CN106893698A - One kind restructuring Taq archaeal dna polymerases and its encoding gene and expression - Google Patents
One kind restructuring Taq archaeal dna polymerases and its encoding gene and expression Download PDFInfo
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Abstract
The present invention relates to one kind restructuring ZHB Taq archaeal dna polymerases and its encoding gene and expression, the amino acid sequence such as SEQ ID NO of the ZHB Taq archaeal dna polymerases：Shown in 1, the base sequence such as SEQ ID NO of its encoding gene：Shown in 2.The archaeal dna polymerase that the present invention is provided, with superpower heat endurance and freeze-thaw stability, and the efficient soluble-expression of ZHB Taq archaeal dna polymerase encoding genes energy in Escherichia coli, the ZHB Taq archaeal dna polymerases of higher degree can be obtained by simple purifying process, a kind of reaction buffer and ZHB Taq archaeal dna polymerase kits are provided simultaneously, enables to the sensitivity of ZHB Taq DNA polymerase reactions, amplification efficiency and the fidelity to be all improved.
Description
Technical field
The invention belongs to bioengineering gene field, it is related to a kind of restructuring Taq archaeal dna polymerases and its encoding gene, and its expression, purifying, reaction buffer formula and the application in various nucleic acid amplifications.
Background technology
Archaeal dna polymerase is the crucial sex factor for completing PCR (PCR) process, and the related gene that more than 100 archaeal dna polymerase is had at present is cloned and is sequenced.BROCK in 1969 has isolated Taq archaeal dna polymerases from Thermus aquaticus YT-1, Thermas aquaticus are a kind of Thermus aquaticuas, and the archaeal dna polymerase isolated from the bacterium (Taq archaeal dna polymerases) has function of the catalysis with DNA as templated synthesis DNA.Saiki in 1988 goes out a kind of new Taq archaeal dna polymerases from another Thermus aquaticus YT-1 strain isolation, the discovery of Taq archaeal dna polymerases enables that PCR is widely used, although merchandized handling has been for many years for Taq archaeal dna polymerases, and the crystal structure and the mechanism of action of the enzyme are also clearer, but in order to improve soluble-expression amount of the albumen in Escherichia coli, the trial for simplifying purifying process never has stopping.
Lawyer etc. expresses the albumen using Lac promoters, but expression quantity is very low.Engelke etc. has cloned Taq DNA polymerase genes from T.aquaticus, in recombinating pTTQ18 plasmids, it is transformed into expression in escherichia coli, but yield is relatively low, and purification step will be precipitated by thermal denaturation foreign protein, polyethyleneimine (PEI), then restructuring Taq archaeal dna polymerase sterlings are obtained through the ion-exchange chromatographies of Bio Rex 70.In actual application, Taq archaeal dna polymerases heat endurance, freeze-thaw stability be not good, and the structure, purity and concentration requirement to primer and sample are higher, and reaction sensitivity is relatively low, and amplification efficiency is relatively low, limit using and promoting for Taq archaeal dna polymerases.
The content of the invention
In view of this, the purpose of the present invention is to solve the shortcomings of the prior art, a kind of encoding gene of archaeal dna polymerase is provided, its archaeal dna polymerase for encoding out has superpower heat endurance and freeze-thaw stability, the efficient soluble-expression of Taq archaeal dna polymerase encoding genes energy in Escherichia coli, the Taq DNA polymerase of higher degree can be obtained by simple purifying process, there is provided a kind of reaction buffer simultaneously, the Taq DNA polymerase reaction sensitivity is enabled to, amplification efficiency and fidelity are all improved.
The invention provides one kind restructuring Taq archaeal dna polymerases (hereinafter referred to as " ZHB Taq archaeal dna polymerases "), its amino acid sequence such as SEQ ID NO：Shown in 1.
The invention provides the gene for encoding ZHB Taq archaeal dna polymerases described above, its base sequence such as SEQ ID NO：Shown in 2.The sequence is to aim at escherichia expression system to carry out the sequence that codon optimization is obtained, and can significantly improve expression efficiency of the heterologous gene in Host Strains.
Present invention also offers the carrier of the gene for containing coding ZHB Taq archaeal dna polymerases described above, described carrier is preferably prokaryotic expression carrier pET21b or pDEST14, particularly preferably pET21b as the ZHB efficient soluble-expressions of Taq archaeal dna polymerases carrier.
Present invention also offers the escherichia coli host strain for including carrier described above, preferably, the host strain be selected from e. coli bl21 (DE3) or BL21 (AI) bacterial strain, particularly preferably BL21 (DE3) as the ZHB efficient soluble-expressions of Taq archaeal dna polymerases host strain.
Present invention also offers ZHB Taq archaeal dna polymerases in Escherichia coli efficient soluble-expression method, comprise the following steps：
1. picking one contains recombination bacillus coli single bacterium colony described above, LB nutrient solutions is accessed, in 37 DEG C of overnight incubations；
2. take 5mL overnight cultures access TB or LB nutrient solutions in, in 37 DEG C of concussion and cultivates to mid-log phase (A600=1.0)；
3. IPTG to 1mmol/L is added in culture, in 25 DEG C of overnight induced expressions, the coli somatic containing ZHB Taq archaeal dna polymerases is collected with 6000rpm centrifugations 15min 4 DEG C and is precipitated.
Contain ampicillin 50-100 μ g/mL in TB the or LB nutrient solutions.
Present invention also offers the purification process of ZHB Taq archaeal dna polymerases, comprise the following steps：
1. precipitated what collection was obtained containing induction ZHB Taq archaeal dna polymerases coli somatic, the PBS with precooling is resuspended, and in 4 DEG C of high speed centrifugations treatment.
2. supernatant is sucked, and every gram of thalline adds combination buffer BufferA 3-10ml, and agitation hangs thalline.
3. it is the PMSF of 100mmol/L that every gram of thalline adds 3-10 μ L concentration, and 3-100 μ L concentration is the lysozyme of 100mg/mL, is stirred on ice.
4. thalline is crushed, the ultrasound in ice bath, and supernatant is collected with 12000rpm high speed centrifugations 5min in 4 DEG C.
5. centrifuged supernatant is in 75 DEG C of water-bath insulation 1h, 12000rpm centrifugation 20min removal precipitations.By supernatant bacterial lysate in 0.22um membrane filtrations.
6.IMAC affinity chromatographys, merge in each collection peak sample and standby with 0.22 μm of membrane filtration.
7., further with gel permeation chromatography, required purpose product is obtained.
Preferably, the IMAC affinity chromatographys, the combination buffer BufferA of selection：20mM NaH2PO4, 20mM imidazoles, 300mM NaCl, pH=7.5, elution buffer BufferB：20mM NaH2PO4, 300mM NaCl, 500mM imidazoles, pH=7.5；The gel permeation chromatography, the combination of selection and elution buffer are BufferC:20mM NaH2PO4, 150mM NaCl, pH=7.5,0.22 μm membrane filtration is standby.
Present invention also offers the 10 × reaction buffer BufferD formulas that can be applied to PCR amplifying target genes：100mM Tris-HCl (25 DEG C of pH 8.8at), 500mM KCl, 15mM MgCl2, 0.8% (v/v) Nonidet P40.
Present invention also offers 2 × reaction buffer BufferE formulas that ZHB 2 × Taq archaeal dna polymerase kits are obtained after ratio optimization：20mM Tris-HCl (25 DEG C of pH 8.8at), 100mM KCl, 3mM MgCl2, 0.2% (v/v) Nonidet P40,0.05% (v/v) bromophenol blue, the glycerine of 5~10% (v/v), 400 μM of dNTPs, the ZHB Taq archaeal dna polymerases described in 2.5U/ μ L.The Taq archaeal dna polymerase kits aim at PCR identifications and are optimized, and amplification efficiency is good：Amplification length can carry out efficient amplification up to 8kb to the fragment of 4kb and its following length；Detection sensitivity is high：Specific gene fragment can be amplified from 0.05ng human gene group DNA's templates；It is easy to use, need to only be reacted adding template and primer and being diluted to 1 times of concentration and can enter performing PCR, operating process is significantly simplify, reduce the error in PCR operating process.Simultaneously after the completion of PCR reactions can direct electrophoresis, it is time-consuming.
Brief description of the drawings
Fig. 1 represents nucleotide sequence comparison before and after Taq archaeal dna polymerase codon optimizations
Wherein, odd-numbered line (i.e. " optimization presequence " corresponding row) is Taq archaeal dna polymerase natural gene nucleotide sequences, i.e. sequence before codon optimization；Even number line (i.e. " sequence after optimization " corresponding row) is the gene nucleotide series of ZHB Taq archaeal dna polymerases of the invention, i.e. sequence after codon optimization.
Fig. 2-a, Fig. 2-b are the front and rear CAI indexes in Bacillus coli expression host of Taq archaeal dna polymerases codon optimization.
Wherein, Fig. 2-a represent Taq archaeal dna polymerase natural gene nucleotides sequences be listed in Bacillus coli expression host in CAI indexes be calculated as 0.57 by program；Fig. 2-b represent that the CAI indexes in Bacillus coli expression host of the ZHB Taq archaeal dna polymerases codon of the invention after optimization are calculated as 0.91 by program.
Fig. 3-a, Fig. 3-b are the front and rear optimal codon frequency distribution administrative division map in Bacillus coli expression host of Taq archaeal dna polymerases codon optimization.
Wherein, Fig. 3-a represent that Taq archaeal dna polymerase natural gene nucleotides sequences are listed in optimal codon frequency distribution administrative division map in Bacillus coli expression host, as can be seen from the figure：It is 18% that percentage occurs in the poor efficiency codon of Taq archaeal dna polymerase natural gene nucleotide sequences；Fig. 3-b represent the optimal codon frequency distribution administrative division map in Bacillus coli expression host of the ZHB Taq archaeal dna polymerases codon of the invention after optimization, and it is 0 that percentage occurs in the poor efficiency codon of the ZHB Taq archaeal dna polymerase Codon sequences of the invention after optimization.
Fig. 4-a, Fig. 4-b are GC base contentses distributed areas figure average in Bacillus coli expression host before and after the codon optimization of Taq archaeal dna polymerases.
Wherein, Fig. 4-a represent that Taq archaeal dna polymerase natural gene nucleotides sequences are listed in Bacillus coli expression host average GC base contentses and are：67.95%；Fig. 4-b represent that the average GC base contentses in Bacillus coli expression host of the ZHB Taq archaeal dna polymerases codon of the invention after optimization are：57.29%.
Fig. 5 is the agarose gel electrophoresis figure of ZHB Taq DNA polymerase gene PCR primers.
Wherein, swimming lane 1 is 500bp DNA Ladder；Swimming lane 2 contains the restructuring Taq DNA polymerase gene PCR primers of NdeI and XhoI restriction enzyme sites for two ends.
Fig. 6-a, Fig. 6-b are ZHB Taq archaeal dna polymerase expression plasmid building process figures.
Wherein, Fig. 6-a are building up to expression plasmid pET21b procedure charts for ZHB Taq archaeal dna polymerases；Fig. 6-b are building up to expression plasmid pDEST14 procedure charts for ZHB Taq archaeal dna polymerases.
Fig. 7-a, Fig. 7-b are PAGE gel electroresis appraisal figure before and after the induction of ZHB Taq archaeal dna polymerases.
Wherein, Fig. 7-a swimming lanes 1 are the albumen loading Marker of the pre-dyed of (10-230kDa) wide scope；Swimming lane 2 is ZHB Taq archaeal dna polymerase recombination bacillus coli BL21 (DE3) lysate for not adding IPTG induced expressions；Swimming lane 3 is ZHB Taq archaeal dna polymerase recombination bacillus coli BL21 (DE3) lysate of addition IPTG induced expressions.
Wherein, Fig. 7-b swimming lanes 1 are the albumen loading Marker of the pre-dyed of (10-230kDa) wide scope；Swimming lane 2 is ZHB Taq archaeal dna polymerases e. coli bl21 (AI) lysate for not adding arabinose induced expression；Swimming lane 3 is ZHB Taq archaeal dna polymerases e. coli bl21 (AI) lysate of addition arabinose induced expression.
The PAGE gel electrophoretogram of the different recombinant bacterial strain induced expression ZHB Taq archaeal dna polymerases of Fig. 8 IMAC affinity purifications.
Wherein, Fig. 8-a are the PAGE gel electrophoretogram that IMAC affinity purifications restructuring BL21 (DE3) bacterial strain inducing expresses ZHB Taq archaeal dna polymerases.Swimming lane 1 is the albumen loading Marker of the pre-dyed of (10-230kDa) wide scope；Swimming lane 2-12 is the different collecting pipe samples collected when buffer B ufferB is eluted.
Wherein, Fig. 8-b are the PAGE gel electrophoretogram that IMAC affinity purifications restructuring BL21 (AI) bacterial strain inducing expresses ZHB Taq archaeal dna polymerases.Swimming lane 1 is the albumen loading Marker of the pre-dyed of (10-230kDa) wide scope；Swimming lane 2-12 is the different collecting pipe samples collected when buffer B ufferB is eluted.
The PAGE gel electrophoretogram of gel-filtration purified recombinant bacterial strain BL21 (DE3) the induced expression ZHB Taq archaeal dna polymerases of Fig. 9 Superdex75.
Wherein, swimming lane 1 is the albumen loading Marker of the pre-dyed of (10-230kDa) wide scope；Swimming lane 2-8 is the different collecting pipe samples collected when buffer B ufferC is eluted.
Figure 10 is DNA sequence dna agarose gel electrophoresis figure in the human genome of recombinant Taq DNA polymerase amplification different fragments.
Wherein, swimming lane 1 is 500bp DNA Ladder；Swimming lane 2-7 is DNA fragmentation in ZHB Taq archaeal dna polymerases amplification 1.0-1.5kb human genomes.
Figure 11-a and Figure 11-b are DNA sequence dna agarose gel electrophoresis figure in the ZHB Taq archaeal dna polymerases amplification human genome of high temperature incubation different time.
Wherein, Figure 11-a swimming lanes 1 are 500bp DNA Ladder；Swimming lane 2 is not expand human genome 1.5kb DNA fragmentations by the ZHB Taq archaeal dna polymerases of high temperature incubation；Swimming lane 3 is 1 hour ZHB Taq archaeal dna polymerases amplification human genome 1.5kb DNA fragmentation of high temperature incubation；Swimming lane 4 is 2 hours ZHB Taq archaeal dna polymerases amplification human genome 1.5kb DNA fragmentations of high temperature incubation；Swimming lane 5 is 3 hours ZHB Taq archaeal dna polymerases amplification human genome 1.5kb DNA fragmentations of high temperature incubation；Swimming lane 6 is 4 hours ZHB Taq archaeal dna polymerases amplification human genome 1.5kb DNA fragmentations of high temperature incubation；Swimming lane 7 is 5 hours ZHB Taq archaeal dna polymerases amplification human genome 1.5kb DNA fragmentations of high temperature incubation；Swimming lane 8 is 6 hours ZHB Taq archaeal dna polymerases amplification human genome 1.5kb DNA fragmentations of high temperature incubation.
Figure 11-b swimming lanes 1 are 500bp DNA Ladder；Swimming lane 2 is not expand human genome 1.0kb DNA fragmentations by the ZHB Taq archaeal dna polymerases of high temperature incubation；Swimming lane 3 is 1 hour ZHB Taq archaeal dna polymerases amplification human genome 1.0kb DNA fragmentation of high temperature incubation；Swimming lane 4 is 2 hours ZHB Taq archaeal dna polymerases amplification human genome 1.0kb DNA fragmentations of high temperature incubation；Swimming lane 5 is 3 hours ZHB Taq archaeal dna polymerases amplification human genome 1.0kb DNA fragmentations of high temperature incubation；Swimming lane 6 is 4 hours ZHB Taq archaeal dna polymerases amplification human genome 1.0kb DNA fragmentations of high temperature incubation；Swimming lane 7 is 5 hours ZHB Taq archaeal dna polymerases amplification human genome 1.0kb DNA fragmentations of high temperature incubation；Swimming lane 8 is 6 hours ZHB Taq archaeal dna polymerases amplification human genome 1.0kb DNA fragmentations of high temperature incubation.
Figure 12 is that the ZHB Taq archaeal dna polymerases amplification of the different number of times of alternate freezing and thawing treatment contains 0.5kb DNA sequence dna coli strain agarose gel electrophoresis figures in restructuring human genome.
Swimming lane 1 is 500bp DNA Ladder；Swimming lane 2 is that the ZHB Taq archaeal dna polymerases amplification not processed by alternate freezing and thawing contains 0.5kbDNA sequences in restructuring human genome；Swimming lane 3-22 is that the ZHB Taq Taq DNA polymerases amplification for processing 1-20 times by alternate freezing and thawing contains 0.5kb DNA sequence dnas in restructuring human genome.
The amplification curve diagram of Figure 13 difference Taq archaeal dna polymerases EGFR different mutants genes in ABI7500 real-time fluorescence quantitative PCRs instrument amplification human genome
Figure 14 is that ZHB Taq archaeal dna polymerases obtained 2 × Taq of ZHB archaeal dna polymerases kit after ratio optimization expands DNA sequence dna agarose gel electrophoresis figure in human genome.
Wherein, swimming lane 1 is 500bp DNA Ladder；Swimming lane 2 be ZHB 2 × Taq archaeal dna polymerase kit reaction buffers in contain 10% glycerine amplification human genome in 1.5kb DNA fragmentations；Swimming lane 3 be ZHB 2 × Taq archaeal dna polymerase kit reaction buffers in contain 5% glycerine amplification human genome in 1.5kb DNA fragmentations；Swimming lane 4 be ZHB 2 × Taq archaeal dna polymerase kit reaction buffers in contain 10% glycerine amplification human genome in 1.0kb DNA fragmentations；Swimming lane 5 be ZHB 2 × Taq archaeal dna polymerase kits in contain 5% glycerine amplification human genome in 1.0kb DNA fragmentations.
Figure 15 is that ZHB 2 × Taq archaeal dna polymerases kit amplification of the different number of times of alternate freezing and thawing treatment contains 0.5kb DNA sequence dna recombinant escherichia coli strain agarose gel electrophoresis figures in human genome.
Swimming lane 1 is 500bp DNA Ladder；Swimming lane 2 is that the ZHB 2 × Taq archaeal dna polymerases kit amplification not processed by alternate freezing and thawing contains 0.5kb DNA sequence dna recombinant escherichia coli strains in human genome；Swimming lane 3-22 is that the ZHB 2 × Taq archaeal dna polymerases kit amplification for processing 1-20 times by alternate freezing and thawing contains 0.5kb DNA sequence dna recombinant escherichia coli strains in human genome.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further, it should be appreciated that quote embodiment and be only illustrative of the invention and is not intended to limit the scope of the invention.
Embodiment
1 Taq DNA
Pol gene optimization design
CDNA sequence (GenBank accession number of the inventor according to the published Taq archaeal dna polymerases of GenBank：BAA06775.1), ZHB Taq DNA polymerase genes of the invention, such as SEQ ID No are obtained after codon optimization is carried out to the gene：Shown in 2.
Here is that codon optimization is carried out to Taq archaeal dna polymerases, and each parameter comparison is as follows before and after optimization：
1. codon adaptation indexI (Codon Adaptation Index, CAI)
From Fig. 2-a, before codon does not optimize, Taq archaeal dna polymerase natural genes codon adaptation indexI (CAI) in Escherichia coli is 0.57.From Fig. 2-b, by after codon optimization so that ZHB Taq DNA polymerase genes of the invention CAI indexes in Escherichia coli are 0.91.It is optimal high efficient expression state in the expression system to be considered as the gene during usual CAI=1, CAI indexes are lower to show that gene expression in the host is poorer, it can be seen that the gene order obtained after have passed through codon optimization can improve expression of the Taq DNA polymerase genes in Escherichia coli.
2. optimal codon frequency of use (Frequency of Optimal Codons, FOP)
From Fig. 3-a, based on coli expression carrier, before codon does not optimize, it is 18% that percentage occurs in the poor efficiency codon of Taq archaeal dna polymerase native gene sequences.This gene being not optimized contains series connection rare codon, and these codons may reduce translation efficiency, or even can dismiss translation assemblage.From Fig. 3-b, by the way that after codon optimization, the frequency that poor efficiency codon occur in E. coli system in ZHB Taq DNA polymerase genes of the invention is 0.
3.GC base contentses (GC curve)
G/C content ideal distribution region is 30%-70%, and any peak of appearance outside this region all can to some extent influence transcription and translation efficiency.Contrasted from the GC base average contents distributed areas figure of Fig. 4-a, the Taq DNA polymerase genes of Fig. 4-b, by showing that GC bases average content is 67.95% before optimization in Taq archaeal dna polymerase natural genes in Fig. 4-a, by showing the G/C content all bases outside 30%-70% regions of the sequence elimination after optimization in Fig. 4-b, the GC bases average content for finally giving ZHB Taq archaeal dna polymerases after optimizing is 57.29%.
Embodiment
2 ZHB Taq DNA
The expression vector establishment of pol gene
ZHB Taq archaeal dna polymerases full genomes after optimization are introduced into NdeI restriction enzyme site sequences at 5 ' ends, histidine-tagged and XhoI restriction enzyme sites sequence (such as SEQ ID No are introduced at 3 ' ends：Shown in 2), and full genome synthesis is carried out, the genetic fragment that will synthesize is building up in pUC57 plasmids (being provided by Nanjing Jin Sirui Science and Technology Ltd.s), obtains a kind of long-term preservation plasmid, is designated as pUC57-ZHB Taq DNA plasmids.With pUC57-ZHB Taq DNA plasmids as template, enter performing PCR amplification, the primer sequence is as follows：
Sense primer：
M13F:CGC CAG GGT TTT CCC AGT CAC GAC
Anti-sense primer：
M13R:AGC GGA TAA CAA TTT CAC ACA GGA
The reaction μ L of cumulative volume 50, wherein concentration respectively add 2.5 μ L for 10 μm of ol/L primers, and concentration adds 1 μ L for the dNTP of 10mmol/L, and archaeal dna polymerase used is Q5 (being purchased from New England Biolabs companies), 2U/ μ L, plus 0.5 μ L.Reaction condition be 98 DEG C 5 seconds, 55 DEG C 45 seconds, 72 DEG C 30 seconds, 25 circulation after, product analyze through 1.0% agarose gel electrophoresis, as a result show primer size with expection size (2500bp) it is consistent (result is as shown in Figure 5).
Expression vector establishment of the scheme 1.ZHB Taq DNA polymerase genes in pET21b
The gene outcome that will be obtained DNA gel QIAquick Gel Extraction Kit (being purchased from Beijing Tiangeng biochemical technology Co., Ltd) purifying.After purification, with NdeI and XhoI (being purchased from New England Biolabs companies) double digestion, it is connected in pET21b plasmids (purchased from Merck companies) carrier with T4 ligases (being purchased from New England Biolabs companies), it is transformed into DH5 α competent cells (purchased from Beijing Tiangeng biochemical technology Co., Ltd), 37 DEG C of overnight incubations in the LB flat boards of the ampicillin (being purchased from Amresco companies) containing 100 μ g/mL.Screening positive clone bacterium sequencing in second day, compares, completely the same with expected sequence, that is, obtain ZHB a form of expression vectors of Taq archaeal dna polymerases, is designated as pET21b-ZHB Taq DNA (plasmid construction flow is as shown in Fig. 6-a).
Expression vector establishment of the scheme 2.ZHB Taq DNA polymerase genes in pDEST14
The gene outcome that will be obtained DNA gel QIAquick Gel Extraction Kit (being purchased from Beijing Tiangeng biochemical technology Co., Ltd) purifying.After purification, with NdeI and XhoI (being purchased from New England Biolabs companies) double digestion, it is connected to T4 ligases (being purchased from New England Biolabs companies) and introduces NdeI restriction enzyme sites behind attR1 sites respectively, it is introduced into behind attR2 sites in the pDEST14 plasmids of XhoI restriction enzyme sites (purchased from Invitrogen companies), it is transformed into DH5 α competent cells (purchased from Beijing Tiangeng biochemical technology Co., Ltd), 37 DEG C of overnight incubations in the LB flat boards of the ampicillin (being purchased from Amresco companies) containing 100 μ g/mL.Screening positive clone bacterium sequencing in second day, compares, completely the same with expected sequence, that is, obtain recombinating ZHB a form of expression vectors of Taq archaeal dna polymerases, is designated as pDEST14-ZHB Taq DNA (plasmid construction flow is as shown in Fig. 6-b).
Embodiment
3 ZHB Taq DNA
Expression and identification of the polymerase in recombination bacillus coli
Induced expression identification of the scheme 1.ZHB Taq archaeal dna polymerases in BL21 (DE3)
Comprise the following steps that：
1) will be sequenced in the step 1 of embodiment 2 and compare correct pET21b-ZHB Taq DNA plasmids and be transformed into e. coli bl21 (DE3) competent cell in (purchased from Beijing Tiangeng biochemical technology Co., Ltd), incubated overnight in 37 DEG C of ampicillin plates.
2) chooses the 1-4 restructuring single bacterium colony containing pET21b-ZHB Taq DNA plasmids for second day, accesses the LB nutrient solutions containing 100 μ g/mL ampicillins, 37 DEG C of overnight incubations.
3) take 25mL overnight cultures access 500mL containing 100 μ g/mL ampicillins TB nutrient solutions in, 37 DEG C of shaken cultivations.
4) bacterium solution OD was surveyed every 1 hour after inoculations600Value, treats OD600During ≈ 1.0, induced expression is carried out with the IPTG (being purchased from Amresco companies) of 1mmol/L.Negative control is done with the E. coli broth for not adding IPTG simultaneously.
5) bacterium solution is collected after .4 hours, 12000rpm high speed centrifugation 3min are precipitated with the PBS of precooling, adds 5 × sds gel sample-loading buffer, 100 DEG C of heating 5min, 12000rpm high speed centrifugation 1min take supernatant.The E. coli broth of IPTG is not added by this step process yet.
6) respectively takes 5 μ L and does not add IPTG and add the culture suspension of IPTG inductions, 10%SDS-PAGE gel electrophoresis analysis.
7) .8-15V/cm electrophoresis, separation gel bottom is moved to bromophenol blue.
8) coomassie brilliant blue stainings, observe expression product band, see Fig. 7-a.
Induced expression identification of the scheme 2.ZHB Taq archaeal dna polymerases in BL21 (AI)
Comprise the following steps that：
1) will be sequenced the correct pDEST14-ZHB Taq DNA plasmids of comparison and be transformed into e. coli bl21 (AI) competence bacterial strain (purchased from Invitrogen companies) in the step 2 of embodiment 2, incubated overnight in 37 DEG C of ampicillin plates.
2) chooses the 1-4 restructuring single bacterium colony containing pDEST14-ZHB Taq DNA plasmids for second day, accesses the LB nutrient solutions containing 100 μ g/mL ampicillins, 37 DEG C of overnight incubations.
3) take 25mL overnight cultures access 500mL containing 100 μ g/mL ampicillins LB nutrient solutions in, 37 DEG C of shaken cultivations.
4) bacterium solution OD was surveyed every 1 hour after inoculations600Value, treats OD600During ≈ 1.0, induced expression is carried out with the arabinose (being purchased from Amresco companies) of 0.2% (V/V).Negative control is done with the E. coli broth for not adding arabinose simultaneously.
5) collects bacterium solution after 4 hours, and 12000rpm high speed centrifugation 3min are precipitated with the PBS of precooling, adds 5 × sds gel sample-loading buffer, and 100 DEG C of heating 5min, 12000rpm high speed centrifugation 1min take supernatant.The E. coli broth that arabinose is induced is not added by this step process yet.
6) respectively takes 5 μ L and does not add arabinose and add the culture suspension of arabinose induction, 10%SDS-PAGE gel electrophoresis analysis.
7) 8-15V/cm electrophoresis, separation gel bottom is moved to bromophenol blue.
8) coomassie brilliant blue stainings, observe expression product band, see Fig. 7-b.
Embodiment
4 ZHB Taq DNA
Polymerization enzyme purification
Step 1. recombinates bacterial cell disruption
1) recombinates BL21 (AI) thalline during BL21 (DE3) thalline or the step 5 of scheme 2 will be recombinated in the scheme 1 of embodiment 3, the Escherichia coli precipitation containing induction ZHB Taq archaeal dna polymerases that pre-cooled PBS is obtained respectively, PBS with precooling is resuspended, and 15min is centrifuged with 12000rpm in 4 DEG C；It is repeated once.
2) sucks supernatant, claims bacterial sediment weight, and every gram of thalline adds 5mL combination buffer BufferA, stirred with polished glass rod, hangs thalline.
3) every gram of thalline of adds 5 μ L 100mmol/L PMSF, 5 μ L 100mg/mL lysozymes to stir 20min on ice.
4) crushes thalline with probe type ultrasonication ripple instrument, and sample is placed on ice, ultrasound 120 times, is spaced 5 seconds within 5 seconds every time, circulates three times, and each intercycle waits 5min.12000rpm, 4 DEG C of high speed centrifugation 15min.
5) will be centrifuged the supernatant that obtains respectively at being incubated 1h in 75 DEG C of water-baths, 12000rpm centrifugation 20min removal precipitations.Cross 0.22 μm of membrane filtration standby.
Step 2.IMAC affinity purification bacterial lysates
1) chromatographic column that .IMAC affinity chromatographys are selected is HisTrap crude FF 5mL (purchased from GE healthcare), combination buffer BufferA：20mM NaH2PO4, 20mM imidazoles, 300mM NaCl, pH=7.5, elution buffer BufferB：20mM NaH2PO4, 300mM NaCl, 500mM imidazoles, pH=7.5,0.22 μm of membrane filtration is standby.
2) during HisTrap FF crude 5mL are accessed AKTA avant150 purifying instrument post position valve by, successively with distilled water cleaning robot and pillar after, then with combination buffer BufferA balance pillars.And loading is carried out to the restructuring BL21 (DE3) and BL21 (AI) bacterial lysate that are processed in step 1 with sample pump.After having gone up sample, pillar is first cleaned with combination buffer BufferA, then eluted with elution buffer BufferB and collected eluting peak.
3) sample of each collecting pipe that is collected to IMAC affinity chromatographys carries out SDS-PAGE proteins gel electrophoresis detections, as shown in Fig. 8-a and 8-b, the amount of the ZHB Taq archaeal dna polymerases of recombinant bacterial strain BL21 (DE3) induction soluble-expressions is far above restructuring BL21 (AI) bacterial strain.
The gel permeation chromatographies of step 3.Superdex 75
1) chromatographic column that gel permeation chromatographies are selected is HiLoad 16/60superdex75pg (purchased from GE healthcare), and buffer B ufferC is 20mM NaH2PO4, 150mM NaCl, pH=7.5,0.22 μm membrane filtration is standby.
2) during Superdex75 gel permeation chromatographies post is accessed AKTA avant150 purifying instrument post position valve by, successively with distilled water cleaning robot and pillar after, then balance pillar with buffer B ufferC, each loading cumulative volume is no more than the 5% of column volume.After having gone up sample, eluted with BufferC, and collect eluting peak.
3) carries out PAGE gel electrophoresis detection to each collecting pipe sample that Superdex75 gel permeation chromatographies are collected (result is as shown in Figure 9).
Embodiment
5 ZHB Taq DNA
Application of the polymerase in nucleic acid amplification
The ZHB Taq archaeal dna polymerases that embodiment 4 is prepared are applied to nucleic acid amplification.First, test the reaction condition of the ZHB Taq archaeal dna polymerases in nucleic acid amplification, using in human genome 1.0kb and 1.5kb genetic fragments as template, PCR instrument (Applied Biosystems,96-Well Thermal Cycler) expand the optimum pH and K for determining its reaction+、Mg2+Ion concentration.Prepare 10 × PCR initial reaction buffer solutions:100mmol/L Tris-HCl, pH value is 7.4~8.8,15mmol/L MgCl2Various concentrations KCl, Nonidet P40, Triton X-100 are added respectively, reaction system is 50 μ L, the wherein μ L of 10 × PCR reaction buffers 5 containing different condition, the dNTPs1 μ L of 10mmol/L, each 1 μ l of upstream and downstream primer of 10 μm of ol/L, the μ L of ZHB Taq archaeal dna polymerases 1 (2.5U/ μ L), the μ L of template 0.5 of 50ng/ μ L, by 94 DEG C of 1min, (94 DEG C of 15s, 55 DEG C of 45s, 72 DEG C of 1min) 25cycles, last 72 DEG C extend 5min, and electrophoresis detection determines its optimal reaction buffer solution.According to result, determine that ZHB Taq DNA PCR optimal reaction buffer Bs ufferD (10 × ZHB Taq DNA PCR buffer) are：100mM Tris-HCl (25 DEG C of pH 8.8at), 500mM KCl, 15mM MgCl2, 0.8% (v/v) Nonidet P40.
The ZHB Taq archaeal dna polymerases that above-described BufferD and embodiment 4 are prepared into expand the DNA sequence dna of human genome different fragments respectively, and agarose gel electrophoresis result as shown in Figure 10, can well be expanded and obtain purpose fragment.Above-mentioned PCR primer is delivered into Nanjing Jin Sirui Science and Technology Ltd.s carries out DNA sequence dna sequencing, and sequence alignment result shows, only one of which coding mutation, fidelity has reached 99.3%.
Embodiment
6 ZHB Taq DNA
Polymerization enzyme heat stability
The ZHB Taq archaeal dna polymerases that embodiment 4 is prepared are in 90 DEG C of high temperature incubations 0,1,2,3,4,5,6h；And the ZHB Taq archaeal dna polymerases of the different time obtained after incubation are surveyed into its people with BufferD in embodiment 5 in PCR instrument activity is expanded in genome 1.0-1.5kb DNA fragmentations, 1% agarose gel electrophoresis detects its expanding effect.Result is as shown in Figure 11-a and Figure 11-b, swimming lane 2-8 is ZHB Taq archaeal dna polymerases in after 90 DEG C of high temperature incubations 0,1,2,3,4,5,6h, amplification target DNA fragment is carried out with this polymerase respectively, after ZHB Taq archaeal dna polymerases are incubated 5h in 90 DEG C of high temperature, still amplify genes of interest, illustrate that ZHB Taq archaeal dna polymerases have heat endurance higher, its 90 DEG C of half-life period are 5h.
Embodiment
7 ZHB Taq DNA
Polymerase freeze-thaw stability
The ZHB Taq archaeal dna polymerases kits that embodiment 4 is prepared are freezed with dry ice, 37 DEG C of water-bath dissolvings carry out alternate treatment 20 times, and the ZHB Taq archaeal dna polymerases of the different number of times for obtaining alternate treatment survey it in PCR instrument and activity is expanded in human genome 0.5kb DNA fragmentations are expanded with the BufferD in embodiment 5,1% agarose gel electrophoresis detects its expanding effect.Result ZHB Taq archaeal dna polymerases as shown in figure 12 freeze by dry ice, can well expand after 37 DEG C of water-bath dissolving alternate treatments 20 times and obtain purpose fragment, ZHB Taq archaeal dna polymerases are illustrated by multigelation more than 20 times, still with stability higher.
Embodiment
8 ZHB Taq DNA
Polymerase is in fluorescent quantitation
PCR
In application
The ZHB Taq archaeal dna polymerases that embodiment 4 is prepared survey its different mutant activity of EGFR gene in amplification human genome in quantitative real time PCR Instrument (ABI 7500) with the BufferD in embodiment 5, while and with the AmpliTaq of ABIThe kit (DRR001A) of the EX Taq of DNA Polymerase kits (N8080241) and Takara uses each supporting reaction buffer, Mg as control, and when the preparation of performing PCR reaction solution is entered2+And dNTP is ensureing amplification efficiency.PCR reaction systems are 20 μ l, and each composition is respectively：ZHB Taq/ABI/Takara archaeal dna polymerases, 0.5 μ l, dNTP (10mM) 1 μ l, Mg2+(25mM) 1 μ l, 10 × buffer 2 μ l, the μ l of forward primer (1 μM) 1, the μ l of reverse primer (1 μM) 1, fluorescence probe (1 μM) 1 μ l, DNA profiling 50ng, ddH2O complements to 20 μ l.Each reagent is prepared into mixed liquor according to above-mentioned requirements, in packing to each PCR pipe, and lid is covered, is slightly centrifuged, with 95 DEG C of 10min, (95 DEG C of 10s, 60 DEG C of 1min) 40cycles, the program that fluorescence is collected at 60 DEG C is expanded.As shown in figure 13, the EGFR different mutant in amplification human genome in ABI quantitative real time PCR Instruments of ZHBTaq archaeal dna polymerases, various reaction amplification curve CT values are respectively less than other two kinds of enzymes to amplification, and fluorescent value is higher than other two kinds of enzymes；Illustrate that ZHB Taq archaeal dna polymerases have the activity of more preferable expanding effect and Geng Gao.
Embodiment
9 ZHB 2
×
Taq DNA
The preparation of polymerase kit
The ZHB Taq archaeal dna polymerases that embodiment 4 is prepared are applied to the preparation of ZHB 2 × Taq archaeal dna polymerase kits.With 1.0kb and 1.5kb genetic fragments in human genome as template, ZHB 2 × Taq archaeal dna polymerase kit optimum response buffer solutions are determined.ZHB 2 × Taq archaeal dna polymerase kit initial reaction buffer solutions are prepared first：20mmol/L Tris-HCl, pH value is 7.4~8.8,100mmol/L KCl, and various concentrations MgCl is then added respectively2, Nonidet P40, Triton X-100, glycerine etc., reaction system is 50 μ L, the wherein μ L of 2 × PCR reaction buffers 25 containing different condition, the dNTPs1 μ L of 10mmol/L, each 0.5 μ L of upstream and downstream primer of 10 μm of ol/L, the μ L of ZHB Taq archaeal dna polymerases 1 (2.5U/ μ L), by 94 DEG C of 1min, (94 DEG C of 15s, 55 DEG C of 45s, 72 DEG C of 1min) 25cycles, last 72 DEG C of extensions 5min, agarose electrophoresis electrophoresis detection (result is shown in Figure 14) determines optimal reaction buffer solution.According to result, determine that ZHB 2 × Taq archaeal dna polymerase kit optimal reaction buffer Bs ufferE is：20mM Tris-HCl (25 DEG C of pH 8.8at), 100mM KCl, 3mM MgCl2, 0.2% (v/v) Nonidet P40,0.05% (v/v) bromophenol blue, the glycerine of 5~10% (v/v), 400 μM of dNTPs, 2.5U/ μ L ZHB Taq archaeal dna polymerases.
Embodiment
10 ZHB 2
×
Taq DNA
Polymerase kit freeze-thaw stability
ZHB 2 × Taq archaeal dna polymerases kits that embodiment 9 is prepared are freezed with dry ice, 37 DEG C of water-bath dissolvings carry out alternate treatment 20 times, and ZHB 2 × Taq archaeal dna polymerases kits of the different number of times for obtaining alternate treatment survey it in PCR instrument and activity is expanded in 0.5kb DNA fragmentations in expanding human genome, 1% agarose gel electrophoresis detects its expanding effect.Result 2 × Taq of ZHB archaeal dna polymerases kit as shown in figure 15 freezes by dry ice, can well expand after 37 DEG C of water-bath dissolving alternate treatments 20 times and obtain purpose fragment, ZHB 2 × Taq archaeal dna polymerases kits are illustrated by multigelation more than 20 times, still with stability higher.
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as limitation of the present invention, protection scope of the present invention should be defined by claim limited range.For those skilled in the art, without departing from the spirit and scope of the present invention, some improvements and modifications can also be made, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (10)
1. it is a kind of to recombinate Taq archaeal dna polymerases, its amino acid sequence such as SEQ ID NO：Shown in 1.
2. a kind of encoding gene of archaeal dna polymerase as claimed in claim 1, its base sequence such as SEQ ID NO：Shown in 2.
3. a kind of carrier for containing encoding gene as claimed in claim 2.
4. carrier as claimed in claim 3 is pET21b or pDEST14.
5. a kind of coli strain comprising carrier as claimed in claim 4.
6. bacterial strain as claimed in claim 5 is e. coli bl21 (DE3) or BL21 (AI) bacterial strain.
7. a kind of expression of Taq archaeal dna polymerases, comprises the following steps：
The E. coli clones as claimed in claim 6 of step 1. picking one, access LB nutrient solutions, in 37 DEG C of overnight incubations；
Step 2. is taken in 5mL overnight cultures access TB or LB nutrient solutions, in 37 DEG C of concussion and cultivates to mid-log phase (A600=1.0)；
Step 3. adds IPTG to 1mmol/L in culture, and in 37 DEG C, overnight induced expression, is centrifuged in 4 DEG C with 6000rpm
15min collects the coli somatic precipitation containing Taq archaeal dna polymerases；
Contain ampicillin 50-100 μ g/mL in TB the or LB nutrient solutions.
8. a kind of purification process of the recombination expression of Taq archaeal dna polymerases, comprises the following steps：
Step 1. will collect the coli somatic precipitation containing induction Taq archaeal dna polymerases as claimed in claim 7 for obtaining,
PBS with precooling is resuspended, and in 4 DEG C of high speed centrifugation treatment；
Step 2. sucks supernatant, and every gram of thalline adds combination buffer BufferA 3-10ml, and agitation hangs thalline；
It is the PMSF of 100mmol/L that every gram of thalline of step 3. adds 3-10 μ L concentration, and 3-100 μ L concentration is molten for 100mg/mL's
Bacterium enzyme, stirs on ice；
Step 4. crushes thalline, the ultrasound in ice bath, and collects supernatant with 12000rpm high speed centrifugations 5min in 4 DEG C；
Step 5. centrifuged supernatant splits supernatant bacterium in 75 DEG C of water-bath insulation 1h, 12000rpm centrifugation 20min removal precipitations
Solution liquid is in 0.22um membrane filtrations；
Step 6.IMAC affinity chromatographys, merge in each collection peak sample and standby with 0.22 μm of membrane filtration；
Step 7. obtains required purpose product further with gel permeation chromatography；
The IMAC affinity chromatographys, the combination buffer BufferA of selection：20mM NaH2PO4, 20mM imidazoles, 300mM
NaCl, pH=7.5, elution buffer BufferB：20mM NaH2PO4, 300mM NaCl, 500mM imidazoles, pH=7.5；
The gel permeation chromatography, the combination of selection and elution buffer are BufferC:20mM NaH2PO4, 150mM NaCl,
PH=7.5,0.22 μm of membrane filtration is standby.
9. a kind of reaction buffer for being applied to PCR amplifying target genes, its formula：100mM Tris-HCl(pH 8.8at
25 DEG C), 500mM KCl, 15mM MgCl2, 0.8% (v/v) Nonidet P40.
10. a kind of Taq archaeal dna polymerases kit, its reaction buffer formula：20mM Tris-HCl (25 DEG C of pH 8.8at),
100mM KCl, 3mM MgCl2, 0.2% (v/v) Nonidet P40,0.05% (v/v) bromophenol blue, 5~10% (v/v)
Glycerine, 400 μM of dNTPs, and the Taq archaeal dna polymerase 2.5U/ μ L as described in any one of claim 1 to 9.
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