CN107828758A

CN107828758A - Recombinate ura DNA glycosidase and its encoding gene, preparation method and application

Info

Publication number: CN107828758A
Application number: CN201711112394.XA
Authority: CN
Inventors: 马永; 王安良; 赵百学
Original assignee: ZONHON BIOPHARMA INSTITUTE Inc
Current assignee: ZONHON BIOPHARMA INSTITUTE Inc
Priority date: 2017-11-13
Filing date: 2017-11-13
Publication date: 2018-03-23

Abstract

The invention belongs to bioengineering gene field, is related to a kind of restructuring ura DNA glycosidase and its encoding gene, and its expression, purifying and the application in various nucleic acid amplifications.Present invention restructuring ura DNA glycosidase encoding gene efficient soluble-expression of energy in Escherichia coli, can obtain the higher restructuring UNG enzymes of purity, and recombinate UNG enzymes to have preferable biological activity and larger application value by two-step purifying technique.

Description

Recombinate uracil-DNA glycosidase and its encoding gene, preparation method and application

Technical field

The invention belongs to bioengineering gene field, is related to a kind of restructuring uracil-DNA glycosidase and its encoding gene, And its expression, purifying and the application in various nucleic acid amplifications.

Background technology

In PCR operating process, it is easiest to " pollution " problem occur, reason mainly there are following several situations：1. template Cross pollution：In sample collection or processing, foam or other reasonses when aerosol, sample injector sample cause sample or template Cross pollution, cause false positive, belong to and pollute caused by normal DNA (by tetra- kinds of base compositions of A, T, G, C)；2. amplified production Pollution：In amplification procedure, base T is replaced as U so that containing dU-DNA (by tetra- kinds of bases of A, U, G, C in amplified production Composition), because product amount is especially big, easy spill and leakage causes pollution, causes false positive；3. the pollution of nuclease, protease causes False positive；It is the most seriously second in these three situations, that is, is referred to as：" residual contamination ", because the times magnification of PCR amplifications Number is up to thousands of times, and expands be repeated always in actual applications, causes the bulk deposition of amplified production, it is difficult to will Its control degraded, even quantitative fluorescent PCR also is difficult to avoid the pollution of amplified production, in order that PCR results it is relatively reliable, Accurately, it is necessary to reaction tube it is completely enclosed after in pipe by enzyme or other method, it is residual by what may be brought into operating process Stay pollutant to destroy, prevent it from the template as new round amplification.

UNG enzymes, i.e. uracil-DNA glycosidase (Uracil-DNA Glycocasylase).UNG action principle is choosing Selecting property hydrolytic cleavage contain dU double-strand or single stranded DNA in uracil glycosidic bond, formed with missing base DNA, in alkali Property medium and high temperature under can further hydrolytic cleavage, so as to be eliminated.UNG enzymes are one in intracellular DNA damage repair system The important component of kind, is widely present, including have UNG expression of enzymes in bacterium, eucaryote, people's cell in nature.

Currently used way is the addition UNG enzymes in PCR reaction systems, and substitutes dTTP, such PCR primer with dUTP All it is the DNA containing dUTP.Increase the incubation step of a step lower temperature before PCR starts, UNG enzymes can be by reaction system In uracil base degraded in the last round of amplified production containing U-DNA that may be present, and be then denatured this step Under the conditions of DNA be broken, eliminate due to pollution DNA caused by amplification, so as to ensure the specificity of amplification, accuracy.Together When UNG enzymes be inactivated, will not degrade the product U-DNA newly expanded again, thus can effectively prevent in laboratory expand production The pollution of thing, avoids the appearance of false positive, and this technology is called UNG/dUTP pollution prevention technologies.Presently commercially available UNG enzymes are main With technique for gene engineering prepare restructuring UNG enzymes based on, but in the prevalence of expression quantity it is not high or restructuring UNG enzymatic activitys it is not high The problem of.

The content of the invention

It is an object of the invention to provide a kind of encoding gene of UNG enzymes, its UNG enzyme encoded out has stronger work Property, it is higher can to obtain purity by two-step purifying technique for the UNG enzyme coding genes efficient soluble-expression of energy in Escherichia coli Restructuring UNG enzymes.

The invention provides one kind restructuring UNG enzymes (hereinafter referred to as " rUNG "), its amino acid sequence such as SEQ ID NO：1 It is shown.

The invention provides the gene for encoding rUNG enzymes described above, its base sequence such as SEQ ID NO：Shown in 2.The sequence Row are to aim at escherichia expression system to carry out the sequence that codon optimization obtains, and can significantly improve heterologous gene in Host Strains In expression efficiency.

Present invention also offers the carrier for the gene for containing coding rUNG described above, described carrier is preferably protokaryon The carrier of expression vector pET21b, pDEST14, pET28a, particularly preferably pET28a as the efficient soluble-expressions of rUNG.

Present invention also offers the escherichia coli host strain for including carrier described above, it is preferable that the Host Strains Select good strains in the field for seed and be used as the efficient soluble-expressions of rUNG from e. coli bl21 (DE3) or BL21 (AI) bacterial strain, particularly preferably BL21 (DE3) Host strain.

Present invention also offers rUNG 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 are accessed, in 37 DEG C, 220rpm Overnight incubation；

2. take in 10mL overnight cultures access TB or LB nutrient solutions, in 37 DEG C of concussion and cultivates to mid-log phase (A₆₀₀= 1.0)；

3. adding IPTG to 1mmol/L in culture, in 25 DEG C of overnight induced expressions, 4 DEG C are centrifuged with 12000rpm 3min collects the coli somatic precipitation containing rUNG enzymes.

Contain kanamycins 25-50 μ g/mL in TB the or LB nutrient solutions.

Present invention also offers the purification process of rUNG enzymes, comprise the following steps：

1. precipitated what collection obtained containing induction rUNG enzymes coli somatic, with the combination buffer of precooling BufferA is resuspended, and is handled in 4 DEG C of high speed centrifugations.

2. sucking supernatant, every gram of thalline adds combination buffer BufferA 3-10ml, agitation, hangs thalline.

3. every gram of thalline adds the PMSF that 3-10 μ L concentration is 100mmol/L, 3-100 μ L concentration is 100mg/mL bacteriolyze Enzyme, stirred on ice.

4. broken thalline, ultrasonic in ice bath, and supernatant is collected with 12000rpm high speed centrifugations 5min in 4 DEG C, 0.22 μm Membrane filtration is standby.

5.IMAC affinity chromatographys, merge sample and standby with 0.22 μm of membrane filtration in each collection peak.

6. required purpose product is further obtained with gel permeation chromatography.

Preferably, the IMAC affinity chromatographys, the combination buffer BufferA of selection：20mM Tris-HCl, 20mM miaows Azoles, 300mMNaCl, pH=8.0；Elution buffer BufferB：20mM Tris-HCl, 300mM NaCl, 300mM imidazoles, pH =8.0；The gel permeation chromatography, the combination of selection and elution buffer are BufferC:20mM Tris-HCl, 50mM NaCl, pH=8.0,0.22 μm of membrane filtrations are standby.

Brief description of the drawings

Fig. 1 is nucleotide sequence comparison figure before and after rUNG enzyme codon optimizations.

Wherein, odd-numbered line (i.e. row corresponding to " optimization presequence ") is rUNG enzyme natural gene nucleotide sequences, i.e. password Sequence before son optimization；Even number line (i.e. row corresponding to " sequence after optimization ") is the gene nucleotide sequence of the rUNG enzymes of the present invention Row, 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 rUNG enzymes codon optimization.

Wherein, Fig. 2-a represent that rUNG enzyme natural gene nucleotides sequences are listed in CAI indexes in Bacillus coli expression host and passed through Program is calculated as 0.53；Fig. 2-b represent that the CAI in Bacillus coli expression host of the rUNG enzymes codon of the invention after optimization refers to Number is calculated as 0.96 by program.

Fig. 3-a, Fig. 3-b are the front and rear optimal codon frequency point in Bacillus coli expression host of rUNG enzymes codon optimization Cloth administrative division map.

Wherein, Fig. 3-a represent that rUNG enzyme natural gene nucleotides sequences are listed in optimal codon in Bacillus coli expression host Frequency distribution administrative division map, as can be seen from the figure：The poor efficiency codon of rUNG enzyme natural gene nucleotide sequences occurs hundred Divide than being 25%；Fig. 3-b represent the optimal password in Bacillus coli expression host of the rUNG enzymes codon of the invention after optimization There is percentage and are in sub- frequency distribution administrative division map, the poor efficiency codon of the rUNG enzyme Codon sequences of the invention after optimization 0。

Fig. 4-a, Fig. 4-b are the front and rear GC base contentses point average in Bacillus coli expression host of rUNG enzymes codon optimization Cloth administrative division map.

Wherein, Fig. 4-a represent that rUNG enzyme natural gene nucleotides sequences are listed in Bacillus coli expression host average GC bases Content is：52.66%；Fig. 4-b represent that the rUNG enzymes codon of the invention after optimization is average in Bacillus coli expression host GC base contentses are：59.54%.

Fig. 5 is the agarose gel electrophoresis figure of rUNG enzyme gene PCR primers.

Wherein, swimming lane 1 is 1000bp DNA Ladder；Swimming lane 2 is the rUNG that NdeI and XhoI restriction enzyme sites are contained at both ends Enzyme gene PCR primer.

Fig. 6 is that rUNG enzymes are building up to expression plasmid pET28a procedure charts；

Fig. 7 is PAGE gel electroresis appraisal figure before and after rUNG enzyme inductions.

Wherein, swimming lane 1 is the albumen loading Marker of the pre-dyed of (10-230kDa) wide scope；Swimming lane 2 does not add for clone 1 Enter rUNG enzyme recombination bacillus colis BL21 (DE3) lysate of IPTG induced expressions；Swimming lane 3 adds IPTG for clone 1 and induces table RUNG enzyme recombination bacillus colis BL21 (DE3) lysate reached；Swimming lane 4 does not add the rUNG enzymes of IPTG induced expressions for clone 2 Recombination bacillus coli BL21 (DE3) lysate；Swimming lane 5 adds the rUNG enzyme recombination bacillus colis of IPTG induced expressions for clone 2 BL21 (DE3) lysate.

Fig. 8 is that IMAC affinity purifications recombinate rUNG enzyme bacterial lysate chromatograms.

Fig. 9 is the PAGE gel electrophoretogram that IMAC affinity purifications affinity purification recombinates rUNG enzyme bacterial lysates.

Wherein, swimming lane 1 is the albumen Marker of the pre-dyed of (10-230kDa) wide scope；Swimming lane 2-5 is buffer solution The different collecting pipe samples that BufferB is collected when eluting.

Figure 10 is the gel-filtration purified rUNG enzymes chromatograms of Superdex75.

Figure 11 is the PAGE gel electrophoretogram of the gel-filtration purified rUNG enzymes of Superdex75.

Wherein, swimming lane 1 is the albumen Marker of the pre-dyed of (10-230kDa) wide scope；Swimming lane 2-4 is buffer solution The different collecting pipe samples that BufferC is collected when eluting.

Figure 12 is rUNG enzyme purification sample HPLC purity detecting chromatograms.

After Figure 13 is addition rUNG enzymes/dUTP or rUNG enzymes/dTTP, PCR primer agarose gel electrophoresis figure.

Wherein, swimming lane 1 is 1000bp DNA Ladder；Swimming lane 2 is addition rUNG enzymes/dUTP to PCR system PCR amplifications Product；Swimming lane 3 is addition rUNG enzymes/dTTP to PCR system pcr amplification product.

Figure 14 rUNG enzymes remove the PCR primer ability detection containing dUTP, and wherein 14-a represents to remove 10¹⁰Copy contains dUTP PCR primer ability, 14-b remove 10⁸The ability of the PCR primer containing dUTP of copy.

Figure 15 rUNG enzymes expanding effect in quantitative fluorescent PCR double reaction.

Embodiment

With reference to specific embodiment, the present invention is expanded on further, it should be appreciated that quote embodiment and be merely to illustrate the present invention Rather than limitation the scope of the present invention.

Embodiment 1rUNG enzyme gene optimization designs

Inventor is according to cDNA sequence (the GenBank accession number of the published UNG enzymes of GenBank：NC_000913.3), The rUNG enzyme genes of the present invention are obtained after codon optimization is carried out to the gene, such as SEQ ID No：Shown in 2.

Here is to carry out codon optimization to rUNG enzymes, 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, UNG enzyme natural genes codon adaptation indexI in Escherichia coli (CAI) it is 0.53.From Fig. 2-b, after codon optimization so that rUNG enzyme genes of the invention are in Escherichia coli CAI indexes are 0.96.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 after have passed through codon optimization To gene order can improve expression of the rUNG enzyme 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, UNG enzyme native gene sequences There is percentage for 25% in poor efficiency codon.This gene being not optimized contains series connection rare codon, and these are close Numeral may reduce translation efficiency, or even can dismiss translation assemblage.From Fig. 3-b, after codon optimization, this hair The frequency that poor efficiency codon occur in E. coli system in bright rUNG enzyme genes 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 be to some extent Influence transcription and translation efficiency.Contrasted from the GC base average contents distributed areas figure of Fig. 4-a, Fig. 4-b UNG enzymes, by Show that GC bases average content is 52.66% before optimization in UNG enzyme natural genes in Fig. 4-a, by showing optimization in Fig. 4-b The G/C content all bases outside 30%-70% regions of sequence elimination afterwards, the GC bases for finally giving rUNG enzymes after optimization are put down Equal content is 59.54%.

The expression vector establishment of embodiment 2UNG enzyme genes

By rUNG enzymes full genome (such as SEQ ID No after optimization：Shown in 2) 5 ' end introduce XbaI enzyme cutting site sequences, Histidine-tagged and XhoI restriction enzyme site sequences are introduced at 3 ' ends, and carry out full genome synthesis, by the genetic fragment of synthesis, structure Into pUC57 plasmids (being provided by Nanjing Jin Sirui Science and Technology Ltd.s), a kind of long-term preservation plasmid is obtained, is designated as pUC57- RUNG plasmids.Using pUC57-rUNG 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 μ L of cumulative volume 50 are reacted, wherein concentration is that 10 μm of ol/L primers respectively add 2.5 μ L, and concentration is that 10mmol/L dNTP adds 1 μ L, archaeal dna polymerase used are Q5 (#M0491L, purchased from New England Biolabs companies), 2U/ μ L, add 0.5 μ L.Reaction Condition is 98 DEG C of 30 seconds pre-degenerations, thermal cycle 30 times (98 DEG C 10 seconds, 55 DEG C 15 seconds, 72 DEG C 30 seconds), 72 DEG C extend 2 minutes, 4 DEG C Preserve, product is analyzed through 1.0% agarose gel electrophoresis, is as a result shown that primer size is consistent with expected size (750bp) and (is tied Fruit is as shown in Figure 5).

Obtained gene outcome is purified into QIAquick Gel Extraction Kit (DP214-02, purchased from Beijing Tiangeng biochemistry section with universal DNA Skill Co., Ltd) purifying.After purification, with XbaI (#R0145S, purchased from New England Biolabs companies) and XhoI (# R0146S, purchased from New England Biolabs companies) double digestion, with T4 ligases (#M0202S, purchased from New England Biolabs companies) it is connected in pET28a plasmids (being purchased from Merck companies) carrier, it is transformed into DH5 α competent cells (CB101- 02, purchased from Beijing Tiangeng biochemical technology Co., Ltd) in, containing 50 μ g/mL kanamycins (0408, it is public purchased from Amresco Department) LB flat boards in 37 DEG C of overnight incubations.Screening positive clone bacterium sequencing in second day, is compared, completely the same with expected sequence, i.e., Obtain a form of expression vector of rUNG enzymes, be designated as pET28a-rUNG (plasmid construction flow is as shown in Figure 6).

Expression and identification of the embodiment 3rUNG enzymes in recombination bacillus coli

Comprise the following steps that：

1) sequencing in embodiment 2 is compared correct pET28a-rUNG plasmids and is transformed into e. coli bl21 (DE3) sense by In by state cell in (CB105-02, purchased from Beijing Tiangeng biochemical technology Co., Ltd), trained overnight in 37 DEG C of kanamycins flat boards Support.

2) chooses the 1-4 restructuring single bacterium colonies containing pET28a-rUNG plasmids for second day, and containing 50 μ g/mL cards, that is mould for access The LB nutrient solutions of element, 37 DEG C of overnight incubations.

3) is taken in TB nutrient solutions of the 10mL overnight cultures access 500mL containing 50 μ g/mL kanamycins, 37 DEG C of vibration trainings Support.

4) bacterium solution OD600 values were surveyed every 1 hour after inoculations, when OD600 ≈ 1.0, with 1mmol/L IPTG (0487, Purchased from Amresco companies) carry out induced expression.Simultaneously negative control is done not add IPTG E. coli broth.

5) bacterium solution is collected after .25 DEG C of overnight induced expression, takes 1mL bacterium solutions, 12000rpm high speed centrifugation 3min, with precooling PBS precipitates, and adds 5 × sds gel sample-loading buffer, 100 DEG C of heating 5min, 12000rpm high speed centrifugation 1min, takes Clearly.IPTG E. coli broth is not added by this step process yet.

6) respectively takes 5 μ L not add IPTG and adds the culture suspension of IPTG inductions, 10%SDS-PAGE gel electrophoresises point Analysis.

7) .8-15V/cm electrophoresis, separation gel bottom is moved to bromophenol blue.

8) coomassie brilliant blue stainings, expression product band is observed, sees Fig. 7.

Embodiment 4rUNG enzyme purifications

Step 1. recombinates bacterial cell disruption

1) will recombinate BL21 (DE3) thalline in embodiment 3, and what pre-cooled combination buffer BufferA cleaned to obtain contains There is induction rUNG Escherichia coli precipitation, be resuspended with the combination buffer BufferA of precooling, centrifuged in 4 DEG C with 12000rpm 15min；It is repeated once.

2) sucks supernatant, claims bacterial sediment weight, and every gram of thalline adds 5mL combination buffer BufferA, with polishing glass Glass rod stirs, and hangs thalline.

3) every gram of thalline of adds 5 μ L 100mmol/L PMSF, 5 μ L 100mg/mL lysozymes, stirs 20min on ice.

4) crushes thalline with probe type ultrasonication ripple instrument, and sample is placed on ice, ultrasound 60 times, is spaced 3 seconds within 4 seconds every time, circulation Three times, each intercycle waits 5min.12000rpm, 4 DEG C of high speed centrifugation 15min.

5) will centrifuge the supernatant that obtains to 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 that HisTrap crude FF 5mL (17-5286-01, are purchased from GEhealthcare), combination buffer BufferA：20mM Tris-HCl, 20mM imidazoles, 300mMNaCl, pH=7.5, elution Buffer B ufferB：20mM Tris-HCl, 300mM NaCl, 300mM imidazoles, pH=8.0,0.22 μm of membrane filtration are standby.

2) accesses HisTrap FF crude 5mL in AKTA avant150 purifying instrument, successively with distilled water cleaning machine After device and pillar, then with combination buffer BufferA pillar is balanced, and with sample pump to the restructuring BL21 that is handled in step 1 (DE3) bacterial lysate carries out loading.After having gone up sample, pillar first is cleaned with combination buffer BufferA, then use elution buffer Liquid BufferB is eluted and is collected eluting peak.(see Fig. 8)

3) sample for each collecting pipe that collects to IMAC affinity chromatographys carries out SDS-PAGE proteins gel electrophoresis detections, by Shown in Fig. 9, recombinant bacterial strain BL21 (DE3) can induce the rUNG enzymes for producing higher soluble-expression.

The gel permeation chromatographies of step 3.Superdex 75

1) chromatographic column that gel permeation chromatographies are selected is that HiLoad16/60superdex75pg (17-1068-01, is purchased from GE healthcare), buffer B ufferC is NaCl, pH=8.0,0.22 μm of membrane filtrations of 20mM Tris-HCl, 50mM It is standby.

2) accesses Superdex75 gel permeation chromatographies post in AKTA avant150 purifying instrument, clear with distilled water successively After washing machine device and pillar, then with buffer B ufferC balance pillar, each loading cumulative volume be no more than column volume 5%.Go up After sample, eluted with BufferC, and collect eluting peak.(see Figure 10)

3) each collecting pipe sample that collects to Superdex75 gel permeation chromatographies carries out PAGE gel electrophoresis detection (result is as shown in figure 11).

Step 4.rUNG enzyme purity detectings

Each collecting pipe sample that Superdex75 gel permeation chromatographies are collected merges, and is examined by molecular sieve chromatography Survey rUNG zymoprotein purity, chromatography conditions：HPLC (Waters, e2695HPLC), Superdex200GL increase (purchase From GEhealthcare), mobile phase is Na containing 0.1M₂SO₄PB (pH=6.5), flow velocity 0.8mL/min, Detection wavelength is 280nm, sample size 10 μ L, detection time 35min.

Analysis result is as shown in Figure 12.As can be seen, by after purification, rUNG purity reaches more than 99%, pass through Calculate, above-mentioned 1L culture mediums, the purity that 50~100mg or so can be prepared in shaking flask reaches 99% or so rUNG enzymes.

Step 5.rUNG enzymes preserve

Above-mentioned purifying is merged into sample and adds DTT, TritonX-100 and glycerine, the buffer solution that final rUNG enzymes preserve is： 20mM Tris-HCl, 50mM NaCl, 1mM DTT, 0.1% (v/v) Triton X-100,50%glycerol, pH=8.0, - 20 refrigerators are placed in preserve for a long time.

The rUNG enzymes biological activity of embodiment 5 is verified

The rUNG enzymes that embodiment 4 is prepared are applied in PCR amplifications.First certain weight is extracted with the small extraction reagent kit of plasmid Group escherichia coli plasmid, then prepare reaction group by following program and method：Buffered in 25 μ l reaction solutions containing 2.5 10 × PCR of μ l Liquid, upstream and downstream primer each 0.1mmol of each 1 μ L, 4 × dNTPs (dUTP, dATP, dCTP, dGTP), 25ng recombinant vectors, 1U Taq DNA polymerase (refers to the patent No.：201510946096.5), while prepare control group：Contain 2.5 μ l in 25 μ l reaction solutions 10 × PCR buffer solutions, upstream and downstream primer each 0.1mmol of each 1 μ L, 4 × dNTPs (dTTP, dATP, dCTP, dGTP), 25ng restructuring Plasmid, 1U Taq DNA polymerases, by reaction group and control group be separately added into rUNG enzymes be placed in 37 DEG C reaction 10min after, in PCR Instrument (Applied Biosystems,96-Well Thermal Cycler) amplification determine its reactivity, wherein PCR Reaction condition：94 DEG C of pre-degeneration 2min, thermal cycle 30 times (94 DEG C of denaturation 60s, 55 DEG C of annealing 60s, 70 DEG C of extension 1min), 72 DEG C Extend 5min, 4 DEG C of preservations.Its activity is detected finally by agarose gel electrophoresis, as a result as shown in figure 13, addition rUNG enzymes/ DUTP reaction group pcr amplification products do not have any band, and add rUNG enzymes/purposeful bar of dTTP control group pcr amplification products Band, illustrate that reaction group has obvious uracil-DNA glycosidase specific activity, it is anti-pollution to can be used for DNA in PCR Lab In.

Anti-pollution ability of the rUNG enzymes of embodiment 6 in quantitative fluorescent PCR

PCR system is Taq 0.5U, rUNG 1U, the μ L of 10 × buffer 4,25mM MgCl₂ 4μL、10μM dNTP 0.8 μ L, each 2 μ L of 1 μM of upstream and downstream primer, each 2 μ L of 1 μM of mutation/internal control probe, DNA10ng, ddH₂O complements to 40 μ L, and DNA profiling is PCR recovery products containing dUTP, gradient dilution is carried out, is diluted to different copy numbers：10¹²、10¹⁰、10⁸.PCR response procedures：50 ℃10min；95℃5min；40 circulations of 95 DEG C of 15sec, 60 DEG C of 1min, fluorescence is collected at 60 DEG C.Figure 14-a, 14-b results show Show：The rUNG of the present invention can substantially remove 10⁸The PCR primer containing dUTP of copy number.

Influence of the embodiment 7rUNG enzymes to fluorescent quantitative PCR effect

PCR system is Taq 0.5U, rUNG 1U, the μ L of 10 × buffer 4,25mM MgCl₂ 4μL、10uM dNTP 0.8 μ L, each 2 μ L of 1 μM of upstream and downstream primer, each 2 μ L of 1 μM of mutation/internal control probe, DNA10ng, ddH₂O complements to 40 μ L, and DNA profiling is Contain human genome DNA recombinant vector.PCR response procedures：50℃10min；95℃5min；95 DEG C of 15sec, 60 DEG C of 1min 40 circulations, collect fluorescence at 60 DEG C.Figure 15 results are shown：RUNG of the present invention (is detected simultaneously in quantitative fluorescent PCR double reaction FAM and VIC signals) in, amplification efficiency is high, can specific amplification purpose fragment.

It the above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair The limitation of the present invention, protection scope of the present invention should be defined by claim limited range.For the art For those of ordinary skill, without departing from the spirit and scope of the present invention, some improvements and modifications can also be made, these change Enter and retouch and also should be regarded as protection scope of the present invention.

Sequence table

<110>Jiangsu Zhonghong Biopharma Institute Inc.

<120>Recombinate uracil-DNA glycosidase and its encoding gene, preparation method and application

<130>Recombinate uracil-DNA glycosidase and its encoding gene, preparation method and application

<160> 2

<170> SIPOSequenceListing 1.0

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<211> 229

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<213> Escherichia coli

<400> 1

Met Ala Asn Glu Leu Thr Trp His Asp Val Leu Ala Glu Glu Lys Gln

1 5 10 15

Gln Pro Tyr Phe Leu Asn Thr Leu Gln Thr Val Ala Ser Glu Arg Gln

20 25 30

Ser Gly Val Thr Ile Tyr Pro Pro Gln Lys Asp Val Phe Asn Ala Phe

35 40 45

Arg Phe Thr Glu Leu Gly Asp Val Lys Val Val Ile Leu Gly Gln Asp

50 55 60

Pro Tyr His Gly Pro Gly Gln Ala His Gly Leu Ala Phe Ser Val Arg

65 70 75 80

Pro Gly Ile Ala Ile Pro Pro Ser Leu Leu Asn Met Tyr Lys Glu Leu

85 90 95

Glu Asn Thr Ile Pro Gly Phe Thr Arg Pro Asn His Gly Tyr Leu Glu

100 105 110

Ser Trp Ala Arg Gln Gly Val Leu Leu Leu Asn Thr Val Leu Thr Val

115 120 125

Arg Ala Gly Gln Ala His Ser His Ala Ser Leu Gly Trp Glu Thr Phe

130 135 140

Thr Asp Lys Val Ile Ser Leu Ile Asn Gln His Arg Glu Gly Val Val

145 150 155 160

Phe Leu Leu Trp Gly Ser His Ala Gln Lys Lys Gly Ala Ile Ile Asp

165 170 175

Lys Gln Arg His His Val Leu Lys Ala Pro His Pro Ser Pro Leu Ser

180 185 190

Ala His Arg Gly Phe Phe Gly Cys Asn His Phe Val Leu Ala Asn Gln

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Trp Leu Glu Gln Arg Gly Glu Thr Pro Ile Asp Trp Met Pro Val Leu

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Pro Ala Glu Ser Glu

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atggcgaacg agctgacctg gcacgacgtg ctggcggagg aaaagcagca accgtacttc 60

ctgaacaccc tgcagaccgt ggcgagcgag cgtcaaagcg gcgttaccat ctatccgccg 120

cagaaggatg tgtttaacgc gttccgtttt accgaactgg gcgacgttaa agtggttatt 180

ctgggtcagg acccgtacca cggtccgggt caagcgcacg gcctggcgtt cagcgttcgt 240

ccgggtatcg cgattccgcc gagcctgctg aacatgtaca aggagctgga aaacaccatc 300

ccgggtttta cccgtccgaa ccacggctat ctggaaagct gggcgcgtca gggtgtgctg 360

ctgctgaaca ccgtgctgac cgttcgtgcg ggccaagcgc acagccacgc gagcctgggt 420

tgggagacct tcaccgacaa agttatcagc ctgattaacc aacaccgtga aggtgtggtt 480

tttctgctgt ggggcagcca cgcgcagaag aaaggtgcga tcattgataa gcaacgtcac 540

cacgtgctga aagcgccgca cccgagcccg ctgagcgcgc accgtggttt ctttggctgc 600

aaccacttcg tgctggcgaa ccagtggctg gagcaacgtg gcgaaacccc gattgactgg 660

atgccggttc tgccagcgga gagcgaacac caccaccacc accactgata a 711

Claims

1. one kind restructuring UNG enzymes, its amino acid sequence such as SEQ ID NO：Shown in 1.

2. a kind of encode the gene that UNG enzymes are recombinated described in claim 1, its base sequence such as SEQ ID NO：Shown in 2.

3. a kind of carrier for containing coding restructuring UNG gene described in claim 2, the carrier is pET28a.

4. a kind of escherichia coli host strain for containing carrier described in claim 3, the escherichia coli host strain are BL21(DE3)。

5. one kind restructuring UNG enzymes efficient soluble-expression method in Escherichia coli, comprises the following steps：

1) pickings one access LB nutrient solutions, in 37 DEG C of cultures containing the coli strain single bacterium colony described in claim 4 Overnight；

2) is taken in 10mL overnight cultures access TB or LB nutrient solutions, in 37 DEG C of concussion and cultivates to mid-log phase (A₆₀₀=1.0)；

3) adds IPTG to 1mmol/L in culture, stays overnight induced expression in 25 DEG C of concussions, 4 DEG C are centrifuged with 12000rpm 3min collects the coli somatic precipitation containing restructuring UNG enzymes.

Contain kanamycins 25-50 μ g/mL in TB the or LB nutrient solutions.

6. a kind of purification process of restructuring UNG enzymes, comprises the following steps：

1) precipitates what collection obtained containing induction restructuring UNG enzymes coli somatic, with the combination buffer of precooling BufferA is resuspended, and is handled in 4 DEG C of high speed centrifugations.

2) sucks supernatant, and every gram of thalline adds combination buffer BufferA 3-10mL, agitation, hangs thalline.

3) every gram of thalline of adds the PMSF that 3-10 μ L concentration is 100mmol/L, and 3-100 μ L concentration is 100mg/mL lysozyme, Stirred on ice.

4) crushes thalline, ultrasonic in ice bath, and collects supernatant, 0.22 μm of filter membrane in 4 DEG C with 12000rpm high speed centrifugations 5min Filter standby.

5) .IMAC affinity chromatographys, sample and standby with 0.22 μm of membrane filtration in each collection peak is merged.

6) obtains required purpose product further with gel permeation chromatography.

7. purification process according to claim 6, the combination buffer that the IMAC affinity chromatographys are selected is BufferA： 20mM Tris-HCl, 20mM imidazoles, 300mMNaCl, pH=8.0；Elution buffer is BufferB：20mM Tris-HCl, 300mM NaCl, 300mM imidazoles, pH=8.0；The gel permeation chromatography, the combination of selection and elution buffer are BufferC:NaCl, pH=8.0,0.22 μm of membrane filtrations of 20mM Tris-HCl, 50mM are standby.

8. restructuring UNG enzymes are preventing non-specific PCR amplification and pollution, are ensureing PCR result accuracys as claimed in claim 1 In application.