CN114015672B - Pfu DNA polymerase - Google Patents
Pfu DNA polymerase Download PDFInfo
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- CN114015672B CN114015672B CN202111511759.2A CN202111511759A CN114015672B CN 114015672 B CN114015672 B CN 114015672B CN 202111511759 A CN202111511759 A CN 202111511759A CN 114015672 B CN114015672 B CN 114015672B
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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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
- C12Q1/686—Polymerase chain reaction [PCR]
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- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of biological engineering, in particular to Pfu DNA polymerase, the amino acid sequence of which is shown as Seq ID No.1, and the concentration of dialyzed protein is 1.2 mg/mL; the nucleotide sequence of the gene for coding the Pfu DNA polymerase is shown as Seq ID No.2, an expression vector carrying the gene is transferred into host bacteria to construct an expression strain, and the Pfu DNA polymerase is obtained through induced expression. The Pfu DNA polymerase can amplify DNA fragments within 6000bp without mismatch, and has high fidelity, rapid extension speed of 42bp/s and higher denaturation temperature of 100 ℃; the protein can still keep a relatively complete protein structure and an excellent amplification effect after being stored for one week at 37 ℃, and the stability of the protein is remarkably improved compared with that of wild unmodified enzyme. Therefore, the Pfu DNA polymerase of the present invention can save more time in PCR reaction, has stronger stability, and increases recovery efficiency after purification.
Description
Technical Field
The invention relates to Pfu DNA polymerase, and belongs to the technical field of biological engineering.
Background
PCR procedures are the most basic and common technique in molecular biology. The PCR reaction is catalyzed by DNA polymerase, and the selection of an appropriate polymerase for the purpose of the experiment is a matter of consideration before the PCR experiment is carried out. Many DNA polymerases differ primarily in several factors, such as specificity, fidelity, amplification rate, and amplified fragment length.
The most commonly used DNA polymerase at present is Taq enzyme, which has high amplification efficiency but no correction function. Pfu DNA polymerase has a correction effect because Pfu enzyme has excellent thermal stability, has 5 '-3' DNA polymerase activity and 3 '-5' exonuclease activity, and the main function of 3 '-5' exonuclease activity is a correction effect, and is cleaved by 3 '-5' exonuclease to re-polymerize the corresponding nucleotide at this position when the added nucleotide is not complementary to the template nucleotide, thereby effectively reducing the base misincorporation rate and improving the authenticity of the amplification result, but Pfu DNA polymerase has a correction effect, but one disadvantage of Pfu enzyme is that the amplification rate is not as high as that of ordinary Taq enzyme.
Disclosure of Invention
In order to solve the technical problems, the invention provides Pfu DNA polymerase, which solves the problems of slow extension speed and poor stability of the conventional Pfu DNA polymerase, and additionally, 8 His tags are added at the C end of the novel Pfu DNA polymerase, so that the subsequent purification steps are greatly facilitated.
The first object of the present invention is to provide a Pfu DNA polymerase whose amino acid sequence is shown in Seq ID No. 1.
It is a second object of the present invention to provide a gene encoding the Pfu DNA polymerase.
Further, the nucleotide sequence of the gene is shown in Seq ID No. 2.
The third purpose of the invention is to provide an expression vector carrying the gene.
The fourth object of the present invention is to provide a host cell carrying the expression vector.
Further, the host cell is Escherichia coli.
Preferably, the E.coli is a competent cell of Ecoli BL21, Rosetta-gami, OrigamiB or Rosetta series.
The fifth purpose of the present invention is to provide a reaction buffer solution, which is suitable for the pfu DNA polymerase to perform DNA amplification reaction, and the reaction buffer solution is composed of the following components: 20-50mM Tris-HCl, 5-10mM (NH4)2SO4, 2-4mM MgSO4, 10-20mM KCl, 5-10mg/mL BSA and 0.5-1% TritonX-100.
Preferably, the reaction buffer consists of: 30-50mM Tris-HCl, 5-10mM (NH4)2SO4, 2-3mM MgSO4, 10-15mM KCl, 5-8mg/mL BSA, and 0.5-1% TritonX-100.
Further, the construction method of the Pfu DNA polymerase comprises the following steps:
(1) the plasmid vector is subjected to EcoRI and NotI double enzyme digestion, after metal bath for 3-4h at 37 ℃, the detection is carried out by 1% agarose gel electrophoresis, and then the vector fragment is recovered by using an agarose gel recovery kit. Connecting a gene segment (the sequence is shown as Seq ID No. 1) of Pfu DNA polymerase coded by a target segment with a vector segment, standing overnight at 16 ℃, and transferring the constructed recombinant plasmid into a host bacterium to obtain an expression strain;
(2) A1L liquid shake flask was filled with 200mL of LB medium, 50-100. mu.g/mL of kanamycin was added, and 10-20. mu.L of overnight-shaken strain was inoculated, and the broth was shaken at 37 ℃ to OD600(iii) adding IPTG at a molar concentration of 0.1-0.25mM to 0.4-0.6, and inducing protein expression overnight at 16 ℃;
(3) centrifuging at 12000g for 10min to collect thallus, resuspending with 20mL PBS, adding lysozyme with the mass percentage concentration of 400-. Crushing thallus with ultrasonic cell crusher, placing the bacterial liquid on ice, crushing at 300W for 2s, stopping crushing for 2s, crushing for 3min at intervals, centrifuging at 4 ℃ at 12000g for 10min, and collecting supernatant.
Compared with the prior art, the technical scheme of the invention has the following advantages:
(1) according to the Pfu DNA polymerase, the C end is provided with 8 His tags, and the protein is more easily combined on a Ni column due to the multiple His tags in purification, so that the recovery rate after purification is increased;
(2) the Pfu DNA polymerase can amplify DNA fragments within 6000bp without mismatch, has high fidelity and rapid extension speed, the extension speed of the wild-type Pfu DNA polymerase is 10bp/s, and the extension speed of the Pfu DNA polymerase reaches 42bp/s, so that more time can be saved in PCR reaction;
(3) compared with wild unmodified enzyme, the Pfu DNA polymerase provided by the invention has the advantages that the stability is remarkably improved, a relatively complete protein structure can be kept after the Pfu DNA polymerase is stored for one week at 37 ℃, DNA fragments within 6000bp can be amplified, and the amplification speed and the fidelity are unchanged;
(4) the Pfu DNA polymerase also has higher denaturation temperature, the common wild-type Pfu DNA polymerase is basically inactivated at the temperature of more than 60 ℃, the activity of the Pfu DNA polymerase is not obviously changed, the denaturation temperature can even reach 100 ℃, the template double-stranded DNA is effectively promoted to be converted into single-stranded DNA, and the PCR amplification efficiency is further enhanced;
(5) the optimized reaction buffer solution provides an optimal reaction environment for the Pfu DNA polymerase, so that the enzymatic reaction efficiency is greatly improved.
Drawings
In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the present disclosure taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a protein electrophoretogram of Pfu DNA polymerase of the present invention.
FIG. 2 is a nucleic acid electrophoresis chart of an amplification product of Pfu DNA polymerase of the present invention.
FIG. 3 is a diagram showing the sequencing alignment of the Pfu DNA polymerase amplification products of the present invention.
FIG. 4 is a graph showing the stability analysis of Pfu DNA polymerase of the present invention.
FIG. 5 is a nucleic acid electrophoresis chart of an amplification product of Pfu DNA polymerase of the present invention after storage at 37 ℃ for one week.
FIG. 6 is a graph showing the results of sequencing alignment of Pfu DNA polymerase of the present invention after storage at 37 ℃ for one week.
The specification reference numbers indicate: in the HPLC analysis chart of the stability of Pfu DNA polymerase, 1 is a peak pattern of the Pfu DNA polymerase of the present invention stored at-80 ℃ for 1 week, 2 is a peak pattern of the Pfu DNA polymerase of the present invention stored at 37 ℃ for 1 week, and 3 is a peak pattern of the wild-type unmodified Pfu DNA polymerase stored at 37 ℃ for 1 week; in the gel electrophoresis chart, lane 1 shows the protein degradation of wild-type unmodified Pfu DNA polymerase after storage at 37 ℃ for 1 week, and lane 2 shows the protein degradation of Pfu DNA polymerase of the present invention after storage at 37 ℃ for 1 week.
Detailed Description
The present invention is further described below with reference to specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
The invention adopts the conventional test method, and the materials and the reagents are all commercial products.
Carrying out EcoRI and NotI double digestion on pET-30(a) + plasmid to obtain a plasmid large fragment, carrying out metal bath at 37 ℃ for 3-4h, detecting by using 1% agarose gel electrophoresis, and finally recovering by using an agarose gel recovery kit to obtain a vector fragment;
connecting a gene fragment encoding Pfu DNA polymerase with a vector fragment, standing overnight at 16 ℃, and transferring the constructed recombinant plasmid into Ecoli BL21(DE3) to obtain an expression strain;
A1L liquid shake flask was charged with 200mL LB medium, 100. mu.g/mL kanamycin was added, and 10. mu.L of overnight shaken strain was inoculated, and the broth was shaken at 37 ℃ to OD600(iv) 0.6, final concentration of 0.25mM IPTG was added to induce protein expression overnight at 16 ℃;
the cells were collected by centrifugation at 12000g for 10min, resuspended in 20mL PBS, lysozyme was added to a final concentration of 400. mu.g/mL and lysed at 4 ℃ for 30 min. Crushing thallus with ultrasonic cell crusher, placing the bacterial liquid on ice, crushing at a power of 300W for 2s every 2s, continuing for 3min, centrifuging at 4 ℃ at 12000g for 10min, and collecting supernatant.
Protein purification was performed using an AKTA pure protein purification system: after the supernatant sample was injected completely, the column was washed with 50 column volumes of binding buffer (20mM phosphate buffer, 0.5M NaCl, pH 7.4); then wash away the proteins not bound to the column with 50 column volumes of wash buffer (binding buffer +20mM imidazole); finally, the column was washed with Elution buffer (20mM phosphate buffer, 0.5M NaCl, 500mM imidazole), and after the peak was detected by the instrument, 15mL of the effluent fraction was collected and dialyzed to a stock solution (20mM Tris-HCl, 1mM DTT, 0.1mM EDTA, 0.1M KCl, 0.1% Tween 20, 20% glycerol). The protein electrophoretogram is shown in FIG. 1.
Determination of concentration of Pfu DNA polymerase
The Pfu DNA polymerase concentration was measured by the BCA method using Bovine Serum Albumin (BSA) as a control.
As can be seen from the data analysis, the concentration of the protein dialyzed by Pfu DNA polymerase of the present invention was 1.2 mg/mL.
Amplification Effect of Pfu DNA polymerase
PCR amplification was performed using pLenti CRISPR v2 vector as a DNA template and a commercially available Pfu DNA polymerase as a control.
The amplification reaction system is shown in Table 1, the amplification reaction conditions are shown in Table 2, and the amplification results are shown in FIGS. 2 and 3.
TABLE 1 PCR amplification reaction System
Wherein, the 10x Pfu enzyme buffer solution consists of the following components: 200mM Tris-HCl (pH 8.8, 25 ℃), 100mM (NH)4)2SO4、20mM MgSO4100mM KCl, 5mg/mL BSA and 1% TritonX-100.
TABLE 2 PCR reaction conditions
Analysis of results
As can be seen from FIG. 1, the Pfu DNA polymerase prepared by the present invention has 8 His tags at the C-terminus, and the multi-His tag makes the protein more easily bind to the Ni column during purification, thereby increasing the recovery rate after purification.
FIG. 2 is a nucleic acid electrophoresis chart showing that the Pfu DNA polymerase of the present invention can amplify a DNA fragment of 0.4-6kb and has a rapid extension rate, the extension rate of the wild-type Pfu DNA polymerase is 10bp/s, and the extension rate of the Pfu DNA polymerase of the present invention reaches 42bp/s, thereby saving more time in the PCR reaction.
FIG. 3 shows the sequencing comparison of the Pfu DNA polymerase amplification products of the present invention, in which the DNA fragments are randomly selected for sequencing, and the novel Pfu DNA polymerase of the present invention can amplify the 2000-6000bp DNA fragment without mismatch by comparison with the template sequence.
FIG. 4 HPLC analysis chart result of Pfu DNA polymerase stability of the present invention shows: compared with wild unmodified enzyme, the novel Pfu DNA polymerase provided by the invention has the advantages that the stability is remarkably improved, and a relatively complete protein structure can be maintained after the novel Pfu DNA polymerase is stored for one week at 37 ℃.
As shown in FIGS. 5 and 6, the Pfu DNA polymerase prepared according to the present invention can amplify DNA fragments of 0.4-6kb after storage at 37 ℃ for one week, and the amplification rate and fidelity are not changed.
It was determined that Pfu DNA polymerase of the present invention also has a higher denaturation temperature. The common wild type Pfu DNA polymerase is basically inactivated at the temperature of more than 60 ℃, while the activity of the Pfu DNA polymerase is not obviously changed, the denaturation temperature can even reach 100 ℃, the template double-stranded DNA is effectively promoted to be converted into single-stranded DNA, and the PCR amplification efficiency is further enhanced.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> a Pfu DNA polymerase
<130> 2021.12.06
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 773
<212> PRT
<213> (Artificial Synthesis)
<400> 1
Met Ile Leu Asp Val Asp Tyr Ile Thr Glu Glu Gly Lys Pro Val Ile
1 5 10 15
Arg Leu Phe Lys Lys Glu Asn Gly Lys Phe Lys Ile Glu His Asp Arg
20 25 30
Thr Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Arg Asp Asp Ser Lys Ile
35 40 45
Glu Glu Val Lys Lys Ile Thr Gly Glu Arg His Gly Lys Ile Val Arg
50 55 60
Ile Val Asp Val Glu Lys Val Glu Lys Lys Phe Leu Gly Lys Pro Ile
65 70 75 80
Thr Val Trp Lys Leu Tyr Leu Glu His Pro Gln Asp Gln Pro Thr Ile
85 90 95
Arg Glu Lys Val Arg Glu His Pro Ala Val Val Asp Ile Phe Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125
Met Glu Gly Glu Glu Glu Leu Lys Ile Leu Ala Phe Ala Ile Ala Thr
130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Gly Lys Gly Pro Ile Ile Met Ile
145 150 155 160
Ser Tyr Ala Asp Glu Asn Glu Ala Lys Val Ile Thr Trp Lys Asn Ile
165 170 175
Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met Ile Lys
180 185 190
Arg Phe Leu Arg Ile Ile Arg Glu Lys Asp Pro Asp Ile Ile Val Thr
195 200 205
Tyr Asn Gly Asp Ser Phe Asp Phe Pro Tyr Leu Ala Lys Arg Ala Glu
210 215 220
Lys Leu Gly Ile Lys Leu Thr Ile Gly Arg Asp Gly Ser Glu Pro Lys
225 230 235 240
Met Gln Arg Ile Gly Asp Met Thr Ala Val Glu Val Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Tyr His Val Ile Thr Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu
275 280 285
Lys Val Tyr Ala Asp Glu Ile Ala Lys Ala Trp Glu Ser Gly Glu Asn
290 295 300
Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Ala Thr Tyr
305 310 315 320
Glu Leu Gly Lys Glu Phe Leu Pro Met Glu Ile Gln Leu Ser Arg Leu
325 330 335
Ile Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Val Ala
355 360 365
Pro Asn Lys Pro Ser Glu Glu Glu Tyr Gln Arg Arg Leu Arg Glu Ser
370 375 380
Tyr Thr Gly Gly Phe Val Lys Glu Pro Glu Lys Gly Leu Trp Asp Asp
385 390 395 400
Ile Val Tyr Leu Asp Phe Ile Ala Leu Tyr Pro Ser Ile Ile Ile Thr
405 410 415
His Asn Val Ser Pro Asp Thr Leu Asn Leu Glu Gly Cys Lys Asn Tyr
420 425 430
Asp Ile Ala Pro Gln Val Gly His Lys Phe Cys Lys Asp Ile Pro Gly
435 440 445
Phe Ile Pro Ser Leu Leu Gly His Leu Leu Glu Glu Arg Gln Lys Ile
450 455 460
Lys Thr Lys Met Lys Glu Thr Gln Asp Pro Ile Glu Lys Ile Leu Leu
465 470 475 480
Asp Tyr Arg Gln Lys Ala Ile Lys Leu Leu Ala Asn Ser Phe Tyr Gly
485 490 495
Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu
500 505 510
Ser Val Thr Ala Trp Gly Arg Lys Tyr Ile Glu Leu Val Trp Lys Glu
515 520 525
Leu Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly
530 535 540
Leu His Ala Thr Ile Pro Gly Gly Glu Ser Glu Glu Ile Lys Lys Lys
545 550 555 560
Ala Leu Glu Phe Val Lys Tyr Ile Asn Ser Lys Leu Pro Gly Leu Leu
565 570 575
Glu Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys
580 585 590
Lys Arg Tyr Ala Val Ile Asp Glu Glu Gly Lys Val Ile Thr Arg Gly
595 600 605
Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln
610 615 620
Ala Arg Val Leu Glu Thr Ile Leu Lys His Gly Asp Val Glu Glu Ala
625 630 635 640
Val Arg Ile Val Lys Glu Val Ile Gln Lys Leu Ala Asn Tyr Glu Ile
645 650 655
Pro Pro Glu Lys Leu Ala Ile Tyr Glu Gln Ile Thr Arg Pro Leu His
660 665 670
Glu Tyr Lys Ala Ile Gly Pro His Val Ala Val Ala Lys Lys Leu Ala
675 680 685
Ala Lys Gly Val Lys Ile Lys Pro Gly Met Val Ile Gly Tyr Ile Val
690 695 700
Leu Arg Gly Asp Gly Pro Ile Ser Asn Arg Ala Ile Leu Ala Glu Glu
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Tyr Asp Pro Lys Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn
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Gln Val Leu Pro Ala Val Leu Arg Ile Leu Glu Gly Phe Gly Tyr Arg
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Lys Glu Gly Ser Ser Leu Glu Val Leu Phe Gln Gly Pro His His His
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His His His His His
770
<210> 2
<211> 2322
<212> DNA
<213> (Artificial Synthesis)
<400> 2
atgatactag acgtagatta tattacagaa gagggcaagc cggtcattcg cctgttcaaa 60
aaagagaacg gcaaattcaa aattgaacat gatcgtacct ttcgtccgta tatttacgcc 120
ctgttgcgtg acgacagcaa aattgaagag gtgaaaaaga tcactggtga acgtcatggt 180
aagatcgtgc gtatcgttga tgtggaaaag gttgagaaga agttcctggg taagccgatc 240
accgtttgga aactgtacct ggagcacccg caggatcagc cgactatccg tgagaaggtt 300
cgtgagcatc cggcagttgt tgatatcttt gaatacgaca ttccgtttgc gaaacgttat 360
ctgatcgaca aaggcctgat accgatggaa ggcgaagagg aacttaaaat actggcgttt 420
gcaattgcta ccctatacca cgaaggcgag gagttcggta agggtccgat catcatgatt 480
agctacgcgg atgagaacga agcaaaggtg atcacttgga aaaacattga cctgccgtat 540
gttgaagtcg tgagcagcga gcgtgaaatg attaagcgct ttctgcgtat tatccgtgag 600
aaggaccctg atatcattgt aacctataac ggtgactctt ttgattttcc gtatctggcg 660
aagcgagctg agaagctggg tattaagttg accattggcc gcgacggttc ggaaccgaaa 720
atgcagcgta ttggtgacat gaccgcggtt gaagttaaag gtcgcattca tttcgatctg 780
taccacgtga tcactcgcac catcaacctg ccaacgtaca ccctggaagc ggtgtacgaa 840
gcgatcttcg gtaagccgaa ggagaaggtg tatgcggatg aaatcgccaa ggcatgggaa 900
tctggtgaaa acctggagcg cgtggcgaag tacagcatgg aagatgcgaa agctacctat 960
gaattaggca aggaatttct cccgatggag atccaactga gccgtctgat tggccaaccg 1020
ctgtgggacg ttagcagaag ctccaccggt aatttggttg aatggtttct gctgcggaag 1080
gcgtatgaac gcaacgaagt agctccgaat aaaccgtccg aggaagagta ccagcgtcgt 1140
ctgagagaaa gctataccgg cggtttcgtg aaggagccgg aaaaagggct gtgggatgac 1200
attgtttact tagacttcat cgcgttatac ccgagcatta ttatcacgca taatgtttcc 1260
ccggataccc tgaatttgga gggctgcaag aactatgaca tcgctccgca agtgggtcac 1320
aaattctgca aagatattcc gggttttatc ccgagcctgc tgggccatct gttggaggag 1380
cgtcaaaaaa tcaagaccaa aatgaaagaa acccaggatc cgatcgagaa gattttgctg 1440
gattatcgtc aaaaagctat caagctcttg gcgaactctt tttatggcta ttacggctat 1500
gcaaaggcgc gttggtactg caaagaatgt gcagaatcag ttacggcttg gggtcgtaaa 1560
tacatcgagt tggtgtggaa agagctggag gagaagttcg gtttcaaagt tctgtacatc 1620
gacaccgatg gtctccacgc aaccattccg ggtggtgaat cggaagagat caagaagaag 1680
gcgttggagt tcgttaagta catcaactcc aaactgcctg gcctgctgga gcttgagtac 1740
gaaggcttct ataaacgtgg tttcttcgtg accaaaaagc gctacgcggt gatcgacgaa 1800
gaaggcaaag tgattacccg tggtttggag atagtgcgcc gtgattggtc tgagatcgct 1860
aaagaaacgc aagcgcgtgt tttggagacg atcctaaaac acggcgacgt cgaggaggcc 1920
gtgcgcatcg tcaaagaggt catccagaaa ctggccaatt atgagatccc accggaaaag 1980
ctcgcgatct acgagcagat tacgcgtccg ctgcacgagt acaaagcgat tggccctcat 2040
gtcgctgtgg ccaagaaact ggcggctaag ggtgttaaaa ttaaaccggg tatggttatc 2100
ggctatattg tgttgcgcgg tgacggccca atcagcaatc gtgcgatcct ggcagaggag 2160
tacgacccga aaaagcacaa atacgacgca gaatactaca ttgagaacca ggtactgccg 2220
gcagtgctgc gtattttgga gggattcggc taccgcaagg aaggctctag cctggaggtt 2280
ttatttcagg gtccgcatca ccatcaccac caccaccact aa 2322
Claims (7)
1. A Pfu DNA polymerase, wherein the amino acid sequence of said Pfu DNA polymerase is Seq ID No. 1.
2. A gene encoding the Pfu DNA polymerase of claim 1.
3. The gene of claim 2, wherein the nucleotide sequence of the gene is shown in Seq ID No. 2.
4. An expression vector carrying the gene of claim 2 or 3.
5. A host cell carrying the expression vector of claim 4.
6. The host cell of claim 5, wherein the host cell is E.coli.
7. The host cell of claim 6, wherein the E.coli is a competent cell of Ecoli BL21, Rosetta-gami, OrigamiB, or Rosetta series.
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