CN105176946A - Uracil DNA (deoxyribonucleic acid) glycosidase, and preparation method and application thereof - Google Patents
Uracil DNA (deoxyribonucleic acid) glycosidase, and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of gene engineering, and particularly relates to a uracil DNA (deoxyribonucleic acid) glycosidase, and a preparation method and application thereof. The amino acid sequence of the uracil DNA glycosidase provided by the invention is disclosed as SEQ ID NO:3 or SEQ ID NO:4. The invention has the advantages that the technical universality is favorable and the uracil base in the DNA of the uracil DNA glycosidase can be ablated to relieve the inhibiting action on the B-type DNA polymerases and improve PCR (polymerase chain reaction) of all the B-type DNA polymerases, thereby enhancing the PCR amplification yield by 2-6 times and increasing the effective amplification length of the DNA segment by 2-3 times. The reaction system has favorable compatibility; and the uracil DNA glycosidase and DNA polymerase are thermally-stable proteins and have the same heat stability, and thus maximally displaying the activities of the two enzymes, thereby achieving the optimal PCR amplification effect.
Description
Technical field
The invention belongs to gene engineering technology field, particularly relate to a kind of ura DNA glycosidase, preparation method and application thereof.
Background technology
Progress of Nucleic Acid Amplification Technologies, as the foundation stone of molecular biology and genetic engineering technique, facilitates the development of present life science greatly.PCR is as a kind of high-efficiency nucleic acid amplification technique the most common, it occurs making the specific DNA molecular of external quick preparation become possibility, be widely used in various nucleic acid amplification and detection field at present, greatly facilitate the development of nucleic acid diagnostic techniques, modern genetic engineering, recombinant protein engineering.In order to improve PCR performance further, have now been developed various new pcr amplification technology.Wherein PCR reaction system optimization, such as by adding dimethyl sulfoxide (DMSO) (DMSO), optimizing magnesium ion concentration etc., PCR performance can be improved to a certain extent, but also there is the problem such as versatility, poor repeatability, after particularly changing goal gene fragment, usually need again to optimize these PCR reaction conditionss.In addition, warm start archaeal dna polymerase, by suppressing the enzymic activity under normal temperature, weakens the by products such as non-specific amplification product and primer dimer, increases target DNA amplification output.
In PCR process, high temperature can make deoxy cytidine triphosphate (dCTP) hydrolytic deaminization in 4 kinds of deoxynucleoside triphosphates, generate deoxyuridine triphosphoric acid (dUTP), it is mixed after in the DNA molecular of new synthesis by archaeal dna polymerase exists with uridylic base form, uridylic base seriously suppresses B-form DNA polymerase activity, the effective amplification length of the final DNA of reduction and the output that increases.In view of the B-form DNA polysaccharase of various high fidelity widely uses in PCR, therefore must address this problem, thus improve the amplification capability of B archaeal dna polymerase.
Summary of the invention
In order to overcome the problem that B-form DNA polymerase activity seriously suppresses by uridylic base, the object of the present invention is to provide a kind of ura DNA glycosidase, preparation method and application thereof; Ura DNA glycosidase of the present invention makes it be widely used in the pcr amplification example of various B-form DNA polysaccharase.This pcr amplification technology has the features such as versatility is wide, high comprehensive performance; The PCR output of multiple commercialization archaeal dna polymerase can not only be significantly improved, the effective amplification length of pcr amplified fragment can also be increased.
The improvement principle of the present invention to the pcr amplification technology of various B-form DNA polysaccharase is: ura DNA glycosidase can by the uridylic base in DNA molecular from ribodesose hydrolysis excision, thus uridylic, to the restraining effect of archaeal dna polymerase, improves PCR output and the effective amplification length of DNA in elimination DNA.
Technical scheme of the present invention is specific as follows.The invention provides a kind of ura DNA glycosidase, it is the ura DNA glycosidase of thermophilic microorganism; Described thermophilic microorganism is fireball bacterium (Pyrococcusfuriosus) or Aeropyrum pernix (Aeropyrumpernix); Wherein: the aminoacid sequence of the ura DNA glycosidase of fireball bacterium is as shown in SEQIDNO:3; The aminoacid sequence of the ura DNA glycosidase of Aeropyrum pernix is as shown in SEQIDNO:4.
The present invention also provides a kind of gene of above-mentioned ura DNA glycosidase of encoding, and the gene of the ura DNA glycosidase of coding fireball bacterium is as shown in SEQIDNO:1; The gene of the ura DNA glycosidase of coding Aeropyrum pernix is as shown in SEQIDNO:2.
The present invention further provides a kind of preparation method of ura DNA glycosidase, concrete steps are as follows:
(1) recombinant expression plasmid of ura DNA glycosidase is built
Based on the ura DNA glycosidase gene order of thermophilic microorganism coding, design primer, amplification gene, and by gene clone to prokaryotic expression carrier pET28, build ura DNA glycosidase recombinant expression plasmid;
(2) recombinant expressed ura DNA glycosidase
By ura DNA glycosidase recombinant expression plasmid transformation of E. coli expressive host BL21 (DE3) built, obtain ura DNA glycosidase recombinant strains; Inductor IPTG is used to carry out bacterium ura DNA glycosidase abduction delivering again;
(3) ura DNA glycosidase of affinity purification expression
Intestinal bacteria after collected by centrifugation abduction delivering ura DNA glycosidase, thalline is resuspended in non-denatured protein lysate, ultrasonic disruption thalline, heated and inactivated intestinal bacteria oneself protein, collected by centrifugation contains the intestinal bacteria cracking supernatant liquor of ura DNA glycosidase; Recycling immobilization nickel ion affinity purification resin difference purifying ura DNA glycosidase from supernatant liquor;
(4) enzymic activity and the thermostability of ura DNA glycosidase is detected.
Ura DNA glycosidase after purifying is carried out enzymic activity and heat stability test, and the enzymic activity transformation period therefrom filtering out specific excision uridylic base at 95 DEG C is not less than the ura DNA glycosidase of 30 minutes.
In above-mentioned steps (1), described thermophilic microorganism is fireball bacterium or Aeropyrum pernix.
In above-mentioned steps (2) with the actual conditions that inductor IPTG carries out inducing culture be: first ura DNA glycosidase recombinant strains is cultured to OD
600=0.4-1.0, then add 0.5mM inductor IPTG cultivate at 37 DEG C of temperature 3 hours or at 22 DEG C of temperature cultivate within 12 hours, carry out abduction delivering.
In above-mentioned steps (3), described non-denatured protein lysate composed as follows: 20mMpH value is the Tris-HCl of 8.0,300mMNaCl, 0.5mMDTT, 10vol% glycerine.
The present invention also provides the above-mentioned application of ura DNA glycosidase in the pcr amplification technology of B-form DNA polysaccharase further; Preferably, described B-form DNA polysaccharase is PfuDNA polysaccharase, KODDNA polysaccharase or VentDNA polysaccharase.
Concrete grammar during above-mentioned application is as follows: in the PCR reaction system that B-form DNA is polymerase catalysed, add ura DNA glycosidase, amplification target gene.Utilize 1% sepharose to detect pcr amplification result, measure ura DNA glycosidase to the improved effect of PCR.
Compared with prior art, beneficial effect of the present invention is:
(1) versatility is good, because ura DNA glycosidase is for the suppression defect of uridylic base pair DNA polymerase activity in DNA, carries out improvement design; Therefore, it is possible to significantly improve the pcr amplification performance of all B-form DNA polysaccharases, be not limited to a certain archaeal dna polymerase, significantly can improve pcr amplification output and the effective amplification length of DNA of multiple commercialization B-form DNA polysaccharase.
(2) ura DNA glycosidase can not only increase pcr amplification output, can also increase the effective amplification length of DNA, be specially adapted to the pcr amplification of DNA long fragment.
Accompanying drawing explanation
Fig. 1 is the enzyme activity determination figure of fireball bacterium ura DNA glycosidase.
Fig. 2 is the thermostability figure of fireball bacterium ura DNA glycosidase.
Fig. 3 is that fireball bacterium ura DNA glycosidase illustrates the promoter action of the polymerase catalysed PCR of PfuDNA.
Fig. 4 is that fireball bacterium ura DNA glycosidase illustrates the promoter action of the polymerase catalysed PCR of KODDNA.
Fig. 5 is that Aeropyrum pernix ura DNA glycosidase illustrates the promoter action of the polymerase catalysed PCR of VentDNA.
Embodiment
By the following examples technical scheme of the present invention is described in further detail.Following examples do not form limitation of the invention.
The preparation of embodiment 1 fireball bacterium ura DNA glycosidase
The first step, design and synthesis fireball bacterium ura DNA glycosidase genes, and insert pET28 expression vector, build the recombinant expression plasmid of ura DNA glycosidase.Restructuring fireball bacterium ura DNA glycosidase N end band has 6 the continuous Histidine affinity purification labels deriving from pET28 carrier, for immobilization nickel ion affinity chromatograph purifying.
The gene order of fireball bacterium ura DNA glycosidase is as shown in SEQIDNO:1.
Second step, recombinant expressed fireball bacterium ura DNA glycosidase.By fireball bacterium ura DNA glycosidase recombinant expression plasmid transformation of E. coli expressive host BL21 (DE3), obtain fireball bacterium ura DNA glycosidase recombinant strains.Expression strain is cultured to OD
600=0.6, add 0.5mM inductor IPTG, cultivate 3 hours at 37 DEG C of temperature, induction ura DNA glycosidase is expressed.
The aminoacid sequence (nitrogen end → carbon teminal) of fireball bacterium ura DNA glycosidase recombinant protein is as shown in SEQIDNO:3.
3rd step, fireball bacterium ura DNA glycosidase affinity purification.After intestinal bacteria collected by centrifugation after step 2 being induced, thalline Eddy diffusion is in protein lysate (20mMTris-HCl, pH8.0,300mMNaCl, 0.5mMDTT, 10vol% glycerine).Ultrasonic disruption thalline, heated and inactivated e. coli protein at 70 DEG C of temperature, collected by centrifugation contains the cellular lysate supernatant liquor of fireball bacterium ura DNA glycosidase.Utilize immobilization nickel ion affinity purification resin purification fireball bacterium ura DNA glycosidase.
4th step, detects enzymic activity and the thermostability of fireball bacterium ura DNA glycosidase.Utilize the oligonucleotide containing uridylic base as substrate, measure the activity of glycosidic link between fireball bacterium ura DNA glycosidase hydrolysis uridylic and ribodesose.Concrete Activity Results is shown in Fig. 1.Result shows that fireball bacterium ura DNA glycosidase has the activity of glycosidic link between effectively hydrolyzing uridylic and ribodesose, and the ura DNA glycosidase of 8ng just can remove the uridylic base on oligonucleotide completely in 5 minutes.
On this basis, the thermostability of fireball bacterium ura DNA glycosidase is detected.Fireball bacterium ura DNA glycosidase is incubated 30 minutes in differing temps, then measures its remaining enzymic activity size.The thermal stability results of fireball bacterium ura DNA glycosidase recombinant protein is shown in Fig. 2, and result shows that it is 65% at the remaining activity of 95 DEG C of insulations after 30 minutes, can remove the uridylic base in DNA in PCR.
Embodiment 2 is based on the High Efficiency PC R amplification technique of fireball bacterium ura DNA glycosidase and PfuDNA polysaccharase
Utilize the fireball bacterium ura DNA glycosidase of purifying to improve the polymerase catalysed PCR reaction of PfuDNA, set up the High Efficiency PC R amplification technique of PfuDNA polysaccharase.Amplifying target genes is Escherichia coli nucleic acid enzyme IV gene, PCR reaction buffer: 20mMTris-HCl (pH8.8), 10mM (NH
4)
2sO
4, 10mMKCl, 0.1mg/mLBSA, 0.1% (v/v) TritonX-100,2mMMgSO
4; Other component is 0.2mMdNTP, 0.3 μM of primer, 20ng genome of E.coli DNA, 2.5 units Pfu DNA polymerase, 0,50,200ng ura DNA glycosidase.Fig. 3 is shown in by the agarose gel electrophoresis picture of pcr amplification result, and result shows that fireball bacterium ura DNA glycosidase makes pcr amplification output increase 3-4 doubly.
Embodiment 3 is based on the High Efficiency PC R amplification technique of fireball bacterium ura DNA glycosidase and KODDNA polysaccharase
Utilize the fireball bacterium ura DNA glycosidase of purifying to improve the polymerase catalysed PCR reaction of KODDNA, set up the High Efficiency PC R amplification technique of KODDNA polysaccharase.Amplifying target genes is intestinal bacteria exonuclease I II gene, PCR reaction buffer: 20mMTris-HCl (pH8.8), 10mM (NH
4)
2sO
4, 10mMKCl, 0.1mg/mLBSA, 0.1% (v/v) TritonX-100,2mMMgSO
4; Other component is 0.2mMdNTP, 0.3 μM of primer, 20ng genome of E.coli DNA, 2.5 unit K ODDNA polysaccharases, 200ng ura DNA glycosidase.Fig. 4 is shown in by the agarose gel electrophoresis picture of pcr amplification result, and result shows that fireball bacterium ura DNA glycosidase makes pcr amplification output increase 5-6 doubly.
Embodiment 4 is based on the High Efficiency PC R amplification technique of Aeropyrum pernix ura DNA glycosidase and KODDNA polysaccharase
According to the technique of embodiment 1, prepare Aeropyrum pernix ura DNA glycosidase, as shown in SEQIDNO:4, the gene order of this zymoprotein of encoding is as shown in SEQIDNO:2 for this protein amino acid sequence (nitrogen end → carbon teminal).Utilize the Aeropyrum pernix ura DNA glycosidase of purifying to improve the polymerase catalysed PCR reaction of VentDNA, set up the High Efficiency PC R amplification technique of VentDNA polysaccharase.Amplifying target genes selects Long fragment gene---intestinal bacteria ung gene, PCR reaction buffer: 20mMTris-HCl (pH8.8), 10mM (NH
4)
2sO
4, 10mMKCl, 0.1mg/mLBSA, 0.1% (v/v) TritonX-100,2mMMgSO
4; Other component is 0.2mMdNTP, 0.3 μM of primer, 20ng genome of E.coli DNA, 2.5 units Vent DNA polymerase, 200ng ura DNA glycosidase.Fig. 5 is shown in by the agarose gel electrophoresis picture of pcr amplification result, and result shows that Aeropyrum pernix ura DNA glycosidase makes length be that the pcr amplification output of the intestinal bacteria ung gene of 0.6kb about increases 2-3 doubly.
Claims (8)
1. a ura DNA glycosidase, is characterized in that, it is the ura DNA glycosidase of thermophilic microorganism; Described thermophilic microorganism is fireball bacterium or Aeropyrum pernix; Wherein: the aminoacid sequence of the ura DNA glycosidase of fireball bacterium is as shown in SEQIDNO:3; The aminoacid sequence of the ura DNA glycosidase of Aeropyrum pernix is as shown in SEQIDNO:4.
2. encode the gene of ura DNA glycosidase described in claim 1, it is characterized in that, the gene of the ura DNA glycosidase of coding fireball bacterium is as shown in SEQIDNO:1.
3. encode the gene of ura DNA glycosidase described in claim 1, it is characterized in that, the gene of the ura DNA glycosidase of coding Aeropyrum pernix is as shown in SEQIDNO:2.
4. a preparation method for ura DNA glycosidase, is characterized in that, concrete steps are as follows:
(1) recombinant expression plasmid of ura DNA glycosidase is built
Based on the ura DNA glycosidase gene order of thermophilic microorganism coding, design primer, amplification gene, and by gene clone to prokaryotic expression carrier pET28, build ura DNA glycosidase recombinant expression plasmid;
(2) recombinant expressed ura DNA glycosidase
By ura DNA glycosidase recombinant expression plasmid transformation of E. coli expressive host BL21 (DE3) built, obtain ura DNA glycosidase recombinant strains; Inductor IPTG is used to carry out bacterium ura DNA glycosidase abduction delivering again;
(3) ura DNA glycosidase of affinity purification expression
Intestinal bacteria after collected by centrifugation abduction delivering ura DNA glycosidase, thalline is resuspended in non-denatured protein lysate, ultrasonic disruption thalline, heated and inactivated intestinal bacteria oneself protein, collected by centrifugation contains the intestinal bacteria cracking supernatant liquor of ura DNA glycosidase; Recycling immobilization nickel ion affinity purification resin difference purifying ura DNA glycosidase from supernatant liquor;
(4) enzymic activity and the thermostability of ura DNA glycosidase is detected.
Ura DNA glycosidase after purifying is carried out enzymic activity and heat stability test, and the enzymic activity transformation period therefrom filtering out specific excision uridylic base at 95 DEG C is not less than the ura DNA glycosidase of 30 minutes.
5. preparation method according to claim 4, is characterized in that: in step (1), and described thermophilic microorganism is fireball bacterium or Aeropyrum pernix.
6. preparation method according to claim 4, is characterized in that: in step (2) with the actual conditions that inductor IPTG carries out inducing culture be: first ura DNA glycosidase recombinant strains is cultured to OD
600=0.4-1.0, then add 0.5mmol/L inductor IPTG cultivate at 37 DEG C of temperature 3 hours or at 22 DEG C of temperature cultivate within 12 hours, carry out abduction delivering.
7. the application of ura DNA glycosidase according to claim 1 in the pcr amplification technology of B-form DNA polysaccharase.
8. application according to claim 7, is characterized in that, described B-form DNA polysaccharase is PfuDNA polysaccharase, KODDNA polysaccharase or VentDNA polysaccharase.
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| CN106636161A (en) * | 2016-12-28 | 2017-05-10 | 苏州旷世骏弛生物科技有限公司 | Preparation method of low-temperature protease |
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| CN106754823A (en) * | 2016-12-28 | 2017-05-31 | 苏州旷世骏弛生物科技有限公司 | A kind of preparation method and applications of ura DNA glycosidase |
| 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 |
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| 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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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107868774A (en) * | 2016-09-23 | 2018-04-03 | 上海紫众生物科技有限公司 | A kind of thermal starting archaeal dna polymerase, preparation method and applications |
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Application publication date: 20151223 |