CN101948853A - Thermophilic fat bacillus DNA polymerase - Google Patents

Abstract

The invention relates to a thermophilic fat bacillus DNA polymerase, a gene, a plasmid, a fusion protein, applications and a preparation method of the thermophilic fat bacillus DNA polymerase. The thermophilic fat bacillus DNA polymerase can carry out nucleic acid amplification reaction, and leads the reaction to be carried out easily with high detection sensitivity. The fusion protein is beneficial for the purification of the thermophilic fat bacillus DNA polymerase.

Description

The bacstearothermophilus archaeal dna polymerase

Technical field

The present invention relates to archaeal dna polymerase, be specifically related to the preparation method of a kind of bacstearothermophilus archaeal dna polymerase, gene, plasmid, fusion rotein, purposes and bacstearothermophilus archaeal dna polymerase.

Background technology

DNA is the genetic material of most of living species.The function of archaeal dna polymerase mainly is to carry out dna replication dna and reparation.Clone at present the archaeal dna polymerase of multiple living species, comprised some thermophilic archaeal dna polymerases.The separated DNA polysaccharase has higher thermostability from thermophile, and the Taq archaeal dna polymerase that for example obtains from Thermus aquaticus is 1.6 hours 95 ℃ transformation period.

Loop-mediated isothermal amplification technology (loop-mediated isothermal amplication of DNA is called for short LAMP) is to finish in 45～90 minutes under constant temperature (63～65 ℃) condition.This comparatively gentle temperature condition and do not have temperature cycle that required instrument is oversimplified, overcome conventional P CR inherent detection time long, pollute easily and detect shortcoming such as cost height.And this detection method is lower to testing staff's technical quality requirement, and actually operating is very easy, does not need special reagent and plant and instrument, helps setting up rapid screening system with low cost.Therefore need to seek a kind of archaeal dna polymerase and adapt to the LAMP detection method.

Summary of the invention

A goal of the invention of the present invention is to solve the deficiencies in the prior art part and gene that a kind of bacstearothermophilus archaeal dna polymerase is provided.

A kind of gene of bacstearothermophilus archaeal dna polymerase, the nucleotide coding sequence of described bacstearothermophilus dna polymerase gene is shown in SEQ ID NO 1.

Another goal of the invention of the present invention is to solve the deficiencies in the prior art part and a kind of plasmid is provided.

A kind of plasmid pMal-c5x/bst that contains the gene of above-mentioned bacstearothermophilus archaeal dna polymerase.

The pMal system is a kind of protein fusion expression efficiently and purification system.The pMal carrier contains the coding maltose binding protein, and (multiple clone site in its downstream is convenient to the goal gene insertion for Maltose Binding Protein, intestinal bacteria mal E gene MBP), expresses the fusion rotein (1,2,3) that the N end has MBP.Make clone gene obtain to efficiently express by " Ptac " strong promoter and mal E translation initiation signal, and utilize MBP that the specificity of maltose is gone on foot affinity purification in conjunction with one of realization fusion rotein.

The pMal carrier of this system contains the sequence of one section coding specific proteins enzyme recognition site at contiguous multiple clone site place, thereby is convenient to behind the purifying target protein and MBP cutting and separating (5).In order to achieve this end, there is a restriction enzyme enzyme recognition site just in time to be positioned at specific proteins enzyme recognition site place in the multiple clone site, also be simultaneously the site that goal gene inserts.

The pMal serial carrier can give expression to the fusion rotein of 100mg from 1 liter nutrient solution.Under most of situation, the fusion rotein that gives expression to is soluble, can improve the proteic solubility of expression in escherichia coli (6) because MBP has been proved.Although there is not a kind of expression system to be applicable to all gene clones, pMal protein fusion expression and purification system are verified: in detected known protein, have above 75% to give expression to stable rate.There is a chapter that making of pMal carrier has been used as in-depth analysis among the Current Protocols in Molecular Biology (3).

The present invention adopts this expression system can express the fusion rotein of maltose binding protein and bacstearothermophilus archaeal dna polymerase.Expressed fusion protein can be carried out purifying with the amylose starch affinity chromatography, and products therefrom purity is very high.In addition, it is reported that maltose binding protein can improve the solubility of fusion rotein, helps the purifying of fusion rotein.

Another goal of the invention of the present invention is to solve the deficiencies in the prior art part and a kind of archaeal dna polymerase is provided.

A kind of bacstearothermophilus archaeal dna polymerase, the aminoacid sequence of described bacstearothermophilus archaeal dna polymerase is shown in SEQ ID NO 2.

Because the thermostability of bacstearothermophilus archaeal dna polymerase is stronger, and has higher strand displacement activity so can be used in the high temperature order-checking and the nucleic acid isothermal amplification.Bacstearothermophilus archaeal dna polymerase of the present invention is 8 minutes 65 ℃ transformation period.

The present invention also provides the application at nucleic acid amplification reaction of above-mentioned bacstearothermophilus archaeal dna polymerase.

Another goal of the invention of the present invention is to solve the deficiencies in the prior art part and a kind of fusion rotein is provided.

A kind of fusion rotein, it has above-mentioned bacstearothermophilus archaeal dna polymerase, and described fusion rotein also has maltose binding protein.

Expressed fusion protein can be carried out purifying with the amylose starch affinity chromatography, and products therefrom purity is very high.In addition, maltose binding protein can improve the solubility of fusion rotein, helps the purifying of fusion rotein.

The present invention also provides the application at bacstearothermophilus archaeal dna polymerase purifying of above-mentioned fusion rotein.

The present invention also provides a kind of preparation method of bacstearothermophilus archaeal dna polymerase, and described purification process comprises the steps:

(1) nucleotide coding sequence of the bacstearothermophilus dna polymerase gene of amplification shown in SEQ ID NO 1;

(2) nucleotide coding sequence that step (1) is obtained is cloned in the pMal-c5x expression vector, obtains recombinant plasmid pMal-c5x/bst;

(3), obtain maltose binding protein and the big segmental fusion rotein of bacstearothermophilus archaeal dna polymerase at expression in escherichia coli recombinant plasmid pMal-c5x/bst;

(4) fusion rotein that adopts amylose starch affinitive layer purification step (3) to obtain;

(5) fusion rotein behind the purifying is carried out protease hydrolyzed, obtain the bacstearothermophilus archaeal dna polymerase shown in SEQ ID NO 2.

Embodiment

For making the present invention easier to understand, will further set forth specific embodiments of the invention below.

LAMP loop-mediated isothermal amplification technique (Loop-Mediated Isothermal Amplification)

DNA thymus nucleic acid (Deoxyribonucleic acid)

PCR polymerase chain reaction (Polymerase Chain Reaction)

EDTA ethylenediamine tetraacetic acid (EDTA) (Ethylene Diamine Tetraacetic Acid)

DTT dithiothreitol (DTT) (Dithiothreitol)

ATCC US mode bacterial classification is collected center (American Type Culture Collection)

OD optical density(OD) (Optical Density)

DNTP deoxynucleoside triphosphate (deoxyribonucleoside triphosphate)

Bacstearothermophilus strains A TCC12980 is available from Chinese industrial microbial strains preservation center.

The big fragment gene of embodiment 1. usefulness PCR method amplification bacstearothermophilus archaeal dna polymerase

Having designed primer according to the bacstearothermophilus dna polymerase gene in the ncbi database, is that template increases with the bacstearothermophilus genomic dna.Employed condition be 94 ℃ 3 minutes; 94 ℃ of 30 round-robin 0.5 minute, 50 ℃ 0.5 minute, 72 ℃ 3 minutes; Last 72 ℃ 7 minutes.Identify through agarose gel electrophoresis, obtained the amplified fragments of about 1.8kb.Through order-checking, shown in SEQ ID NO 1, be 1761bp.

Embodiment 2. clonal expansion fragments are in the pMal-c5x expression vector

The amplified fragments of embodiment 1 is cut with FspI and BamHI enzyme, and pMal-c5x carrier (available from NEB company) is cut with XmnI and BamHI enzyme.Add the T4 dna ligase after fragment and carrier mix, connect product transformed into escherichia coli DH5a bacterial strain.Cut evaluation and order-checking through enzyme, obtained required clone pMal-c5x/bst.Plasmid is transformed into carries out protein expression in the TBl bacterial strain.

The big segmental expression of embodiment 3. bacstearothermophilus archaeal dna polymerases

Is that 0.5 o'clock adding IPTG induces centrifugal collection thalline after 2 hours with the intestinal bacteria that contain expression plasmid pMal-c5x/bst of embodiment 2 growing to OD600.

The big segmental purifying of embodiment 4. bacstearothermophilus archaeal dna polymerases

Embodiment 3 is collected the thalline that obtains at 20mM Tris PH6.8,100mM NaCl, 1mM EDTA, 1mM DTT, 0.1 volume %Triton X100, cracking is 30 minutes in the 1mg/ml N,O-Diacetylmuramidase damping fluid, and 12000rpm mixed with amylose resin after collecting supernatant liquor in centrifugal 30 minutes, and 4 ℃ adsorbed 60 minutes.Amylose resin is with 20mM Tris PH6.8,100mM NaCl, after the washing of 0.3mM DTT damping fluid, with recombinant protein with 20mM Tris PH6.8,100mM NaCl, 10mM maltose elutes.Add 1mMCaCl in the albumen of wash-out₂And 2ug Protease F actor Xa, 4 ℃ of cuttings promptly obtained the big fragment of bacstearothermophilus archaeal dna polymerase in 20 hours.The big fragment of described bacstearothermophilus archaeal dna polymerase has the aminoacid sequence shown in SEQ ID NO 2.

Embodiment 5. nucleic acid isothermals amplifications (LAMP)

Reaction conditions is 20mM Tris-HCl pH 8.8,10mM (NH₄)₂SO₄, 10mM KCl, 8mMMgSO₄, 0.1% Triton X-100,0.8M trimethyl-glycine, 1.6mM dNTP, each 1.6 μ M of primer BIP and FIP, each 0.2 μ M of primer B3 and F3, big fragment of bacstearothermophilus archaeal dna polymerase and template DNA that 33ng embodiment 4 obtains.65 ℃ of reactions added 2 μ l 1000x Sybr Green I dyestuffs in 60 minutes afterwards, and it is then positive for amplification that reaction solution presents green.The result shows that the big fragment of bacstearothermophilus archaeal dna polymerase that embodiment 4 obtains is the same with the Bst polysaccharase (big fragment) that commercial NEB company produces, and can both be used for nucleic acid isothermal increase (LAMP).But compared with commercial product, the big fragment of mentioning among the present invention of bacstearothermophilus archaeal dna polymerase, high 2 orders of magnitude of detection sensitivity.Be example to detect Salmonellas below.

Cultivate Salmonellas, through the bacterium colony plate count, select the 9th extent of dilution to carry out reading, average colony number is 126cfu, calculates that the bacterium original liquid concentration is 1.26 * 10¹¹Cfu/mL, with commercial Bst polysaccharase (big fragment), other all conditions is constant, can detect the 5th extent of dilution, is 1.26 * 10⁶Cfu/mL.With the big fragment of mentioning among the present invention of bacstearothermophilus archaeal dna polymerase, detection sensitivity improves 2 orders of magnitude, is 1.26 * 10⁴Cfu/mL.Sensitivity improving is obvious.

Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although the present invention has been done detailed description with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can make amendment or be equal to replacement technical scheme of the present invention, and not break away from the essence and the scope of technical solution of the present invention.

Claims (7)

1. the gene of a bacstearothermophilus archaeal dna polymerase is characterized in that, the nucleotide coding sequence of described bacstearothermophilus dna polymerase gene is shown in SEQ ID NO 1.

2. plasmid pMal-c5x/bst who contains the gene of the described bacstearothermophilus archaeal dna polymerase of claim 1.

3. a bacstearothermophilus archaeal dna polymerase is characterized in that, the aminoacid sequence of described bacstearothermophilus archaeal dna polymerase is shown in SEQ ID NO 2.

4. bacstearothermophilus archaeal dna polymerase as claimed in claim 3 is in the application of nucleic acid isothermal amplified reaction.

5. fusion rotein, it has the described bacstearothermophilus archaeal dna polymerase of claim 3, and described fusion rotein also has maltose binding protein.

6. fusion rotein as claimed in claim 5 is in the application of bacstearothermophilus archaeal dna polymerase purifying.

7. the preparation method of a bacstearothermophilus archaeal dna polymerase is characterized in that, described purification process comprises the steps:

(1) nucleotide coding sequence of the bacstearothermophilus dna polymerase gene of amplification shown in SEQ ID NO 1;

(2) nucleotide coding sequence that step (1) is obtained is cloned in the pMal-c5x expression vector, obtains recombinant plasmid pMal-c5x/bst;

(3), obtain maltose binding protein and the big segmental fusion rotein of bacstearothermophilus archaeal dna polymerase at expression in escherichia coli recombinant plasmid pMal-c5x/bst;

(4) fusion rotein that adopts amylose starch affinitive layer purification step (3) to obtain;

(5) fusion rotein behind the purifying is carried out protease hydrolyzed, obtain the bacstearothermophilus archaeal dna polymerase shown in SEQ ID NO 2.