CN109022386A - Recombinate Taq direct expansion enzyme and preparation method thereof, recombinant plasmid and engineering bacteria - Google Patents
Recombinate Taq direct expansion enzyme and preparation method thereof, recombinant plasmid and engineering bacteria Download PDFInfo
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Abstract
The invention belongs to technical field of bioengineering, and in particular to a kind of recombination Taq direct expansion enzyme and preparation method thereof, recombinant plasmid and engineering bacteria.The gene order of the recombination Taq direct expansion enzyme is as shown in SEQ ID NO.1.The improved recombination Taq direct expansion enzyme strengthens anti-interference ability, the effect of various inhibiting factors in reaction system can be especially resisted, to improve amplification efficiency.Therefore, recombination Taq direct expansion enzyme of the invention can less nucleic acid purification step or be not necessarily to nucleic acid purification step under the conditions of, can directly carry out the amplification of template complex, Direct PCR reaction can be carried out.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of recombination Taq direct expansion enzyme and preparation method thereof.
Background technique
Early in 1969, people isolated a kind of thermophilic aquatic true from the Volcanic Thermal Spring of U.S. Huangshi National forest park
Bacterium Thermus aquaticus, the bacterium can grow at 70~75 DEG C, isolate and purify to obtain a kind of heat-resisting, dependence DNA from the bacterium
Archaeal dna polymerase, abbreviation Taq archaeal dna polymerase.Then, it has been found that various other hot resistant DNA polymerases, these enzymes are usual
The multiple nucleic acid amplification (PCR) being used in scientific experiment.
Currently, the hot resistant DNA polymerase of application is broadly divided into plain edition and high-fidelity type.
Plain edition:
1.Taq archaeal dna polymerase is separated and extracted by a kind of thermus aquaticus yT1 plants, is the hot resistant DNA polymerase of discovery
Middle highest one kind of activity, up to 200,000 unit/mg.With 5'-3' 5 prime excision enzyme activity, but do not have the circumscribed enzyme activity of 3'-5'
Property, thus there is no calibration function to certain single nucleotide mismatch in synthesis.Taq DNA polymerase will also have non-template dependence
Property activity, can by each chain 3' of PCR double-stranded products be added mononucleotide tail, therefore can make PCR product have 3' list A outstanding
Nucleotide tail；On the other hand, in the presence of only dTTP, list T nucleotide tail can be added in the end 3' of the plasmid of flush end by it, generated
The end 3' list T nucleotide tail outstanding.Using this characteristic, it can be achieved that the T-A PCR cloning PCR of PCR product.
2.Tth archaeal dna polymerase is extracted from Thermus thermophilus HB8 and is obtained, change enzyme in high temperature and
MnCl2Under the conditions of, it can effectively reverse transcription RNA；As addition Mg2+Afterwards, the polymerization activity of the enzyme greatly increases, so that cDNA be made to close
A kind of enzymatic can be used at amplification.
High-fidelity type:
1.pfu archaeal dna polymerase is that refined high-fidelity high temperature-resisting DNA is poly- from Pyrococcus furiosis
Synthase, it does not have 5'-3' 5 prime excision enzyme activity, but has 3'-5' 5 prime excision enzyme activity, generates during recoverable PCR amplification
Mistake keeps the base mispairing rate of product extremely low.PCR product is flush end, the no end 3' list A nucleotide outstanding.
2.Vent archaeal dna polymerase, the enzyme are to dwell on isolating in hot-bulb bacterium from Litoralis, and it is circumscribed not have 5'-3'
Enzymatic activity, but there is 3'-5' 5 prime excision enzyme activity, the base of mispairing can be removed, there is proofreading function.
But the type of existing hot resistant DNA polymerase is limited.
Summary of the invention
It is an object of the invention to overcome the above-mentioned deficiency of the prior art, a kind of recombination Taq direct expansion enzyme and its preparation are provided
Method, it is intended to solve the limited technical problem of the selection of existing hot resistant DNA polymerase.
For achieving the above object, The technical solution adopted by the invention is as follows:
One aspect of the present invention provides a kind of recombination Taq direct expansion enzyme, the gene order such as SEQ ID of the recombination Taq direct expansion enzyme
Shown in NO.1.
Another aspect of the present invention provides a kind of preparation method of above-mentioned recombination Taq direct expansion enzyme, includes the following steps:
Construct the recombinant plasmid containing sequence shown in SEQ ID NO.1；
The recombinant plasmid is transferred in Escherichia coli, the engineering bacteria for expressing the recombination Taq direct expansion enzyme is obtained；
After the engineering bacteria is carried out inducing expression, purifying is extracted, the recombination Taq direct expansion enzyme is obtained.
The present invention also provides a kind of recombinant plasmid, the recombinant plasmid contains the sequence as shown in SEQ ID NO.1.
The last present invention provides a kind of engineering bacteria, and the engineering bacteria contains above-mentioned recombinant plasmid of the invention.
Recombination Taq direct expansion enzyme provided by the invention carries out gene work on the basis of the DNA sequence dna of existing general T aq enzyme
Journey is transformed to obtain: specifically, becoming CTG from GGG at 1824-1827bp, the amino acid of coding becomes Asp from Pro；?
GAG is become from TTC at 1875-1878bp, the amino acid of coding becomes Leu from Lys；Become at 2118-2121bp from GAT
The amino acid of GAG, coding are constant, but improve the expression efficiency of recombination Taq direct expansion enzyme.The improved recombination Taq direct expansion
Enzyme strengthens anti-interference ability, can especially resist the effect of various inhibiting factors in reaction system, to improve amplification effect
Rate.Therefore, recombination Taq direct expansion enzyme of the invention can less nucleic acid purification step or be not necessarily to nucleic acid purification step under the conditions of,
The amplification that template complex can directly be carried out, can carry out Direct PCR reaction.In addition, compared to general T aq enzyme, recombination Taq
Direct expansion enzyme has higher amplification fidelity；Compared to other high fidelity enzymes, recombination Taq direct expansion enzyme can have relatively high again
Amplification efficiency.
The preparation method of above-mentioned recombination Taq direct expansion enzyme provided by the invention, it is simple for process, large scale equipment is not needed,
During the preparation method, corresponding expression vector is constructed by genetic engineering transformation, then carries out luring for recombination Taq direct expansion enzyme
Expression is led, then expression product is extracted and purified, the recombination Taq direct expansion enzyme of strong antijamming capability can be obtained.This hair
The recombination Taq direct expansion enzyme that bright preparation method obtains can be in less nucleic acid purification step or without nucleic acid purification step condition
Under, the amplification of template complex is directly carried out, Direct PCR reaction can be carried out.
Recombinant plasmid of the invention contains the gene order of recombination Taq direct expansion enzyme of the invention, and it is straight to can be used for recombination Taq
Expand the vector construction of enzyme.Engineering bacteria of the invention contains above-mentioned recombinant plasmid of the invention, can be used for expressing and extracting the recombination
Taq direct expansion enzyme.
Detailed description of the invention
Fig. 1 is the Activity determination agarose electrophoresis figure that Taq direct expansion enzyme is recombinated in the embodiment of the present invention 5；Wherein, from a left side in figure
Into 13 right tracks, 1-3 is 750bp segment, and 4-6 is 1000bp segment, and 7-9 is 2000bp segment, and 10-12 is
4000bp segment, 13 be DNA maker.
Specific embodiment
In order to which technical problems, technical solutions and advantageous effects to be solved by the present invention are more clearly understood, below in conjunction with
Embodiment, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used to explain
The present invention is not intended to limit the present invention.
On the one hand, the embodiment of the invention provides a kind of recombination Taq direct expansion enzyme, the gene sequences of the recombination Taq direct expansion enzyme
Column are as shown in SEQ ID NO.1.
Recombination Taq direct expansion enzyme provided in an embodiment of the present invention carries out on the basis of the DNA sequence dna of existing general T aq enzyme
Genetic engineering is transformed to obtain: specifically, becoming CTG from GGG at 1824-1827bp, the amino acid of coding becomes Asp from Pro；
GAG is become from TTC at 1875-1878bp, the amino acid of coding becomes Leu from Lys；Become at 2118-2121bp from GAT
The amino acid of GAG, coding are constant, but improve the expression efficiency of recombination Taq direct expansion enzyme.The improved recombination Taq direct expansion
Enzyme strengthens anti-interference ability, can especially resist the effect of various inhibiting factors in reaction system, to improve amplification effect
Rate.Therefore, the recombination Taq direct expansion enzyme of the embodiment of the present invention can be in less nucleic acid purification step or without nucleic acid purification step item
Under part, the amplification of template complex can be directly carried out, Direct PCR reaction can be carried out.In addition, this is heavy compared to general T aq enzyme
Group Taq direct expansion enzyme has higher amplification fidelity；Compared to other high fidelity enzymes, recombination Taq direct expansion enzyme can have again
Relatively high amplification efficiency.
On the other hand, the embodiment of the invention also provides a kind of preparation method of above-mentioned recombination Taq direct expansion enzyme, including it is as follows
Step:
S01: recombinant plasmid of the building containing sequence shown in SEQ ID NO.1；
S02: the recombinant plasmid is transferred in Escherichia coli, obtains the engineering bacteria for expressing the recombination Taq direct expansion enzyme；
S03: after the engineering bacteria is carried out inducing expression, purifying is extracted, the recombination Taq direct expansion enzyme is obtained.
The preparation method of above-mentioned recombination Taq direct expansion enzyme provided in an embodiment of the present invention, it is simple for process, do not need large size
Equipment during the preparation method, constructs corresponding expression vector by genetic engineering transformation, then carries out recombination Taq direct expansion
The inducing expression of enzyme is then extracted and is purified to expression product, and the recombination Taq direct expansion of strong antijamming capability can be obtained
Enzyme.The recombination Taq direct expansion enzyme that the preparation method of the embodiment of the present invention obtains can be in less nucleic acid purification step or without core
Under the conditions of sour purification step, the amplification of template complex is directly carried out, Direct PCR reaction can be carried out.
Further, in above-mentioned steps S01: the recombinant plasmid of the building containing sequence shown in SEQ ID NO.1
Step includes: the protection alkali that sequence both ends shown in SEQ ID NO.1 are added to restriction enzyme EcoR I and Not I respectively
Base is then attached on the pET-32A carrier through restriction enzyme EcoR I and Not I digestion, obtains the recombination matter
Grain.Preferably, the primer of sequence shown in SEQ ID NO.1 is expanded as shown in SEQ ID NO.2 and SEQ ID NO.3.Specifically,
The guarantor of restriction enzyme EcoR I and Not I are added at the recombination Taq direct expansion enzyme gene segment both ends after modifying of offer
Base is protected, segment is synthesized, then by segment needed for the primer amplification, is connected to by ligase through EcoR after segment recycling
On the pET-32A carrier of I and Not I digestion, the recombinant plasmid transformed after connection is entered into E.coli BL21 (DE3) expression and is carried
Body can be obtained the engineered strain of production recombination Taq direct expansion enzyme by plasmid screening.
Further, in above-mentioned steps S02: the step of engineering bacteria is carried out inducing expression includes:
S021: by engineering bacteria switching in the LB Liquid Culture containing the ampicillin that concentration is 50-100 μ g/ml
Base carries out activation culture；
S022: the culture solution after the activation culture is transferred to containing the ampicillin that concentration is 100-200 μ g/ml
LB liquid medium carries out shaken cultivation, makes final OD600For 0.4-1.0.
Preferably, the condition of the activation culture are as follows: 36-37 DEG C of temperature, revolving speed 200-250rpm, time 10-16h；Institute
State the condition of shaken cultivation are as follows: 36-37 DEG C of temperature, revolving speed 200-250rpm, time 12-22h.It is highly preferred that the concussion training
It include: in OD in supporting600When for 0.2-0.4, the IPTG (isopropylthiogalactoside) of final concentration of 0.4-0.8mM is added.
IPTG can preferably induce engineering bacterium expression to recombinate Taq direct expansion enzyme under this condition as a kind of extremely strong inducer.One
In specific embodiment, inducing expression process are as follows: take the positive monoclonal converted, switching is in ampicillin concentration
The 2-3ml LB liquid medium of 50-100 μ g/ml, 37 DEG C, 200rpm activation culture 10-16h；Then 50-100 μ l is therefrom taken
Culture medium is transferred to the 50-100ml LB liquid medium that ampicillin concentration is 100-200 μ g/ml, 37 DEG C, 200rpm oscillation
2-6h is cultivated, OD is made600About 0.2-0.4 or so is added IPTG (50-100mM), makes the final concentration of 0.4-0.8mM of IPTG,
37 DEG C, 200rpm shaken cultivation 10-16h, make final OD600Reach 0.4-1.0 or so.
Further, in above-mentioned steps S03: the step of extraction purification includes: according to hot method of purification, using buffering
Liquid A, lysozyme, buffer solution B, DNase I, DTT (dithiothreitol (DTT)), buffer C neutralization buffer D, from the engineering bacteria into
The isolated recombination Taq direct expansion enzyme in culture solution after row inducing expression.
In a specific embodiment, hot the step of purifying, includes:
(1) inoculum after inducing expression is transferred to 50-100ml centrifuge tube, 4 DEG C, 7000rpm centrifugation 5-10min.
(2) remove supernatant, 1-2ml buffer solution A is added, 4-6mg lysozyme (4-6mg/ml) is added after being resuspended, room temperature is incubated
Educate 10-15min.
(3) 1-2ml buffer solution B is added, mixes, 70-90 DEG C of water-bath 50-60min.
(4) 4 DEG C, 12000rpm centrifugation 5-10min, remove precipitating.
(5) 2-5ul DNase I, 37 DEG C of incubations 1-2h, 70-90 DEG C of water-bath 20-30min are added in supernatant.
(6) 4 DEG C, 12000rpm centrifugation 5-10min, remove precipitating.
(7) DTT that 1-5 μ l concentration is 1M is added in 2-5ml buffer C, DTT final concentration is made to reach 0.4-0.5mM.
(8) it in supernatant that the 2-5ml pre-cooling buffer C for being added to DTT is added in step (6) and will mix.
(9) DTT that 1-5 μ l concentration is 1M is added in 5-8ml buffer D, DTT final concentration is made to reach 0.4-0.5mM.
(10) it in mixed liquor that the 5-8ml pre-cooling buffer D for being added to DTT is added in step (8) and will mix；Then
Mixed liquor is dispensed into 0.5-1.5ml centrifuge tube, -80 DEG C of preservations.
The formula of required reagent is as follows:
The buffer solution A are as follows: 50mM Tris-HCl, pH 7.9,50mM glucose, 1mM EDTA；(lysozyme then adds)
The buffer solution B are as follows: 10mM Tris-HCl, pH 7.9,50mM KCl, 1mM EDTA, 0.5%Tween-20,
0.5%nonidet P-40；
The buffer C are as follows: 50% glycerol, 50mM Tris-HCl, pH 8.0,100mM NaCl, 0.1mM EDTA, 1%
Triton X-100；(DTT then adds)
The buffer D are as follows: 75% glycerol, 50mM Tris-HCl, pH 8.0,100mM NaCl, 0.1mM EDTA, 1%
Triton X-100；(DTT then adds)
The embodiment of the present invention also provides a kind of recombinant plasmid, and the recombinant plasmid contains the sequence as shown in SEQ ID NO.1.
The last embodiment of the present invention provides a kind of engineering bacteria, and the engineering bacteria contains the above-mentioned recombination matter of the embodiment of the present invention
Grain.
The recombinant plasmid of the embodiment of the present invention contains the gene order of the recombination Taq direct expansion enzyme of the embodiment of the present invention, can use
In the vector construction of recombination Taq direct expansion enzyme.The engineering bacteria of the embodiment of the present invention contains the above-mentioned recombination matter of the embodiment of the present invention
Grain, can be used for expressing and extracting recombination Taq direct expansion enzyme.
The present invention successively carried out test of many times, and it is further detailed as reference pair invention progress now to lift A partial experiment result
Thin description, is described in detail combined with specific embodiments below.
Embodiment 1
Recombinate the synthesis of Taq direct expansion enzyme fragment
Restriction enzyme is added at the modification Taq enzyme genetic fragment both ends (shown in following SEQ ID NO.1) of offer
The protection base of EcoR I and Not I send Synbiotics AB's synthesis recombination Taq direct expansion enzyme sequence segment.
SEQ ID NO.1:
Embodiment 2
Recombinate the building of Taq direct expansion expression of enzymes carrier
Design segment needed for suitable primer (shown in following SEQ ID NO.2 and SEQ ID NO.3) is expanded, segment recycling
It is connected on the pET-32A carrier through EcoR I and Not I digestion later, connection plasmid is transformed into E.coli BL21
(DE3) expression vector can be obtained the engineered strain of production recombination Taq direct expansion enzyme by plasmid screening.
SEQ ID NO.2:5'-CCGGAATTCCGGATGAGGGGGATGCTGCCCCT-3 '；
SEQ ID NO.3:5'-GGCTCTCCGCCAAGGAGTGATTGCGGCCGCAA-3 '.
Embodiment 3
Recombinate the inducing expression of Taq direct expansion enzyme gene
The positive monoclonal converted is taken, transfers and is trained in the 1ml LB liquid that ampicillin concentration is 100 μ g/ml
Support base, 37 DEG C, 200rpm activation culture 12h.Then taking 100 μ l culture mediums to be transferred to ampicillin concentration is 200 μ g/ml's
50ml LB liquid medium, 37 DEG C, 200rpm shaken cultivation 4h, makes OD600About 0.3 or so, it is added IPTG (100mM),
Make the final concentration of 0.6mM of IPTG, 37 DEG C, 200rpm shaken cultivation 16h make final OD600Reach 0.6 or so.
Embodiment 4
Recombinate the extraction and purification of Taq direct expansion enzyme
(1) inoculum after inducing expression is transferred to 50ml centrifuge tube, 4 DEG C, 7000rpm centrifugation 10min.
(2) remove supernatant, 1.5ml buffer solution A is added, 6mg lysozyme (4mg/ml) is added after being resuspended, incubation at room temperature
15min。
(3) 1.5ml buffer solution B is added, mixes, 75 DEG C of water-bath 60min.
(4) 4 DEG C, 12000rpm centrifugation 10min, remove precipitating.
(5) 5ul DNase I, 37 DEG C of incubations 2h, 75 DEG C of water-bath 30min are added in supernatant.
(6) 4 DEG C, 12000rpm centrifugation 10min, remove precipitating.
(7) DTT that 1.5 μ l concentration are 1M is added in 3ml buffer C, DTT final concentration is made to reach 0.5mM.
(8) it in supernatant that the 3ml pre-cooling buffer C for being added to DTT is added in step (6) and will mix.
(9) DTT that 3 μ l concentration are 1M is added in 6ml buffer D, DTT final concentration is made to reach 0.5mM.
(10) it in mixed liquor that the 6ml pre-cooling buffer D for being added to DTT is added in step (8) and will mix.It will mixing
Liquid is dispensed into 1.5ml centrifuge tube, -80 DEG C of preservations.
Embodiment 5
Recombinate the Activity determination of Taq direct expansion enzyme
Suitable primer and sample are selected, using common commercialization Taq enzyme as control, carries out PCR reaction, detection recombination
The enzymatic activity of Taq direct expansion enzyme.Detailed process is as follows:
1. sample is centrifuged 5min in 1500rpm, supernatant is abandoned；
2. the distilled water of 9 times of precipitating quality and 10 × Buffer B of 1 times of precipitating quality are added into precipitating, piping and druming is mixed
It is even, until precipitating is completely dissolved, and (if not being completely dissolved, distilled water and lysate can be properly added without block objects.
3. liquid is thick at this time, distilled water is added and dilutes 10-20 times, can be used as the template of PCR reaction.
4. carrying out PCR amplification according to following PCR system.
PCR system:
10 × buffer (500mM KCl, 100mM Tris-HCl pH8.3,15mM MgCl2): 2 μ l
Dntp (2.5mM): 1.6 μ l
Template: 1 μ l
Primer (10mM): 0.5 × 2 μ l
Recombinate Taq direct expansion enzyme: 0.5 μ l
Distilled water: 13.9 μ l.
PCR response procedures:
(1) 94 DEG C: 3min；
(2) 94 DEG C: 30s；45-65 DEG C (being arranged according to primer): 30s；72 DEG C: 1-2kb/min；35 circulations；
(3) 72 DEG C: 10min
(4) 4 DEG C: ∞.
Product after reaction is subjected to agarose electrophoresis figure, testing result is as shown in Figure 1.In Fig. 1, the amplification of 1-3 swimming lane
Sample be human genome, 4-6 swimming lane amplified sample be the amplified sample people's cell of the preliminary lysate of human blood, 7-12 swimming lane at the beginning of
Walk lysate；
In 1-3, amplification obtains 750bp segment, the primer sequence of corresponding amplification are as follows:
SEQ ID No.4:5'-CTGTCATGCCATGAACCCAC-3 '；
SEQ ID No.5:5'-CTTTTACTTGGCTTTTAGGAA-3 '.
In 4-6, amplification obtains 1000bp segment, the primer sequence of corresponding amplification are as follows:
SEQ ID No.6:5'-AGTCCCCGGTGGATATCCGC-3 '；
SEQ ID No.7:5'-CATGATTAGAGGAGGCCAGT-3 '.
In 7-9, amplification obtains 2000bp segment, the primer sequence of corresponding amplification are as follows:
SEQ ID No.8:5'-AGTCCCCGGTGGATATCCGC-3 '；
SEQ ID No.9:5'-AGGAGGGCCCGGAAGAAAAC-3 '.
In 10-12, amplification obtains 4000bp segment, the primer sequence of corresponding amplification are as follows:
SEQ ID No.10:5'-ACCTGTGAGACTTTGGCTCCA-3 '；
SEQ ID No.11:5'-AGGAGGGCCCGGAAGAAAACA-3 '.
Known to the result of the present embodiment: recombination Taq direct expansion enzyme can less nucleic acid purification step or without nucleic acid it is pure
Under the conditions of changing step, the amplification of template complex is directly carried out, and recombination Taq direct expansion enzyme strengthens anti-interference ability, especially
The effect of various inhibiting factors in reaction system can be resisted, to improve amplification efficiency.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>Shenzhen Central China biology medical instruments Co., Ltd
Wuhan University
<120>Taq direct expansion enzyme and preparation method thereof, recombinant plasmid and engineering bacteria are recombinated
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2500
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atgaggggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac 60
cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg gggggagccg 120
gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac 180
gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg 240
tacaaggcgg gccgggcccc cacgccggag gactttcccc ggcaactcgc cctcatcaag 300
gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac 360
gtcctggcca gcctggccaa gaaggcggaa aaggagggct acgaggtccg catcctcacc 420
gccgacaaag acctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgagggg 480
tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc 540
gactaccggg ccctgaccgg ggacgagtcc gacaaccttc ccggggtcaa gggcatcggg 600
gagaagacgg cgaggaagct tctggaggag tgggggagcc tggaagccct cctcaagaac 660
ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag 720
ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa 780
aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc 840
ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc 900
ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat 960
cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa 1020
gccctcaggg acctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc 1080
ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg 1140
gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag 1200
gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt 1260
gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc 1320
ctggcccaca tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc 1380
ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct ggccggccac 1440
cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt 1500
cccgccatcg gcaagacgga gaagaccggc aagcgctcca ccagcgccgc cgtcctggag 1560
gccctccgcg aggcccaccc catcgtggag aagatcctgc agtaccggga gctcaccaag 1620
ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc 1680
cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc cgatcccaac 1740
ctccagaaca tccccgtccg caccccgctt gggcagagga tccgccgggc cttcatcgcc 1800
gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc 1860
cacctctccg gcgacgagaa cctgatccgg gtcttccagg aggggcggga catccacacg 1920
gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgccgg 1980
gcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccg cctctcccag 2040
gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc 2100
cccaaggtgc gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg 2160
gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt gaagagcgtg 2220
cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctc 2280
atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc 2340
cttcaggtcc acgacgagct ggggcccagg ccttcattga gcgctaagag agggcggagg 2400
ccgtggcccg gctggccaag gaggtcatgg agggggtgta tcccctggcc gtgcccctgg 2460
aggtggaggt ggggataggt ggctctccgc caaggagtga 2500
<210> 2
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ccggaattcc ggatgagggg gatgctgccc ct 32
<210> 3
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ggctctccgc caaggagtga ttgcggccgc aa 32
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ctgtcatgcc atgaacccac 20
<210> 5
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cttttacttg gcttttagga a 21
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
agtccccggt ggatatccgc 20
<210> 7
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
catgattaga ggaggccagt 20
<210> 8
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
agtccccggt ggatatccgc 20
<210> 9
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
aggagggccc ggaagaaaac 20
<210> 10
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
acctgtgaga ctttggctcc a 21
<210> 11
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
aggagggccc ggaagaaaac a 21
Claims (10)
1. a kind of recombination Taq direct expansion enzyme, which is characterized in that the gene order such as SEQ ID NO.1 institute of the recombination Taq direct expansion enzyme
Show.
2. the preparation method of recombination Taq direct expansion enzyme as described in claim 1, which comprises the steps of:
Construct the recombinant plasmid containing sequence shown in SEQ ID NO.1；
The recombinant plasmid is transferred in Escherichia coli, the engineering bacteria for expressing the recombination Taq direct expansion enzyme is obtained；
After the engineering bacteria is carried out inducing expression, purifying is extracted, the recombination Taq direct expansion enzyme is obtained.
3. preparation method as claimed in claim 2, which is characterized in that the building contains sequence shown in SEQ ID NO.1
The step of recombinant plasmid includes:
Sequence both ends shown in SEQ ID NO.1 are added to the protection base of restriction enzyme EcoR I and Not I respectively, then
It is connected on the pET-32A carrier through restriction enzyme EcoR I and Not I digestion, obtains the recombinant plasmid.
4. preparation method as claimed in claim 3, which is characterized in that the primer such as SEQ of sequence shown in amplification SEQ ID NO.1
Shown in ID NO.2 and SEQ ID NO.3.
5. preparation method as claimed in claim 2, which is characterized in that the step of engineering bacteria is carried out inducing expression packet
It includes:
Engineering bacteria switching is activated in the LB liquid medium containing the ampicillin that concentration is 50-100 μ g/ml
Culture；
Culture solution after the activation culture is transferred to the LB liquid training containing the ampicillin that concentration is 100-200 μ g/ml
Base is supported, shaken cultivation is carried out, makes final OD600For 0.4-1.0.
6. preparation method as claimed in claim 5, which is characterized in that the condition of the activation culture are as follows: 36-37 DEG C of temperature,
Revolving speed 200-250rpm, time 10-16h；And/or
The condition of the shaken cultivation are as follows: 36-37 DEG C of temperature, revolving speed 200-250rpm, time 12-22h；And/or
It include: in OD in the shake culture600When for 0.2-0.4, the IPTG of final concentration of 0.4-0.8mM is added.
7. preparation method as claimed in claim 2, which is characterized in that the step of extraction purification includes: to be purified according to heat
Method is carried out using buffer solution A, lysozyme, buffer solution B, DNase I, DTT, buffer C neutralization buffer D from the engineering bacteria
The isolated recombination Taq direct expansion enzyme in culture solution after inducing expression.
8. preparation method as claimed in claim 7, which is characterized in that the buffer solution A are as follows: 50mM Tris-HCl, pH
7.9,50mM glucose, 1mM EDTA；And/or
The buffer solution B are as follows: 10mM Tris-HCl, pH 7.9,50mM KCl, 1mM EDTA, 0.5%Tween-20,0.5%
nonidet P-40；And/or
The buffer C are as follows: 50% glycerol, 50mM Tris-HCl, pH 8.0,100mM NaCl, 0.1mM EDTA, 1%
Triton X-100；And/or
The buffer D are as follows: 75% glycerol, 50mM Tris-HCl, pH 8.0,100mM NaCl, 0.1mM EDTA, 1%
Triton X-100。
9. a kind of recombinant plasmid, which is characterized in that the recombinant plasmid contains the sequence as shown in SEQ ID NO.1.
10. a kind of engineering bacteria, which is characterized in that the engineering bacteria contains recombinant plasmid as claimed in claim 9.
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| CN112175786A (en) * | 2019-07-01 | 2021-01-05 | 申翌生物科技(杭州)有限公司 | PCR reaction system |
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| CN109022386B (en) | 2020-11-03 |
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