/DB104 suspensions and the pure enzyme sample of OPH are taken ,-
20 DEG C of incubation 1h in the above way detect its activity after dissolution, repeat three groups and record.Take 100 μ l Recombinant spores
PHY300PLK-cotG-opdcb/DB104 suspension and the pure enzyme sample of OPH are separately added into 0.25% trypsase, 37 DEG C of incubations
1h in the above way detects its activity, repeats three groups and records.Take 100 μ l Recombinant spores pHY300PLK-cotG-opdcb/
DB104 suspension and the pure enzyme sample of OPH, are separately added into 50U Proteinase K, and 37 DEG C of incubation 1h in the above way detect its activity, weight
It answers three groups and records.100 μ l Recombinant spores pHY300PLK-cotG-opdcb/DB104 suspensions and the pure enzyme sample of OPH are taken, it is above
It states method and detects its activity in the Gly-NaOH buffer solution of pH 10, repeat three groups and record.Take 100 μ l Recombinant spores
PHY300PLK-cotG-opdcb/DB104 suspension and the pure enzyme sample of OPH, the in the above way Na in pH 42HPO4-Citri
Its activity is detected in Acid buffer solution, repeats three groups and is recorded.After its activity of spectrophotometry, residual is calculated
Activity accounts for the percentage of original activity, and tolerance testing result is shown in Fig. 9.The experimental results showed that showing the Recombinant spores of OPH
PHY300PLK-cotG-opdcb/DB104 enzyme activity is in methanol, ether, freeze thawing, high temperature, pH 4, trypsase, Proteinase K
Tolerance in environment significantly increases (P < 0.001).
Claims (2)
1. a kind of Ko subtilis for showing organophosphor hydrolytic enzyme, it is characterised in that the Ko subtilis surface display organophosphor hydrolytic enzyme
The fusion protein of OPH and capsid protein CotG, the bioactivity with OPH degrading organic phosphor compound, have complex environment
Excellent resistance；Wherein, the amino acid sequence of organophosphor hydrolytic enzyme OPH are as follows: MSAQAMRSIRARPITISEAGFTLTHEDI
SAARQDSCVLGQSSSVAQSSSGKGCERIARQSGWRANDCRCVDFRYRSRRQFIGRGFAGCRRSYLAATGLWFDPPL
SMRLRYVEELTLVLPAVRFNMASKYTGIRAGIIKVATTGKATPFQELVLKAAARASLATGVPVTTHTAASQRDGER
GRPPFLSPKLEPSRVCIGHSDDTDDLSYLTALLRGYLIGLDHIPHSAIGLEDNASASPLLGIRSWQTRALLIKALI
DQGYMKQILVSNDWLFGFSSYVTNIMDVMDRVNPDGMAFIH, the amino acid sequence of capsid protein CotG are as follows: MGHYSHS
DIEEAVKSAKKEGLKDYLYQEPHGKKRSHKKSHRTHKKSRSHKKSYCSHKKSRSHKKSFCSHKKSRSHKKSYCSHK
KSRSHKKSYRSHKKSRSYKKSYRSYKKSRSYKKSCRSYKKSRSYKKSYCSHKKKSRSYKKSCRTHKKSYRSHKKYY
KKPHHHCDDYKRHDDYDSKKEYWKDGNCWVVKKKYK。
2. a kind of Ko subtilis preparation method for showing organophosphor hydrolytic enzyme, it is characterised in that the Ko subtilis preparation method step
It is as follows:
(1) it extracts Bacillus subtillis genome as template, passes through PCR amplification capsid protein CotG and its own promoter
Nucleotide sequence；Use the restriction enzyme nucleotide sequence to above-mentioned amplification and large intestine-withered grass shuttle plasmid respectively
Carrier carries out double digestion；Two kinds of digestion products are mixed with 1: 3~1: 5 ratio, are connected using nucleic acid ligase, building includes
The recombinant plasmid of capsid protein CotG and its own promoter nucleotide sequence；Above-mentioned recombinant plasmid is led by chemical transformation
Enter escherichia coli cloning bacterial strain host, by selecting monoclonal after antibiotic-screening culture；Culture expansion is carried out to above-mentioned monoclonal
Increase, and extracts recombinant plasmid and carry out restriction enzyme digestion and electrophoresis verifying and gene sequencing identification；
(2) according to the codon preference of Escherichia coli and Bacillus subtillis to the gene code sequence of organophosphor hydrolytic enzyme OPH
Column optimize, and the nucleotide sequence of organophosphor hydrolytic enzyme OPH: atgagcgcgc is synthesized by artificial synthesized mode
aggcgatgcg tagcattcgt gcgcgtccga ttaccattag cgaagcgggc tttaccctga cccatgaaga
tattagcgcg gcgcgtcagg atagctgcgt gctgggccag agcagcagcg tggcgcagag cagcagcggc
aaaggctgcg aacgtattgc gcgtcagagc ggctggcgtg cgaacgattg ccgttgcgtg gattttcgtt
atcgtagccg tcgtcagttt attggccgtg gctttgcggg ctgccgtcgt agctatctgg cggcgaccgg
cctgtggttt gatccgccgc tgagcatgcg tctgcgttat gtggaagaac tgaccctggt gctgccggcg
gtgcgtttta acatggcgag caaatatacc ggcattcgtg cgggcattat taaagtggcg accaccggca
aagcgacccc gtttcaggaa ctggtgctga aagcggcggc gcgtgcgagc ctggcgaccg gcgtgccggt
gaccacccat accgcggcga gccagcgtga tggcgaacgt ggccgtccgc cgtttctgag cccgaaactg
gaaccgagcc gtgtgtgcat tggccatagc gatgataccg atgatctgag ctatctgacc gcgctgctgc
gtggctatct gattggcctg gatcatattc cgcatagcgc gattggcctg gaagataacg cgagcgcgag
cccgctgctg ggcattcgta gctggcagac ccgtgcgctg ctgattaaag cgctgattga tcagggctat
atgaaacaga ttctggtgag caacgattgg ctgtttggct ttagcagcta tgtgaccaac attatggatg
Tgatggatcg tgtgaacccg gatggcatgg cgtttattca ttaa, and pass through PCR amplification；Use restriction enzyme
The nucleotide sequence to above-mentioned amplification and the middle recombinant plasmid constructed of step (1) carry out double digestion to enzyme respectively；Two kinds of digestions are produced
Object is mixed with 1: 3 to 1: 5 ratio, and organophosphor hydrolytic enzyme gene is connected to capsid protein CotG gene using nucleic acid ligase
C-terminal, recombination of the building comprising capsid protein CotG, CotG own promoter and organophosphor hydrolytic enzyme OPH nucleotide sequence
Plasmid；Above-mentioned recombinant plasmid is imported into escherichia coli cloning bacterial strain host by chemical transformation, passes through antibiotic-screening culture
After select monoclonal；Culture amplification is carried out to above-mentioned monoclonal, and extracts recombinant plasmid and carries out restriction enzyme digestion and electrophoresis verifying and gene survey
Sequence identification；
(3) recombinant plasmid constructed in step (2) is imported into Bacillus subtillis expressive host by electrotransformation, passes through antibiosis
Monoclonal is selected after plain screening and culturing；Culture amplification is carried out to above-mentioned monoclonal, and extracts recombinant plasmid progress restriction enzyme digestion and electrophoresis and tests
Card；
(4) the Bacillus subtillis expressive host constructed in step (3) is subjected to raw born of the same parents' culture by nutrition failure method, in DSM
Culture solution, DSM culture medium, that is, 8g nutrient broth medium, 1g potassium chloride, seven water of 0.25g are collected after cultivating 1~2 day in culture medium
Conjunction magnesium sulfate, tetra- chloride hydrate manganese of 2mg, sodium hydroxide tune pH to 7.0, distilled water are settled to 1L, 121 DEG C of high pressure sterilization 20min,
The 1ml 1M calcium nitrate solution and 1ml 1mM ferric sulfate of filter membrane degerming is added；
(5) using aqua sterilisa to above-mentioned culture solution dilution 106Afterwards, 100~200 microlitres are taken to be coated on screening flat board, after being incubated overnight
Total bacteria count is counted, separately takes above-mentioned culture solution in 80 DEG C of incubation 15min or more, dilution 106After take 100~200 microlitres to be coated on sieve
Plate is selected, brood cell's number is counted after being incubated overnight, the results showed that brood cell's production rate of engineered strain is up to 60~70%；
(6) brood cell in step (4) culture solution is purified, 4000~20000g centrifugation collection bacterium abandons after supernatant with 2mg/ml
Lysozyme soln is resuspended, and brood cell is collected by centrifugation in 37 DEG C of incubations 30min~1h, 4000~20000g, uses 1M NaCl and 1M respectively
KCl washing, uses pure water 3~5 times, brood cell is collected by centrifugation in 4000~20000g, is resuspended in 10ml PBS buffer solution later；
(7) brood cell in step (6) after purification is resuspended in the strip buffer of 1/10th volumes, 0.1M DTT, 0.5%
SDS, 0.1M NaCl；70 DEG C or more high temperature incubation 30min or more, brood cell's capsid protein of acquisition after dialysis desalination by using
Column purification that histidine is affine, purifying protein are analyzed by protein immunoblot and point Blot experiment, are weighed compared with the control group
The extraction albumen of group brood cell has significant chemiluminescence, the results showed that the fusion protein molecule of each brood cell expression is up to 105Quantity
Grade；
(8) using the brood cell in source of mouse polyhistidine antibody incubation step (6) after purification, with sheep anti mouse-FITC secondary antibody after PBS washing
It is incubated for, after PBS is washed, is added dropwise to glass slide, is placed in observation analysis under laser confocal microscope, recombinates compared with the control group
Brood cell has significant fluorescence, shows fusion protein by successful presentation on the surface of Ko subtilis；
(9) using paraoxon as reaction substrate, in 75mM Tris-HCl, 50 μM of CoCl2, pH=8.0 reaction environment in be added
The spore suspension of 20% step (6) preparation, UV spectrophotometer measuring reaction solution A is used after 37 DEG C of reaction 2min410, knot
Fruit shows: showing that the Ko subtilis of organophosphor hydrolytic enzyme has significant hydrolysing activity, reaches 15.81U/mg；
Ko subtilis detects activity after organic reagent, high temperature, freeze thawing, acidity, alkalinity, Protease Treatment is respectively adopted, as a result table
It is bright: to show the Ko subtilis hydrolysis vigor of organophosphor hydrolytic enzyme in organic reagent methanol or ether, 40~60 DEG C of high temperature, -15
Tolerance in~-20 DEG C of freeze thawing, pH3~5, pH9~11, trypsase or Proteinase K environment significantly increases, and P is less than
0.001。
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| CN101475954A (en) * | 2008-12-05 | 2009-07-08 | 江苏大学 | Preparation of recombinant spore with surface for displaying lipase having catalytic activity |
| CN103937821A (en) * | 2014-04-22 | 2014-07-23 | 江苏大学 | Nitrilase gene and prokaryotic expression and immobilization technology thereof |
| CN105505846A (en) * | 2016-01-07 | 2016-04-20 | 南京工业大学 | Recombined spore with surface displaying glutamate dehydrogenase and construction method and application thereof |
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|---|---|---|---|---|
| CN101475954A (en) * | 2008-12-05 | 2009-07-08 | 江苏大学 | Preparation of recombinant spore with surface for displaying lipase having catalytic activity |
| CN103937821A (en) * | 2014-04-22 | 2014-07-23 | 江苏大学 | Nitrilase gene and prokaryotic expression and immobilization technology thereof |
| CN105505846A (en) * | 2016-01-07 | 2016-04-20 | 南京工业大学 | Recombined spore with surface displaying glutamate dehydrogenase and construction method and application thereof |
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