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CN113186175B - Mutant Taq DNA polymerase, coding DNA sequence, recombinant vector, recombinant expression cell and application thereof - Google Patents

Mutant Taq DNA polymerase, coding DNA sequence, recombinant vector, recombinant expression cell and application thereof Download PDF

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CN113186175B
CN113186175B CN202110629006.5A CN202110629006A CN113186175B CN 113186175 B CN113186175 B CN 113186175B CN 202110629006 A CN202110629006 A CN 202110629006A CN 113186175 B CN113186175 B CN 113186175B
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CN113186175A (en
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程克文
顾玲
柴常升
王雨薇
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Magnesium Futai Biotechnology Shanghai Co ltd
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Yisheng Biotechnology Shanghai Co ltd
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Abstract

The invention provides mutant Taq DNA polymerase, the protein sequence of which is shown as SEQ ID NO:3 is shown in the figure; or for SEQ ID NO:3, and still have the amino acid sequence shown in SEQ ID NO:3 and high concentration protein tolerance and high concentration salt ion tolerance. And discloses a coding DNA sequence, a recombinant vector, a recombinant expression cell and application thereof. Compared with wild type, the mutant Taq DNA polymerase has 5 amino acid mutations, has better protein and salt ion tolerance and SNP typing capability, and simultaneously shows better uniformity in the aspect of multiplex PCR library establishment.

Description

Mutant Taq DNA polymerase, coding DNA sequence, recombinant vector, recombinant expression cell and application thereof
Technical Field
The invention relates to mutant Taq DNA polymerase, coding DNA sequence, recombinant vector, recombinant expression cell and application thereof, belonging to the field of biotechnology.
Background
The DNA polymerase is derived from thermophilic bacteria (Thermus aquaticus), and the good heat resistance is widely used in the medical fields of disease diagnosis and treatment, infectious disease detection, medicine action mechanism and the like. In-vitro diagnosis, human whole blood is directly used for molecular diagnosis, so that the nucleic acid extraction step is omitted, the pollution risk is greatly reduced, the time is shortened, and the expenditure is saved. In cells, the primary activity of DNA polymerase is to catalyze the replication of DNA (in the case of templates, primers, dntps, etc.) from the 5 'end to the 3' end. However, the blood components are complex, and have great influence on the fidelity, thermal stability and polymerization performance of the wild type Taq enzyme.
Disclosure of Invention
The invention aims to provide mutant Taq DNA polymerase which has high tolerance to blood.
The invention adopts the technical scheme that:
a mutant Taq DNA polymerase, the protein sequence of which is shown in SEQ ID NO:3 is shown in the figure; or for seq id NO:3, and still have the amino acid sequence shown in SEQ ID NO:3 and high concentration protein tolerance and high concentration salt ion tolerance.
The invention also discloses a coding DNA sequence of the mutant Taq DNA polymerase, and the sequence of the coding DNA sequence is shown as SEQ ID NO:4 is shown in the figure; or in seq id No:4, and can still code the derivative protein with the mutant Taq DNA polymerase activity after one or more bases are substituted, deleted or added in the nucleotide sequence shown in the formula 4.
The invention also discloses a recombinant vector containing the coding nucleotide.
Preferably, the recombinant vector is a pET-28a plasmid.
The invention also discloses a recombinant expression cell, which is characterized in that: the mutant Taq DNA polymerase described above was expressed.
Preferably, the cell is BL21 or DE3.
The invention also discloses application of the mutant Taq DNA polymerase in PCR amplification of blood samples.
The invention also discloses application of the mutant Taq DNA polymerase in DNA sequence polymorphism SNP typing detection.
The mutant Taq DNA polymerase is obtained by mutating wild DNA polymerase with E130N, D188K, F306T, A472I and V669Q, wherein the E130N represents that the 130 th amino acid is changed from glutamic acid to asparagine; D188K represents that amino acid at position 188 is changed from aspartic acid to lysine; f306T represents that the amino acid at position 306 has changed from phenylalanine to threonine; A472I represents that the 472 th amino acid is changed from alanine to isoleucine; V669Q indicates that the amino acid at position 669 is changed from valine to glutamine.
Compared with wild type, the mutant Taq DNA polymerase has 5 amino acid mutations, has better protein and salt ion tolerance and SNP typing capability, and simultaneously shows better uniformity in the aspect of multiplex PCR library establishment.
Drawings
FIG. 1 shows the results of agarose gel electrophoresis in example 3, wherein Lane1 is a marker and Lane 2-4BSA concentrations are 0.8mg/ml, 1mg/ml and 1.2mg/ml, respectively, and Lane5-7 BSA concentrations are 0.8mg/ml, 1mg/ml and 1.2mg/ml, respectively.
FIG. 2 shows the agarose gel electrophoresis results of example 4, lane1 is marker, lane 2-3K 2 SO 4 The concentration is 80mM, 100mM, and lane 4-5K respectively 2 SO 4 The concentrations were 80mM and 100mM, respectively.
FIG. 3 is the real-time fluorescent quantitative PCR results of example 4, wherein line 1 and line 2 are mutant Taq DNA polymerase amplification results, line 1 target fragment amplification results, and line 2 corresponds to nonspecific amplification; line 3 and line 4 are wild-type Taq DNA polymerase amplification results, line 3 target fragment amplification results, line 4 corresponding nonspecific amplification.
FIG. 4 shows the results of multiplex PCR pool-building sequencing of example 6, with two lines representing sequencing reads for wild-type and mutant types, respectively.
Detailed Description
Example 1
Example 1: constructing a vector of Taq DNA polymerase containing a mutant DNA sequence:
E130N-1：CATATAGAGGAAGAAATGAGGGGGATGCTGCCCCT
E130N-2：CGATAGTGGTACCTGGTTCGCCTTCTTGGCCAGGC
D188K-1：GCTATCACCATGGACAAGGAGGGCTACGAGGTCCG
D188K-2：ATCGCGATTAGTCTATTTCCCGGTCAGGGCCCGGT
F306T-1：TAGCGCTAATCAGATGAGTCCGACAACCTTCCCGG
F306T-2：AGTTGACGTAGTCGAGGTGGCCCCTTCCGGCGGGG
A472I-1：TCAACTGCATCAGCTGTGGGCTTTGTGCTTTCCCG
A472I-2：GTACGTAGCTAGAATAATCTCGAGGCGGGCGATCT
V669Q-1：CATGCATCGATCTTACTCCAGAAGGCGGACCGGCC
V669Q-2：TGACTAGCTATAGTTTTGCCCGAAGTTGATGGTCT
KONG-1：ACTGATCGATATCAAGAGATGCCGTACAGCCGGGT
KONG-2：TGTCGACGGAGCTCGTCACTCCTTGGCGGAGAGCC
the sequence of SEQ ID NO:2, the PCR amplification was divided into 4 reactions, and primers E130M-1, E130M-2, D188K-1, D188K-2, F306T-1, F306T-2, A472I-1, A472I-2, V669Q-1, V669Q-2 were added to a 20. Mu.l reaction system, 1. Mu.l primer (5. Mu.M) was used, mix was 2X Hieff Canace et al Gold PCR Master Mix (product number: 10149ES01, hi-san Co., ltd., of the next 25 cycles) and the amplification procedure was followed: 95 ℃ for 1min;95 ℃ for 10s; 20s at 60 ℃;72 ℃ for 1min; stored at 72℃for 5min and at 4 ℃. The amplified reaction system was digested with 0.5. Mu.l DpnI (NEB, cat# R0176V) at 37℃for 1h,2% agarose gel, 150V, electrophoresis for 30min, to obtain the desired strip gel recovery (MolPure's Gel Extraction Kit, cat# 19101ES50, molPure's) for pET-28a vector, ecoRI and NcoI treatment, plasmid vector and recovered product were ligated with (Shanghai's St. Biotechnology Co., ltd., cat# 10912ES 10), 50℃for 20min, 10. Mu.l of cooled recombinant product was added to 100. Mu.L chemically competent cells BL21 (DE 3) (Saint Bio (Shanghai) Co., cat# 11804ES 80), heat shock for 90 seconds at 42℃for 2min, ice bath incubation, 900. Mu.l of SOC or LB medium was added, and incubation at 37℃for 10 min for full resuscitation. Shaking at 37℃and 200rpm for 45 min.5000 Centrifuge at rpm for 3 min and discard 900. Mu.l supernatant. The bacteria were resuspended in the remaining medium and gently spread on plates containing the correct resistance with sterile spreading bars. After the bacterial solution was absorbed, the plate was inverted and incubated overnight at 37 ℃. The next day clone sequencing was picked up and the resulting sequence SEQ ID NO-4.
EXAMPLE 2 Taq DNA polymerase obtaining mutant form
Plasmid vector was introduced into BL21 (DE 3) (product number 11804ES80, product name: shanghai, inc. of the next san Biotech Co., ltd.),
LB culture medium (containing 100 mug/mL ampicillin) is cultured overnight at 37 ℃ for 160 r/min, escherichia coli thalli obtained after cloning and induced expression are selected, and mutant Taq DNA polymerase is obtained after purification, wherein the specific sequence is shown in SEQ ID NO-3.
EXAMPLE 3 high protein tolerance experiment
Mutant and wild-type DNA polymerases were tested for their ability to amplify a 1kb fragment of interest in a PCR reaction using human whole blood as template in the presence of high concentrations of protein BSA. The amplification system and time used for the reaction are shown in tables 1-3 below:
the primer sequences (direction 5 '-3') were as follows:
Primer-1：CCAGTGTAAGTTAACCCCACAGT
Primer-2：AGCTTTGATAGTCCTGCATTCTC
TABLE 1 PCR mix component concentrations
Component (A) Concentration in PCR System
Tris-HCl（pH9.0） 40mM
MgCl2 2mM
Tween-20 1%
dNTPs 0.2mM
K2SO4 50mM
BSA 0.8、1、1.2mg/ml
Taq DNA polymerase 0.25U/μl
TABLE 2 PCR reaction System
Component (A) Volume of Final concentration
Human whole blood 0.5μl -
Primer-F（10μM） 0.5μl 0.25μM
Primer-R（10μM） 0.5μl 0.25μM
mix 10μl -
ddH 2 O To 20μl -
TABLE 3 PCR reaction procedure
And finally, carrying out electrophoresis judgment on the PCR product, wherein the conditions are as follows: 2% agarose gel, 150V,30min, to determine if the band is specific and if the target band is to be maintained. The results are shown in fig. 1. As can be seen from FIG. 1, wild-type Taq enzyme was significantly weaker than mutant Taq enzyme at 0.8mg/ml, 1mg/ml, and 1.2mg/ml BSA, indicating that mutant Taq enzyme was tolerant to higher concentrations of protein.
EXAMPLE 4 salt ion tolerance experiment
At a high concentration of salt ion K 2 SO 4 In the presence, the ability of mutant and wild-type DNA polymerases to amplify a 1kb target fragment was tested in a PCR reaction using human whole blood as a template. The amplification system and time used for the reaction are shown in tables 4-6:
the primer sequences (direction 5 '-3') were as follows:
Primer-1：GAGGATTTCCCTAACCTGACAGT
Primer-2：CCTCAACTAGGAGGTTCCAAACA
TABLE 4 PCR mix component concentrations
Component (A) Concentration in PCR System
Tris-HCl（pH9.0） 40mM
MgCl2 2mM
Tween-20 1%
dNTPs 0.2mM
K 2 SO 4 80, 100mM
BSA 0.4mg/ml
Taq DNA polymerase 0.25U/μl
TABLE 5 PCR reaction System
Component (A) Volume of Final concentration
Human whole blood 0.5μl -
Primer-F（10μM） 0.5μl 0.25μM
Primer-R（10μM） 0.5μl 0.25μM
mix 10μl -
ddH2O To 20μl -
TABLE 6 PCR reaction procedure
Final PCR product electrophoresis determinations, results are shown in fig. 2, conditions: 2% agarose gel, 150V,30min, judging whether the band is specific or not, and whether the band is the target band or not. From the electrophoresis results, wild-type Taq enzyme was weaker than mutant Taq enzyme under conditions of 80mM K2SO4 and 100mM K2SO4, indicating that mutant Taq enzyme is more tolerant to high concentrations of salt ions.
Example 5 SNP typing Capacity experiment
PCR reactions mutant and wild type DNA polymerase amplifications were tested using human whole blood as template. The amplification system and time used for the reaction are shown in tables 7-9:
exemplary primer sequences (directions 5 '-3') are as follows:
Primer-1：CTGGTGGACCTGATGCACC
Primer-2：TCACCTGGTCGAAGCAGTATG
Probe：FAM-CACGCTACCCACCA-MGB
TABLE 7 PCR mix component concentrations
Component (A) Concentration in PCR System
Tris-HCl（pH9.0） 40mM
MgCl2 2mM
Tween-20 1%
dNTPs 0.2mM
K2SO4 60mM
BSA 0.4mg/ml
DMSO 1%
Taq DNA polymerase 0.25U/μl
TABLE 8 PCR reaction System
Component (A) Volume of Final concentration
Human whole blood 0.5μl -
Primer-F（10μM） 0.5μl 0.4μM
Primer-R（10μM） 0.5μl 0.4μM
probe（5μM） 0.5μl 0.2μM
mix 10μl -
ddH2O To 20μl -
TABLE 9 PCR reaction procedure
FIG. 3 is a graph of fluorescence quantitative data, wherein the abscissa indicates cycle number and the ordinate indicates fluorescence value, and FIG. 3 shows that mutant Taq DNA polymerase has better discrimination effect on SNP than wild-type Taq DNA polymerase.
Example 6 PCR homogeneity assay
PCR reactions mutant and wild type DNA polymerase amplifications were tested using human whole blood as template. The amplification system and time used for the reaction are shown in tables 10-14:
TABLE 10 PCR mix component concentrations
Component (A) Concentration in PCR System
Tris-HCl（pH9.0） 40mM
MgCl2 2mM
Tween-20 1%
dNTPs 0.2mM
K2SO4 70mM
BSA 0.4mg/ml
DMSO 2%
Taq DNA polymerase 0.25U/μl
TABLE 11 first round amplification reaction System
Mix 10μl 1×
Primer mix（0.25μM） 6μl 0.05μM
Human whole blood 0.5μl -
Sterile ultrapure water To 20μl -
Table 12 first round amplification procedure
First round of product bead screening (first round of primer removal):
1. adding 10 μl of magnetic beads (0.5 x) into the PCR product, mixing, standing for 5min, and placing on a magnetic rack; taking a new centrifuge tube, and adding 6 mu l of magnetic beads for standby;
2. after the solution was clear, 30 μl of supernatant was pipetted into a new centrifuge tube with 10 μl (0.5 x) of magnetic beads and mixed well;
3. standing for 5min, and placing the magnetic beads on a magnetic frame;
4. after the solution is clarified, discarding the supernatant;
5. 200 μl of 80% ethanol was added to wash the beads;
6. ethanol was aspirated with a 20 μl pipette;
7. the instant centrifugation is carried out, the magnetic beads are placed on a magnetic rack, and the magnetic rack is kept stand for 30 seconds, and then 20 mu l of pipettor is used for sucking residual ethanol;
8. when the surface of the magnetic beads is not reflective and loses luster, adding 15 mu l of ultrapure water into each hole to re-suspend uniformly, and standing for 2min;
9. placing the magnetic beads on a magnetic frame, and standing for 2min;
10. mu.l of supernatant was pipetted into a new centrifuge tube.
TABLE 13 second round amplification reaction System
Component (A) Volume (mu L) Final concentration
Mix 10μl 1×
Primer mix（2μM） 2μl 0.2μM
Supernatant of the previous step 8μl -
Total volume of To 20μl -
TABLE 14 second round amplification reaction procedure
Second round of product magnetic bead purification:
1. adding 20 μl of magnetic beads (1 x) into the PCR product, and mixing well;
2. standing for 5min, and placing the magnetic beads on a magnetic frame;
3. after the solution is clarified, sucking and discarding the supernatant;
4. 200 μl of 80% ethanol was added;
5. the ethanol was pipetted and discarded with a 200 μl pipette;
6. sucking the residual ethanol in the previous step by using a 20-mu l pipette and discarding the residual ethanol;
7. when the surface of the magnetic beads is not reflective and loses luster, adding 20 mu l of ultrapure water into each hole and blowing and sucking the suspended magnetic beads;
8. standing for 2min, and placing on a magnetic rack;
9. after the solution was clear, 20 μl of supernatant was aspirated and transferred to a new tube;
10. the library samples obtained after purification were sequenced.
FIG. 4 is a graph of sequencing results, the abscissa is the target site, 60 total sites are sequenced, the ordinate is the number of sequencing reads corresponding to each site, and FIG. 4 shows that mutant Taq enzyme has more sequencing reads than wild-type Taq enzyme pairs, and the uniformity of each site is also better.
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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 acgccctcca 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 ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2400
cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag 2460
gtggggatag gggaggactg gctctccgcc aaggagtga 2499
<210> 3
<211> 832
<212> PRT
<213> artifical sequence
<400> 3
Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu
1 5 10 15
Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly
20 25 30
Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala
35 40 45
Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val
50 55 60
Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly
65 70 75 80
Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu
85 90 95
Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu
100 105 110
Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys
115 120 125
Ala Asn Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp
130 135 140
Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Ala Leu His Pro Glu Gly
145 150 155 160
Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro
165 170 175
Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Lys Glu Ser Asp Asn
180 185 190
Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu
195 200 205
Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu
210 215 220
Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys
225 230 235 240
Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val
245 250 255
Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe
260 265 270
Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu
275 280 285
Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly
290 295 300
Ala Thr Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp
305 310 315 320
Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro
325 330 335
Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu
340 345 350
Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro
355 360 365
Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn
370 375 380
Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu
385 390 395 400
Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu
405 410 415
Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu
420 425 430
Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly
435 440 445
Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala
450 455 460
Glu Glu Ile Ala Arg Leu Glu Ile Glu Val Phe Arg Leu Ala Gly His
465 470 475 480
Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp
485 490 495
Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg
500 505 510
Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile
515 520 525
Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr
530 535 540
Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu
545 550 555 560
His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser
565 570 575
Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln
580 585 590
Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala
595 600 605
Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly
610 615 620
Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr
625 630 635 640
Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro
645 650 655
Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Gln Leu Tyr Gly
660 665 670
Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu
675 680 685
Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg
690 695 700
Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val
705 710 715 720
Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg
725 730 735
Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro
740 745 750
Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu
755 760 765
Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His
770 775 780
Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala
785 790 795 800
Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro
805 810 815
Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu
820 825 830
<210> 4
<211> 2499
<212> DNA
<213> artifical sequence
<400> 4
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 gaaggcgaac aaggagggct acgaggtccg catcctcacc 420
gccgacaaag acctttacca gctcctttcc gaccgcatcc acgccctcca ccccgagggg 480
tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc 540
gactaccggg ccctgaccgg gaaagagtcc 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 gggccaccgt 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 gagattgagg 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 cgggcaactc 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 ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2400
cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag 2460
gtggggatag gggaggactg gctctccgcc aaggagtga 2499

Claims (9)

1. A mutant Taq DNA polymerase, the protein sequence of which is shown in SEQ ID NO: 3.
2. The mutant Taq DNA polymerase of claim 1 encoding DNA.
3. The coding DNA of claim 2, wherein: the sequence of the polypeptide is shown in SEQ ID NO: 4.
4. A recombinant vector comprising the coding DNA of claim 2 or 3.
5. The recombinant vector according to claim 4, wherein: the recombinant vector is pET-28a plasmid.
6. A recombinant expression cell, characterized in that: the mutant Taq DNA polymerase of claim 1 is expressed.
7. The recombinant expression cell of claim 6, wherein: the cells are BL21 or DE3.
8. Use of a mutant Taq DNA polymerase of claim 1 in PCR amplification of blood samples, which is not directly used for diagnosis or treatment.
9. Use of a mutant Taq DNA polymerase of claim 1 in DNA sequence polymorphism SNP typing detection, said use not being directly used for diagnosis or treatment.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101528919A (en) * 2006-10-18 2009-09-09 霍夫曼-拉罗奇有限公司 Mutant DNA polymerases and related methods
CN108192907A (en) * 2018-01-26 2018-06-22 遵义医学院 A kind of heat-stable DNA amplification fusion enzyme
CN108265039A (en) * 2016-12-30 2018-07-10 天津强微特生物科技有限公司 A kind of mutation T aqDNA polymerases and its purification process
CN108350087A (en) * 2015-11-27 2018-07-31 国立大学法人九州大学 Archaeal dna polymerase variant
CN110684752A (en) * 2019-10-08 2020-01-14 南京诺唯赞生物科技有限公司 Mutant Taq DNA polymerase with improved tolerance as well as preparation method and application thereof
CN112513262A (en) * 2018-07-13 2021-03-16 宝生物工程株式会社 DNA polymerase mutants suitable for nucleic acid amplification from RNA

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040109853A1 (en) * 2002-09-09 2004-06-10 Reactive Surfaces, Ltd. Biological active coating components, coatings, and coated surfaces
US20100210745A1 (en) * 2002-09-09 2010-08-19 Reactive Surfaces, Ltd. Molecular Healing of Polymeric Materials, Coatings, Plastics, Elastomers, Composites, Laminates, Adhesives, and Sealants by Active Enzymes
EP2357227B1 (en) * 2010-02-11 2015-07-15 Süd-Chemie IP GmbH & Co. KG Optimized cellulase enzymes
JP5824205B2 (en) * 2010-10-27 2015-11-25 アークレイ株式会社 Mutant glucose dehydrogenase

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101528919A (en) * 2006-10-18 2009-09-09 霍夫曼-拉罗奇有限公司 Mutant DNA polymerases and related methods
CN108350087A (en) * 2015-11-27 2018-07-31 国立大学法人九州大学 Archaeal dna polymerase variant
CN108265039A (en) * 2016-12-30 2018-07-10 天津强微特生物科技有限公司 A kind of mutation T aqDNA polymerases and its purification process
CN108192907A (en) * 2018-01-26 2018-06-22 遵义医学院 A kind of heat-stable DNA amplification fusion enzyme
CN112513262A (en) * 2018-07-13 2021-03-16 宝生物工程株式会社 DNA polymerase mutants suitable for nucleic acid amplification from RNA
CN110684752A (en) * 2019-10-08 2020-01-14 南京诺唯赞生物科技有限公司 Mutant Taq DNA polymerase with improved tolerance as well as preparation method and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Ishino,Y.等.Thermus aquaticus gene for DNA polymerase, complete cds.《GenBank Database》.2008,Accession No.D32013.1. *

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