CN106754823A - A kind of preparation method and applications of ura DNA glycosidase - Google Patents
A kind of preparation method and applications of ura DNA glycosidase Download PDFInfo
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- CN106754823A CN106754823A CN201611236142.3A CN201611236142A CN106754823A CN 106754823 A CN106754823 A CN 106754823A CN 201611236142 A CN201611236142 A CN 201611236142A CN 106754823 A CN106754823 A CN 106754823A
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- ura
- dna
- dna glycosidase
- glycosidase
- ura dna
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- 108010072685 Uracil-DNA Glycosidase Proteins 0.000 title claims abstract description 87
- 102000006943 Uracil-DNA Glycosidase Human genes 0.000 title claims abstract description 82
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 238000006243 chemical reaction Methods 0.000 claims abstract description 22
- 238000001514 detection method Methods 0.000 claims abstract description 21
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 12
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 12
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 12
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 9
- 206010057040 Temperature intolerance Diseases 0.000 claims abstract description 7
- 230000008543 heat sensitivity Effects 0.000 claims abstract description 7
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 claims abstract description 6
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims abstract description 6
- 101710163270 Nuclease Proteins 0.000 claims abstract description 4
- 238000003556 assay Methods 0.000 claims abstract description 4
- 230000003301 hydrolyzing effect Effects 0.000 claims abstract description 4
- 230000009849 deactivation Effects 0.000 claims abstract description 3
- 239000002516 radical scavenger Substances 0.000 claims abstract description 3
- 229940035893 uracil Drugs 0.000 claims abstract description 3
- 241001135745 Colwellia psychrerythraea Species 0.000 claims description 27
- 230000014509 gene expression Effects 0.000 claims description 13
- 241000588724 Escherichia coli Species 0.000 claims description 10
- 239000013613 expression plasmid Substances 0.000 claims description 8
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 7
- 238000003259 recombinant expression Methods 0.000 claims description 7
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 6
- 238000001261 affinity purification Methods 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 6
- 241001597008 Nomeidae Species 0.000 claims description 5
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 4
- 229930182478 glucoside Natural products 0.000 claims description 4
- 150000008131 glucosides Chemical class 0.000 claims description 4
- 229910001453 nickel ion Inorganic materials 0.000 claims description 4
- 239000003531 protein hydrolysate Substances 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000005744 Glycoside Hydrolases Human genes 0.000 claims description 3
- 108010031186 Glycoside Hydrolases Proteins 0.000 claims description 3
- 230000006698 induction Effects 0.000 claims description 3
- 239000011347 resin Substances 0.000 claims description 3
- 229920005989 resin Polymers 0.000 claims description 3
- 108020004705 Codon Proteins 0.000 claims description 2
- 238000005336 cracking Methods 0.000 claims description 2
- 238000013461 design Methods 0.000 claims description 2
- 238000012215 gene cloning Methods 0.000 claims description 2
- 238000005215 recombination Methods 0.000 claims description 2
- 230000006798 recombination Effects 0.000 claims description 2
- 238000013112 stability test Methods 0.000 claims description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims 2
- 239000007788 liquid Substances 0.000 claims 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims 1
- 210000002700 urine Anatomy 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 7
- 230000003321 amplification Effects 0.000 abstract description 6
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 6
- 230000002401 inhibitory effect Effects 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 238000011109 contamination Methods 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 30
- 239000003795 chemical substances by application Substances 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108010001132 DNA Polymerase beta Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2497—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing N- glycosyl compounds (3.2.2)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/02—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2) hydrolysing N-glycosyl compounds (3.2.2)
- C12Y302/02003—Uridine nucleosidase (3.2.2.3)
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- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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Abstract
The invention discloses a kind of preparation method and applications of ura DNA glycosidase, the glycosidic bond between uracil base that the ura DNA glycosidase prepared in the present invention can be in 20 37 degree of effectively hydrolyzing DNA and deoxyribose；It has good heat sensitivity simultaneously, its fast deactivation when temperature is higher than 60 degree, reaches 60 degree of 5 minutes enzymatic activitys and loses 99%；The sample DNA that the temperature sensitive ura DNA glycosidase of preparation can be used for preventing in various nuclease assay reactions pollutes.The beneficial effects of the present invention are：In various nuclease assay reactions, temperature sensitive ura DNA glycosidase removes the effect of DNA sample pollution clearly, can either completely remove the DNA of depollution, and do not have any inhibitory action to nucleic acid amplification reaction.Can serve as various nucleic acid amplification reactions（Such as PCR）Sample contamination scavenger, eliminate the DNA pollution caused by a preceding amplified production, the degree of accuracy of increase detection of nucleic acids.
Description
Technical field
The invention belongs to gene engineering technology field, a kind of preparation method more particularly, to ura DNA glycosidase and
Its application.
Background technology
The appearance of round pcr makes it possible quickly to prepare specific DNA molecular in vitro, is now widely used for each seed nucleus
Acid amplification and detection field, are greatly promoted the development of the molecular diagnostic techniques based on nucleic acid.In nucleic acid amplifications such as PCR-baseds
In the molecule diagnosis of technology, the cross pollution of DNA sample, the dirt of particularly former detection sample to subsequent detection experiment sample
Dye, directly affects the accuracy of detection reaction.In order to eliminate the cross pollution that different batches detect DNA sample, use at present
By adding dUTP in PCR reaction systems, so that containing dU bases in the DNA of synthesis, carrying out, PCR detections next time are anti-
Before answering, ura DNA glycosidase is added in the sample so that the DNA that DNA fragmentation is produced come detection reaction before eliminating.
Then pcr amplification reaction is carried out again, carries out the detection of new samples.From
Ura DNA glycosidase in the past has higher stability in high temperature, it is not easy to inactivate, so as in the DNA for causing synthesis
DU be hydrolyzed so that DNA is by fragmentation, amplified reaction is effectively carried out, cause the yield of DNA amplification reaction big
It is big to reduce, reduce detection sensitivity.
The content of the invention
In order to overcome the deficiencies in the prior art, it is an object of the invention to provide a kind of temperature sensitive ura DNA glucosides
The agent of enzyme dna pollution cleanup, preparation method and applications；Ura DNA glycosidase in the present invention has thermo-responsive characteristic good, low
The features such as temperature is active high.The ura DNA glycosidase can effectively remove the pollution DNA molecular in detection sample, improve nucleic acid
Detect the accuracy of reaction.
The action principle of ura DNA glycosidase of the present invention is essentially consisted in：Ura DNA glycosidase being capable of water under middle low temperature
Glycosidic bond between solution uracil base and deoxyribose, so that DNA sample is unable to as the template of PCR because of thermal destruction
Molecule so that pollution DNA molecular does not produce any detection signal.Simultaneously ura DNA glycosidase heat sensitivity cause its
The PCR stages can occur heat inactivation, eliminate its inhibitory action to pcr amplification reaction.Processed through this ura DNA glycosidase, can
To realize the accurate detection of target nucleic acid molecules.
Technical scheme is specific as follows.
The present invention provides a kind of preparation method and applications of temperature sensitive ura DNA glycosidase PCR pollution cleanup agent,
Comprise the following steps that：
（1）Build the recombinant expression plasmid of ura DNA glycosidase
Based on cryophile genomic information, the ura DNA glycosidase gene of its coding is designed, on this basis, modification
Optimize the rare codon of ura DNA glycosidase encoding gene, psychrophile strain is obtained using full genome synthesis or round pcr
Ura DNA glycosidase gene, and by gene cloning to prokaryotic expression carrier pET28 etc., build ura DNA glycosidase weight
Group expression plasmid；
（2）Recombination expression ura DNA glycosidase
The ura DNA glycosidase recombinant expression plasmid conversion Bacillus coli expression host BL21 that will be built（DE3）Deng being urinated
Pyrimidine DNA glycosidase recombinant strains；It is carried out into ura DNA glycosidase induced expression with derivant IPTG again；
（3）The ura DNA glycosidase of affinity purification expression
The Escherichia coli after induced expression ura DNA glycosidase are collected by centrifugation, thalline are resuspended in non-denatured protein lysates,
Ultrasonic disruption thalline, is collected by centrifugation the Escherichia coli cracking supernatant containing ura DNA glycosidase；Recycle immobilization nickel
Ion affinity purification resin purifies ura DNA glycosidase from supernatant；
（4）The enzymatic activity and heat sensitivity of the ura DNA glycosidase of purifying are detected, temperature sensitive PCR pollution cleanups are filtered out
Agent albumen.
Enzymatic activity and heat stability test will be carried out by ura DNA glycosidase after purification, therefrom filter out specific for hydrolysis
Glycosidic bond in DNA between dU bases and deoxyribose, and with the ura DNA glycosidase of good heat sensitivity；It is thermo-responsive
The technical parameter of type is：5 minutes are incubated more than 60 degree, the residual activity of ura DNA glycosidase is less than 1%.
（5）Temperature sensitive ura DNA glycosidase is used to remove the DNA pollution thing in the detection of nucleic acids of PCR-based.
The present invention furthermore provides the application of the protein type PCR pollution cleanup agent based on ura DNA glycosidase.Should
PCR pollution cleanup agent is applied to the PCR detection of nucleic acids systems of archaeal dna polymerase.Specific method is as follows：Add in PCR reaction systems
Enter sample DNA and ura DNA glycosidase, process 15 minutes, remove the dU bases in DNA pollution thing, then enter performing PCR amplification
Target dna molecule, PCR amplifications are detected using 1% Ago-Gel.
In the present invention, step（1）In, the cryophile is including Colwellia psychrerythraea etc..
In the present invention, step（2）It is middle Fiber differentiation is carried out with derivant IPTG actual conditions be：First by ura DNA
Glycosidase recombinant strains culture adds 0.5mM derivants IPTG and is trained at a temperature of 30-37 DEG C to OD600=0.4-1.0
Culture carries out induced expression in 12 hours at a temperature of supporting 3 hours or 10-30 DEG C.
In the present invention, step（3）In, the composition of the non-denatured protein lysates is as follows：20 mM pH value are 8.0
Tris-HCl, 300 mM NaCl, 0.5mM DTT, 10vol% glycerine.
In the present invention, temperature sensitive ura DNA glycosidase PCR pollution cleanups agent is Colwellia
Psychrerythraea ura DNA glycosidases, wherein：The ura DNA glucosides of Colwellia psychrerythraea
The amino acid sequence of enzyme such as SEQ ID NO:Shown in 1.
Compared with prior art, the present invention has marked improvement, and it can be used to remove pollution in the nucleic acid amplification systems such as PCR
DNA sample, accuracy and the sensitivity of nuclease assay reaction can be greatly improved, it is specific as follows：
（1）Detection sensitivity is high.Due to temperature sensitive ura DNA glycosidase rapid deactivation at high temperature, so as in the PCR stages
Inhibitory action will not be caused to amplified reaction, it is thus possible to PCR amplification yield can be increased, detection sensitivity is improved.
（2）Detection accuracy is high.Because ura DNA glycosidase can understand the DNA that former detection reaction is produced（Should
DNA contains dU bases）；Therefore, it is possible to the inspection sun that the DNA molecular of detection reaction generation before being obviously reduced is detected to target molecule
Property phenomenon.
（3）Operating condition is simple, without optimization, directly uses.Because ura DNA glycosidase is in numerous nucleic acid such as PCR
There is activity very high in amplification system, without optimizing reaction system, only ura DNA glycosidase need to be directly added into, before PCR
Plus previous step normal temperature incubation step.
Brief description of the drawings
Fig. 1 is psychrophilic bacteria Colwellia psychrerythraea ura DNA glycosidases to dU bases in DNA
Hydrolysing activity.
Fig. 2 is the heat endurance of psychrophilic bacteria Colwellia psychrerythraea ura DNA glycosidases.
Fig. 3 is that psychrophilic bacteria Colwellia psychrerythraea ura DNA glycosidases are catalyzed to archaeal dna polymerase
The elimination effect of DNA pollution thing in the sample of PCR.
Specific embodiment
With reference to the accompanying drawings and examples, specific embodiment of the invention is further described.Following examples are only
For clearly illustrating technical scheme, and can not be limited the scope of the invention with this.
The preparation of the psychrophilic bacteria Colwellia psychrerythraea ura DNA glycosidases of embodiment 1
The first step, design synthesis psychrophilic bacteria Colwellia psychrerythraea ura DNA glycosidase genes, and insert
Enter pET28 expression vectors, build the recombinant expression plasmid of ura DNA glycosidase.Restructuring psychrophilic bacteria Colwellia
Psychrerythraea ura DNA glycosidases N-terminal is with 6 continuous histidine affinity purification marks from pET28 carriers
Sign, for the purifying of immobilization nickel ion affinity chromatograph.
Second step, recombinantly expresses psychrophilic bacteria Colwellia psychrerythraea ura DNA glycosidases.Will be thermophilic
Cold bacterium Colwellia psychrerythraea ura DNA glycosidases recombinant expression plasmid conversion Bacillus coli expression place
Main BL21（DE3）, obtain psychrophilic bacteria Colwellia psychrerythraea ura DNA glycosidase recombinant strains.
Strain culturing to OD600=0.6 will be expressed, 0.5mM derivant IPTG will be added, cultivated 12 hours at a temperature of 20 DEG C, induced thermophilic
Cold bacterium Colwellia psychrerythraea ura DNA glycosidases expression.
The amino acid sequence of psychrophilic bacteria Colwellia psychrerythraea ura DNA glycosidase recombinant proteins
（Nitrogen end → carbon teminal）Such as SEQ IDNO:Shown in 1.
3rd step, psychrophilic bacteria Colwellia psychrerythraea ura DNA glycosidase affinity purifications.Will step
After Escherichia coli after rapid two induction are collected by centrifugation, thalline is resuspended in protein lysate（20 mM Tris-HCl, pH
8.0,300 mM NaCl, 0.5mM DTT, 10vol% glycerine）.Ultrasonic disruption thalline, is collected by centrifugation and contains psychrophilic bacteria
The cellular lysate supernatant of Colwellia psychrerythraea ura DNA glycosidases.It is affine using immobilization nickel ion
Purification Resin purifies psychrophilic bacteria Colwellia psychrerythraea ura DNA glycosidases.
4th step, detect psychrophilic bacteria Colwellia psychrerythraea ura DNA glycosidases enzymatic activity and
Heat endurance.By the use of the double-stranded DNA containing dU bases as substrate, psychrophilic bacteria Colwellia psychrerythraea are added
Ura DNA glycosidase, determines its removal activity to dU bases.Specific enzymatic activity result is shown in Fig. 1.Result shows psychrophilic bacteria
Glucosides between Colwellia psychrerythraea ura DNA glycosidases specific for hydrolysis dU bases and deoxyribose
Key, and do not have hydrolysing activity to 4 kinds of normal glycosidic bonds between base and deoxyribose.
On this basis, the heat of detection psychrophilic bacteria Colwellia psychrerythraea ura DNA glycosidases is steady
It is qualitative.Psychrophilic bacteria Colwellia psychrerythraea ura DNA glycosidases are incubated 5 minutes in different temperatures, so
After determine its remaining ura DNA glycosidase activity size.Psychrophilic bacteria Colwellia psychrerythraea uracils
The thermal stability results of DNA glycosidase recombinant proteins are shown in Fig. 2, as a result show that its residual activity after being incubated 5 minutes at 60 DEG C is small
In 1%, its can under PCR thermal cycle conditions complete heat inactivation.
The psychrophilic bacteria Colwellia psychrerythraea ura DNA glycosidases of embodiment 2 pollute as PCR
The application of scavenger
It is polymerized as Pfu DNA by the use of the psychrophilic bacteria Colwellia psychrerythraea ura DNA glycosidases of purifying
The pollution cleanup agent of the PCR of enzymatic.Amplifying target genes are e. coli dna polymerase IV genes, PCR reaction buffers：
20 mM Tris-HCl (pH 8.8), 10 mM (NH4)2SO4, 10 mM KCl, 0.1 mg/mL BSA, 0.1% (v/
v) Triton X-100, 2 mM MgSO4；Other components are 0.2 mM dNTP, 0.3 μM of primer, 20ng e. coli dnas
Polymerase IV genetic fragments（Base containing dU）, 2.5 unit Pfu archaeal dna polymerases.The agarose gel electrophoresis of PCR amplifications
Picture is shown in Fig. 3, as a result shows that psychrophilic bacteria Colwellia psychrerythraea ura DNA glycosidases can effectively disappear
Except the DNA pollution thing containing dU bases.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, on the premise of the technology of the present invention principle is not departed from, some improvements and modifications can also be made, these improvements and modifications
Also should be regarded as protection scope of the present invention.
The amino acid sequence of ura DNA glycosidase
SEQ ID NO:1
1 MIKPQWQQLI AGEAKQSYFK NLVKEVATQR ASEISIYPAE GDVFNAFNHV DLAEIKVVIL
61 GQDPYHGKDQ AHGLAFSVQE GIKVPPSLVN IYKELATDVE GFEIPKHGCL THWAEQGVLL
121 LNTVLTVQQA NAHSHAKLGW ETFTEQAINA LNEHNDGCVF ILWGAHAHKK GKNINQEKHL
181 VLAGPHPSPL SAYRGFFGCK HFSQANAWLA KHEVNQVDWH LPLEA
SEQUENCE LISTING
<110>The outstanding Jun Chi bio tech ltd in Suzhou
<120>A kind of preparation method and applications of ura DNA glycosidase
<210> 1
<211> 225
<212> PRT
<213>
<400> 1
1 MIKPQWQQLI AGEAKQSYFK NLVKEVATQR ASEISIYPAE GDVFNAFNHV DLAEIKVVIL
61 GQDPYHGKDQ AHGLAFSVQE GIKVPPSLVN IYKELATDVE GFEIPKHGCL THWAEQGVLL
121 LNTVLTVQQA NAHSHAKLGW ETFTEQAINA LNEHNDGCVF ILWGAHAHKK GKNINQEKHL
181 VLAGPHPSPL SAYRGFFGCK HFSQANAWLA KHEVNQVDWH LPLEA
Claims (6)
1. a kind of preparation method of ura DNA glycosidase, it is characterised in that comprise the following steps that：
（1）Build the recombinant expression plasmid of ura DNA glycosidase
Based on cryophile genomic information, design synthesizes the ura DNA glycosidase gene of its coding, on this basis,
Optimize the rare codon of ura DNA glycosidase gene, and the ura DNA glycosidase gene cloning that will optimize to protokaryon table
Up to carrier pET28 etc., ura DNA glycosidase recombinant expression plasmid is built；
（2）Recombination expression ura DNA glycosidase
The ura DNA glycosidase recombinant expression plasmid conversion Bacillus coli expression host BL21 that will be built（DE3）, obtain urine phonetic
Pyridine DNA glycosidase recombinant strains；It is carried out heat sensitivity ura DNA glycosidase induction table with derivant IPTG again
Reach；
（3）The ura DNA glycosidase of affinity purification expression
The Escherichia coli after induced expression ura DNA glycosidase are collected by centrifugation, thalline are resuspended in non-denatured protein lysates,
Ultrasonic disruption thalline, is collected by centrifugation Escherichia coli cracking supernatant；Immobilization nickel ion affinity purification resin is recycled from upper
Heat sensitivity ura DNA glycosidase is purified in clear liquid；
（4）The enzymatic activity and heat endurance of ura DNA glycosidase are detected, the excellent ura DNA glucosides of heat sensitivity is obtained
Enzyme
The ura DNA glycosidase of purifying is carried out into enzymatic activity and heat stability test, its enzymatic activity and heat endurance is identified；Really
Protecting it can be under middle low temperature with the glycosidic bond in hydrolyzing DNA between uracil base and deoxyribose, while when temperature is high
Its fast deactivation when 60 degree.
2. preparation method according to claim 1, it is characterised in that：Step（1）In, the cryophile includes
Colwellia psychrerythraea etc..
3. preparation method according to claim 1, it is characterised in that：Step（2）It is middle to carry out induction training with derivant IPTG
Foster actual conditions is：First by temperature sensitive ura DNA glycosidase recombinant strains culture to OD600=0.4-1.0, then
Add 0.5mM derivants IPTG to be cultivated at a temperature of culture 3 hours or 10-25 DEG C at a temperature of 25-37 DEG C to be induced for 12 hours
Expression.
4. the temperature sensitive ura DNA glycosidase that the preparation method as described in one of claims 1 to 3 is obtained.
5. DNA pollution scavenger as claimed in claim 4, it is characterised in that it is temperature sensitive ura DNA glycosidase,
Wherein：The amino acid sequence of ura DNA glycosidase such as SEQ ID NO:Shown in 1.
6. the temperature sensitive ura DNA glycosidase that the preparation method as described in one of claims 1 to 3 is obtained is in PCR-based
Deng detection of nucleic acids in application, prevent/remove DNA pollution caused by last nuclease assay reaction.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828758A (en) * | 2017-11-13 | 2018-03-23 | 江苏众红生物工程创药研究院有限公司 | Recombinate ura DNA glycosidase and its encoding gene, preparation method and application |
| CN108048428A (en) * | 2017-12-25 | 2018-05-18 | 苏州旷世骏弛生物科技有限公司 | A kind of preparation method of low temperature ura DNA glycosidase |
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|---|---|---|---|---|
| US20080299609A1 (en) * | 2007-03-12 | 2008-12-04 | Sungkyunkwan University Foundation For Corporate Collaboration | Uracil-DNA glycosylase of psychrobacter sp. HJ147 and use thereof |
| CN105176946A (en) * | 2015-09-16 | 2015-12-23 | 苏州旷世骏弛生物科技有限公司 | Uracil DNA (deoxyribonucleic acid) glycosidase, and preparation method and application thereof |
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2016
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20080299609A1 (en) * | 2007-03-12 | 2008-12-04 | Sungkyunkwan University Foundation For Corporate Collaboration | Uracil-DNA glycosylase of psychrobacter sp. HJ147 and use thereof |
| CN105176946A (en) * | 2015-09-16 | 2015-12-23 | 苏州旷世骏弛生物科技有限公司 | Uracil DNA (deoxyribonucleic acid) glycosidase, and preparation method and application thereof |
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| Title |
|---|
| GUN A KIM等: "Properties of cold-active uracil-DNA glycosylase from Photobacterium aplysiae GMD509, and its PCR application for carryover contamination control", 《ENZYME AND MICROBIAL TECHNOLOGY》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828758A (en) * | 2017-11-13 | 2018-03-23 | 江苏众红生物工程创药研究院有限公司 | Recombinate ura DNA glycosidase and its encoding gene, preparation method and application |
| CN108048428A (en) * | 2017-12-25 | 2018-05-18 | 苏州旷世骏弛生物科技有限公司 | A kind of preparation method of low temperature ura DNA glycosidase |
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