CN102766611B

CN102766611B - Sulfolobus virus dUTP (deoxyuridine triphosphate) pyrophosphatase and polynucleotide coding same

Info

Publication number: CN102766611B
Application number: CN201210240789.9A
Authority: CN
Inventors: 林连兵; 陈美杉; 朱国东; 魏云林; 季秀玲; 张琦
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2012-07-12
Filing date: 2012-07-12
Publication date: 2014-02-12
Anticipated expiration: 2032-07-12
Also published as: CN102766611A

Abstract

The invention discloses a sulfolobus virus dUTP pyrophosphatase and a polynucleotide coding the same. The dUTP pyrophosphatase is coded by sulfolobus acidocaldarius virus STSV2 genome DNA (deoxyribonucleic acid), and has high catalytic activity under 55 DEG C to 80 DEG C. The dUTP pyrophosphatase (dUTPase) is an important DNA modification enzyme, can be used for decreasing the proportion of dUTP/dTTP (deoxythymidine triphosphate) in cells, preventing misincorporation caused by over-high concentration of dUTP in the process of DNA replication and enhancing the stability of DNA replication, and can be applied in the PCR (Polymerase Chain Reaction) amplification technology to increase the efficiency and fidelity of DNA amplification.

Description

The polynucleotide of sulfolobus solfataricus virus deoxyuridine Pyrophosphate phosphohydrolase and coding this kind of enzyme

Technical field

The invention belongs to biological technical field, relate to a kind of deoxyuridine Pyrophosphate phosphohydrolase that derives from sulfolobus solfataricus virus, and the polynucleotide of this enzyme of encoding.

Technical background

Deoxyuridine Pyrophosphate phosphohydrolase (dUTPase) is a kind of important DNA modification enzyme, extensively be present in various biological organisms, dUTP be can specially be hydrolyzed and dUMP and ppi produced, dUMP becomes thymus pyrimidine deoxyribonucleoside triphosphate (dTTP) through methylating, and dTTP is one of main component of synthetic DNA.Therefore, dUTPase has dual basic function in organism: (1) reduces the dUTP/dTTP ratio in cell, prevents dUTP excessive concentration in DNA replication dna process and causes that mistake mixes, and the stability of DNA replication dna is provided; (2) for DNA synthetic, provide the raw material dTTP of essential encoding viral.DUTPase has species specificity, and closely related with viral virulence and efficient replication.

British scholar McGeoch finds after the aminoacid sequence of deoxyuridine Pyrophosphate phosphohydrolase (dUTPase) that has compared different sources, derive from the dUTPase of any species, although molecular size range differs, but the motif that has 5 typical high conservatives that contain dUTPase in its aminoacid sequence, is respectively motif 1 (AGFDL), rnotif 2 (GKSS), motif 3 (GIIDFGYTG), motif 4 (GQKFAQL) and motif 5 (RGDKGFGS).Wherein motif 3 is considered to the enzymic activity catalytic center of dUTPase.McGeehan can be divided into three subclass according to the character of dUTPase, structure: I type comprises the dUTPase (comprise bacterium, fungi, plant and metazoan and a series of virus: poxvirus, retrovirus, the simplexvirus of adenovirus and invertebrates, fish and Amphibians) in nearly all source except most of simplexviruss; II type comprises the dUTPase that the simplexvirus of Mammals and bird is coded.Although I type has 5 similar conservative motif with II type, specific substrate is all dUTP, and they have larger difference on mrna length, motif arrangement, space structure.I type dUTPase is generally 150 amino-acid residues, and the homotrimer (homotrimer) that mineral crystal structure You Sange subunit forms forms, and five conservative motif put in order as 1.2.3.4.5; II type dUTPase total number of atnino acid is the twice of I type, and mineral crystal structure is monomer (monomer), and its motif puts in order as 3.1.2.4.5.In addition, research shows, the dUTPase encoding on the minority biologies such as nematode is structurally very different with the above two, there is no 5 significantly conservative motif, and its hydrolysis substrate is except dUTP also comprises dUDP, belongs to temporarily III type.Analyze and understand the classification of dUTPase and structural performance and contribute to the going deep into the research of functional protein by classification and constructional feature implementation structure albumen.

All biologies have a considerable amount of genes encoding deoxyuridine Pyrophosphate phosphohydrolases, mainly because this enzyme is a kind of important DNA modification enzyme, reduce the dUTP/dTTP ratio in cell, prevent dUTP excessive concentration in DNA replication dna process and cause that mistake mixes, provide the stability of DNA replication dna, so deoxyuridine Pyrophosphate phosphohydrolase has basic and applied research value.

Summary of the invention

The present invention aims to provide a kind of deoxyuridine Pyrophosphate phosphohydrolase of sulfolobus acidocaldarius STSV2 virus, this deoxyuridine Pyrophosphate phosphohydrolase is coded from sulfolobus acidocaldarius's virus STSV2 of Tengchong Rehai by separation, and it is polypeptide, polypeptide analog or the derivative that contains the aminoacid sequence shown in SEQ ID NO.1.

The aminoacid sequence of polypeptide analog described in the present invention or derivative has the homogeny with the aminoacid sequence at least 90% shown in SEQ ID NO.1.

Another object of the present invention is to provide the polynucleotide of deoxyuridine Pyrophosphate phosphohydrolase described in a kind of claim 1 of encoding, and it has nucleotide sequence shown in SEQ ID NO.2 or its complementary sequence or has polynucleotide and the complementary sequence thereof of at least 80% homogeny with nucleotide sequence shown in SEQ ID NO.2.

Another object of the present invention is to provide expression vector and the expression method thereof of the polynucleotide of coding deoxyuridine Pyrophosphate phosphohydrolase.

Beneficial effect of the present invention is as follows:

This enzyme is a kind of important DNA modification enzyme, can prevent dUTP excessive concentration in DNA replication dna process and cause that mistake mixes, provide the stability of DNA replication dna, so deoxyuridine Pyrophosphate phosphohydrolase can be used as the additive of DNA cloning system, to improve DNA cloning efficiency and fidelity.

Accompanying drawing explanation

Fig. 1 is deoxyuridine pyrophosphatase gene conserved structure domain analysis schematic diagram of the present invention.

Fig. 2 is the PCR product of deoxyuridine pyrophosphatase gene of the present invention, wherein: M is Marker; Swimming lane 1 is deoxyuridine pyrophosphatase gene PCR product, and its size is 516bp; Swimming lane is 2 negative contrasts.

Fig. 3 is deoxyuridine pyrophosphatase gene PCR product of the present invention, recombinant plasmid and plasmid enzyme restriction electrophoresis schematic diagram, wherein: M is Marker; Swimming lane 1 is deoxyuridine pyrophosphatase gene PCR product, and its size is 516bp; Swimming lane 2 is that deoxyuridine Pyrophosphate phosphohydrolase recombinant plasmid is through Nco I enzyme and Hind III enzyme double digestion electrophoresis result; Swimming lane 3 is deoxyuridine Pyrophosphate phosphohydrolase recombinant plasmid Nco I restriction enzyme digestion and electrophoresis result swimming lane; Swimming lane 4 is deoxyuridine Pyrophosphate phosphohydrolase recombinant plasmid Hind III restriction enzyme digestion and electrophoresis result; Swimming lane 5 is deoxyuridine Pyrophosphate phosphohydrolase recombinant plasmid electrophoresis result; Swimming lane 6 is pET32a plasmid electrophoresis result.

Fig. 4 is deoxyuridine Pyrophosphate phosphohydrolase purifying schematic diagram of the present invention, and wherein M is Marker; Swimming lane 1,2,3, the 4 elutriant electrophoretic analysis figure that deoxyuridine Pyrophosphate phosphohydrolase elutes successively from nickel post respectively.

Embodiment

Below in conjunction with drawings and Examples, the present invention is described in further detail, but protection domain of the present invention is not limited to described content, the reagent using in embodiment and method, if no special instructions, all adopt conventional reagent and use ordinary method.

Clone and the expression of embodiment 1:STSV2 virus deoxyuridine Pyrophosphate phosphohydrolase

1, the amplification of STSV2 virus deoxyuridine pyrophosphatase gene, (the viral STSV2 DNA of take is template)

(1) the amplification the primer sequence of STSV2 virus deoxyuridine pyrophosphatase gene is as follows:

Forward primer: 5 '-CATG cCATGGaCATGATTTTCAGTGATAGAG-3 '

Reverse primer: 5 '-CCC aAGCTTtGAGAGCTTGGCCGGCGTCAC-3 '

(2) amplification system is as follows:

Table 1: amplification reaction system component

Template	10 ng
		Easy pfu DNA palymerase	1 μl
Primer	Each 1 μ l
		2.5mM dNTPs	4ul
10×Buffer Easypfu	5ul
		Distilled water ddH ₂O	Be supplemented to 50 μ l

(3) amplification condition is as follows:

Reaction system is mixed, first at 94 ℃ of denaturation 4min, then at 94 ℃ of sex change 45s, 55 ℃ of annealing 45 s, 72 ℃ are extended 90 s, and after 30 circulations, 72 ℃ are extended 10min.After having reacted, get product 3 μ l, in 10 g/L sepharoses, carry out electrophoretic analysis (see figure 2).

2, the glue of PCR product reclaims purifying

(1) in electrophoresis apparatus, record 1.0% sepharose;

(2) by the PCR product point sample electrophoresis of purifying to be separated, in appropriate location, stop electrophoresis;

(3) under ultraviolet lamp, cut the gel containing this object segment, transfer in the Ep pipe of 1.5ml;

(4) Yong Tiangen biotech firm glue recovery test kit carries out the recovery of object fragment, and the operation of recovery method by specification is carried out.

Submit to ncbi database Conserved Domain Database protein conserved sequence analysis software to analyze gene order, STSV2-60 analytical results (see figure 1), the important Motif that shows deoxyuridine Pyrophosphate phosphohydrolase in figure, comprises avtive spot and some Trimer interface.

3, the structure of recombinant expression vector

For goal gene segment is connected to expression vector pET32a, just need to make object segment with the segment of sticky end, with restriction enzyme site.

(1) enzyme of deoxyuridine pyrophosphatase gene fragment is cut evaluation

it is as follows that enzyme is cut system:

Table 2: reaction system component

DUTPase gene fragment	5 μ l (about 200ng)
		10 * H damping fluid	1 μl
NcoⅠ	0.3 μl
		HindⅢ	0.3 μl
Distilled water	3.4 μl
		Total amount	10 μl

enzyme tangent condition: 37 ℃, spend the night, reclaim deoxyuridine pyrophosphatase gene fragment.

(2) with the preparation of sticky end linear carrier pET32a

Upper for goal gene segment being connected to expression vector pET32a, just need to make object segment with the segment of sticky end, with restriction enzyme site.Equally, in order to make, in object segment energy insertion vector, also to need to make belt carrier toughness end, and make their restriction enzyme site identical.

A, plasmid extraction: with plasmid extraction kit (Bo Ya), operation steps is as follows:

actication of culture: aseptic inoculation ring dips-80 ℃ of frozen bacterial classifications and preserves liquid, it is dull and stereotyped that trilinear method is inoculated in ammonia benzyl LB, cultivates 12-16 hour for 37 ℃;

2. increase bacterium and collect thalline: getting penbritin 5 μ l (final concentration 100 μ g/ml) and add in 5ml LB substratum; With transfering loop picking positive colony, be inoculated in Amp ⁺in-LB substratum; Then put into 37 ℃ of incubators, shaking table is cultivated, and spends the night.And the bacterium liquid of cultivation is joined in the centrifuge tube of 5 ml, centrifugal 5 min of room temperature, 5000 rpm, make bacterial sediment, abandon supernatant liquor;

3. in centrifuge tube, add solution S1(containing RNA enzyme) 250 μ l, vibrate to thalline and thoroughly suspend, proceed in the EP pipe of 1.5ml;

4. at EP pipe, add solution S 2 250 μ l, put upside down gently several times, room temperature is placed 5 min, makes the RNA enzyme of solution I after RNA degraded wherein, adds solution S3 400 μ l, and room temperature is centrifugal, 13000 rpm, 10 min;

5. the supernatant liquor of centrifugal rear gained is proceeded in DNA adsorption column, room temperature is centrifugal, 5000 rpm, and 1 min, reclaims twice, abandons lower clear;

6. in adsorption column, add 500 μ l rinsing liquids (W1 solution), room temperature is centrifugal, 5000 rpm, 1 min;

7. then add 750 μ l rinsing liquids (W2 solution), room temperature is centrifugal, 5000rpm, and 1min, abandons lower clear (repeating once); Room temperature is once centrifugal again, 13000 rpm, 2 min;

8. adsorption column is moved in the centrifuge tube of 1.5 new ml, in adsorption film central authorities, add 70 μ l ddH ₂o water, places 1 min, and then room temperature is centrifugal, 13000 rpm, and 2 min, abandon supernatant, obtain plasmid.

9. the plasmid of gained is got to 1 μ l and carried out electrophoresis detection, and quantitatively.

B, plasmid pET32a enzyme are cut evaluation.

it is as follows that enzyme is cut system:

Table 3: reaction system component

Plasmid	50 μ l (about 2000ng)
		10 * H damping fluid	10μl
NcoⅠ	3 μl
		HindⅢ
	3 μl
		Distilled water	34 μl
Total amount	100 μl

2. reaction conditions: 37 ℃, spend the night.

(3) expression and purity of the structure of recombinant expression vector, STSV2 viral protein

1. by the linear carrier pET32a with sticky end that experiment obtains above and STSV2 virus deoxyuridine pyrophosphatase gene fragment, by connection, transform and use bacterium colony PCR, enzyme to cut and identify (see figure 3) and sequence verification, can obtain recombinant expression vector.

2. the abduction delivering of STSV2 virus deoxyuridine Pyrophosphate phosphohydrolase albumen in intestinal bacteria

The recombinant vectors building is transformed to e. coli bl21, contain the bacterial strain of recombinant plasmid through overnight incubation, bacterium liquid is inoculated in 30 ml Amp+-LB (penbritin final concentration 100 μ g/ml) nutrient solution in 1% ratio, and putting into 37 ℃ of shaking tables, to be cultured to its OD value be 1.0; Take out 4ml bacterium liquid as control experiment; All the other bacterium liquid add inductor IPTG(final concentration 100 μ g/ml) put into 37 ℃, 80 rpm shaking tables are cultivated abduction delivering 9 hours (after induction 3h, start sampling, get 4ml bacterium liquid at every turn, within later every two hours, get one time sample, after induction, thalline is got sample altogether 4 times).

4, STSV2 virus deoxyuridine Pyrophosphate phosphohydrolase protein SDS-PAGE detects

From step 3(2) take out a small amount of induction in sample 2. after bacterium liquid survey OD value, according to bacterium liquid OD value difference, get different bacterium liquid measures, make its biomass equal, 5000rpm, centrifugal 10min, abandons supernatant; Add 80 μ l distilled waters, 20 μ l 5 * sample-loading buffers, after vibration is scattered thalline; At 98 ℃, place 10min, make cellular lysate, discharge protein; Get the bacterium liquid after abduction delivering, prepare sample; Sample is splined in electrophoresis apparatus at 120V, and 70mA, after electrophoresis 2h, adds the dyeing of 100ml R250 coomassie brilliant blue staining liquid, shaking table vibration, and 100 rpm, after 30min; Staining fluid is poured out, used clear water rinsing, then add appropriate destainer 100 rpm, 10h, finally uses scanner scanning gel, detects protein expression situation.

5, the purifying of deoxyuridine Pyrophosphate phosphohydrolase albumen

Utilize a large amount of inductions of aforesaid method containing the BL21 bacterial strain of recombinant plasmid, bacterium liquid is through centrifugal collection coli somatic (4 ℃, 5,000x g, 10 min).Thalline is suspended in right amount and (makes the OD of bacteria suspension ₆₀₀reach 20) in buffer A [30 mM imidazoles, pH 7.4 for 20 mM sodium phosphates, 500 mM NaCl], ultrasonication cell on ice, 4 ℃, centrifugal 10 min of 20,000x g, supernatant carries out manual purifying with nickel post, first uses 10 times of column volume ddH ₂o cleans pillar, then uses 10 times of column volume 30mM imidazoles balance pillars, sample upper prop, with 10 times of column volume 30mM imidazoles wash-out pillars, then use 10 times of column volume 150mM imidazoles wash-out pillars, then use 10 times of column volume 500mM imidazoles wash-out pillars, with 10 times of column volume ddH ₂o cleans pillar, finally with 20% dehydrated alcohol, fills pillar.By damping fluid (50 mM Tris-HCl, pH7.4) dialysed overnight, the recombinant protein component of purifying is carried out SDS-PAGE(as shown in Figure 4), result shows, deoxyuridine Pyrophosphate phosphohydrolase molecular weight of albumen is about 38KD, expresses correct.

Embodiment 2: the detection of deoxyuridine PPase Activity

1, experiment reagent is as follows:

(1) tetra-sodium (PPi) storage liquid: take 4.46g trisodium phosphate crystallization (Na ₄p ₂o ₇.10H ₂o) be dissolved in 100ml distilled water,

Being made into 100 times of storage liquid. 4 ℃ of preservations, 1:100 dilutes use.

(2) sulphuric acid soln (10N): slowly add the 27ml vitriol oil in 73ml distilled water, because diluting concentrated sulfuric acid is violent thermopositive reaction, so operation must slowly and in time mix.

(3) molybdic acid reagent: by 2.5g ammonium molybdate (NH ₄) ₆mo ₇o ₂₄.4H20 be dissolved in 100ml sulphuric acid soln (10N H ₂s0 ₄), 4 ℃ save backup.

(4) sulfurous acid ammonium salt reagent: A solution, by 10g sodium bisulfite (NaHS03) and O.5g S-WAT (Na2SO3) be dissolved in 100ml distilled water, B solution: by distilled water 1: 15 dilution for A solution.

(5) sulfhydryl reagent: 10ml beta-mercaptoethanol adds in 90ml distilled water, fresh preparation during use was finished in 24 hours.

2, the drafting of PPi typical curve

Measure different concns PPi standardized solution at the absorbance value of 575nm, and the amount of drawing OD value and tetra-sodium, particular content is as follows:

(1) tetra-sodium (PPi) storage liquid 1:100 is diluted to work body lotion;

(2) get 10 Ep pipes, add respectively tetra-sodium working fluid (1mmol/L), 0,10,20 ... 70 μ 1, every pipe is added distilled water to 800 μ 1 again, mixes;

(3) every pipe adds 50ul molybdic acid reagent successively, mixes;

(4) every pipe adds sulfurous acid reagent (A) reagent more successively, mixes;

(5) every pipe adds 50ul sulfhydryl reagent successively, mixes;

(6) the standing colour developing of room temperature 10min;

(7) every pipe adds 100ul sulfurous acid reagent (B), mixes;

(8) every pipe adds 50ul sulfhydryl reagent successively, carries even;

(9) every pipe adds 100ul dehydrated alcohol successively again, and rod is even;

(10) every pipe is got respectively the mensuration that 10ul carries out 575nmPPi absorbance;

(11) acquired results carries out systems analysis with Excel, calculates relation conefficient, draws regression curve.

3, detect the substrate specificity of deoxyuridine Pyrophosphate phosphohydrolase dUTPase

Measure the catalytic capability of dUTPase to the different substrates of dUTP, dATP, dGTP, dCTP or dTTP, establish various negative controls simultaneously, particular content is as follows:

(1) by 60 ℃ of preheating 5min of dUTPase, add in different substrate solutions, establish negative control simultaneously;

(2) get 24 Eppendorf pipes, add respectively substrate (3.5ul) and the dUTPase (5ul) of various various combinations, wherein 12 Eppendorf pipes add 3ul Mg ²⁺other 12 pipes do not add Mg ²⁺, the concrete composition of enzymatic reaction sees the following form;

(3) with Tris-HCI (0.05 mol/L, pH8.0) polishing, react cumulative volume to 60ul, mix;

(4) 60 ℃ of water-bath 15min, then add distilled water to 700ul and mix;

(5) measure 575nmOD value, calculate the output of PPi, as shown in (as table 4).

The substrate specificity reaction of table 4: dUTPase

Specificity of the recombined dUTPase for different Nucleotides

Result shows, deoxyuridine Pyrophosphate phosphohydrolase can be take dUTP and be produced phosphate radical as substrate hydrolysis dUTP, and Mg ²⁺catalysis to this enzyme plays a driving role, and has proved that restructuring deoxyuridine Pyrophosphate phosphohydrolase of the present invention has biologic activity simultaneously.

4, the impact of temperature on dUTPase activity

The dUTPase concentration of purifying is 0.143mg/ml, will add Mg ²⁺dUTPase respectively at 37 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 93 ℃ preheating 5min, add respectively 3.5ul dUTP, the Tris-HCl polishing that is 7 by 0.05mol/L pH value is to 60ul, in corresponding water-bath, react 15min, then carry out the detection that enzyme is lived, as shown in acquired results (as table 5);

DUTPase activity unit is defined as: 60 ℃ of needed enzyme amounts of (optimum temperuture) per minute hydrolysis 1umoldUTP, be 1 enzyme activity unit; The specific activity of enzyme is the quantity (U/mg) of contained unit of enzyme in every milligram of albumen.

Table 5: the impact of temperature on dUTPase

Result shows, restructuring deoxyuridine Pyrophosphate phosphohydrolase has higher catalytic activity at 55-75 ℃.

Sequence table

<110> Kunming University of Science and Technology

The polynucleotide of <120> sulfolobus solfataricus virus deoxyuridine Pyrophosphate phosphohydrolase and coding this kind of enzyme

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 172

<212> PRT

<213> sulfolobus solfataricus virus

<400> 1

Met Ile Phe Ser Asp Arg Asp Leu Lys Tyr Tyr Leu Glu Lys Gly Trp Ile Lys Ile Glu

1 10 20

Pro Leu Arg Glu Asp Thr Ile Arg Glu Asn Gly Val Asp Leu Arg Ile Gly Asn Glu Ile

21 30 40

Ala Arg Phe Lys Lys Asn Arg Ile Phe Asp Pro Asp Lys Asp Ser Ile Asp Asp Phe Ile

41 50 60

Glu Lys Glu Val Gly Asn Glu Phe Ile Ile Asn Pro His Glu His Ala Leu Leu Thr Thr

61 70 80

Glu Glu Tyr Val Arg Leu Pro Asn Asp Val Met Ala Phe Val Asn Leu Arg Ser Thr Phe

81 90 100

Ala Arg Leu Gly Leu Phe Ile Pro Pro Thr Ile Val Asp Ala Gly Phe Glu Gly Gln Leu

101 110 120

Thr Ile Glu Leu Val Gly Ser Glu Phe Pro Ile Lys Leu Lys Tyr Gly Met Arg Phe Ile

121 130 140

His Leu Ile Phe Ala Lys Thr Leu Thr Pro Val Glu Lys Pro Tyr Asn Gly Lys Tyr Gln

141 150 160

Lys Gln Met Gly Val Thr Pro Ala Lys Leu Ser Ser

161 170 172

<210> 2

<211> 516

<212> DNA

<213> sulfolobus solfataricus virus

<400> 2

atgattttca gtgatagaga tttaaaatat tacctggaaa agggatggat 50

aaagattgag ccattaagag aagacactat tagggaaaat ggggtagatt 100

taaggattgg taacgagatt gctagattca agaaaaatag gatttttgat 150

cctgacaaag attccataga tgattttata gagaaagagg tggggaatga 200

gtttataata aatccccatg agcatgcatt attaactaca gaagaatatg 250

taagattgcc taatgatgta atggcattcg taaatcttag atcaacattt 300

gctaggttag gtcttttcat tcctccaact atcgtagatg caggatttga 350

agggcaatta actatagagt tagtaggatc agaattccca ataaaactaa 400

aatatggaat gagatttatc cacctaatct tcgcaaaaac actaacacca 450

gttgaaaagc catataatgg aaaatatcag aaacaaatgg gtgtgacgcc 500

ggccaagctc tcaagt 516

Claims

1. sulfolobus solfataricus virus deoxyuridine Pyrophosphate phosphohydrolase, is characterized in that: its aminoacid sequence is as shown in SEQ ID NO.1.

2. polynucleotide for deoxyuridine Pyrophosphate phosphohydrolase described in the claim 1 of encoding, is characterized in that: its nucleotide sequence is as shown in SEQ ID NO.2.