CN102304500B - Thermus siphoviridae phage 4 (TSP4) DNA helicase and polynucleotide coding same - Google Patents

Abstract

The invention discloses thermus siphoviridae phage 4 (TSP4) DNA helicase and a polynucleotide coding the same. The DNA helicase which is separated thermus siphoviridae phage 4 is critical in polymerase chain reaction (PCR) technique, and can improve the efficiency of the amplification of the DNA fragment by the PCR technique so as to improve the efficiency of amplification.

Description

The polynucleotide of phage TSP4 dna helicase and this helicase of coding

Technical field

The present invention relates to the polynucleotide of phage TSP4 dna helicase and coding helicase, belong to biological technical field.

Background technology

Helicase, i.e. the double-stranded class of enzymes of isolating nucleic acid, its participates in all biological most cellular pathways.Its catalytic core reaction generally is very conservative.Helicase is in the hydrolysis of the simultaneous ribonucleoside triphosphote (NTP, normally ATP) of the two strands that reduces nucleic acid (DNA-DNA, DNA-RNA or RNA-RNA).Helicase is participated in most of nucleic acid metabolism processes in the cell, comprises duplicating of karyomit(e) and plasmid, transcribes translation, the recombinational repair of DNA and the processing of RNA etc.Many enzymes that the helicase function is arranged are arranged in the organism, former nuclear dimension, the helicase of at least 12 supposition has at present been identified out in colibacillary genome; The eucaryon aspect, the gene of coding helicase GAP-associated protein GAP surpasses genomicly 2% in the yeast saccharomyces cerevisiae, can infer that thus helicase plays an important role for the survival and development of cell.

Through carrying out the comparison of Argine Monohydrochloride sequence, can divide five big types with helicase, wherein the hugest is I and II helicase superfamily (SFI and SFII); Great majority have 3 '-5 ' polarity in these two types of helicases, wherein comprise seven (I, Ia; II, III, IV; V, and VI) so-called helicase characteristic motif (motif); In these two superfamilies, characteristic sequence Walker A and the B sequence of conservative ATPase are I and II motif, and in SFI and SF II albumen, other motifs are not high conservative.The SF3 helicase derives from DNA or RNA viruses usually, only contains I, three conservative motifs of II and III, and other helicase numbers are less, as E.ColiIn six the gathering helicase and constitute a class by itself of DnaB-like; The 5th type of helicase, albumen is less, and the Rho factor is transcribed in the termination that wherein comprises in the bacterium.

The dna double spiral of mechanism at present untie to(for) helicase also is not very clear, but all helicases still have certain common characteristic, is in particular in to untie duplex and on strand, move.There is the mechanism of report helicase can be divided into passive (passive) and active (aetive) two kinds of forms.Passive form thinks that helicase and ssDNA are that passive interaction and travel direction is uncertain; And it has been generally acknowledged that at present the poly helicase adopts active mechanisms to untwist, it can both combine with dsDNA and ssDNA.Everybody universally recognized helicase action model is " active roling " and " inchworm " model now.

High temperature bacterium phage TSP4 is for infecting the high temperature bacteria strain ThermusThe phage of TC16, the Master's thesis of Li Qiupeng has carried out preliminary parsing to this phage genome in (Kunming University of Science and Technology, 2009); And announced the partial sequence of the dna helicase of TSP4; But this part sequence is only arranged, and the helicase structure is imperfect, non-activity.

All biologies all have a considerable amount of genes encoding helicases; Mainly be since they duplicate, recombinate, repair, transcribe, translation, montage, mRNA are edited, due to the critical function that risen when chromosome reconstitution, transhipment and degraded, so helicase also becomes the focus that scientific circles are studied.

A kind of dependence helicase isothermal PCR technology of development at present, its principle is the dna replication dna of simulation organism, in the PCR system, adds helicase, has saved high-temperature denatured step, PCR is reflected under the temperature carries out.Therefore this round pcr can return in a waters pot and carry out, and makes the PCR reaction not rely on the PCR appearance and carries out.But because the helicase major part that present clonal expression comes out is from the normal temperature mikrobe; Its stability and thermotolerance are not fine; Affect the reaction efficiency of PCR, therefore from high temperature microbe, seeking heat-stable helicase is an important channel that addresses the above problem.Dwell hot bacterium as the model animals of high temperature bacterium, and the research of its phage is also increasing, from the hot bacterium phage that dwells, obtains heat-stable helicase and has important value relying on helicase isothermal PCR technical field.

Summary of the invention

The present invention aims to provide TSP4 phage DNA helicase, promptly includes the polypeptide of the aminoacid sequence shown in the SEQ ID NO.1 or active fragments, analogue or the verivate of its conservative property variation polypeptide or its polypeptide; This dna helicase separates from high temperature phage TSP4, comprises the active fragments of its conservative property variation polypeptide, polypeptide.This patent provides the gene and the protein sequence thereof of complete high temperature bacterium phage TSP4 helicase.

The aminoacid sequence of polypeptide described in the present invention, analogue or verivate has the homogeny with the aminoacid sequence at least 70% shown in the SEQ ID NO.1.

Dna helicase described in the present invention has the aminoacid sequence shown in the SEQ ID NO.1.

Another object of the present invention provides the polynucleotide of coding TS P4 phage DNA helicase, its comprise be selected from down the group in a kind of:

(a). coding has polypeptide or its fragment, the analogue of aminoacid sequence shown in the SEQ ID NO.1, the polynucleotide of verivate;

(b). with polynucleotide (a) complementary polynucleotide; Or

(c). the polynucleotide of at least 70% homogeny with (a) or (b) are arranged.

Polynucleotide encoding described in the present invention has aminoacid sequence shown in the SEQ ID NO.1.

Polynucleotide sequence described in the present invention includes the sequence of 1-1224 position among sequence or the SEQ ID NO.2 of 200-451 position among the SEQ ID NO.2.

Another object of the present invention provides the recombinant vectors of the polynucleotide that contain the coding DNA helicase, and it is the recombinant vectors that is formed by the described polynucleotide of preamble and plasmid, virus or vector expression vector establishment.

Helicase protein sequence of the present invention is carried out the conservative region prediction, find to have a plurality of conserved domains.Wherein 150 ~ 360 aa belong to P-loop NTPase superfamily member.And conservative ATP binding site is arranged near 165 aa, 187 aa and 285 aa; Near Walker A motif 165 aa is a Walker B motif at 285 aa.

Characteristics of P-loop NTPase superfamily member are exactly the binding site that contains Triphosaden; Be called Walker A motif (GxxxxGK [S/T] respectively; Wherein x represents any residue) and Walker B motif (hhhh [D/E], wherein h represents hydrophobic residue).Wherein Walker A motif and Walker B motif combine ATP, GTP and Mg respectively ²⁺, this is the important conservative region distribution situation of helicase.

Beneficial effect of the present invention:

The dna helicase that the present invention obtains; Prove the additive that can be used as the pcr amplified dna reaction system through experiment, improve the efficient of unwinding of DNA, to reach the result who improves amplification efficiency; Can realize improving the segmental efficient of pcr amplified dna; Also can be used in the isothermal PCR technology, under constant temperature, realize a kind of technology of DNA cloning, in this reaction system key component also be dna helicase.

Description of drawings

Fig. 1 is that isotope-labelling method is surveyed helicase enzyme schematic diagram alive.

(helicase is added in "+" expression among the right figure, and helicase is not added in "-" expression, and this figure is illustrated under the situation of adding helicase, and the probe of mark breaks away from from DNA)

Fig. 2 is a unwindase gene tsp4-h42 conserved structure domain analysis synoptic diagram, shows the important Motif of helicase among the figure, comprises ATP-binding site, Walker A and Walker B.

Fig. 3 is cloned sequence and the relevant restriction enzyme mapping of unwindase gene tsp4-h42.M is Marker, and swimming lane 1 is a tsp4-h42 gene PCR product, and its size is 1224bp; Swimming lane 2 is the pET-23b plasmid; Swimming lane 3 is the tsp4-h42-pET-23b plasmid, and swimming lane 4 is a pET-23b plasmid double digestion electrophoresis result, and swimming lane 5 is a tsp4-h42-pET-23b double digestion electrophoresis result.

Fig. 4 is the SDS-PAGE analyzing and testing electrophorogram of unwindase gene tsp4-h42 expressing protein, and its molecular weight is about 46490 dalton.

Fig. 5 is the mensuration synoptic diagram that reorganization helicase ATP enzyme is lived.1 is contrast: damping fluid+dsDNA+ ATP+Mg ²⁺, 2 are reorganization helicase+ATP, and 3 are reorganization helicase+dsDNA+ATP, and 4 are reorganization helicase+dsDNA+ATP+Mg ²⁺, helicase has the activity of ATP enzyme simultaneously, measures the release of phosphate radical through fixing phosphorus method, thereby proves the existence of helicase activity, proves that simultaneously the vigor of helicase needs Mg ²⁺Activation.

Fig. 6 is the DNA of the reorganization helicase tsp4-h42 active detection figure that untwists.("+" among the figure and "-" represent the reactant that " interpolation " and " not adding " right side is corresponding respectively) is at reorganization tsp4-h42 helicase+dsDNA+ATP+Mg ²⁺Under the simultaneous condition, the tsp4-h42 helicase shows the double-stranded DNA vigor that untwists.

Fig. 7 is the DNA of the reorganization helicase tsp4-h42 active atomic force microscope observation figure that untwists.At reorganization helicase+dsDNA+ATP+Mg ²⁺Under the simultaneous condition, the tsp4-h42 helicase shows double-stranded DNA is untwisted, and forms replication bubble and these two kinds of typical DNA of replication fork form of untwisting.

Embodiment

The clone and the expression of instance 1:DNA helicase

One, the amplification of TSP4 phage DNA unwindase gene, (is template with phage TSP4DNA)

(1) TSP4 phage DNA unwindase gene amplification the primer sequence is following:

Forward primer: 5 '- CATATGACGGACATAAAGCTGGAG-3 '

Reverse primer: 5 '- CTCGAGGGTAAGAGAGAAATCAAAGTT-3 '

(2) amplification system is following:

Table 1: amplification reaction system component

Template	10 ng
		Premix exTaq	25μl
Primer	Each 1 μ l
		Distilled water ddH 2O	Be supplemented to 50 μ l

(3) amplification condition is following:

With the reaction system mixing, earlier at 94 ℃ of preparatory sex change 4min, then at 94 ℃ of sex change 30 s, 60 ℃ of annealing 30 s, 72 ℃ are extended 30 s, and after 30 circulations, 72 ℃ are extended 10min.Get product 5 μ l after having reacted, in 10 g/L sepharoses, carry out the electrophoretic analysis (see figure 3).

Two, the glue of PCR product reclaims purifying

1. in electrophoresis apparatus, record 1.0% sepharose;

2. will treat the PCR product point sample electrophoresis of separation and purification, stop electrophoresis in the appropriate location;

3. cutting-out contains the pulsating gel of this purpose under uv lamp, transfers in the Ep pipe of 1.5ml;

4. reclaim test kit with day root biotech firm glue and carry out the segmental recovery of purpose, 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, unwindase gene tsp4-h42 analytical results is seen Fig. 2.

Three, the structure of recombinant expression vector

In order to be connected to expression vector pET23b to the goal gene segment, just need make the purpose segment have the segment of sticky end, promptly have restriction enzyme site.But on the goal gene that pcr amplification obtains, only have polyA; And have polyT on the pMD18-T carrier; Therefore, can earlier the purpose segment be connected on the pMD18-T carrier, carry out corresponding enzyme again and cut; Finally obtain having the purpose fragment of sticky end, for next step expression vector establishment experiment is prepared.Concrete experimental procedure is following:

(1) TSP4 phage DNA unwindase gene fragment is connected on the pMD18-T carrier, obtains recombinant plasmid.

ligation system is following:

Table 2: reaction system component

PCR glue reclaims product	7.5 μl
		10 * T4 DNA ligase enzyme damping fluid	1μl
The pMD18-T linear carrier	0.5μl
		T4 DNA ligase enzyme	1μl
Total amount	10μl

condition: 16 ℃, connection is spent the night.

(2) recovery of recombinant plasmid.

1. get connection product 10 μ l, add 40 μ l ddH ₂O;

2. transfer in EP (effpendorf) pipe of 1.5ml;

3. add 5 μ l sodium-acetates (pH 5.2);

4. add 2.5 times of absolute ethyl alcohols with upper volume;

5. at-80 ℃ of held 15min;

6. 4 ℃ of centrifugal 5min, 13000rpm abandons supernatant;

7. 70% ethanol is washed twice, 100% ethanol and is washed once;

8. after drying naturally, add 10 μ l ddH ₂The O dissolving.

(3) conversion of recombinant plasmid.

100 μ L competent cells ( E.coliBL21) lining adds 10 μ L and connects product, ice bath 30min, and 42 ℃ are heated 1 ~ 2min then, and ice bath 2min adds 400 μ L LB liquid nutrient mediums afterwards again, and in 37 ℃, the 150rpm shaking table is cultivated 1h.Coating contains penbritin (Amp then ⁺) the LB solid plate, be inverted to cultivate 12h in 37 ℃.

(4) detect the bacterial strain that contains recombinant plasmid through bacterium colony PCR, filter out positive bacterium colony.

Observe transforming flat board, the several bacterium colonies of random choose rise with 10 μ l rifle choicests, are dissolved in 20 μ l ddH ₂Among the O, behind the mixing, shift 10 μ l bacterium liquid in the LB liquid nutrient medium, remain 10 μ l bacterium liquid and be used for thermo-cracking, 98 ℃ of cracking temperatures, time 10min.

2. gently get rid of with the palm whizzer after the cracking, lysate is as the bacterium colony pcr template.

3. the colony PCR amplification reaction system is following:,

Table 3: reaction system component

Template	2μl
		Premix rTaq	12.5μl
Primer (forward and reverse primer)	Each 1 μ l
		ddH 2O	Be supplemented to 25 μ l

bacterium colony PCR reaction conditions:

Behind the reaction system mixing, at 94 ℃ of preparatory sex change 4min; Then at 94 ℃ of sex change 30 s, 60 ℃ of annealing 30 s, 72 ℃ are extended 30 s, 30 circulations; 72 ℃ are extended 10min.Reaction is got product 5 μ l after accomplishing, and in 1% sepharose, carries out the electrophoretic analysis (see figure 3).

identifies positive bacterium colony.

(5) positive colony increases bacterium.

1. getting penbritin 5 μ l (final concentration 100 μ g/ml) adds in the 5ml LB substratum;

2. use transfering loop picking positive colony, be inoculated in Amp ⁺In-LB the substratum;

3. put into 37 ℃ of incubators then, shaking table is cultivated, and spends the night.

(6) recombinant plasmid extracting (method that adopts the plasmid extraction test kit to provide).

1. collect thalline: join the bacterium liquid of cultivating in the step (5) in the centrifuge tube of 5ml, centrifugal collection bacterial sediment is abandoned supernatant;

2. add solution I (containing the RNA enzyme) 100 μ l, vibrating to thalline thoroughly is uniformly dispersed, and changes in the EP pipe of 1.5ml;

3. add solution II 150 μ l, mixing, room temperature is placed 5min, makes RNA in the RNA enzymic digestion bacterium of solution I;

adds solution III 150 μ l; 4 ℃ centrifugal; 13000rpm, 10min;

5. supernatant is changed in the DNA adsorption column, add 420 μ l binding buffer liquid again, mixing, room temperature is centrifugal, 5000rpm, 1 min reclaims twice, abandons down clear liquid;

6. add 750 μ l rinsing liquids in adsorption column, room temperature is centrifugal, 5000rpm, and 1min abandons down clear filtrate (repeating once);

7. room temperature 13000rpm, 2min is once centrifugal again;

8. adsorption column is moved in the centrifuge tube of new 1.5ml, central authorities add 70 μ l ddH at adsorption film ₂O places 1min, and room temperature is centrifugal then, 13000rpm, 2min;

9. resulting plasmid is got 1 μ l electrophoretic analysis.

(6) enzyme of recombinant plasmid is cut the evaluation (see figure 3).

It is following that

enzyme is cut system:

Table 4: reaction system component

Plasmid	5 μ l (about 200ng)
		10 * H damping fluid	1 μl
NdeⅠ	0.3μl
		XhoⅠ	0.3μl
Distilled water	3.4μl
		Total amount	10μl

enzyme tangent condition: 37 ℃; Spend the night, reclaim the dna helicase gene fragment.

(7) have the preparation of sticky end linear carrier pET23b.

For the goal gene segment is connected on the expression vector pET23b, just need make the purpose segment have the segment of sticky end, promptly have restriction enzyme site.Equally, can insert in the carrier, also need make carrier have sticky end, and make their restriction enzyme site identical in order to make the purpose segment.

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

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

2. increase bacterium and collect thalline: increase the bacterium method with step 5, and the bacterium liquid of cultivating is joined in the centrifuge tube of 5 ml, centrifugal 5 min of room temperature, 5000 rpm make bacterial sediment, abandon supernatant;

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

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

5. the supernatant with centrifugal back gained changes in the DNA adsorption column, and room temperature is centrifugal, 5000 rpm, and 1 min reclaims twice, abandons down clear;

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

7. add 750 μ l rinsing liquids (W2 solution) then, room temperature is centrifugal, 5000rpm, and 1min abandons down 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, central authorities add 70 μ l ddH at adsorption film ₂O water is placed 1 min, and room temperature is centrifugal then, 13000 rpm, and 2 min abandon supernatant, obtain plasmid.

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

B, plasmid pET23b enzyme are cut evaluation.(see figure 3)

It is following that

enzyme is cut system:

Table 5: reaction system component

Plasmid	5 μ l (about 200ng)
		10 * H damping fluid	1 μl
NdeⅠ	0.3 μl
		XhoⅠ	0.3 μl
Distilled water	3.4 μl
		Total amount	10 μl

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

(8) structure of recombinant expression vector, TSP4 phage DNA helicase induction expression of protein and purifying

1. the linear carrier pET23b that has sticky end and the TSP4 phage DNA unwindase gene fragment that the front experiment are obtained through connecting conversion and cutting evaluation and sequence verification with bacterium colony PCR, enzyme, can obtain recombinant expression vector.

2. the abduction delivering of TSP4 phage DNA helicase albumen in intestinal bacteria

With the recombinant vectors tsp4-helicase/pET23b transformed into escherichia coli BL21 that builds; The bacterial strain that contains recombinant plasmid is through overnight cultures; Bacterium liquid is inoculated in 30 ml Amp+-LB (the 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 1ml bacterium liquid as control experiment; All the other bacterium liquid are put into 37 ℃, and 60 rpm shaking tables were cultivated abduction delivering 6 hours.

3. TSP4 phage DNA helicase protein SDS-PAGE detects

Take out and induce back bacterium liquid to survey the OD value on a small quantity, get different bacterium liquid measures, its biomass is equated according to bacterium liquid OD value is different, 5000rpm, centrifugal 5min abandons supernatant; Add 80 μ l distilled waters, 20 μ l, 2 * sample-loading buffer is after vibration is scattered thalline; At 98 ℃ of held 15min, make cellular lysate, discharge protein; Get the bacterium liquid behind the abduction delivering, the preparation sample; Sample is splined in the electrophoresis apparatus at 100V, and 60mA behind the electrophoresis 2h, adds the dyeing of 100ml R250 coomassie brilliant blue staining liquid, the shaking table vibration, and 100 rpm are behind the 20min; Staining fluid is poured out, used the clear water rinsing, add an amount of destainer 100 rpm again, 10h uses the scanner scanning gel at last, detects protein expression situation (see figure 4), and the result is the target protein band that obtains 46kD.

4. the proteic purifying of helicase of recombinating

The BL21 bacterial strain that utilizes aforesaid method to induce in a large number to contain recombinant plasmid tsp4-h42/pET23b, bacterium liquid is through centrifugal collection coli somatic (4 ^oC, 5,000x g, 10 min).Thalline is suspended in right amount and (makes the OD of bacteria suspension ₆₀₀Reach 20) buffer A [20 mM sodium phosphates, 500 mM NaCl, 30 mM imidazoles; PH 7.4] in, ultrasonication cell on ice, 4 ℃; 20, centrifugal 10 min of 000x g, supernatant are splined on HisTrap HP post (1 ml that uses the buffer A balance good; GE Healthcare) in, it is constant until the UV value to wash unconjugated albumen with buffer A again; Bonded albumen contains buffer B [20 mM sodium phosphates, 500 mM NaCl, the 30 mM imidazoles of imidazoles gradient (0-500 mM) with 20 ml; PH 7.4] wash-out, the Fractional Collections elutriant, with histidine-tagged recombinant protein elution peak at 475 mM; Merge the elution peak component, use damping fluid [50 mM Tris-HCl, 25 mM Mg2+ at last; PH7.4] dialysed overnight, the purified recombinant protein ingredient carries out SDS-PAGE and analyzes.

The mensuration that the proteic ATP enzyme of instance 2 reorganization helicases enzyme is lived

ATP enzyme enzyme activity determination adopts malachite green ammonium molybdate method, when ATP is hydrolyzed into ADP by helicase, discharges the free phosphoric acid root, can produce blue complex with molybdate, through measuring absorbancy, can confirm that its ATP enzyme enzyme is alive, and method is following:

Malachite green dyestuff liquid storage preparation method is: the vitriol oil (1.84 g/mL) 60 mL are slowly added 300 mL H ₂Among the O, be cooled to room temperature, add 0.44 g malachite green dyestuff.

Sample is: 1 is damping fluid+dsDNA+ ATP+Mg ²⁺(blank), 2 are reorganization tsp4-h42 helicase+ATP, and 3 are reorganization tsp4-h42 helicase+dsDNA+ATP, and 4 are reorganization tsp4-h42 helicase+dsDNA+ATP+Mg ²⁺

PH 7.5 Tris-HCl that contain 50 mmol/L in the mixed liquid (200 mL) of the reaction that atpase activity is measured, testing protein sample, 20 ng DNA, 25 mmol/L MgCl ₂, mixed solution is behind reaction 1 h under 60 ℃; The perchloric acid termination reaction that adds 800 mL 20% adds 1 mL H then successively ₂O, 0.5 mL dye liquor (2.5 mL, 15 % ammonium molybdates, 10 mL dyestuff liquid storages; 0.2 mL 11% polysorbas20), after room temperature was placed 10 min, 630 nm surveyed light absorption value; Light absorption value increases explanation under the effect of helicase, and ATP is decomposed and discharges phosphate radical (seeing table 6).

The light absorption value of each sample under the table 6:630 nm

Sample number	1 (contrast)	2	3	4
					△OD	0	0.037	0.029	0.132

Instance 3 isotope-labelling methods are surveyed the helicase enzyme and are lived

1, measuring principle is seen Fig. 1.

2, untwist template preparation

A. adopt T4 polynucleotide kinase (Takara) and [γ- ³²P] 3 ' sequence of the synthetic good primer sequence p42 of ATP mark, the p42 primer sequence is:

5’-CAGTGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGA

B. p42 that labelled with radioisotope is good and the genome of M13 phage are annealed, and form partially double stranded.

C. annealing forms the template of untwisting of mark:

Annealing process is following: corresponding primer and template are boiled 3 min in the ratio of 1:1.5 in annealing buffer (20 mM Tris-HCl, pH 7.5,100 mM NaCl), naturally cool to room temperature, generally need 4 h, form end-labelled DNAp42/M13.

3, the mensuration of helicase activity

The helicase activity mensuration system of standard is 20 μ l, and reaction mixture comprises 50 mM Tris-HCl, pH 7.5,10 mM MgCl ₂, 2 mM ATP, the reorganization helicase albumen of 7.5 μ g and the end-labelled DNAp42/M13 of 10f mol; Mixed solution is at 60 ℃ of reaction 60 min, after reaction finishes, place 15 min on ice after; Sample adds 10 μ l sample-loading buffers (10 mM EDTA and 0.1% tetrabromophenol sulfonphthalein) behind centrifugal 15 min of 16,000 x g; Be splined in the electrophoresis apparatus, electrophoresis uses the polyacrylamide gel electrophoresis of 15% non-sex change, electrophoresis in 0.5 x tbe buffer liquid; After accomplishing, reclaims electrophoresis glue; And shield quantitative (see figure 6) to blob of viscose to X-ray film exposure or with phosphorus, visible under the effect of helicase, the probe of part mark disintegrates down from the bonded plasmid because of the effect of helicase.

Instance 4 AFMs are measured helicase activity

The concrete grammar of measuring is: with Nde I and Xho I digestion pET-23b plasmid; The big fragment of plasmid on 1% sepharose; The substrate that untwists as the helicase of recombinating. the helicase of reorganization and the dna fragmentation of above-mentioned recovery are mixed; Make both concentration be 0.5 ng/L, mixed solution is added in the reaction system, reaction system is 20 mmol/L Tris-HC1 and the 10 mmol/L MgC1 of pH 7.5 ₂, in reaction system, behind the adding 1 microlitre 10 mmol/L ATP, be added to the fresh mica surface that strips at once at last; Room temperature is placed 10min, make DNA and protein molecular be adsorbed onto mica surface after, water washes; Remove loose molecule, mica surface is dried up with nitrogen, subsequent use; Utilize AFM Nanoscope III a Multimode-AFM (Digital Instruments) under the pattern of rapping, to scan; Needle point uses the super-sharp silicon tips (Silicon.MDT Ltd.) of resonant frequency 106 kHz; Sweep velocity is 1～2 Hz, and scanning result is seen Fig. 7.

Sequence table

SEQUENCE LISTING

< 110>Kunming University of Science and Technology

< 120>polynucleotide of phage TSP4 dna helicase and this helicase of coding

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 408

<212> PRT

< 213>TSP4 phage

<400> 1

Met Thr Asp Ile Lys Leu Glu Arg Ser Phe Val Ala Ala Cys Tyr His Asn Arg Arg Leu

1 10 20

Val Arg Lys Phe Pro Asp Leu Ser Pro Asp Asp Phe Gln Asp Phe Asp Ser Arg Arg Tyr

21 30 40

Trp Gln Ala Leu Val Ala Gly Glu Pro Ile Val Asp Phe Thr Ile Tyr Glu Pro Pro Leu

41 50 60

Ala Glu Asp Glu Ala Pro Ala Ala Val Gln Arg Ile Arg Lys Asn Ala Trp Leu Arg Arg

61 70 80

Val His Ser Trp Ala Asp Arg Leu Lys Val Leu Ala Glu Arg Lys Asp Glu Glu Lys Leu

81 90 100

Ser Asp Leu Leu Ala Lys Ser Pro Leu Leu Ser Glu Glu His Gly Val Gln Val Glu Asp

101 110 120

Ala Leu Asn Glu Ile Gln Ala Ala Leu Leu Thr Asp Thr Leu Val Trp Glu Leu Lys Asp

121 130 140

Pro Lys Leu Ala Pro Leu Gly Tyr Leu Phe Tyr Pro Gly Ser Ile Thr Val Ile Gly Ala

141 150 160

Thr Pro Gly Thr Gly Lys Ser Ala Leu Ala Glu Gln Ile Leu Leu Asp Leu Thr Glu Gln

161 170 180

Gly Ala Met Ala Leu Asp Ile Ser Val Glu Leu Ser Ala Glu Val Arg Thr Ser Arg Tyr

181 190 200

Met Gln His Leu Val Gly Ser Gly Val Thr Tyr Arg Lys Leu Leu Ala Lys Thr Tyr Asp

201 210 220

Pro Glu Ala Leu Arg Gln Gly Met Gln Leu Leu Arg Ala Thr Ser Ser Gly Ala Ala Arg

221 230 240

Lys Leu Ile Ile Asp Pro Ser Ser Val Ser Leu Asp Thr Val Leu Ala Ser Val Gln Ala

241 250 260

Phe His Gln Gln Tyr Leu Ala His Lys Thr Gln Leu Glu Ala Gln Gly Met Arg Ile Gly

261 270 280

Pro Pro Val Val Leu Val Asp Phe Leu Gln Ala Val Ser Val Glu Thr Lys Thr Glu Gly

281 290 300

Ile Tyr Glu Arg Met Ser Leu Leu Ala Glu Arg Leu Tyr Ala Leu Gly Lys His Leu Gly

301 310 320

Leu Ala Met Ile Trp Leu Ser Gln Leu Lys Lys Arg Asp Tyr Ser Arg Arg Met Pro Pro

321 330 340

Asn Arg Ser Asp Ile Glu Gly Ser Gly Arg Ile Glu Gln Tyr Ala His Thr Leu Ser Leu

341 350 360

Leu Phe Thr Pro Pro Asp Ala Ala Ile Thr Pro Ala Gly Arg Glu Ile His Ile Tyr Thr

361 370 380

Pro Lys Ala Arg Leu Gly Asn Val Gln Pro Phe Leu Thr Gly Val Phe Asn Gly Asp Thr

381 390 400

Leu Asn Phe Asp Phe Ser Leu Thr

401 408

<210> 2

<211> 1224

<212> DNA

< 213>TSP4 phage

<400> 2

ATGACGGACA TAAAGCTGGA GCGCTCCTTC GTTGCGGCCT GCTACCACAA 50

CCGGAGGCTG GTGCGGAAGT TTCCTGACCT CAGTCCGGAC GACTTCCAGG 100

ACTTTGACTC CCGGCGTTAC TGGCAGGCGC TTGTGGCCGG GGAGCCCATT 150

GTGGATTTCA CCATCTACGA ACCGCCGCTG GCGGAGGACG AGGCCCCGGC 200

GGCGGTCCAG CGCATCCGCA AGAACGCCTG GCTCCGTCGG GTGCACTCAT 250

GGGCTGACCG GCTCAAGGTA CTGGCCGAGC GTAAGGACGA GGAGAAGCTC 300

TCTGACCTGC TGGCTAAAAG CCCCCTGCTC TCTGAAGAAC ACGGAGTACA 350

GGTGGAGGAC GCTCTGAACG AAATCCAGGC GGCACTTCTG ACCGATACCC 400

TGGTGTGGGA GCTCAAGGAC CCAAAGCTAG CCCCATTGGG TTATCTCTTC 450

TATCCAGGAT CCATCACGGT CATCGGCGCT ACTCCTGGAA CCGGTAAGAG 500

CGCCCTGGCG GAGCAGATAC TCCTGGACCT CACCGAGCAA GGGGCCATGG 550

CCCTGGACAT CAGCGTGGAG CTCTCGGCGG AAGTACGCAC TTCCCGCTAC 600

ATGCAGCACC TGGTGGGAAG TGGGGTTACC TACCGCAAGC TCCTGGCCAA 650

GACCTATGAC CCGGAGGCTT TGCGCCAGGG AATGCAGCTT CTACGTGCTA 700

CTTCCAGCGG AGCTGCCCGT AAGCTTATCA TTGACCCGTC CTCGGTATCC 750

TTGGACACGG TGCTGGCCTC AGTGCAGGCC TTTCACCAGC AATACTTGGC 800

CCACAAGACG CAGCTGGAGG CTCAAGGTAT GCGCATTGGT CCTCCGGTGG 850

TGCTTGTGGA CTTCCTGCAG GCGGTCAGCG TGGAGACCAA GACCGAGGGT 900

ATCTACGAAC GCATGAGCTT GCTCGCCGAG AGGCTTTACG CTCTGGGTAA 950

GCATCTTGGG CTGGCCATGA TTTGGCTGTC CCAGCTCAAG AAGCGGGACT 1000

ACTCCCGCAG GATGCCGCCC AACCGCTCGG ACATTGAGGG TTCTGGACGC 1050

ATTGAGCAGT ACGCTCATAC CCTTTCTTTG CTCTTTACGC CGCCGGACGC 1100

TGCCATCACT CCAGCAGGTC GTGAGATTCA CATCTACACG CCTAAGGCTA 1150

GGCTGGGCAA TGTCCAGCCG TTTCTGACCG GAGTGTTCAA CGGAGACACC 1200

CTGAACTTTG ATTTCTCTCT TACC 1224

Claims (2)

1. phage TSP4 dna helicase is characterized in that: have the aminoacid sequence shown in the SEQ ID NO.1.

2. according to the said phage TSP4 of claim 1 dna helicase, it is characterized in that: this helicase is coded by nucleotide sequence shown in the SEQ ID NO.2.

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