CN1948495A - Saliva streptococcus 57.I urase gene and its urase - Google Patents

Abstract

This invention relates to a urase gene of streptococcus salivarius, concretely, concerning about a 57.I urase gene of streptococcus salivarius and its urase. A 57.I urase gene of streptococcus salivarius , whose nucleotide sequence is what is described in SEQ ID NO.1. Nickel ion bonding in the gene and nucleotide sequence of transit-related gene MQO are what are described in SEQ ID NO.2. The advantage of this invention is that gaining the cloning of urase gene of streptococcus salivarius U35248 by the method of segment cloning and gradually enzyme-cutting connection ,and its object gene length proceeds to 8Kb, increasing Nickel ion bonding of the gene and transit-related gene MQO compared with existing technology , therefore, expressing ureolytic activity without adding exogenous NiCl2.Attained cloning can be used for future replacement therapy in anti-caries research building without adding exogenous NiCl2,more suitable for clinical application of production alkali effect germ.

Description

A kind of streptococcus-salivarius 57.I urease gene and urease thereof

Technical field

The present invention relates to a kind of streptococcus-salivarius urease gene, specifically about a kind of streptococcus-salivarius 57.I urease gene and urease thereof.

Background technology

The dental caries disease is one of modal bacterial infection disease that threatens human health, it also is the one of the main reasons that causes human teeth pain and disappearance, oral health epidemiological survey data according to China's nineteen eighty-three and nineteen ninety-five shows that the morbidity of China's dental caries disease is in rising trend.Though the prevention for the dental caries disease has several different methods at present, does not find very ideal method so far as yet.

Alternative medicine (Replacement therapy), be to exist naturally or nonadverse effect bacterium (effector strain) that the laboratory makes up for good and all is colonizated in the human micropopulation with a kind of, replace and get rid of certain specific pathogen microbial growth, thus the method that the prevention specified disease takes place.Early in the twentieth century, external doctor just attempts with bacterial infection diseases such as some harmless resident bacterize tuberculosis, anthrax, diphtheria, because antibiotic extensive application, this bacteriotherapy does not have large-scale promotion, but becoming increasingly conspicuous along with the bacterial drug resistance problem in recent years, people recognize that microbiotic is not omnipotent, with bacterium itself antibacterial alternative medicine is more and more come into one's own, and obtained certain progress in the Prevention Research of antagonism bacterial infection disease.For example: discover that children inoculate the generation that Alpha-hemolytic streptococcus can reduce acute secretory otitis media afterwards; The bacteriocin that streptococcus-salivarius K12 produces to streptococcal pharyngitis main pathogenic bacterium--streptococcus pyogenes is inhibited, be expected to become the effect bacterium of prevention streptococcal pharyngitis.

The dental caries disease is as a kind of bacterial infection disease, might use alternative medicine to prevent equally, promptly adopt certain technique means to obtain harmless mutant strain from resident bacterium, this mutant strain is inoculated in the predilection site of dental caries disease in the oral cavity, occupying the position of deleterious wild-type cariogenicity bacterial strain adhesion and aggregation, thus prevention and treatment dental caries disease.

Adopt the pre-preventing decayed tooth disease of alternative medicine easy and simple to handle, need not the good compliance of patient, and preventing decayed tooth effect bacterium also is expected to realize mother-to-baby transmission in the crowd.

Adopt alternative medicine to eliminate or reduce the virulence factor of dental caries disease, come the theory of pre-preventing decayed tooth disease to be accepted by more and more scholars gradually by the ecology that changes oral cavity bacterium, the research prophesy is arranged in from now on 20 years, some new methods such as alternative medicine might become the method for the pre-preventing decayed tooth disease of more being paid close attention to.At present, external research is being devoted to the normal microflora in the oral cavity is transformed, but does not obtain ideal preventing decayed tooth effect bacterium so far as yet.

Early stage research is devoted to weaken the product acidity of streptococcus mutans more, adopt means such as ultraviolet ray, X ray, rapid neutron and chemical mutagen to bring out the streptococcus mutans sudden change, filter out serum lactic dehydrogenase (lactate dehydrogenase, LDH) mutant strain of defective, these streptococcus mutanses do not produce lactic acid, but output is little, reverse mutation rate is high, be unsuitable for the large-scale groups inoculation, so people begin to have in mind from gene level transformation streptococcus mutans.Hillman etc. have knocked out the gene 1dh of coding streptococcus mutans serum lactic dehydrogenase, insert again from movable yeast Zymomonas mobilis (Zymomonas mobilis) coding ethanol dehydrogenase (alcohol dehydrogenase, ADH) gene adh, to remedy the lethality that the LDH defective causes, the BCS3-L1 bacterial strain that constructs produces acid and cariogenicity all weakens to some extent.But studies show that of vitro culture, though rejected 1dh, after the BCS3-L1 bacterial strain was cultivated 1 hour in containing the substratum of glucose, the pH value was 5.1, still was acid; The intravital cariogenicity research of animal is also found, SPF (specific pathogen free) rat even without the streptococcus mutans field planting, raise so that dental caries food after 8 weeks, still have certain caries prevalence rate, show other bacteriums in the oral cavity, as lactobacillus and actinomycetes, also have the possibility of acid of producing and generation dental caries disease, the product acidity that as seen reduces streptococcus mutans may be not enough to stop the generation of dental caries disease.So people turn one's attention to the product alkali ability that improves plaque, make up product alkali effect bacterium with ureolytic activity.

The advantage of producing the alkali effect bacterium is not only at a certain cariogenic bacteria, but produces the reduction that ammonia resists pH value in the whole bacterial plaque environment by decomposing urea.The secretory volume of urea remains on 3～10mM in healthy people's saliva and the level in gingival sulcus fluid, the urea that the urease of product alkali effect bacterium can decompose in the oral cavity produces ammonia, in and the acid that produces of glucose glycolysis, alleviate the reduction of pH value, and promote the remineralization of dental surface, and also may influence the ecotope of whole bacterial plaque simultaneously, help bacterium field planting in bacterial plaque that some are not acidproof, cariogenicity is more weak, by influencing the generation that the bacterial plaque microbial film reduces the dental caries disease, has bigger research potential.

The building process that produces the alkali effect bacterium mainly is to clone urease gene from the oral cavity bacterium with ureolytic activity, is inserted in the resident bacterium of bacterial plaque of cariogenicity or non-cariogenicity again.Urease is a kind of many subunits enzyme that utilizes nickel ion, can be decomposed into ammonia and carbonic acid gas by catalyzing urea, and the pH value in the rising environment is the conservative protein of plant, bacterium, fungi and algae camber.

The bacterium that has ureolytic activity in the oral cavity has streptococcus-salivarius (Streptococcussalivarius), actinomyces naeslundii (Actinomyces naeslundii), helicobacter pylori various bacteria such as (Halicobacter. pylori), wherein streptococcus-salivarius extensively is present in soft tissue and the lingual surface in the oral cavity, and its most bacterial strains can hydrolyze urea.Chen etc. reported the molecule and the biochemical characteristic of streptococcus-salivarius 57.I urease gene in 1996, and the expression in intestinal bacteria and Ge Deng suis (Streptococcus gordonii).This research uses one section sequence of ure C gene inside as probe, identifies two sections clones from the subgene library, and connects, and has obtained the ure IABCEFGD gene fragment of the about 5.6Kb of length.Contain the activity that intestinal bacteria that the plasmid of this gene fragment transforms and Ge Deng suis can both give expression to urease, but regrettably, must in the substratum of recombinant bacterial strain growth, add nickelous chloride (Nickel chloride, the NiCl of certain level ₂) can detect the activity of urease, otherwise the level of urease is just very low, in addition detect less than, this may be owing to there is not complete nickel ion movement system in resulting clone, adds ectogenic NiCl so must rely in substratum ₂

Clancy etc. is according to the principle of alternative medicine in research subsequently, the urease gene ure IABCEFGD that will separate from streptococcus-salivarius 57.I genome inserts in the genome of streptococcus mutans UA159, reorganization streptococcus mutans strains A CO series and ACUS series have successively been made up with ureolytic activity, but same, the expression of recombinant bacterial strain urease activity still needs to add ectogenic nickel ion in substratum.In vitro tests shows: the reorganization streptococcus mutans that carries ure IABCEFGD plasmid is containing urea and NiCl ₂Substratum in when growing, even have more than 10 times mole glucose, the decline that also is enough to resist the pH value; In vivo test also finds, the rat of inoculation reorganization streptococcus mutans ACUS6 is raised so that dental caries food and add the NiCl of 50mM urea and 50 μ M in drinking-water ₂After 5 weeks, the incidence of smooth surface caries and pit and fissure caries is all very low, significantly is lower than the rat of field planting streptococcus mutans UA159.

This shows that the open defect of at present constructed product alkali effect bacterium is that recombinant bacterial strain needs ectogenic NiCl ₂Join substratum and can give expression to ureaclastic activity, this may be owing to lack and the relevant gene of exogenous nickel ion combination in the urease gene that inserts bunch, and streptococcus mutans itself is a kind of bacterium of non-ureolytic activity, also do not possess the high-affinity translocator that absorbs enough nickel ions from environment, this defective brings difficulty will for the clinical application from now on of preventing decayed tooth effect bacterium.

At this defective, the foreign scholar has carried out deep research again.Thermophilus streptococcus (Streptococcus thermophilus) is a kind of and the very approaching bacterium with ureolytic activity of streptococcus-salivarius, Chan etc. are in order to verify the gene that whether has one section coding and nickel ion absorption and transfer related protein in the streptococcus-salivarius, sequences Design primer according to thermophilus streptococcus LMG18311, use PCR (Polymerase Chain Reaction, the polymerase chain amplified reaction) method has amplified a fragment gene sequence of the streptococcus-salivarius urease gene ure IABCEFGD 3 ' end that is positioned at traditional concept, sequential analysis finds that this fragment gene comprises three opening code-reading frames, reverse transcription PCR studies confirm that this three parts that opening code-reading frame also is the urease operon.When after one section kalamycin resistance gene of the inner insertion of first opening code-reading frame makes its inactivation, find that this mutant strain can not absorb and gather nickel ion in process of growth from environment, the activity of urease also completely loses, and has only an amount of NiCl of interpolation in substratum ₂Can partly recover the activity of urease, it is extremely important to illustrate that this fragment gene absorbs nickel ion for bacterium from environment.Final these three opening code-reading frames are confirmed as the part of urease operon, difference called after ure M, and ure Q and ure O, effect is a kind of nickel ion specificity boxlike ATP binding transport albumen of coding.Thereby the complete sequence of streptococcus-salivarius urease gene to be redefined be 11 genes, be ure IABCEFGDMQO, sequence number in GenBank is U35248, so far people for urease gene in recombinant bacterial strain expression and with being combined with of exogenous nickel ion more deep understanding, may for further making up that even more ideal product alkali effect bacterium provides.

But up to now, the clone of streptococcus-salivarius urease gene U35248 complete sequence does not appear in the newspapers as yet.

Summary of the invention

The objective of the invention is to:

(1) provides a kind of streptococcus-salivarius 57.I urease gene;

(2) provide the method for preparing the streptococcus-salivarius urease gene;

(3) provide a kind of streptococcus-salivarius urease;

(4) provide the method for preparing the streptococcus-salivarius urease;

(5) provide the application of a kind of 57.I urease gene in preparation treatment or the sick medicine of prevention oral cavity dental caries;

(6) provide the application of a kind of streptococcus-salivarius urease in preparation treatment or the sick medicine of prevention oral cavity dental caries.

The objective of the invention is to realize: before, during and after urease gene U35248 is divided into three sections by following technological method, design primer respectively, the evaluation of cloning and check order of the method for using PCR, and then the method for employing double digestion, segmentation clone's product is sheared again, made up, progressively be connected to complete urease gene; The plasmid transformed competence colibacillus intestinal bacteria that will contain complete urease gene, and detect its ureolytic activity through phenol red urease test, and whether the expression of urease activity needs to add ectogenic NiCl ₂

A kind of streptococcus-salivarius 57.I urease gene, its nucleotide sequence is as described in the SEQ ID NO.1.The nucleotide sequence of nickel ion combination in this gene and the relevant gene M QO of transhipment is as described in the SEQ IDNO.2, and its protein sequence is as described in the SEQ ID NO.4.

Prepare the method for streptococcus-salivarius urease gene, comprise the following steps:

(1) segmentation of liquid suis 57.I urease gene clone;

(2) segmentation clone's product progressively is connected to intactly urease gene.

In (1) step, segmentation place of streptococcus-salivarius 57.I urease gene is: be positioned at the single endonuclease digestion site-Bam H I of the restriction endonuclease at 2038bp place, be positioned at the single endonuclease digestion site-Xho I of the restriction endonuclease at 4812bp place.(1) in the step, the primer Uf1 that amplified reaction is used,

Sequence: TGC CTT CAC CTA CCT TTA CTC AG;

Primer Ur1, sequence: CAA CAA CGC ATG GCC ACT TC AC;

Primer Uf2, sequence: ACC CAA TCA ACC CAT ACA CCA C;

Primer Ur2, sequence: CAC CAT CAC CAA ATA GCA AAG C;

Primer Uf3, sequence: AAT GTT AAG GAA GTG GAA GAA TGG;

Primer Ur3, sequence: ATG ATG AAG GTG CTT GAA GTG ATG.

A kind of streptococcus-salivarius urease, its aminoacid sequence is as described in the SEQ ID NO.3.

The method for preparing the streptococcus-salivarius urease comprises with 57.I urease gene plasmid U ₁₊₂₊₃The transformed competence colibacillus intestinal bacteria obtain the streptococcus-salivarius urease.

Disclosed a kind of streptococcus-salivarius 57.I urease gene of the present invention and urease thereof, its advantage shows: cut the method that is connected by segmentation clone with enzyme progressively, obtained the clone of streptococcus-salivarius urease gene U35248, gained goal gene length reaches 8Kb, compared with prior art, increased the nickel ion combination gene M QO relevant, therefore need not to add exogenous NiCl with transhipment ₂Can give expression to ureaclastic activity.Institute's DCRP can be used for from now on making up in the research of alternative medicine preventing decayed tooth and need not to add exogenous NiCl ₂, be more suitable for the product alkali effect bacterium of clinical application; For the urea decomposition mechanism of further studying oral cavity bacterium has been set up new experiment porch.

Description of drawings

Fig. 1: the agarose electrophoresis of streptococcus-salivarius 57.I genomic dna.

Fig. 2: the agarose electrophoresis swimming lane 1 of the 16s rDNA amplified production of streptococcus-salivarius 57.I: the 16s rDNA amplified production of streptococcus-salivarius 57.I.

Fig. 3: the agarose gel electrophoresis of three sections pcr amplification products.

Fig. 4: the enzyme of three sections segmentation cloned plasmids is cut the evaluation photo.

Fig. 5: three sections technological lines that segmentation clone product connects.

Fig. 6: U ₂₊₃The enzyme of plasmid is cut the evaluation photo, and swimming lane 1:U2+3 plasmid is through the result (2.8Kb+6.1Kb) of Sph I and Xho I double digestion.

Fig. 7: U ₁₊₂₊₃The enzyme of plasmid is cut the evaluation photo, swimming lane 1:U ₁₊₂₊₃Plasmid is through the result (2Kb+9Kb) of Sph I and Bam H I double digestion.

Fig. 8: use phenol red urease test qualitative detection to contain the plasmid U of urease gene U35248 ₁₊₂₊₃Expression in intestinal bacteria.

Embodiment

Below in conjunction with embodiment the present invention is further described.

Embodiment 1

The extracting and the evaluation of streptococcus-salivarius 57.I genomic dna

The purpose of whole research is the clone who obtains activated streptococcus-salivarius 57.I urease gene U35248, primary the first step work is exactly the template that obtains this goal gene, the complete genome DNA of donor bacterium just, therefore, this experiment will be according to the principle of molecular cloning, use commercially available bacterial genomes extraction agent box to extract the genomic dna of streptococcus-salivarius 57.I, as template, so that in follow-up research, adopting the method for PCR, is to clone required urease gene in the template with the genomic dna.

Simultaneously, because in procaryotic genome, the dna molecular function-stable of coding 16S ribosome-RNA(rRNA), form by high conservative region and variable region, mutation rate in the conserved regions is extremely low, and the variable sequence of its characteristic area has significant species specificity between different Pseudomonas different strains, can be used for the analysis of microorganism hereditary feature, is the ideal material of division bacteria.Therefore this experiment will be adopted 16S rDNA microorganism identification universal primer, utilize the method for PCR to amplify 16S rrna characteristic area sequence in the experimental strain genome, through after the sequencing again with gene library in the characteristic area 16S rDNA fragment of streptococcus-salivarius compare, with the hereditary feature of identification experiment bacterial strain.

1.1 material and method

1.1.1 experimental strain and substratum

Experimental strain: streptococcus-salivarius 57.I, according to people such as Sissons (Sissons C.H., HancockE.M., Perinpanayagam H.E.R., et al.The bacteria responsible forureolysis in artificial dental plaque.Arch Oral Biol, 1988,33 (10): method 727-734.) is separated acquisition, and lyophilize is preserved.Concrete separation method is as follows: collect after 37 ℃ of 1ml human salivas hatched 7 days, getting one of about 10-100mg adding weighs in advance, contain in the 1.5ml centrifuge tube of 1ml fluid thioglycolate medium, carry out ultra-sonic dispersion after weighing, get a copy of it and in the anaerobism glove box, use the fluid thioglycolate medium serial dilution.Get the 0.1ml extent of dilution and be about 10 ⁴-10 ⁷Diluent be inoculated on brain-heart-infusion (BHI) nutrient agar, anaerobism was cultivated 5 days, waited to grow after the clone, got a plurality ofly to be cloned on the TSA substratum that contains urea and indicator streak culturely, detected its ureolytic activity.Being cloned on the new TSA substratum that the picking ureolytic activity is the strongest is streak culture, isolates mono-clonal and observes by gramstaining and microscopically afterwards, obtains required streptococcus-salivarius.

Substratum: TSB liquid nutrient medium, compound method be as testing as described in one, be stored in 4 ℃ standby; TSA blood agar substratum, compound method be as testing as described in one, be stored in 4 ℃ standby.

1.1.2 main agents

N,O-Diacetylmuramidase: Sangon Biotech (Shanghai) Co., Ltd. produces, and uses aseptic double-distilled water to be mixed with the lysozyme soln of 50mg/ml, in-20 ℃ of preservations, is diluted to 20mg/ml during use after the packing.

Tutofusin tris (tris hydroxymethyl aminomethane, Tris), 1mol/l, pH 8.0: get 800ml dissolved in distilled water 121.1g Tris alkali, add concentrated hydrochloric acid and transfer pH to 8.0, adding distil water is settled to 1L, high pressure steam sterilization after the packing, be stored in after the cooling 4 ℃ standby.

Ethylenediamine tetraacetic acid (EDTA) (ethylene diaminetetraacetic acid, EDTA), 0.5mol/l, pH=8.0: get 800ml dissolved in distilled water 186.1g EDTA-Na.2H ₂O, vigorous stirring, with the pH value to 8.0 of sodium hydrate regulator solution, adding distil water is settled to 1L, high pressure steam sterilization after the packing, cooling be stored in afterwards 4 ℃ standby.

Triton X-100: Sangon Biotech (Shanghai) Co., Ltd. produces, and adding distil water is mixed with 1% solution, be stored in 4 ℃ standby.

Genome extraction agent box, Germany MACHEREY-NAGEL company (being called for short M-N company) produces, include: Proteinase K (the 6mg Proteinase K is provided in the proteolytic enzyme damping fluid that provides in the 260 μ l test kits, vibration dissolving, be stored in-20 ℃ standby), the B3 damping fluid, the B5 damping fluid, the BW damping fluid, BE elutriant, centrifugal adsorption column.

RNA enzyme A (RNase A): the import packing of Sigma company, RNase A crystal powder is mixed with the solution of 10mg/ml with aseptic double-distilled water dissolving, in 100 ℃ of heating 10min deactivation DNase, be stored in after the packing-20 ℃ standby.

Dehydrated alcohol (analytical pure): Shanghai development chemical industry one factory produces.

The common agar Icing Sugar of using: Shanghai Shun's biotechnology company limited produces.

Tris-boric acid (TBE) electrophoretic buffer, 5 * stock solution: 54g Tris alkali, 27.5g boric acid, 20ml 0.5mol/L EDTA (pH8.0), adding distil water be settled to 1L be stored in 4 ℃ standby, during use with the stock solution of dilution in 1: 10 as using liquid.

The precious biotechnology (Dalian) of DNA Marker:TaKaRa company limited produces.

The precious biotechnology (Dalian) of 10 * sample-loading buffer (Loading Buffer): TaKaRa company limited produces.

Ethidium bromide (10mg/ml): add the 1g ethidium bromide in 100ml distilled water, magnetic agitation a few hours dissolve fully to guarantee it, then with the tinfoil paper wrapping container or be transferred in the brown bottle, are stored in room temperature.(annotate: band gloves and mouth mask during operation.)

Taq archaeal dna polymerase (5U/ μ l), dNTP mixed solution (10mM), 10 * Taq damping fluid: Shanghai Shenergy Biocolor BioScience ﹠ Technology Company produces.

1.1.3 key instrument equipment

Electronic analytical balance: Japanese Tokyo company produces.

Portable High pressure steam sterilizer: produce in Jiangsu.

Electric heating constant temperature air dry oven: go up the grand experimental installation of Nereid company limited and produce.

Heraus 28RS low-temperature and high-speed desk centrifuge: Germany produces.

Electric heating constant temperature water bath: go up marine products.

Ultralow Temperature Freezer: U.S. HARRIS produces.

Gene Quant RNA/DNA ultraviolet spectrometry survey meter: Sweden Pharmacia Corp produces.

PE 2400 DNA cloning instrument: the U.S. produces.

Tanon UV-2000 ultraviolet gel analysis instrument: go up marine products.

EPS 600 voltage stabilizing electrophoresis apparatuses and horizontal strip electrophoresis groove: Sweden Pharmacia Corp produces.

Mould and comb that the perfusion gel is used: go up marine products.

Microwave oven: Glanz company produces.

The Eppendorf micropipet: Britain produces.

Mineral oil: Beijing ancient cooking vessel state biotech development center produces.

Consumptive material: 15ml glass test tube (aseptic), Eppendorf centrifuge tube (1.5ml, 0.5ml, 0.2ml), micropipet TIP head (1000 μ l, 200 μ l, 20 μ l), microbial culture plate.

1.1.4 experimental technique

The extracting of streptococcus-salivarius 57.I genomic dna (the genome extraction agent box that uses German M-N company to produce carries out):

With being inoculated in the 3ml TSB liquid nutrient medium after the conventional recovery cultivation of the streptococcus-salivarius 57.I bacterial classification that is stored in-20 ℃, in 37 ℃, 90%N ₂, 10%CO ₂Cultivate 24h under the condition in the anaerobism incubator, plate coating checking is after the pure growth, gets 1ml bacterium liquid, in 4 ℃, 8000rpm, centrifugal 5min, the mycetocyte of collecting precipitation, again be suspended in 180 μ l 20mM Tris/Cl, 3mM EDTA among the 1%TritonX-100 (pH 8.0), and adds the 20mg/ml N,O-Diacetylmuramidase, hatch 1h in 37 ℃ of gentle down vibrations, with the cell wall of reduction mycetocyte; Add 25 μ l Proteinase Ks, violent mixing is hatched 3h in 56 ℃ of shaking baths; Add 5 μ l 10mg/ml RNase A, hatch 5min in 37 ℃ after the mixing; Add 200 μ l B3 damping fluids, after the violent mixing, hatch 10min in 70 ℃, limpid until liquid, cracking is complete; Add 210 μ l dehydrated alcohols, after the mixing complete soln is transferred on the adsorption column that has the 2ml collection tube, 12, the centrifugal 1min of 000rpm discards filtrate, and adsorption column is put back to collection tube; Add 500 μ l BW damping fluids, 12, the centrifugal 1min of 000rpm discards filtrate, and adsorption column is put back to collection tube; Add 600 μ l B5 damping fluids, 12, the centrifugal 1min of 000rpm discards filtrate, and adsorption column is put back to collection tube; In 12, the centrifugal 1min of 000rpm thoroughly abandons clean filtrate, and adsorption column is put back to collection tube once more; Adsorption column is transferred in the new aseptic 1.5ml centrifuge tube, and the aseptic double-distilled water in that the central authorities of adsorption column film add 100 μ l preheatings leaves standstill under the room temperature and hatches 3min, 12, the centrifugal 2min of 000rpm collects filtrate, the streptococcus-salivarius genomic dna that promptly extracts.

Getting 5 μ l filtrates uses Gene Quant RNA/DNA ultraviolet spectrometry survey meter to detect the concentration and the purity of genome DNA sample, working method is as follows: the start back is used the aseptic double-distilled water suppressed zero of 50 μ l earlier, then with behind 5 μ l filtrates and the 45 μ l aseptic double-distilled water mixings (being equivalent to dilute 10 times), transfer in the quartz cuvette of survey meter, read optical density value respectively at 260nm and 280nm place respectively, and calculating concentration and purity.

Other gets 5 μ l filtrates and carries out 0.7% agarose electrophoresis (all the other sample retention are standby in-20 ℃), operation mill method is as follows: put into 0.7g agar Icing Sugar and 100ml 0.5 * TBE electrophoretic buffer in Erlenmeyer flask, seal bottleneck with preservative film, and leave a little slit, place middle-grade heating in the microwave oven, agarose is fully dissolved, in the heating for dissolving process otherwise the time shaking flasks gently, in order to avoid bumping; Treat to take out cooling after agarose dissolves fully, when agarose gel solution is cooled to 50～60 ℃ (feel heat is non-scald on hand still), adds ethidium bromide dye liquor (10mg/ml) and make final concentration reach 0.5 μ g/ml; The agarose solution that will be added with ethidium bromide is then poured in the mould of cleaning, and gel thicknesses is generally 0.3～0.5cm, assigns comb at mould one end rapidly, and scrutiny has or not bubble, then uses micropipet to shift out carefully if any bubble; Room temperature is placed 30～45min, agarose solution is finished solidify, and takes out comb carefully, and gel is placed electrophoresis chamber (the negative electrode place of a side of comb jack near electrophoresis chamber arranged); Add 0.5 * TBE electrophoretic buffer to electrophoresis chamber, make the high plastic emitting of the liquid level about 1～2mm in plane; In 5 μ l DNA samples, add 0.5 μ l, 10 * sample-loading buffer, use the centrifugal 5sec of Eppendorf centrifuge after the mixing, solution is concentrated on manage at the end, sample is added carefully in the well of sepharose with micropipet; Connect the power supply of electrophoresis chamber and electrophoresis apparatus, carry out electrophoresis with the 1.5V/cm constant voltage power supply; According to the position of bromjophenol blue indicator migration, estimate the position that the DNA sample moves, when the bromjophenol blue indicator moves to a half of about gel, cut off the electricity supply, take out gel, in ultraviolet gel analysis instrument, observe electrophoretic result and Taking Pictures recording.

Strain identification (amplification of 16S rDNA characteristic area):

16S rDNA characteristic area universal primer: the general conservative primer of 16S ribosome-RNA(rRNA) that uses bibliographical information: upstream sequence is corresponding to the 8th～27 bit base (8F) of intestinal bacteria 16S rDNA, be 8F-AGA GTT TGA TCM TGG CTC AG, downstream sequence is corresponding to the 519th～536 bit base (519R) of intestinal bacteria 16S rDNA, i.e. 519R-GWA TTA CCG CGG CKGCTG; Wherein M, W, K are degenerated primer, and its based composition is as follows:

M=A or C W=A or T K=G or T

Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, 2OD, HAP purifying, vacuum lyophilization transportation; Receive after the primer that the centrifuge tube that will fill primer is in 12 earlier, the centrifugal 1min of 000rpm, tube used for bottom pouring lid carefully adds an amount of distilled water and shake fully up and down then, makes it thoroughly to be dissolved as the primer solution of 20 μ M, and packing is afterwards standby in-20 ℃ of preservations.

Adopt 100 μ l pcr amplification systems, the application of sample process is operated on ice bath:

Taq archaeal dna polymerase (5U/ μ l) 1 μ l

Upstream primer 8F (20 μ M) 2.5 μ l

Downstream primer 519R (20 μ M) 2.5 μ l

DNTP mixed solution (10mM) 1 μ l

10 * Taq damping fluid, 10 μ l

Streptococcus-salivarius 57.I genomic dna template 1 μ l

With aseptic double-distilled water final volume is mended to 100 μ l; After the mixing, centrifugal 5sec concentrates on reacted constituent and manages the end and centrifugal mixing, adds an amount of mineral oil again and covers liquid level.

The PCR reaction conditions is as follows:

94 ℃ of pre-sex change 4min

94 ℃ of sex change 30sec

55 ℃ of annealing 30sec

72 ℃ are extended 1min (carrying out 30 circulations)

Mend flat 10min for 72 ℃

Get 10 μ l pcr amplification products and carry out 1% agarose gel electrophoresis (operation steps as previously mentioned), in ultraviolet gel analysis instrument, observe electrophoresis result and Taking Pictures recording, all the other products send Invitrogen Bioisystech Co., Ltd to carry out sequencing, with 16S ribosome-RNA(rRNA) universal primer 8F or 519R as sequencing primer.

1.2 experimental result

1.2.1 the genomic extracting of streptococcus-salivarius 57.I

The genome extraction agent box that uses German M-N company to produce extracts the complete genome DNA of streptococcus-salivarius 57.I, get the solution that 5 μ l contain genomic dna and carry out 0.7% agarose gel electrophoresis, in ultraviolet gel analysis instrument, observe, electrophoresis result as shown in Figure 1:

The concentration of genomic dna and purity: use Gene Quant RNA/DNA ultraviolet spectrometry survey meter to detect streptococcus-salivarius 57.I genomic dna respectively in the optical density value at 260nm and 280nm wavelength place, the result shows OD _260nm=0.518, OD _280nm=0.295.Because under the ultraviolet ray of wavelength 260nm, it is 50 μ g/ml that the optical density(OD) of 1OD value is equivalent to double-stranded DNA concentration, draws concentration (μ g/ μ the l)=OD of DNA sample to be measured _260nm* nucleic acid extension rate * 50/1000, the extension rate of genome DNA sample is 10 times in this experiment, the concentration that therefore calculates genomic dna is 0.26 μ g/ μ l.

Spectrophotometry can also be by being determined at the ratio of 260nm and 280nm place ultraviolet radiation absorption value, i.e. OD _260nm/ OD _280nm, the purity of estimation nucleic acid, wherein the ratio of DNA should be 1.8, if be higher than 1.8, the RNA not Ex-all as yet in the prompting preparation if be lower than 1.8, has phenol or proteinic pollution in the prompting solution, records OD in this experiment _260nm/ OD _280nm=1.756, very near 1.8, can tentatively think extractive genomic dna purity ideal can be used for follow-up experiment.

1.2.2 strain identification (amplification of 16S rDNA characteristic area)

Genome with streptococcus-salivarius 57.I is a template, after 16S rDNA characteristic area increased, get 10 μ l pcr amplification products and carry out 1% agarose gel electrophoresis, in ultraviolet gel analysis instrument, observe, electrophoresis result as shown in Figure 4, product is to be positioned at the single band of locating about 500bp.

16S rDNA pcr amplification product order-checking: with 8F or 519R 16S ribosome-RNA(rRNA) universal primer as sequencing primer, by Invitrogen Bioisystech Co., Ltd the pcr amplification product of experimental strain 16S rDNA is carried out sequencing analysis, sequencing result has 489bp, carry out after the BLAST comparison with known 16s rDNA in the gene library, the homology of finding experimental strain and streptococcus-salivarius 16s rDNA characteristic area is 100%, confirms that hereditary feature is accurate.The sequencing result, the order-checking collection of illustrative plates consistent with the result who in gene library, compares.

Embodiment 2

The segmentation clone of streptococcus-salivarius 57.I urease gene U35248

Polymerase chain reaction (polymerase chain reaction, PCR) can be used for increasing DNA section between two sections known arrays, yet the length of streptococcus-salivarius urease gene reaches 8Kb, carry out disposable amplification if directly adopt the method for PCR, be difficult to guarantee to reach the length of expection, and more sudden change might in the process of amplification, occur.This experiment intends adopting segmentation clone's method, the full length sequence of streptococcus-salivarius urease gene U35248 is analyzed discovery afterwards, single endonuclease digestion site-Bam H I (being positioned at the 2038bp place) and Xho I (being positioned at the 4812bp place) that two restriction endonucleases are arranged in the inside of this gene, therefore before, during and after design is divided into urease gene three sections, clone respectively, some overlap all between every two neighboring sections, and comprise the site of a single endonuclease digestion in each overlap.Each segmental clone adopts classical molecular biological method, to contain the segmental complete genome DNA of purpose as template, and under the guiding of Auele Specific Primer, synthetic specific dna fragmentation; Adopt T/A clone's method then,, connect with identical direction with purpose fragment and identical carrier; Next change resulting recombinant DNA molecules over to competent intestinal bacteria; And by resistance screening and incision enzyme map evaluation, preliminary screening goes out recon; By determined dna sequence, goal gene is confirmed to analyze at last.

1.1 material and method

1.1.1 main agents

Taq archaeal dna polymerase (5U/ μ l), dNTP mixed solution (10mM), 10 * Taq damping fluid: available from Shanghai Shenergy Biocolor BioScience ﹠ Technology Company.

KOD-Plus archaeal dna polymerase (1U/ μ l), KOD Plus special use 10 * PCR damping fluid, MgSO ₄Solution (25mM), dNTP mixed solution (2mM): spin (Shanghai) bio tech ltd available from TOYOBO Japan.

The quick glue of miniprep dna fragment reclaims test kit (including DE-A solution, DE-B solution, W1 rinsing liquid, W2 rinsing liquid, centrifugal adsorption column): available from V-gene company.

PGEM-T Easy carrier: U.S. Promega company produces.

T4DNA ligase enzyme (3Weiss U/ μ l), 2 * fast connect damping fluid: U.S. Promega company produces.

Competence intestinal bacteria TG-1: this laboratory self-control ,-70 ℃ of preservations are standby.

Penbritin sodium salt: available from the rich photo bio Science and Technology Ltd. (import packing) in Shanghai.Add the storage liquid that aseptic double-distilled water is mixed with 50mg/ml, after the filtration sterilization, place 4 ℃ of preservations standby.

LB (Luria-Bertani) liquid nutrient medium:

Tryptone (Britain OXOID company) 10g

Yeast extractives (Britain OXOID company) 5g

Sodium-chlor (Shanghai chemical reagents corporation of Chinese Medicine group) 10g

Adding distil water is settled to 1L, and the pH value is transferred to 7.0, and high pressure steam sterilization after the packing places 4 ℃ of preservations standby after cooling.

Amicillin resistance LB agar culture plate: in 1L LB liquid nutrient medium, add the 15g agar powder, high pressure steam sterilization, to be cooled to about 55 ℃ when (but feel heat non-scald on hand), adding penbritin solution to final concentration is 60 μ g/ml, after the mixing, pour in the microbial culture plate of 90mm diameter, room temperature is placed about 30min and is treated that agar solidifies the back and uses, or standby with being inverted in 4 ℃ of preservations after the freshness protection package sealing.

Type B plasmid sample rapid extraction test kit (production of vast Tyke, Beijing biological gene technology limited liability company), include: solution 1 (50mmol/L glucose, 25mmol/L Tris-Cl pH value 8.0,10mmol/L EDTA pH value 8.0, add RNase A and reach 0.6mg/ml to final concentration, be stored in 4 ℃ standby), solution 2 (0.2mol/L NaOH, 1%SDS, room temperature is placed), solution 3 (3mol/L potassium acetate, the 5mol/L glacial acetic acid, room temperature is placed), binding buffer liquid (checks in binding buffer liquid and the solution 2 whether high salt crystallization is arranged earlier before extracting the plasmid test, if crystallization is arranged then place the middle-grade continuous heating 10～15sec of microwave oven after bottle cap unscrewed at every turn, high salt crystallization is fully dissolved to be re-used afterwards), concentrated bleaching liquid (ratio according to 1: 3 before using adds dehydrated alcohol, and room temperature is placed after the mixing), centrifugal adsorption column.

Restriction enzyme and corresponding damping fluid thereof: Bam H I, Sph I, Xho I, Nco I, 10 * H enzyme cutting buffering liquid, 10 * T enzyme cutting buffering liquid, 10 * K enzyme cutting buffering liquid and 10 * BSA damping fluid: available from the precious biotechnology (Dalian) of TaKaRa company limited.

1.1.2 key instrument equipment

Heraus 28RS low-temperature and high-speed desk centrifuge: Germany produces.

The electric heating constant temperature water bath: Shanghai produces.

The HZ-9211K constant temperature oscillator: Taicang science and education equipment factory produces.

Ultralow Temperature Freezer: U.S. HARRIS company produces.

PE 2400 DNA cloning instrument: the U.S. produces.

Tanon UV-2000 ultraviolet gel analysis instrument: Chinese Shanghai production.

The water isolation type electro-heating standing-temperature cultivator: the Shanghai medical apparatus and instruments factory of making a leapleap forward produces.

CA-920-3 vertical laminar flow clean bench: go up marine clean treating plant company limited and produce.

Ice-making machine: Shanghai produces.

The vortex oscillation device: Suzhou, Jiangsu produces.

Electronic analytical balance: Japanese Tokyo company produces.

Portable High pressure steam sterilizer: Jiangsu produces.

Electric heating constant temperature air dry oven: go up the grand experimental installation of Nereid company limited and produce.

Consumptive material: 15ml glass test tube (aseptic), Eppendorf centrifuge tube (1.5ml, 0.5ml, 0.2ml), micropipet TIP head (1000 μ l, 200 μ l, 20 μ l), microbial culture plate.

1.1.3 experimental technique

The primer that uses and sequence thereof in this experiment: used primer all uses PrimerPremier 5 PCR primer-design softwares to design voluntarily in this experiment, and primer sequence and corresponding expanding fragment length are as shown in table 2.Synthetic after design of primers is finished by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, 2OD, HAP purifying, vacuum lyophilization transportation; Receive after the primer that the centrifuge tube that will fill primer is in 12 earlier, the centrifugal 1min of 000rpm, tube used for bottom pouring covers carefully then, add an amount of aseptic double-distilled water and shake fully up and down, make it thoroughly to be dissolved as the primer solution of 10 μ M or 20 μ M, standby after the packing in-20 ℃ of preservations.

Table 2 experiment the primer and sequence thereof

Primer	Sequence	Amplified fragments (bp)
			Uf1 Ur1 Uf2 Ur2 Uf3 Ur3	TGC CTT CAC CTA CCT TTA CTC AG CAA CAA CGC ATG GCC ACT TC AC ACC CAA TCA ACC CAT ACA CCA C CAC CAT CAC CAA ATA GCA AAG C AAT GTT AAG GAA GTG GAA GAA TGG ATG ATG AAG GTG CTT GAA GTG ATG	2107 3905 3268

Adopt 50 μ l pcr amplification systems, respectively to before, during and after three sections sequences carry out amplified reaction, the application of sample process is operated on ice bath:

First section:

KOD-plus archaeal dna polymerase (5U/ μ l) 1 μ l

Upstream primer Uf1 (10 μ M) 2 μ l

Downstream primer Ur1 (10 μ M) 2 μ l

DNTP mixed solution (2mM) 5 μ l

MgSO ₄Solution 4 μ l

10 * damping fluid, 5 μ l

Streptococcus-salivarius 57.I genomic dna template 1 μ l

With aseptic double-distilled water final volume is mended to 50 μ l, centrifugal 5sec after the mixing concentrates on reacted constituent and manages the end and centrifugal mixing, adds an amount of mineral oil again and covers liquid level.

Second section:

KOD-plus archaeal dna polymerase (5U/ μ l) 1 μ l

Upstream primer Uf2 (20 μ M) 1 μ l

Downstream primer Ur2 (20 μ M) 1 μ l

DNTP mixed solution (2mM) 5 μ l

MgSO ₄Solution 4 μ l

10 * damping fluid, 5 μ l

Streptococcus-salivarius 57.I genomic dna template 1 μ l

With aseptic double-distilled water final volume is mended to 50 μ l, centrifugal 5sec after the mixing concentrates on reacted constituent and manages the end and centrifugal mixing, adds an amount of mineral oil again and covers liquid level.

The 3rd section:

KOD-plus archaeal dna polymerase (5U/ μ l) 1 μ l

Upstream primer Uf3 (20 μ M) 1 μ l

Downstream primer Ur3 (20 μ M) 1 μ l

DNTP mixed solution (2mM) 5 μ l

MgSO ₄Solution 4 μ l

10 * damping fluid, 5 μ l

Streptococcus-salivarius 57.I genomic dna template 1 μ l

With distilled water final volume is mended to 50 μ l, centrifugal 5sec after the mixing concentrates on reacted constituent and manages the end and centrifugal mixing, adds an amount of mineral oil again and covers liquid level.

The pcr amplification reaction condition of each section is as follows:

First section:

94 ℃ of pre-sex change 4min

94 ℃ of sex change 1min

56 ℃ of annealing 1min

72 ℃ are extended 2min 20 sec (carrying out 35 circulations)

Mend flat 10min for 72 ℃

Second section:

94 ℃ of pre-sex change 4min

94 ℃ of sex change 1min

56 ℃ of annealing 1min

72 ℃ are extended 4min (carrying out 35 circulations)

Mend flat 10min for 72 ℃

The 3rd section:

94 ℃ of pre-sex change 4min

94 ℃ of sex change 1min

56 ℃ of annealing 1min

72 ℃ are extended 3min 30 sec (carrying out 30 circulations)

Mend flat 10min for 72 ℃

The PCR reaction is respectively got 5 μ l amplified productions and is carried out 1% agarose gel electrophoresis (operation steps as previously mentioned) after finishing, and observes electrophoresis result in ultraviolet gel analysis instrument, Taking Pictures recording after the confirmation electrophoresis result is single expection band.Respectively remaining 45 μ l PCR product is directly reclaimed (using the quick glue recovery of the miniprep dna fragment test kit of V-gene company to carry out) through the adsorption column purifying then, the concrete operations step is as follows:

Add 200 μ l DE-A solution and 100 μ l DE-B solution respectively in remaining 45 μ l pcr amplification products, fully be transferred in the centrifugal adsorption column after the mixing, room temperature leaves standstill after the 1min, and the centrifugal 1min of 5400rpm abandons clean filtrate; Add 500 μ l W1 rinsing liquid room temperatures and leave standstill after the 1min, the centrifugal 1min of 5400rpm abandons clean filtrate; Add 700 μ l W2 rinsing liquid room temperatures and leave standstill after the 1min, the centrifugal 1min of 5400rpm abandons clean filtrate; Add 700 μ l W2 rinsing liquid room temperatures once more and leave standstill after the 1min the centrifugal 1min of 5400rpm; Abandon clean filtrate; Again in 12, the centrifugal 1min of 000rpm thoroughly removes clean residual rinsing liquid afterwards; At last adsorption column is transferred in the aseptic 1.5ml centrifuge tube, added the aseptic double-distilled water of 30 μ l preheatings in the central authorities of adsorption column film, room temperature leaves standstill after the 1min, and 12, the centrifugal 1.5min of 000rpm collects filtrate.

Pcr amplification product behind the purifying adds the A tail: use KOD Plus archaeal dna polymerase to carry out the product end that pcr amplification obtains and be flush end, to carry out the T-A clone in order in next step experiment, being connected, need to utilize the Taq archaeal dna polymerase to hold and add a dependent deoxyadenylic acid A of non-template at 3 ' of the PCR of purifying product with pGEM-T Easy carrier.Reaction process adopts 50 μ l systems, and specific operation process is as follows:

Taq archaeal dna polymerase (5U/ μ l) 1.5 μ l

DNTP mixed solution (10mM) 1 μ l

10 * Taq damping fluid, 5 μ l

The pcr amplification product 30 μ l of purifying

With aseptic double-distilled water final volume is mended to 50 μ l, centrifugal 5sec after the mixing concentrates on reacted constituent and manages the end and centrifugal mixing, add an amount of mineral oil again and cover liquid level, subsequently with mixed solution in 72 ℃ of incubation 30min; The quick glue of miniprep dna fragment that re-uses V-gene company afterwards reclaims test kit and directly reclaims through adsorption column, and the concrete operations step uses 50 μ l aseptic double-distilled waters to reclaim as previously mentioned.

Add the pcr amplification product of purifying and being connected of carrier behind the A tail, adopt 20 μ l linked systems, the concrete operations step is as follows:

The pcr amplification product 8 μ l that add purifying behind the A tail

PGEM-T Easy carrier (50ng/ μ l) 0.5 μ l

2 * fast connect damping fluid 10 μ l

T4DNA ligase enzyme (3Weiss U/ μ l) 1.5 μ l

Use micropipet rifle head to blow and beat gently, after the careful mixing, will connect liquid and place 16 ℃ of connections of spending the night.

(competence intestinal bacteria TG-1 is made by oneself by this testing laboratory transformed competence colibacillus intestinal bacteria TG-1, be stored in-70 ℃), the concrete operations step is as follows: use ice chest to take out 100 μ l competence intestinal bacteria TG-1 in-70 ℃ cryogenic refrigerator, transfer in the aseptic technique platform, add fragment, the carrier that 10 μ l spend the night and connect liquid, ice bath 30min after gentle mixing on ice; Move to thermal treatment 90sec in 45 ℃ of constant water bath box fast, move to cooled on ice 2min immediately fast; Mixed solution is joined in the 400 μ l LB liquid nutrient mediums, be fixed on the shaking table, 37 ℃ of about 45min of shaking culture; Get 200 μ l bacterium liquid, be coated on the LB culture plate that contains penbritin (60 μ g/ml), spread evenly with aseptic triangle glass rod gently, room temperature is placed after the 20min, moves into and is inverted the about 9h of cultivation in 37 ℃ of constant incubators.

The picking positive colony increases bacterium and cultivates: the bacterium colony of visible adularescent grows behind the dull and stereotyped 8～9h of cultivation of the LB of conversion, immediately in the aseptic technique platform with 200 μ l micropipet rifle heads some single bacterial clones of picking from the LB flat board that transforms, be seeded in the LB liquid nutrient medium that about 3ml contains penbritin (60 μ g/ml), break up mixing, be fixed on the shaking table, 37 ℃ of shaking culture are spent the night.

The quick extracting of a small amount of of plasmid (using the Type B plasmid sample rapid extraction test kit of vast Imtech to carry out), the concrete operations step is as follows: take out about 1.5ml bacterium liquid and pack in the Eppendorf centrifuge tube of 1.5ml from the muddy bacterium liquid of incubated overnight, in 12, the centrifugal 30sec of 000rpm, remove supernatant liquor, be upside down on the toilet paper of testing laboratory's desktop cleaning to control the nutrient solution in the dried centrifuge tube; Add 100 μ l solution 1 (containing RNAse A 0.6mg/ml), use vortex oscillation device thermal agitation, or use micropipet rifle head repeatedly pressure-vaccum the bacterial precipitation thing is fully suspended; Add 150 μ l solution 2, softly put upside down centrifuge tube abundant mixing for several times behind the tight pipe lid of lid immediately, make the abundant cracking of thalline, the liquid in the centrifuge tube is limpid, transfers to and places 1～2min on ice with being about to centrifuge tube; Add 150 μ l solution 3, behind the tight pipe lid of lid immediately gentleness put upside down centrifuge tube for several times, fully transfer to behind the mixing and place 5min on ice, then 12, the centrifugal 12min of 000rpm; Supernatant liquor is transferred in the centrifugal adsorption column (note will precipitate move into adsorption column), added 420 μ l binding buffer liquid, with pipettor pressure-vaccum mixing afterwards 12, the centrifugal 30sec of 000rpm abandons clean filtrate; Add 750 μ l rinsing liquids, room temperature leaves standstill after the 1min, and 12, the centrifugal 15sec of 000rpm abandons clean filtrate; Repeat to add 750 μ l rinsing liquids, room temperature leaves standstill after the 1min, and 12, the centrifugal 15sec of 000rpm abandons clean filtrate; Once more in 12, the centrifugal 2min of 000rpm thoroughly eliminates rinsing liquid afterwards; At last adsorption column is transferred in the aseptic 1.5ml centrifuge tube, added the aseptic double-distilled water of 50 μ l preheatings in the central authorities of adsorption column film, room temperature is placed after 2～3min, and in 12, the centrifugal 1.5min of 000rpm collects filtrate.

Extractive plasmid is carried out preliminary enzyme cut evaluation: first section plasmid selects for use Sph I single endonuclease digestion site on the carrier and the Bam H I single endonuclease digestion site on the fragment to carry out double digestion, and enzyme is cut the application of sample process and operated on ice chest, and the endonuclease reaction system is as follows:

Extractive plasmid DNA 5 μ l

Restriction enzyme Sph I 1 μ l

Restriction enzyme Bam H I 1 μ l

10 * K enzyme cutting buffering liquid, 2 μ l

Become 20 μ l reaction systems with the aseptic aqueous solution trim that contains 20 μ g/ml RNAse A, centrifugal 5sec after the mixing concentrates on reacted constituent and manages the end and centrifugal mixing, places 37 ℃ constant incubator endoenzyme to cut 4～5h.

Second section plasmid selects for use Sph I single endonuclease digestion site on the carrier and the Xho I single endonuclease digestion site on the fragment to carry out double digestion, and enzyme is cut the application of sample process and operated on ice chest, and the endonuclease reaction system is as follows:

Plasmid DNA 5 μ l

Restriction enzyme Sph I 1 μ l

Restriction enzyme Xho I 1 μ l

10 * H enzyme cutting buffering liquid, 2 μ l

Become 20 μ l systems with the aseptic aqueous solution trim that contains 20 μ g/ml RNAse A, centrifugal 5sec after the mixing concentrates on reacted constituent and manages the end and centrifugal mixing, places 37 ℃ constant incubator endoenzyme to cut 4～5h.

The 3rd section plasmid selects for use Nco I to carry out single endonuclease digestion, and Nco I is respectively having a single endonuclease digestion site on the carrier He on the fragment, and enzyme is cut the application of sample process and operated on ice chest, and the endonuclease reaction system is as follows:

Plasmid DNA 5 μ l

Restriction enzyme Nco I 2 μ l

10 * BSA damping fluid, 2 μ l

10 * K enzyme cutting buffering liquid, 2 μ l

Become 20 μ l systems with the aseptic aqueous solution trim that contains 20 μ g/ml RNAse A, centrifugal 5sec after the mixing concentrates on reacted constituent and manages the end and centrifugal mixing, places 37 ℃ constant incubator endoenzyme to cut 4～5h.

Add 2 μ l, 10 * sample-loading buffer in the mixed solution after enzyme is cut, identify positive colony through 1% agarose gel electrophoresis after the mixing, the concrete operations step as previously mentioned.Electrophoresis is observed electrophoresis result after finishing in ultraviolet gel analysis instrument, and Taking Pictures recording.

Get 5 μ l enzymes respectively and cut the correct plasmid of evaluation, mix (diluting 10 times) with 45 μ l aseptic double-distilled waters, use Gene Quant RNA/DNA ultraviolet spectrometry survey meter to detect the optical density value of plasmid DNA under 260nm and 280nm wavelength UV-light respectively, and calculate its concentration and purity, specifically detect step as previously mentioned.

Preliminary enzyme cut identify that correct plasmid send Invitrogen Bioisystech Co., Ltd to carry out sequencing, use T7 promotor on the carrier and SP6 promotor respectively as sequencing primer.

1.2 experimental result

1.2.1 three sections pcr amplification products of urease gene segmentation clone

Respectively get three sections pcr amplification products of 5 μ l and carry out 1% agarose gel electrophoresis, electrophoresis is observed in ultraviolet gel analysis instrument after finishing, and as shown in Figure 3, is single band.According to design, the length of three sections amplified productions should be respectively 2107bp, 3905bp and 3268bp, and the position of visible electrophoretic band is consistent with expected results.

1.2.2 the enzyme of urease gene segmentation clone product is cut evaluation

Use specific restriction endonuclease respectively three sections plasmids to be digested, screening positive clone is identified recon.And the plasmid of segmentation being cloned owing to the method that needs to adopt enzyme to cut in follow-up experiment progressively couples together, so the direction that requires these three sections PCR products to insert in carrier is identical, therefore these three sections PCR products and plasmid after carrier is connected are being carried out enzyme when cutting evaluation, the purpose that enzyme is cut not only needs to determine to have inserted the corresponding target fragment in the plasmid, and needs to determine that the direction of three sections purpose fragments insertions is identical.

According to design, the length of first section pcr amplification product is 2.1Kb, and the carrier size is 3Kb, should be 5.1Kb so first section goal gene inserts the plasmid size that forms behind the carrier, selects the Sph on the carrier for use

Double digestion is carried out in Bam H I single endonuclease digestion site on I single endonuclease digestion site and the fragment, and expected result should be to locate respectively to have a single band about 2Kb and 3Kb; The length of second section pcr amplification product is 3.9Kb, the carrier size is 3Kb, so second section goal gene inserts the plasmid size that forms behind the carrier and should be 6.9Kb, select for use Sph I single endonuclease digestion site on the carrier and the Xho I single endonuclease digestion site on the fragment to carry out double digestion, expected result should be to locate respectively to have a single band about 2.9Kb and 4Kb; The length of the 3rd section pcr amplification product is 3.3Kb, the carrier size is 3Kb, so the 3rd section goal gene inserts the plasmid size that forms behind the carrier and should be 6.3Kb, select for use Nco I to carry out single endonuclease digestion, Nco I is respectively having a single endonuclease digestion site on the carrier He on the fragment, and expected result should be to locate respectively to have a single band about 2.5Kb and 3.8Kb.

Fig. 4 is that three sections plasmids carry out 1% agarose gel electrophoresis after corresponding digestion with restriction enzyme, and in ultraviolet gel analysis instrument observed electrophoresis result, visible endonuclease reaction is complete, the position of electrophoretic band is all consistent with expected results.

Concentration and the purity of three sections segmentation cloned plasmids DNA: get 5 μ l enzymes respectively and cut and identify that correct plasmid mixes (diluting 10 times) afterwards with 45 μ l aseptic double-distilled waters, use Gene Quant RNA/DNA ultraviolet spectrometry survey meter to detect the optical density value of plasmid DNA under 260nm and 280nm wavelength UV-light respectively, the OD value that records is as shown in table 3.

As previously mentioned, under the ultraviolet ray of wavelength 260nm, the concentration that the optical density(OD) of 1OD value is equivalent to double-stranded DNA is 50 μ g/ml, according to formula: the concentration of DNA sample to be measured (μ g/ μ l)=OD _260nm* nucleic acid extension rate * 50/1000 calculates the concentration (as shown in table 3) of three sections plasmid DNA respectively.

In like manner, according to the ratio of 260nm and 280nm place ultraviolet radiation absorption value, i.e. OD _260nm/ OD _280nm, the purity of estimation nucleic acid, wherein the ratio of DNA should be 1.8.Calculate the OD of three sections plasmid DNA that obtain in this experiment _260nm/ OD _280nmRatio, the result as shown in the table 3, as seen very near 1.8, tentatively thinks resulting plasmid DNA purity ideal can be used for follow-up experiment respectively.

The concentration and the purity of three sections plasmid DNA of table 3

	OD 260nm	OD 280nm	OD 260nm/OD 280nm	Concentration (μ g/ μ l)
					The 3rd section plasmid of second section plasmid of first section plasmid	0.648 0.786 0.819	0.368 0.441 0.461	1.761 1.782 1.776	0.32 0.39 0.41

Use T7 promotor on the carrier and SP6 promotor as sequencing primer respectively, the insertion fragment of each section plasmid is carried out two-way sequencing analysis, and carry out the BLAST comparison, determine that insertion sequence is correct in gene library by Invitrogen Bioisystech Co., Ltd.Wherein first section plasmid comprises the sequence of the 85bp among the urease gene U35248～2195bp, second section plasmid comprises the sequence of the 1967bp among the urease gene U35248～5872bp, the 3rd section plasmid comprises the sequence of the 4662bp among the urease gene U35248～7929bp, and three sections direction unanimities that sequence is inserted in carrier.Three sections are inserted fragments sequence measurement result, order-checking collection of illustrative plates and carry out the result of BLAST comparison in gene libraries consistent.

Embodiment 3

Segmentation clone's product progressively is connected to complete urease gene.

When the urease gene U35248 to streptococcus-salivarius 57.I carries out initial step-by-step design, between first section and second section and second section and the 3rd section target gene fragment one section overlap is arranged all, a single endonuclease digestion site is respectively arranged in the overlap.In above experiment, three fragments have been connected into identical carrier with identical direction respectively, this experiment will utilize on the carrier and each single endonuclease digestion site of sheet intersegmental part, adopt the method for double digestion, respectively goal gene and carrier are sheared, reconfigure again, progressively be connected to complete urease gene thereby will test three segmentation clone products that checked order correct in three.

1.1 material and method

1.1.1 main agents

The quick glue of miniprep dna fragment reclaims test kit (including DE-A solution, DE-B solution, W1 rinsing liquid, W2 rinsing liquid, centrifugal adsorption column): available from V-gene company.

Competence intestinal bacteria TG-1: this laboratory self-control ,-70 ℃ of preservations are standby.

The penbritin sodium salt: available from the rich photo bio Science and Technology Ltd. (import packing) in Shanghai, compound method as previously mentioned, be stored in 4 ℃ standby.

The LB liquid nutrient medium: collocation method as previously mentioned, be stored in 4 ℃ standby.

Amicillin resistance LB agar culture plate: collocation method as previously mentioned, be stored in 4 ℃ standby.

Type B plasmid sample rapid extraction test kit (available from Beijing vast Qin Ke biological gene technology limited liability company): include: solution 1 (contains RNase A 0.6mg/ml, be stored in 4 ℃ standby), solution 2 (room temperature placement), solution 3 (room temperature placement), (ratio according to 1: 3 before using adds dehydrated alcohol for binding buffer liquid (room temperature placement), concentrated bleaching liquid, room temperature is placed after the mixing), centrifugal adsorption column.

Restriction endonuclease and corresponding damping fluid thereof: Bam H I, Sph I, Xho I, 10 * H enzyme cutting buffering liquid, 10 * K enzyme cutting buffering liquid and 10 * BSA damping fluid: available from the precious biotechnology (Dalian) of TaKaRa company limited.

The T4 dna ligase, 350 U/ μ l, 10 * connect damping fluid: available from the precious biotechnology (Dalian) of TaKaRa company limited.

1.1.2 key instrument equipment

Electronic analytical balance: Japanese Tokyo company produces.

Portable High pressure steam sterilizer: produce in Jiangsu.

Electric heating constant temperature air dry oven: go up the grand experimental installation of Nereid company limited and produce.

Heraus 28RS low-temperature and high-speed desk centrifuge: Germany produces.

Electric heating constant temperature water bath: go up marine products.

The HZ-9211K constant temperature oscillator: Taicang science and education equipment factory produces.

Ultralow Temperature Freezer: U.S. HARRIS company produces.

Tanon UV-2000 ultraviolet gel analysis instrument: go up marine products.

The water isolation type electro-heating standing-temperature cultivator: the Shanghai medical apparatus and instruments factory of making a leapleap forward produces.

CA-920-3 vertical laminar flow clean bench: go up marine clean treating plant company limited and produce.

Ice-making machine: go up marine products.

The vortex oscillation device: produce in Suzhou, Jiangsu.

Consumptive material: 15ml glass test tube (aseptic), Eppendorf centrifuge tube (1.5ml, 0.5ml, 0.2ml), micropipet TIP head (1000 μ l, 200 μ l, 20 μ l), microbial culture plate.

1.1.3 experimental technique

The technological line of three sections segmentation clone products connection as shown in Figure 5

Connect experimental procedure:

For the correct second section plasmid of order-checking, select for use Sph I single endonuclease digestion site on the carrier and the Xho I single endonuclease digestion site on the fragment to carry out double digestion respectively, enzyme is cut the application of sample process and is operated on ice chest, and it is as follows that enzyme is cut system:

Second section plasmid DNA 8 μ l

Restriction enzyme Sph I 1 μ l

Restriction enzyme Xho I 1 μ l

10 * H enzyme cutting buffering liquid, 2 μ l

Become 20 μ l reaction systems with the aqueous solution trim that contains 20 μ g/ml RNAse A, centrifugal 5sec after the mixing concentrates on reacted constituent and manages the end and centrifugal mixing, cuts 7～8h in 37 ℃ of following enzymes.

For correct the 3rd section plasmid of order-checking, select for use Sph I single endonuclease digestion site on the carrier and the Xho I single endonuclease digestion site on the fragment to carry out double digestion respectively, enzyme is cut the application of sample process and is operated on ice chest, and it is as follows that enzyme is cut system:

The 3rd section plasmid DNA 2.5 μ l

Restriction enzyme Sph I 1 μ l

Restriction enzyme Xho I 1 μ l

10 * H enzyme cutting buffering liquid, 2 μ l

Become 20 μ l reaction systems with the aqueous solution trim that contains 20 μ g/ml RNAse A, centrifugal 5sec after the mixing concentrates on reacted constituent and manages the end and centrifugal mixing, cuts 7～8h in 37 ℃ of following enzymes.

Agarose gel electrophoresis reclaims: above-mentioned enzyme is cut product carry out 1% conventional agarose gel electrophoresis, operation steps as previously mentioned.In ultraviolet gel analysis instrument, observe then, the position of determining electrophoretic band is with after expected results is consistent, use respectively the quick glue of the miniprep dna fragment of V-gene company reclaim test kit after with second section plasmid enzyme restriction fragment (about 2.8Kb) and the carrier (about 6.1Kb) behind the 3rd section plasmid enzyme restriction cut the glue recovery, the concrete operations step is as follows:

Get an aseptic 1.5ml Eppendorf centrifuge tube, weigh up weight and record, under ultraviolet lamp, downcut the sepharose (comprising under the segmental prerequisite of all purposes, the gel volume that is downcut should be as much as possible little) that contains target DNA fragment carefully then with clean knife blade; Exhaust the liquid of gel surface and chopping with paper handkerchief and pack in the Eppendorf centrifuge tube that weighs up weight, weigh once more and the weight of calculated for gel, with this weight as a gel volume; According to the concentration (1%) of the volume and the gel of gel, add the DE-A solution of 3 times of volumes, after the mixing on desk centrifuge centrifugal 5sec, make the gel piece of chopping and DE-A solution all focus on the pipe end and centrifugal mixing; Then centrifuge tube is put into 75 ℃ of constant water bath box and heated, continuous gentle mixing melts fully until gel piece in the heat-processed; Take out the DE-B solution that centrifuge tube adds the 1/2DE-A liquor capacity, use micropipet rifle head pressure-vaccum mixing gently; Draw whole mixed solutions and be transferred in the centrifugal adsorption column, static 1min under the room temperature, the centrifugal 1min of 5400rpm abandons clean filtrate; Add 500 μ l W1 rinsing liquids, static 1min under the room temperature, the centrifugal 1min of 5400rpm abandons clean filtrate; Add 700 μ l W2 rinsing liquids, static 1min under the room temperature, the centrifugal 1min of 5400rpm abandons clean filtrate; Add 700 μ l W2 rinsing liquids once more, static 1min under the room temperature, the centrifugal 1min of 5400rpm abandons clean filtrate, and then in 12, the centrifugal 1min of 000rpm thoroughly abandons clean rinsing liquid; At last adsorption column is transferred in the aseptic 1.5ml centrifuge tube, added the aseptic double-distilled water of 30 μ l preheatings in the central authorities of adsorption column film, static 1min under the room temperature, in 12, the centrifugal 1.5min of 000rpm collects filtrate.

After reclaiming fragment is connected with carrier, adopts 20 μ l linked systems:

Fragment (2.8Kb) the 11 μ l that agarose electrophoresis reclaims behind second section plasmid enzyme restriction

Carrier (6.1Kb) the 1.5 μ l that agarose electrophoresis reclaims behind the 3rd section plasmid enzyme restriction

10 * connection damping fluid, 2 μ l

T4 dna ligase (350U/ μ l) 1 μ l

Add aseptic double-distilled water trim to 20 μ l, use micropipet rifle head pressure-vaccum gently, after the gentle mixing, will connect liquid and place 16 ℃ of connections of spending the night.

With above-mentioned connection liquid transformed competence colibacillus intestinal bacteria TG-1, be tiled on the LB agar plate that contains penbritin (60 μ g/ml) after spending the night, be inverted flat board down in 37 ℃ and cultivate 8～9h, the concrete operations step as previously mentioned.

Treat to grow on the LB agar plate after the bacterium colony of white, the some bacterial clones of picking are seeded in the LB nutrient solution that contains penbritin (60 μ g/ml), be fixed on the shaking table after breaing up mixing, concussion overnight incubation under 37 ℃, concrete operation method is as previously mentioned.

After the overnight incubation, the quick extracting of a small amount of of using the quick extraction agent box of vast Tyke Type B plasmid sample to carry out plasmid uses 50 μ l aseptic double-distilled waters to reclaim, and concrete operation method as previously mentioned.

Extractive plasmid is carried out preliminary enzyme cut evaluation: select for use Sph I single endonuclease digestion site on the carrier and the Xho I single endonuclease digestion site on the fragment to carry out double digestion, enzyme is cut the application of sample process and is operated on ice chest, and it is as follows that enzyme is cut system:

Plasmid DNA 6 μ l

Restriction enzyme Sph I 1 μ l

Restriction enzyme Xho I 1 μ l

10 * H enzyme cutting buffering liquid, 2 μ l

Become 20 μ l reaction systems with the aseptic aqueous solution trim that contains 20 μ g/ml RNAse A, centrifugal 5sec after the mixing concentrates on reacted constituent and manages the end and centrifugal mixing, cuts 4～5h in 37 ℃ of following enzymes.

Mixed solution after enzyme cut is identified positive colony through 1% agarose electrophoresis, observes and Taking Pictures recording in ultraviolet gel analysis instrument, and working method is cut enzyme the result and expected that consistent plasmid is labeled as U as previously mentioned ₂₊₃

Get 5 μ l enzymes and cut the correct U of evaluation ₂₊₃Plasmid and 45 μ l aseptic double-distilled water mixings (diluting 10 times) are afterwards, use Gene Quant RNA/DNA ultraviolet spectrometry survey meter to detect the optical density value of plasmid DNA under 260nm and 280nm wavelength UV-light respectively, and calculate its concentration and purity, specifically detect step as previously mentioned.

For the correct first section plasmid of order-checking, select for use Sph I single endonuclease digestion site on the carrier and the Bam H I single endonuclease digestion site on the fragment to carry out double digestion respectively, enzyme is cut the application of sample process and is operated on ice chest, and it is as follows that enzyme is cut system:

First section plasmid DNA 10 μ l

Restriction enzyme Sph I 1 μ l

Restriction enzyme Bam H I 1 μ l

10 * K enzyme cutting buffering liquid, 2 μ l

Become 20 μ l reaction systems with the aqueous solution trim that contains 20 μ g/ml RNAse A, centrifugal 5sec after the mixing concentrates on reacted constituent and manages the end and centrifugal mixing, cuts 7～8h in 37 ℃ of following enzymes.

Cut the correct U of evaluation for enzyme ₂₊₃Plasmid selects for use Sph I single endonuclease digestion site on the carrier and the Bam H I single endonuclease digestion site on the fragment to carry out double digestion respectively, and enzyme is cut the application of sample process and operated on ice chest, and it is as follows that enzyme is cut system:

U ₂₊₃Plasmid DNA 5 μ l

Restriction enzyme Sph I 1 μ l

Restriction enzyme Bam H I 1 μ l

10 * K enzyme cutting buffering liquid, 2 μ l

Become 20 μ l reaction systems with the aqueous solution trim that contains 20 μ g/ml RNAse A, centrifugal 5sec after the mixing concentrates on reacted constituent and manages the end and centrifugal mixing, cuts 7～8h in 37 ℃ of following enzymes.

Agarose gel electrophoresis reclaims: above-mentioned enzyme is cut product carry out 1% conventional agarose gel electrophoresis, operation steps as previously mentioned.Observe in ultraviolet gel analysis instrument then, the position of determining electrophoretic band is with after expected results is consistent, uses the quick glue of miniprep dna fragment of V-gene company to reclaim fragment (about 2Kb) and the U of test kit after with first section plasmid enzyme restriction respectively ₂₊₃Carrier behind the plasmid enzyme restriction (about 8.9Kb) is cut glue and is reclaimed, operation steps as previously mentioned, recovery system is 30 μ l aseptic double-distilled waters.

Carry out being connected of fragment and carrier after gel reclaims, adopt 20 μ l linked systems:

Fragment (2Kb) the 11 μ l that agarose electrophoresis reclaims behind first section plasmid enzyme restriction

U ₂₊₃Carrier (8.9Kb) the 1.5 μ l that agarose electrophoresis reclaims behind the plasmid enzyme restriction

10 * connection damping fluid, 2 μ l

T4 dna ligase (350U/ μ l) 1 μ l

Add aseptic double-distilled water trim to 20 μ l, use micropipet rifle head pressure-vaccum gently, after the gentle mixing, will connect liquid and place 16 ℃ of connections of spending the night, working method as previously mentioned.

With above-mentioned connection liquid transformed competence colibacillus intestinal bacteria TG-1, be tiled on the LB agar plate that contains penbritin (60 μ g/ml) after spending the night, be inverted flat board down in 37 ℃ and cultivate 8～9h, the concrete operations step as previously mentioned.

Treat to grow on the LB agar plate after the bacterium colony of white, the some bacterial clones of picking are seeded in the LB nutrient solution that contains penbritin (60 μ g/ml), be fixed on the shaking table after breaing up mixing, concussion overnight incubation under 37 ℃, concrete operation method is as previously mentioned.

After the overnight incubation, the quick extracting of a small amount of of using the quick extraction agent box of vast Tyke Type B plasmid sample to carry out plasmid uses 50 μ l aseptic double-distilled waters to reclaim, and concrete operation method as previously mentioned.

Extractive plasmid is carried out preliminary enzyme cut evaluation: select for use Sph I single endonuclease digestion site on the carrier and the Bam H I single endonuclease digestion site on the fragment to carry out double digestion, enzyme is cut the application of sample process and is operated on ice chest, and it is as follows that enzyme is cut system:

Plasmid DNA 6 μ l

Restriction enzyme Sph I 1 μ l

Restriction enzyme Bam H I 1 μ l

10 * K enzyme cutting buffering liquid, 2 μ l

Become 20 μ l reaction systems with the aseptic aqueous solution trim that contains 20 μ g/ml RNAse A, centrifugal 5sec after the mixing concentrates on reacted constituent and manages the end and centrifugal mixing, cuts 4～5h in 37 ℃ of following enzymes.

Mixed solution after enzyme cut is identified positive colony through 1% agarose electrophoresis, observes and Taking Pictures recording in ultraviolet gel analysis instrument, and working method is cut enzyme the result and expected that consistent plasmid is labeled as U as previously mentioned ₁₊₂₊₃

Get 5 μ l enzymes and cut the correct U of evaluation ₁₊₂₊₃Plasmid and 45 μ l aseptic double-distilled water mixings (diluting 10 times) are afterwards, use Gene Quant RNA/DNA ultraviolet spectrometry survey meter to detect the optical density value of plasmid DNA under 260nm and 280nm wavelength UV-light respectively, and calculate its concentration and purity, specifically detect step as previously mentioned.Remaining U ₁₊₂₊₃Plasmid solution be stored in-20 ℃ standby.

1.2 experimental result

According to experimental design, second section plasmid enzyme restriction fragment and the 3rd section plasmid U that plasmid enzyme restriction carrier reorganization afterwards is connected to form afterwards ₂₊₃Size should be about 9Kb, select for use Sph I single endonuclease digestion site on the carrier and the Xho I single endonuclease digestion site on the fragment to carry out double digestion afterwards, expected result should be to locate respectively to have a single band about 2.8Kb and 6.1Kb.

Fig. 6 is U ₂₊₃Plasmid carries out 1% agarose gel electrophoresis after restriction enzyme Sph I and Xho I double digestion, and under UV-light observed electrophoresis result, visible endonuclease reaction is complete, the position of electrophoretic band is consistent with expected results.

Get 5 μ l U ₂₊₃After 10 times of the plasmid DNA solution dilutions, use Gene Quant RNA/DNA ultraviolet spectrometry survey meter to detect plasmid DNA respectively in the optical density value at 260nm and 280nm wavelength place, the result shows OD _260nm=0.419, OD _280nm=0.239.Because under the ultraviolet ray of wavelength 260nm, it is 50 μ g/ml that the optical density(OD) of 1OD value is equivalent to double-stranded DNA concentration, according to formula: the concentration of plasmid DNA to be measured (μ g/ μ l)=OD _260nm* nucleic acid extension rate * 50/1000 calculates U ₂₊₃The concentration of plasmid DNA is 0.21 μ g/ μ l.

As previously mentioned, spectrophotometry can also be by being determined at the ratio of 260nm and 280nm place ultraviolet radiation absorption value, i.e. OD _260nm/ OD _280nm, the purity of estimation nucleic acid, wherein the ratio of DNA should be 1.8.

Record OD in this experiment _260nm/ OD _280nm=1.756, very near 1.8, can tentatively think extractive U ₂₊₃Plasmid DNA purity ideal can be used for follow-up experiment.

At last according to design, fragment and U behind first section plasmid enzyme restriction ₂₊₃Carrier reorganization behind the plasmid enzyme restriction is connected to form plasmid U ₁₊₂₊₃Afterwards, three sections segmentation clone products just will testing in three intactly couple together, and have obtained the final clone of streptococcus-salivarius 57.I urease gene.The size of this plasmid is about 11Kb, selects for use Sph I single endonuclease digestion site on the carrier and the Bam H I single endonuclease digestion site on the fragment to carry out double digestion afterwards, and expected result should be to locate respectively to have a single band about 2Kb and 9Kb.

Fig. 7 is U ₁₊₂₊₃Plasmid carries out 1% agarose gel electrophoresis after restriction enzyme Sph I and Bam H I double digestion, and under UV-light observed electrophoresis result, visible endonuclease reaction is complete, the position of electrophoretic band is consistent with expected results.

Get 5 μ l U ₁₊₂₊₃After 10 times of the plasmid DNA solution dilutions, use Gene Quant RNA/DNA ultraviolet spectrometry survey meter to detect plasmid DNA respectively in the optical density value at 260nm and 280nm wavelength place, the result shows OD _260nm=0.185, OD _280nm=0.102.According to formula: the concentration of DNA sample to be measured (μ g/ μ l)=OD _260nm* nucleic acid extension rate * 50/1000 calculates U ₁₊₂₊₃The concentration of plasmid DNA is 0.093 μ g/ μ l.

As previously mentioned, spectrophotometry can also be by being determined at the ratio of 260nm and 280nm place ultraviolet radiation absorption value, i.e. OD _260nm/ OD _280nm, the purity of estimation nucleic acid records OD in this experiment _260nm/ OD _280nm=1.814, very near 1.8, can tentatively think extractive U ₁₊₂₊₃Plasmid DNA purity ideal can be used for follow-up experiment.

Embodiment 4

The Preliminary detection of the complete clone's product characters of streptococcus-salivarius urease gene

Obtained clone's product U of the correct streptococcus-salivarius 57.I urease gene U35248 of sequence by above experiment ₁₊₂₊₃, this experiment will be used plasmid U ₁₊₂₊₃Can the transformed competence colibacillus intestinal bacteria go out ureolytic activity at expression in escherichia coli by this clone of urease test qualitative detection again, and whether the expression of ureolytic activity needs to add exogenous Ni ²⁺

1.1 material and method

1.1.1 experimental strain and substratum

Intestinal bacteria TG-1: preserve in this laboratory, and-70 ℃ frozen.

The LB liquid nutrient medium: compound method as previously mentioned, be stored in 4 ℃ standby.

Amicillin resistance LB culture plate and do not contain antibiotic LB culture plate: compound method as previously mentioned, be stored in 4 ℃ standby.

1.1.2 main agents

The plasmid U that contains streptococcus-salivarius urease gene U35248 ₁₊₂₊₃: obtain in the experiment four ,-20 ℃ are frozen.

2% urea soln, compound method as previously mentioned, filtration sterilization, be stored in 4 ℃ standby.

Phenol red phosphoric acid buffer (be called for short PB solution), pH value 6.0, compound method as previously mentioned, be stored in 4 ℃ standby.

1.1.3 key instrument and equipment

Electronic analytical balance: Japanese Tokyo company produces.

Portable High pressure steam sterilizer: produce in Jiangsu

Electric heating constant temperature air dry oven: go up the grand experimental installation of Nereid company limited and produce.

The water isolation type electro-heating standing-temperature cultivator: the Shanghai medical apparatus and instruments factory of making a leapleap forward produces.

Bechtop: produce in Suzhou, Jiangsu.

The GILSON pipettor: method is homemade.

The HZ-9211K constant temperature oscillator: Taicang science and education equipment factory produces.

Ultralow Temperature Freezer: U.S. HARRIS produces.

Ice-making machine: go up marine products.

Consumptive material: 15ml glass test tube (aseptic), Eppendorf centrifuge tube (1.5ml, 0.5ml, 0.2ml), micropipet TIP head (1000ul, 200ul, 20ul), microbial culture plate.

1.1.4 experimental technique

(competence intestinal bacteria TG-1 is made by oneself by this testing laboratory transformed competence colibacillus intestinal bacteria TG-1, be stored in-70 ℃): use ice chest in-70 ℃ cryogenic refrigerator, to take out 100 μ l competence intestinal bacteria TG-1, transfer in the aseptic technique platform, add the plasmid U that contains urease gene U35248 that obtains in the 0.2 μ l experiment four ₁₊₂₊₃, ice bath 30min after gentle mixing on ice; Move to thermal treatment 90sec in 45 ℃ of constant water bath box fast, move to cooled on ice 2min immediately fast; Mixed solution is joined in the 400 μ l LB liquid nutrient mediums, be fixed on the shaking table 37 ℃ of shaking culture 45min; Get 200 μ l bacterium liquid, be coated on the LB flat board that contains penbritin (60 μ g/ml), even with aseptic triangle glass rod shop, room temperature is placed after the 20min, moves into to be inverted in 37 ℃ of constant incubators and cultivates about 9h.

The picking positive colony increases bacterium and cultivates, preparation test organisms liquid: as seen have the bacterium colony of a large amount of whites to grow behind the dull and stereotyped cultivation of the LB of conversion 8～9h, immediately in the aseptic technique platform with 200 μ l micropipet rifle heads single bacterial clone of picking from the LB culture plate that transforms, be seeded in the L B liquid nutrient medium that about 3ml contains penbritin (60 μ g/ml), break up mixing, be fixed on the shaking table, 37 ℃ of shaking culture are spent the night.After overnight shaking was cultivated, the visible intestinal bacteria that transform increased bacterium in a large number and form dense bacterium liquid, and it is labeled as test organisms liquid.

Preparation contrast bacterium liquid: in-70 ℃ cryogenic refrigerator, take out 100 μ l competence intestinal bacteria TG-1, transfer in the aseptic technique platform, add 400 μ l LB liquid nutrient mediums, be fixed on the shaking table 37 ℃ of shaking culture 45min; Get 200 μ l bacterium liquid, be coated in and do not contain on any antibiotic LB culture plate, even with aseptic triangle glass rod shop, room temperature is placed after the 20min, moves into and is inverted the about 9h of cultivation in 37 ℃ of constant incubators.

As seen there is the bacterium colony of a large amount of whites to grow after cultivating 8～9h, immediately in the aseptic technique platform with 200 μ l micropipet rifle heads single bacterial clone of picking from the LB culture plate, being seeded in about 3ml does not contain in any antibiotic L B liquid nutrient medium, break up mixing, be fixed on the shaking table, 37 ℃ of shaking culture are spent the night.After overnight shaking was cultivated, visible intestinal bacteria increased bacterium in a large number and form dense bacterium liquid, and it is labeled as contrast bacterium liquid.

Get 2 aseptic glass test tubees, add the phenol red PB damping fluid of 300 μ l (pH 6.0) respectively, 300 μ l urea solns, and in developmental tube, add 300 μ l test organisms liquid, in control tube, add 300 μ l contrast bacterium liquid; 37 ℃ leave standstill after the cultivation 18h, observe colour-change.

1.2 experimental result

When initial liquid feeding, developmental tube and control tube all are yellow (because the pH value of phosphoric acid buffer is 6.0, being acidity, so phenol red indicator is shown as yellow); Cultivate after the 18h for 37 ℃, colour-change as shown in Figure 8: experiment tube becomes redness, and the control tube nondiscoloration still is yellow.PH value in the prompting developmental tube has raise, and makes phenol red indicator show redness by showing that yellow becomes, and the pH value in the control tube does not change, so yet not variation of color.

1.3 discuss

Phenol red urease test is a kind of test of the urease activity of qualitative detection quickly and easily, and in this experiment, transforming has U ₁₊₂₊₃The intestinal bacteria of plasmid are after the process cultivation of 18h, along with colibacillary division growth, U ₁₊₂₊₃Plasmid is also by massive duplication, and synthesized urease, and the urea that decomposes in the mixed-culture medium generates ammonia, makes the pH value rising in the environment, and phenol red indicator becomes redness by yellow.And do not contain U ₁₊₂₊₃The unconverted intestinal bacteria of plasmid do not have ureaclastic activity yet after division growth, can not change the pH value of mixed-culture medium.

Particularly importantly, U ₁₊₂₊₃The urease activity that plasmid goes out at expression in escherichia coli does not need to add ectogenic NiCl in substratum ₂, this point and research in the past differ widely.No matter the streptococcus-salivarius urease gene that obtains in the external research in the past steps on suis when still expressing at intestinal bacteria, lattice in streptococcus mutans, all must add ectogenic NiCl in the substratum of recombinant bacterial strain growth ₂, otherwise just can't detect the activity of urease.This illustrates resulting U in this research ₁₊₂₊₃Plasmid is except the structure gene and auxiliary gene that contain urease gene bunch, also contain nickel ion absorption and transhipment genes involved, be the clone of complete streptococcus-salivarius 57.I urease gene U35248, not only sequence is correct, and do not need to add the activity that ectogenic nickel ion just can go out urease at vivoexpression, decomposing urea produces ammonia, and the pH value in the rising environment can be used for further research and application better.

Also found an interesting phenomenon in this external experiment, the phenol red urease test that streptococcus-salivarius 57.I carries out in the experiment one needed 48 hours can see that just phenol red indicator becomes redness, and in this chapter experiment, contained conversion plasmid U ₁₊₂₊₃Intestinal bacteria only needed 18 hours just can make indicator become redness.This difference may be owing to the fast growth of intestinal bacteria with respect to streptococcus-salivarius, plasmid replication comparatively fast makes the activity of urease show in the shorter time, also may be since the speed that the urease gene that obtains of cloning is transcribed and translated under the effect of promotor more powerful on the carrier faster, measure more cause, these deductions also require further study confirmation.

SEQUENCE LISTING

＜110〉Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University Sch

＜120〉a kind of streptococcus-salivarius 57.I urease gene

<130>/

<160>4

<170>PatentIn version 3.1

<210>1

<211>7822

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>1

atttgccttc acctaccttt actcagctat caatacgatt ttcgattttg atcaacgttt 60

gtatgggtgg tttagtttat ttgtggcaat taatacgcta ccagcaggga ttctttgctt 120

aacatctgga tacggtggta atgcttggta tggtattatt tggttcttgt ggggtattct 180

atggctaact gcctttattg aaattaacct taagaagaac ctaggaaaat ttgtccctta 240

cctagctatt tttgaaggaa ttgtaacagc ttggattccg gggcttttga tgctttgggg 300

caagtggtaa gcattgattt aaggaggaaa aacgatgcaa ttgacaatgc gtgagcagga 360

aaaaatgatg attagccttg cggctatgat tgctcaacga cgtaaagata aaggaatcaa 420

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aggtaaaacg gttgcccaat tgatggatga agctcgcaat cttttaacac gtgaagatgt 540

tatggaaggt attgcggaaa tgattccaat gattcaagtt gaagctactt ttacggacag 600

tacaaaactg gttactgttc atgatcctat tcagtaagga gaaatgtaat gattccaggt 660

gaataccatg tggcgagtga gccaattgat tataacggtg gttacgaagc cattagtctt 720

gaagtgaaaa atgtgggtga ccgtgctgct caagtgggct ctcactacca tttttatgaa 780

gcaaacgaag ctggtcttca gtttgatcgt gaaaaggcgc gaggcaaacg tctagatatt 840

ccagcaggta cagccattcg ttttgagcca ggtgaaacga aaacagtaca cttatgactt 900

ggagggaaac gtcgtatttt tggtttcata acagtcaatg atttcttaga ctagaaagag 960

gacaaatcga tgagttttaa aatggatcgt gaagagtatg ctcaacacta tggaccaact 1020

gtaggtgata gcgtacgtct tggagatacc aatctttttg cagccattga aaaagacttt 1080

actgtttatg gacaggaatc taagttcggt ggcggtaaag ttttgcgtga tggtatgggt 1140

gttagtgcta cggaaacacg tgacaatcca tcagttgttg ataccattat tacaggtgca 1200

accatcattg actatacagg tattattaaa gcagatatcg gtattcgtga tggtaagatt 1260

gttgctatcg gtcgcggtgg taacccaaat acaatggaca atgtggactt tgttgtgggt 1320

gctagtacag aagccattgc tgctgaaggt ttgattgtga ctgctggtgg tattgacctt 1380

cacgtgcact atatttctgc cgaccttcct gaatttggtt tggataacgg gattactacc 1440

ctctttggtg gtggtactgg tcctgctgat ggaagtaatg cgacaacttg tacaccaggt 1500

aaattccata ttactcgtat gttgcaagct gttgatgata tgcctgctaa ctttggtttc 1560

cttgccaaag gtgttggttc tgagactgaa gtgctagaag agcaaattaa ggccggtgca 1620

gcaggaatta aaacacacga ggactggggt gcgacttacg caggtattga taattccctt 1680

aaagttgcgg ataaatacga tgtttccttt gcggttcaca ctgactcttt gaatgagggt 1740

ggatttatgg aaaatacttt ggaatccttc caaggtcgta ctgttcatac cttccacacc 1800

gaaggttcag gtggtggaca tgctccagat atcatggttt ttgctggtaa ggaaaatatt 1860

ttgccatcat caactaaccc aatcaaccca tacaccacaa atgctattgg tgagttgtta 1920

gatatggtta tggtttgcca ccacttggat ccaaaaattc cagaagacgt ctcttttgct 1980

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cttagtatca tgacttcaga tgccatggca atgggacgtg tcggtgaagt ggccatgcgt 2100

tgttggcaac tggctgataa gatgaaggct cagcgtggtc cacttgaagg ggattcagag 2160

tttaacgata ataaccgtat caaacgttac gtggctaaat atacaattaa ccctgccatc 2220

accaatggta ttgcagacta tatcggttct gtagaagttg gtaaatttgc agatttggtt 2280

atctgggaac cagctcaatt tggtgcaaaa cctaagttgg tgcttaaggg tggtatgcta 2340

acttatggtg ttatgggtga cgctggttca agtcttccaa cacctcaacc acgtatcatg 2400

cgtaaattat atggtgctta cggtcaagcg gttcatgaaa caaatcttac atttgtttct 2460

caatatgctt atgatcacgg tatcaaagaa gaaattggtt tgaataagat tgttcttcct 2520

gttaagaata cgcgtaactt gactaagcgt gatatgaagc ttaatgacta cgctccaaaa 2580

acaatccgta tcgatccaca gacctttgat gtcttatgat gatgagttgg ttacttgtga 2640

accaatccat acgacatcat tgtctcaacg ttatttcttg ttctaaggaa gacgctatat 2700

aaatgaggct ggaatttttc ctccaacctc ttttgtattt tatagccata acgtttttag 2760

tgctttatta agttgctata tgagtttgat gctagatttt taaaatgtaa tagaaaagga 2820

aaaagtatga tttttacaaa agtagatgct cttgttaaag atatcgatgt ggacaaatac 2880

catattgaaa cagtcattct ttcgagcgat gaccttaaca aaaaaattat tcgtgtgaag 2940

agtgatcatg ggaatgaatt tggtatcgtc ttgataacgg acaaaattgc aaaatggctc 3000

tggctttttt atcgatgatc acatgtctag ctatggtgat gagtcacaga tttgattgtc 3060

attcacctaa gatatggatg aatgggaatc acagctcaca ttcttgggaa tactcataaa 3120

ccgattgagg tgaaagacgc caagatttat ttagaggttg atccagttgt agagcaagtc 3180

ttgactcaaa aagagattgc ctacacgatt gaagaagtgg tccttgataa gcccctacgc 3240

catgtgaatt taactgccca tgaacattaa tccctttgct aatgtgtctt tgcaagatta 3300

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tgggatggaa aattatctgc gcgaagacac tgtaacagat gataaaggtt acgaggagtg 3420

gcaagaagcc tatttagcta gtcagtttaa atatggtgaa ggtcttgtaa tcaaattggt 3480

ttatgatgct atggctacag acaacttaga gcaggtttgg cattatgata aggtcttgac 3540

agtttcgacg caagcgcgtg aaacaagaca ggggactaaa atgattgcta aacaaatgct 3600

tcgacttatt caaaggctcc atgctattcc ggtattggat gactatcagt ccaaaatacg 3660

taagggtgag gtcttcggca atccagctat tgtctttgca ctctatgtgt ttaacaaggg 3720

cttgggatgt agtgaagcta ttgcacttta tggctatagc gtgatttcga cgatggttca 3780

aaatgctgtt cgtgccattc cacttggaca gtttgctgga caagagattg ttttacgtag 3840

cttttcacaa ttagaaaaaa tgacacaaga aattcaagaa ctggatgcgt cctaccttgg 3900

ggtcaatacg cctggtcttg aattagctca gatgaaacat gaaacacagg tattccgcct 3960

attcatgtcc taaaatatca acaaggttga gaggaaaaaa caatgacaaa acgtactgta 4020

attattggag ttggtggacc tgttggttca ggtaaaaccc ttttgcttga gcgtcttaca 4080

cgacgtatgt ccgacttaaa tttagcagtt attactaacg atatctatac aaaagaagat 4140

gctcttttct tggctaaaaa ttcaagctta gatgaagacc gtatcattgg tgtagaaact 4200

ggtggatgtc ctcatactgc tattcgtgaa gatgcctcta tgaactttga agcgattgaa 4260

actcttcaag agcgctttaa ccatgatttg gatgttattt tccttgaaag tggtggggat 4320

aacttggctg cgaccttcag tcctgatttg gttgatttca ccatttatat tattgacgtt 4380

gctcagggtg aaaaaatccc acgtaaggct ggtcaaggga tgattaagag tgatttgttc 4440

ttgatcaata agactgacct tgctccttat gttggagcca acctagaccg tatgcgtgaa 4500

gatacccttc atttccgtaa cgaagattct ttcattttca caaatttgaa taatgatgac 4560

aatgttaagg aagtggaaga atggattcgt aagaatttcc tactagagga cttgtaagat 4620

gacacaagca tacgatggct ttgtccatct tggattttca aaccgaaatg gtcgtacaat 4680

ttcccacaag aaataccaag aaggcaactc tcgagtatcg gcggataatt cagatgccaa 4740

cggtgttcct tactatttcc tcattaatat gggtggggga tttgtcgagg gtgagcagta 4800

tcaagtgacc attgatgtta ataaagatgc tcatgccttg gtaacaaccc aaacacctac 4860

ctatgtttac aagtgtgaga aaggacagtt gacacatcag aatacgtcca tcacacttga 4920

agaaaatagc tatttggagt acatggctga tgaagtcatt ccctatttga gatcacgcta 4980

tttccaaaca agtcgtattg atatggataa gtctgcccac ttgatttatt cagatggtgt 5040

gacggcaggt tggtctcatg aggatttgcc gtttcatacc attattttcg tatttgacac 5100

aaatctacca agatgatgag cttgtttata gcgatcagac cctcttagag cctcagaaac 5160

aagatatgtt taaacttggt tattttgaag gctggcgtaa ttataatagt ttggtaatgg 5220

tgtcaccaaa tattgacgag gcttttgtta aggctttgca gaagcactta gaaaatctga 5280

atttagagtc tgattttgct atttcatcct tagatatccc gggtctggtg ttacgtatct 5340

taggaaaaac tgctgagatg atcgtcgcgt catttattct tgtgcagact attttagaca 5400

gaaatacatg attaacccct ttgatttgag aaaaaatgat atgaggagat aaaaaatgca 5460

tattcctgaa aattacttaa gccctatgac ttgtgcggca atgggggcag ttatgttgcc 5520

tatttggtat aaggctgtca aggaagtgaa ggtaaaggtt gacactgata aaaaaacgat 5580

tcctatgttg ggaatcggga cttccttgtc cttccttatc atgatgttta atcttccagc 5640

cccaggtgga acgagtgccc atgctgttgg ggcagtgcta attgctatat tattaggacc 5700

ttgggcctcc tgtttagcag ttagtgtggc tctagctatg caggctttgc tatttggtga 5760

tggtgggatt ttggcctttg gtgcgaatgc cttttgtatg gctgttgtca tgccatttgt 5820

gggttatgct gtttataaac tcttgaataa gtggacgaag aacaggataa ttgctagctt 5880

ttttggaggt tatattggaa ttgtagttgc ggccctaact gttgcggttt tactaggaat 5940

tcaaccgatt ctctttaaag atagcagtgg taatccgctt tacaatccat accctttgag 6000

agtgacgctt ccagtaatgg gcttgactca cctgcttatc ggcttggtag aaggattttt 6060

cacagccggt gttcaagaat tcattgaacg tttgaatatt gataatactc aggaaataac 6120

gactaaaaaa ctacgtcctt tattgctctt tatcctagcc ttaattatcc taacgccact 6180

tggtttattg gcgacgggaa cagcttttgc agaatgggat gtcaaagagt tggtagaaaa 6240

attgtctcat taccatgtgg aagcccaagc gccaaaagga atgttgaatg gtttttcatt 6300

caatgccctc ttcccagatt atagtatcgc aggcattcca gaagttttgg gttatatcct 6360

gagcgctgcc tctgctgttt tgattttctt catcctctat cgcttgattt tcggtagaaa 6420

ggttgaaaaa tgattctgcc agattggatg tcggaagagc gcccagtagt cactaaagtc 6480

ggtagaaata actttcttat ccggaatcgt caccatctgg aagctcttct tcaaaagttt 6540

gaaacgcatc ctttaaaagt agcatcagtt tttcatccaa cagctaaggt tttacttctc 6600

tttttcttac ttgtttcagt gggaattagc cgaaatctca cagttttgtg gattgtagcc 6660

ttgtttttag gagctggcgt ggctttttta ccgcattctg ttttagtaag aactttgaaa 6720

aaaactgtag tgttgttgat cttcccttta gttctttatc taccgcatct cttacttagc 6780

ggaggtcaat cgctctttct ttttagactt cctttgattg ctgtagccat tgcttattat 6840

tcagaaacga gtacaataag tgagatgttg gcggcattaa aaggattgca ttttcctgat 6900

cttgttctgc tccagttaga tatcaccata aaatatattg atgtctttgg aaaacaattg 6960

atggatttgc tcaaagggat tgaagcgcga agttttggtg gcaatcatcg tttccggatt 7020

ggaagtaata tctggggaat tttatacctt aaagccatac gctatggtga ggaactgact 7080

caagccatgg aagcacgttg ctttgttggt gagtatgtca agtcatcaca gtcattcaca 7140

tggaaagact ggctggcctt gataagtcta gtagcagtga ttttaggaca gattctgtta 7200

ggaggatgag atgtttcaat tgaatcaagt ggcctgtgcc tatgaacaaa aaaaggtctt 7260

tactggtctt gatttggaga ttagacaagg acaatatgtg atgttgatgg gggaaaatgg 7320

gactgggaaa tcaagtctta tcagtttatt aactggcttc aagcaggaag aatctggacg 7380

tattcttttc ttagggaagg acctcaaaga atggttgaag gacaaacgtc aaaaacgaga 7440

tttttatagc cgcctcggaa tcctctttca ggatgtggat agtcaattat ttaatagtac 7500

tgtctatgat gagattgctt ttggtcctcg tcagctaggt ctgaccgaag aagaggtctc 7560

acagcgggtc caagacacac tgtccctgct taaaattgaa gatttaaggg atcgcgttcc 7620

ctatcaactg tctggtggag aaaagaagaa agtggccttt gccagtatca tggtaacgaa 7680

tccagatgtg tatattcttg atgaaccctt caataatctt tctaaagaat atgaagaatt 7740

ttttagggaa cttctacatg aacttcattc agctgggaaa accattatta tgtctgctca 7800

tcacttcaag caccttcatc at 7822

<210>2

<211>2367

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>2

atgcatattc ctgaaaatta cttaagccct atgacttgtg cggcaatggg ggcagttatg 60

ttgcctattt ggtataaggc tgtcaaggaa gtgaaggtaa aggttgacac tgataaaaaa 120

acgattccta tgttgggaat cgggacttcc ttgtccttcc ttatcatgat gtttaatctt 180

ccagccccag gtggaacgag tgcccatgct gttggggcag tgctaattgc tatattatta 240

ggaccttggg cctcctgttt agcagttagt gtggctctag ctatgcaggc tttgctattt 300

ggtgatggtg ggattttggc ctttggtgcg aatgcctttt gtatggctgt tgtcatgcca 360

tttgtgggtt atgctgttta taaactcttg aataagtgga cgaagaacag gataattgct 420

agcttttttg gaggttatat tggaattgta gttgcggccc taactgttgc ggttttacta 480

ggaattcaac cgattctctt taaagatagc agtggtaatc cgctttacaa tccataccct 540

ttgagagtga cgcttccagt aatgggcttg actcacctgc ttatcggctt ggtagaagga 600

tttttcacag ccggtgttca agaattcatt gaacgtttga atattgataa tactcaggaa 660

ataacgacta aaaaactacg tcctttattg ctctttatcc tagccttaat tatcctaacg 720

ccacttggtt tattggcgac gggaacagct tttgcagaat gggatgtcaa agagttggta 780

gaaaaattgt ctcattacca tgtggaagcc caagcgccaa aaggaatgtt gaatggtttt 840

tcattcaatg ccctcttccc agattatagt atcgcaggca ttccagaagt tttgggttat 900

atcctgagcg ctgcctctgc tgttttgatt ttcttcatcc tctatcgctt gattttcggt 960

agaaaggttg aaaaatgatt ctgccagatt ggatgtcgga agagcgccca gtagtcacta 1020

aagtcggtag aaataacttt cttatccgga atcgtcacca tctggaagct cttcttcaaa 1080

agtttgaaac gcatccttta aaagtagcat cagtttttca tccaacagct aaggttttac 1140

ttctcttttt cttacttgtt tcagtgggaa ttagccgaaa tctcacagtt ttgtggattg 1200

tagccttgtt tttaggagct ggcgtggctt ttttaccgca ttctgtttta gtaagaactt 1260

tgaaaaaaac tgtagtgttg ttgatcttcc ctttagttct ttatctaccg catctcttac 1320

ttagcggagg tcaatcgctc tttcttttta gacttccttt gattgctgta gccattgctt 1380

attattcaga aacgagtaca ataagtgaga tgttggcggc attaaaagga ttgcattttc 1440

ctgatcttgt tctgctccag ttagatatca ccataaaata tattgatgtc tttggaaaac 1500

aattgatgga tttgctcaaa gggattgaag cgcgaagttt tggtggcaat catcgtttcc 1560

ggattggaag taatatctgg ggaattttat accttaaagc catacgctat ggtgaggaac 1620

tgactcaagc catggaagca cgttgctttg ttggtgagta tgtcaagtca tcacagtcat 1680

tcacatggaa agactggctg gccttgataa gtctagtagc agtgatttta ggacagattc 1740

tgttaggagg atgagatgtt tcaattgaat caagtggcct gtgcctatga acaaaaaaag 1800

gtctttactg gtcttgattt ggagattaga caaggacaat atgtgatgtt gatgggggaa 1860

aatgggactg ggaaatcaag tcttatcagt ttattaactg gcttcaagca ggaagaatct 1920

ggacgtattc ttttcttagg gaaggacctc aaagaatggt tgaaggacaa acgtcaaaaa 1980

cgagattttt atagccgcct cggaatcctc tttcaggatg tggatagtca attatttaat 2040

agtactgtct atgatgagat tgcttttggt cctcgtcagc taggtctgac cgaagaagag 2100

gtctcacagc gggtccaaga cacactgtcc ctgcttaaaa ttgaagattt aagggatcgc 2160

gttccctatc aactgtctgg tggagaaaag aagaaagtgg cctttgccag tatcatggta 2220

acgaatccag atgtgtatat tcttgatgaa cccttcaata atctttctaa agaatatgaa 2280

gaatttttta gggaacttct acatgaactt cattcagctg ggaaaaccat tattatgtct 2340

gctcatcact tcaagcacct tcatcat 2367

<210>3

<211>2480

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>3

Ile Cys Leu His Leu Pro Leu Leu Ser Tyr Gln Tyr Asp Phe Arg Phe

1 5 10 15

Ser Thr Phe Val Trp Val Val Phe Ile Cys Gly Asn Tyr Ala Thr Ser

20 25 30

Arg Asp Ser Leu Leu Asn Ile Trp Ile Arg Trp Cys Leu Val Trp Tyr

35 40 45

Tyr Leu Val Leu Val Gly Tyr Ser Met Ala Asn Cys Leu Tyr Asn Pro

50 55 60

Glu Glu Pro Arg Lys Ile Cys Pro Leu Pro Ser Tyr Phe Arg Asn Cys

65 70 75 80

Asn Ser Leu Asp Ser Gly Ala Phe Asp Ala Leu Gly Gln Val Val Ser

85 90 95

Ile Asp Leu Arg Arg Lys Asn Asp Ala Ile Asp Asn Ala Ala Gly Lys

100 105 110

Asn Asp Asp Pro Cys Gly Tyr Asp Cys Ser Thr Thr Arg Arg Asn Gln

115 120 125

Ile Glu Ser Ser Arg Ser Gly Cys Phe Asp Tyr Arg Leu Cys Ala Arg

130 135 140

Cys Lys Arg Arg Asn Gly Cys Pro Ile Asp Gly Ser Ser Gln Ser Phe

145 150 155 160

Asn Thr Arg Cys Tyr Gly Arg Tyr Cys Gly Asn Asp Ser Asn Asp Ser

165 170 175

Ser Ser Tyr Phe Tyr Gly Gln Tyr Lys Thr Gly Tyr Cys Ser Ser Tyr

180 185 190

Ser Val Arg Arg Asn Val Met Ile Pro Gly Glu Tyr His Val Ala Ser

195 200 205

Glu Pro Ile Asp Tyr Asn Gly Gly Tyr Glu Ala Ile Ser Leu Glu Val

210 215 220

Lys Asn Val Gly Asp Arg Ala Ala Gln Val Gly Ser His Tyr His Phe

225 230 235 240

Tyr Glu Ala Asn Glu Ala Gly Leu Gln Phe Asp Arg Glu Lys Ala Arg

245 250 255

Gly Lys Arg Leu Asp Ile Pro Ala Gly Thr Ala Ile Arg Phe Glu Pro

260 265 270

Gly Glu Thr Lys Thr Val His Leu Leu Gly Gly Lys Arg Arg Ile Phe

275 280 285

Gly Phe Ile Thr Val Asn Asp Phe Leu Asp Lys Glu Asp Lys Ser Met

290 295 300

Ser Phe Lys Met Asp Arg Glu Glu Tyr Ala Gln His Tyr Gly Pro Thr

305 310 315 320

Val Gly Asp Ser Val Arg Leu Gly Asp Thr Asn Leu Phe Ala Ala Ile

325 330 335

Glu Lys Asp Phe Thr Val Tyr Gly Gln Glu Ser Lys Phe Gly Gly Gly

340 345 350

Lys Val Leu Arg Asp Gly Met Gly Val Ser Ala Thr Glu Thr Arg Asp

355 360 365

Asn Pro Ser Val Val Asp Thr Ile Ile Thr Gly Ala Thr Ile Ile Asp

370 375 380

Tyr Thr Gly Ile Ile Lys Ala Asp Ile Gly Ile Arg Asp Gly Lys Ile

385 390 395 400

Val Ala Ile Gly Arg Gly Gly Asn Pro Asn Thr Met Asp Asn Val Asp

405 410 415

Phe Val Val Gly Ala Ser Thr Glu Ala Ile Ala Ala Glu Gly Leu Ile

420 425 430

Val Thr Ala Gly Gly Ile Asp Leu His Val His Tyr Ile Ser Ala Asp

435 440 445

Leu Pro Glu Phe Gly Leu Asp Asn Gly Ile Thr Thr Leu Phe Gly Gly

450 455 460

Gly Thr Gly Pro Ala Asp Gly Ser Asn Ala Thr Thr Cys Thr Pro Gly

465 470 475 480

Lys Phe His Ile Thr Arg Met Leu Gln Ala Val Asp Asp Met Pro Ala

485 490 495

Asn Phe Gly Phe Leu Ala Lys Gly Val Gly Ser Glu Thr Glu Val Leu

500 505 510

Glu Glu Gln Ile Lys Ala Gly Ala Ala Gly Ile Lys Thr His Glu Asp

515 520 525

Trp Gly Ala Thr Tyr Ala Gly Ile Asp Asn Ser Leu Lys Val Ala Asp

530 535 540

Lys Tyr Asp Val Ser Phe Ala Val His Thr Asp Ser Leu Asn Glu Gly

545 550 555 560

Gly Phe Met Glu Asn Thr Leu Glu Ser Phe Gln Gly Arg Thr Val His

565 570 575

Thr Phe His Thr Glu Gly Ser Gly Gly Gly His Ala Pro Asp Ile Met

580 585 590

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

595 600 605

Asn Pro Tyr Thr Thr Asn Ala Ile Gly Glu Leu Leu Asp Met Val Met

610 615 620

Val Cys His His Leu Asp Pro Lys Ile Pro Glu Asp Val Ser Phe Ala

625 630 635 640

Glu Ser Arg Val Arg Lys Gln Thr Val Ala Ala Glu Asp Val Leu His

645 650 655

Asp Met Gly Ala Leu Ser Ile Met Thr Ser Asp Ala Met Ala Met Gly

660 665 670

Arg Val Gly Glu Val Ala Met Arg Cys Trp Gln Leu Ala Asp Lys Met

675 680 685

Lys Ala Gln Arg Gly Pro Leu Glu Gly Asp Ser Glu Phe Asn Asp Asn

690 695 700

Asn Arg Ile Lys Arg Tyr Val Ala Lys Tyr Thr Ile Asn Pro Ala Ile

705 710 715 720

Thr Asn Gly Ile Ala Asp Tyr Ile Gly Ser Val Glu Val Gly Lys Phe

725 730 735

Ala Asp Leu Val Ile Trp Glu Pro Ala Gln Phe Gly Ala Lys Pro Lys

740 745 750

Leu Val Leu Lys Gly Gly Met Leu Thr Tyr Gly Val Met Gly Asp Ala

755 760 765

Gly Ser Ser Leu Pro Thr Pro Gln Pro Arg Ile Met Arg Lys Leu Tyr

770 775 780

Gly Ala Tyr Gly Gln Ala Val His Glu Thr Asn Leu Thr Phe Val Ser

785 790 795 800

Gln Tyr Ala Tyr Asp His Gly Ile Lys Glu Glu Ile Gly Leu Asn Lys

805 810 815

Ile Val Leu Pro Val Lys Asn Thr Arg Asn Leu Thr Lys Arg Asp Met

820 825 830

Lys Leu Asn Asp Tyr Ala Pro Lys Thr Ile Arg Ile Asp Pro Gln Thr

835 840 845

Phe Asp Val Leu Val Gly Tyr Leu Thr Asn Pro Tyr Asp Ile Ile Val

850 855 860

Ser Thr Leu Phe Leu Val Leu Arg Lys Thr Leu Tyr Lys Gly Trp Asn

865 870 875 880

Phe Ser Ser Asn Leu Phe Cys Ile Leu Pro Arg Phe Cys Phe Ile Lys

885 890 895

Leu Leu Tyr Glu Phe Asp Ala Arg Phe Leu Lys Cys Asn Arg Lys Gly

900 905 910

Lys Ser Met Ile Phe Thr Lys Val Asp Ala Leu Val Lys Asp Ile Asp

915 920 925

Val Asp Lys Tyr His Ile Glu Thr Val Ile Leu Ser Ser Asp Asp Leu

930 935 940

Asn Lys Lys Ile Ile Arg Val Lys Ser Asp His Gly Asn Glu Phe Gly

945 950 955 960

Ile Val Leu Ile Thr Asp Lys Ile Ala Lys Trp Leu Trp Leu Phe Tyr

965 970 975

Arg Ser His Val Leu Trp Val Thr Asp Leu Ile Val Ile His Leu Arg

980 985 990

Tyr Gly Met Gly Ile Thr Ala His Ile Leu Gly Asn Thr His Lys Pro

995 1000 1005

Ile Glu Val Lys Asp Ala Lys Ile Tyr Leu Glu Val Asp Pro Val

1010 1015 1020

Val Glu Gln Val Leu Thr Gln Lys Glu Ile Ala Tyr Thr Ile Glu

1025 1030 1035

Glu Val Val Leu Asp Lys Pro Leu Arg His Val Asn Leu Thr Ala

1040 1045 1050

His Glu His Ser Leu Cys Cys Val Phe Ala Arg Leu Ser Asn Cys

1055 1060 1065

Ala Asn Cys Arg Phe Asn Leu Ser Asn Trp Ile Ile Pro Leu Phe

1070 1075 1080

Trp Asp Gly Lys Leu Ser Ala Arg Arg His Cys Asn Arg Arg Leu

1085 1090 1095

Arg Gly Val Ala Arg Ser Leu Phe Ser Ser Val Ile Trp Arg Ser

1100 1105 1110

Cys Asn Gln Ile Gly Leu Cys Tyr Gly Tyr Arg Gln Leu Arg Ala

1115 1120 1125

Gly Leu Ala Leu Gly Leu Asp Ser Phe Asp Ala Ser Ala Asn Lys

1130 1135 1140

Thr Gly Asp Asn Asp Cys Thr Asn Ala Ser Thr Tyr Ser Lys Ala

1145 1150 1155

Pro Cys Tyr Ser Gly Ile Gly Leu Ser Val Gln Asn Thr Gly Gly

1160 1165 1170

Leu Arg Gln Ser Ser Tyr Cys Leu Cys Thr Leu Cys Val Gln Gly

1175 1180 1185

Leu Gly Met Ser Tyr Cys Thr Leu Trp Leu Arg Asp Phe Asp Asp

1190 1195 1200

Gly Ser Lys Cys Cys Ser Cys His Ser Thr Trp Thr Val Cys Trp

1205 1210 1215

Thr Arg Asp Cys Phe Thr Leu Phe Thr Ile Arg Lys Asn Asp Thr

1220 1225 1230

Arg Asn Ser Arg Thr Gly Cys Val Leu Pro Trp Gly Gln Tyr Ala

1235 1240 1245

Trp Ser Ile Ser Ser Asp Glu Thr Asn Thr Gly Ile Pro Pro Ile

1250 1255 1260

His Val Leu Lys Tyr Gln Gln Gly Glu Glu Lys Thr Met Thr Lys

1265 1270 1275

Arg Thr Val Ile Ile Gly Val Gly Gly Pro Val Gly Ser Gly Lys

1280 1285 1290

Thr Leu Leu Leu Glu Arg Leu Thr Arg Arg Met Ser Asp Leu Asn

1295 1300 1305

Leu Ala Val Ile Thr Asn Asp Ile Tyr Thr Lys Glu Asp Ala Leu

1310 1315 1320

Phe Leu Ala Lys Asn Ser Ser Leu Asp Glu Asp Arg Ile Ile Gly

1325 1330 1335

Val Glu Thr Gly Gly Cys Pro His Thr Ala Ile Arg Glu Asp Ala

1340 1345 1350

Ser Met Asn Phe Glu Ala Ile Glu Thr Leu Gln Glu Arg Phe Asn

1355 1360 1365

His Asp Leu Asp Val Ile Phe Leu Glu Ser Gly Gly Asp Asn Leu

1370 1375 1380

Ala Ala Thr Phe Ser Pro Asp Leu Val Asp Phe Thr Ile Tyr Ile

1385 1390 1395

Ile Asp Val Ala Gln Gly Glu Lys Ile Pro Arg Lys Ala Gly Gln

1400 1405 1410

Gly Met Ile Lys Ser Asp Leu Phe Leu Ile Asn Lys Thr Asp Leu

1415 1420 1425

Ala Pro Tyr Val Gly Ala Asn Leu Asp Arg Met Arg Glu Asp Thr

1430 1435 1440

Leu His Phe Arg Asn Glu Asp Ser Phe Ile Phe Thr Asn Leu Asn

1445 1450 1455

Asn Asp Asp Asn Val Lys Glu Val Glu Glu Trp Ile Arg Lys Asn

1460 1465 1470

Phe Leu Leu Glu Asp Leu Asp Asp Thr Ser Ile Arg Trp Leu Cys

1475 1480 1485

Pro Ser Trp Ile Phe Lys Pro Lys Trp Ser Tyr Asn Phe Pro Gln

1490 1495 1500

Glu Ile Pro Arg Arg Gln Leu Ser Ser Ile Gly Gly Phe Arg Cys

1505 1510 1515

Gln Arg Cys Ser Leu Leu Phe Pro His Tyr Gly Trp Gly Ile Cys

1520 1525 1530

Arg Gly Ala Val Ser Ser Asp His Cys Arg Cys Ser Cys Leu Gly

1535 1540 1545

Asn Asn Pro Asn Thr Tyr Leu Cys Leu Gln Val Glu Arg Thr Val

1550 1555 1560

Asp Thr Ser Glu Tyr Val His His Thr Arg Lys Leu Phe Gly Val

1565 1570 1575

His Gly Ser His Ser Leu Phe Glu Ile Thr Leu Phe Pro Asn Lys

1580 1585 1590

Ser Tyr Tyr Gly Val Cys Pro Leu Asp Leu Phe Arg Trp Cys Asp

1595 1600 1605

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

1610 1615 1620

Ile His Lys Ser Thr Lys Met Met Ser Leu Phe Ile Ala Ile Arg

1625 1630 1635

Pro Ser Ser Leu Arg Asn Lys Ile Cys Leu Asn Leu Val Ile Leu

1640 1645 1650

Lys Ala Gly Val Ile Ile Ile Val Trp Trp Cys His Gln Ile Leu

1655 1660 1665

Thr Arg Leu Leu Leu Arg Leu Cys Arg Ser Thr Lys Ile Ile Ser

1670 1675 1680

Leu Ile Leu Leu Phe His Pro Ile Ser Arg Val Trp Cys Tyr Val

1685 1690 1695

Ser Glu Lys Leu Leu Arg Ser Ser Arg His Leu Phe Leu Cys Arg

1700 1705 1710

Leu Phe Thr Glu Ile His Asp Pro Leu Phe Glu Lys Lys Tyr Glu

1715 1720 1725

Glu Ile Lys Asn Ala Tyr Ser Lys Leu Leu Lys Pro Tyr Asp Leu

1730 1735 1740

Cys Gly Asn Gly Gly Ser Tyr Val Ala Tyr Leu Val Gly Cys Gln

1745 1750 1755

Gly Ser Glu Gly Lys Gly His Lys Asn Asp Ser Tyr Val Gly Asn

1760 1765 1770

Arg Asp Phe Leu Val Leu Pro Tyr His Asp Val Ser Ser Ser Pro

1775 1780 1785

Arg Trp Asn Glu Cys Pro Cys Cys Trp Gly Ser Ala Asn Cys Tyr

1790 1795 1800

Ile Ile Arg Thr Leu Gly Leu Leu Phe Ser Ser Cys Gly Ser Ser

1805 1810 1815

Tyr Ala Gly Phe Ala Ile Trp Trp Trp Asp Phe Gly Leu Trp Cys

1820 1825 1830

Glu Cys Leu Leu Tyr Gly Cys Cys His Ala Ile Cys Gly Leu Cys

1835 1840 1845

Cys Leu Thr Leu Glu Val Asp Glu Glu Gln Asp Asn Cys Leu Phe

1850 1855 1860

Trp Arg Leu Tyr Trp Asn Cys Ser Cys Gly Pro Asn Cys Cys Gly

1865 1870 1875

Phe Thr Arg Asn Ser Thr Asp Ser Leu Arg Gln Trp Ser Ala Leu

1880 1885 1890

Gln Ser Ile Pro Phe Glu Ser Asp Ala Ser Ser Asn Gly Leu Asp

1895 1900 1905

Ser Pro Ala Tyr Arg Leu Gly Arg Arg Ile Phe His Ser Arg Cys

1910 1915 1920

Ser Arg Ile His Thr Phe Glu Tyr Tyr Ser Gly Asn Asn Asp Lys

1925 1930 1935

Thr Thr Ser Phe Ile Ala Leu Tyr Pro Ser Leu Asn Tyr Pro Asn

1940 1945 1950

Ala Thr Trp Phe Ile Gly Asp Gly Asn Ser Phe Cys Arg Met Gly

1955 1960 1965

Cys Gln Arg Val Gly Arg Lys Ile Val Ser Leu Pro Cys Gly Ser

1970 1975 1980

Pro Ser Ala Lys Arg Asn Val Glu Trp Phe Phe Ile Gln Cys Pro

1985 1990 1995

Leu Pro Arg Leu Tyr Arg Arg His Ser Arg Ser Phe Gly Leu Tyr

2000 2005 2010

Pro Glu Arg Cys Leu Cys Cys Phe Asp Phe Leu His Pro Leu Ser

2015 2020 2025

Leu Asp Phe Arg Lys Gly Lys Met Ile Leu Pro Asp Trp Met Ser

2030 2035 2040

Glu Glu Arg Pro Val Val Thr Lys Val Gly Arg Asn Asn Phe Leu

2045 2050 2055

Ile Arg Asn Arg His His Leu Glu Ala Leu Leu Gln Lys Phe Glu

2060 2065 2070

Thr His Pro Leu Lys Val Ala Ser Val Phe His Pro Thr Ala Lys

2075 2080 2085

Val Leu Leu Leu Phe Phe Leu Leu Val Ser Val Gly Ile Ser Arg

2090 2095 2100

Asn Leu Thr Val Leu Trp Ile Val Ala Leu Phe Leu Gly Ala Gly

2105 2110 2115

Val Ala Phe Leu Pro His Ser Val Leu Val Arg Thr Leu Lys Lys

2120 2125 2130

Thr Val Val Leu Leu Ile Phe Pro Leu Val Leu Tyr Leu Pro His

2135 2140 2145

Leu Leu Leu Ser Gly Gly Gln Ser Leu Phe Leu Phe Arg Leu Pro

2150 2155 2160

Leu Ile Ala Val Ala Ile Ala Tyr Tyr Ser Glu Thr Ser Thr Ile

2165 2170 2175

Ser Glu Met Leu Ala Ala Leu Lys Gly Leu His Phe Pro Asp Leu

2180 2185 2190

Val Leu Leu Gln Leu Asp Ile Thr Ile Lys Tyr Ile Asp Val Phe

2195 2200 2205

Gly Lys Gln Leu Met Asp Leu Leu Lys Gly Ile Glu Ala Arg Ser

2210 2215 2220

Phe Gly Gly Asn His Arg Phe Arg Ile Gly Ser Asn Ile Trp Gly

2225 2230 2235

Ile Leu Tyr Leu Lys Ala Ile Arg Tyr Gly Glu Glu Leu Thr Gln

2240 2245 2250

Ala Met Glu Ala Arg Cys Phe Val Gly Glu Tyr Val Lys Ser Ser

2255 2260 2265

Gln Ser Phe Thr Trp Lys Asp Trp Leu Ala Leu Ile Ser Leu Val

2270 2275 2280

Ala Val Ile Leu Gly Gln Ile Leu Leu Gly Gly Asp Val Ser Ile

2285 2290 2295

Glu Ser Ser Gly Leu Cys Leu Thr Lys Lys Gly Leu Tyr Trp Ser

2300 2305 2310

Phe Gly Asp Thr Arg Thr Ile Cys Asp Val Asp Gly Gly Lys Trp

2315 2320 2325

Asp Trp Glu Ile Lys Ser Tyr Gln Phe Ile Asn Trp Leu Gln Ala

2330 2335 2340

Gly Arg Ile Trp Thr Tyr Ser Phe Leu Arg Glu Gly Pro Gln Arg

2345 2350 2355

Met Val Glu Gly Gln Thr Ser Lys Thr Arg Phe Leu Pro Pro Arg

2360 2365 2370

Asn Pro Leu Ser Gly Cys Gly Ser Ile Ile Tyr Cys Leu Asp Cys

2375 2380 2385

Phe Trp Ser Ser Ser Ala Arg Ser Asp Arg Arg Arg Gly Leu Thr

2390 2395 2400

Ala Gly Pro Arg His Thr Val Pro Ala Asn Arg Phe Lys Gly Ser

2405 2410 2415

Arg Ser Leu Ser Thr Val Trp Trp Arg Lys Glu Glu Ser Gly Leu

2420 2425 2430

Cys Gln Tyr His Gly Asn Glu Ser Arg Cys Val Tyr Ser Thr Leu

2435 2440 2445

Gln Ser Phe Arg Ile Arg Ile Phe Gly Thr Ser Thr Thr Ser Phe

2450 2455 2460

Ser Trp Glu Asn His Tyr Tyr Val Cys Ser Ser Leu Gln Ala Pro

2465 2470 2475

Ser Ser

2480

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Met His Ile Pro Glu Asn Tyr Leu Ser Pro Met Thr Cys Ala Ala Met

1 5 10 15

Gly Ala Val Met Leu Pro Ile Trp Tyr Lys Ala Val Lys Glu Val Lys

20 25 30

Val Lys Val Asp Thr Asp Lys Lys Thr Ile Pro Met Leu Gly Ile Gly

35 40 45

Thr Ser Leu Ser Phe Leu Ile Met Met Phe Asn Leu Pro Ala Pro Gly

50 55 60

Gly Thr Ser Ala His Ala Val Gly Ala Val Leu Ile Ala Ile Leu Leu

65 70 75 80

Gly Pro Trp Ala Ser Cys Leu Ala Val Ser Val Ala Leu Ala Met Gln

85 90 95

Ala Leu Leu Phe Gly Asp Gly Gly Ile Leu Ala Phe Gly Ala Asn Ala

100 105 110

Phe Cys Met Ala Val Val Met Pro Phe Val Gly Tyr Ala Val Tyr Lys

115 120 125

Leu Leu Asn Lys Trp Thr Lys Asn Arg Ile Ile Ala Ser Phe Phe Gly

130 135 140

Gly Tyr Ile Gly Ile Val Val Ala Ala Leu Thr Val Ala Val Leu Leu

145 150 155 160

Gly Ile Gln Pro Ile Leu Phe Lys Asp Ser Ser Gly Asn Pro Leu Tyr

165 170 175

Asn Pro Tyr Pro Leu Arg Val Thr Leu Pro Val Met Gly Leu Thr His

180 185 190

Leu Leu Ile Gly Leu Val Glu Gly Phe Phe Thr Ala Gly Val Gln Glu

195 200 205

Phe Ile Glu Arg Leu Asn Ile Asp Asn Thr Gln Glu Ile Thr Thr Lys

210 215 220

Lys Leu Arg Pro Leu Leu Leu Phe Ile Leu Ala Leu Ile Ile Leu Thr

225 230 235 240

Pro Leu Gly Leu Leu Ala Thr Gly Thr Ala Phe Ala Glu Trp Asp Val

245 250 255

Lys Glu Leu Val Glu Lys Leu Ser His Tyr His Val Glu Ala Gln Ala

260 265 270

Pro Lys Gly Met Leu Asn Gly Phe Ser Phe Asn Ala Leu Phe Pro Asp

275 280 285

Tyr Ser Ile Ala Gly Ile Pro Glu Val Leu Gly Tyr Ile Leu Ser Ala

290 295 300

Ala Ser Ala Val Leu Ile Phe Phe Ile Leu Tyr Arg Leu Ile Phe Gly

305 310 315 320

Arg Lys Val Glu Lys Phe Cys Gln Ile Gly Cys Arg Lys Ser Ala Gln

325 330 335

Ser Leu Lys Ser Val Glu Ile Thr Phe Leu Ser Gly Ile Val Thr Ile

340 345 350

Trp Lys Leu Phe Phe Lys Ser Leu Lys Arg Ile Leu Lys His Gln Phe

355 360 365

Phe Ile Gln Gln Leu Arg Phe Tyr Phe Ser Phe Ser Tyr Leu Phe Gln

370 375 380

Trp Glu Leu Ala Glu Ile Ser Gln Phe Cys Gly Leu Pro Cys Phe Glu

385 390 395 400

Leu Ala Trp Leu Phe Tyr Arg Ile Leu Phe Glu Leu Lys Lys Leu Cys

405 410 415

Cys Ser Ser Leu Phe Phe Ile Tyr Arg Ile Ser Tyr Leu Ala Glu Val

420 425 430

Asn Arg Ser Phe Phe Leu Asp Phe Leu Leu Leu Pro Leu Leu Ile Ile

435 440 445

Gln Lys Arg Val Gln Val Arg Cys Trp Arg His Lys Asp Cys Ile Phe

450 455 460

Leu Ile Leu Phe Cys Ser Ser Ile Ser Pro Asn Ile Leu Met Ser Leu

465 470 475 480

Glu Asn Asn Trp Ile Cys Ser Lys Gly Leu Lys Arg Glu Val Leu Val

485 490 495

Ala Ile Ile Val Ser Gly Leu Glu Val Ile Ser Gly Glu Phe Tyr Thr

500 505 510

Leu Lys Pro Tyr Ala Met Val Arg Asn Leu Lys Pro Trp Lys His Val

515 520 525

Ala Leu Leu Val Ser Met Ser Ser His His Ser His Ser His Gly Lys

530 535 540

Thr Gly Trp Pro Val Gln Phe Asp Arg Phe Cys Glu Asp Glu Met Phe

545 550 555 560

Gln Leu Asn Gln Val Ala Cys Ala Tyr Glu Gln Lys Lys Val Phe Thr

565 570 575

Gly Leu Asp Leu Glu Ile Arg Gln Gly Gln Tyr Val Met Leu Met Gly

580 585 590

Glu Asn Gly Thr Gly Lys Ser Ser Leu Ile Ser Leu Leu Thr Gly Phe

595 600 605

Lys Gln Glu Glu Ser Gly Arg Ile Leu Phe Leu Gly Lys Asp Leu Lys

610 615 620

Glu Trp Leu Lys Asp Lys Arg Gln Lys Arg Asp Phe Tyr Ser Arg Leu

625 630 635 640

Gly Ile Leu Phe Gln Asp Val Asp Ser Gln Leu Phe Asn Ser Thr Val

645 650 655

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

660 665 670

Glu Val Ser Gln Arg Val Gln Asp Thr Leu Ser Leu Leu Lys Ile Glu

675 680 685

Asp Leu Arg Asp Arg Val Pro Tyr Gln Leu Ser Gly Gly Glu Lys Lys

690 695 700

Lys Val Ala Phe Ala Ser Ile Met Val Thr Asn Pro Asp Val Tyr Ile

705 710 715 720

Leu Asp Glu Pro Phe Asn Asn Leu Ser Lys Glu Tyr Glu Glu Phe Phe

725 730 735

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

740 745 750

Ser Ala His His Phe Lys His Leu His His

755 760

Claims (9)

1, a kind of streptococcus-salivarius 57.I urease gene, its nucleotide sequence is as described in the SEQ ID NO.1.

2,57.I urease gene according to claim 1 is characterized in that: the nucleotide sequence of nickel ion combination in this gene and the relevant gene M QO of transhipment is as described in the SEQ ID NO.2.

3, prepare the method for streptococcus-salivarius urease gene, comprise the following steps:

(1) segmentation of streptococcus-salivarius 57.I urease gene clone;

(2) product with the segmentation clone progressively is connected to intactly urease gene.

4, method according to claim 3, it is characterized in that: in (1) step, segmentation place of streptococcus-salivarius 57.I urease gene is: be positioned at the single endonuclease digestion site-BamHI of the restriction endonuclease at 2038bp place, be positioned at the single endonuclease digestion site-XhoI of the restriction endonuclease at 4812bp place.

5, method according to claim 3 is characterized in that: in (1) step, and the primer Uf1 that amplified reaction is used, sequence: TGC CTT CAC CTA CCT TTA CTC AG;

Primer Ur1, sequence: CAA CAA CGC ATG GCC ACT TC AC;

Primer Uf2, sequence: ACC CAA TCA ACC CAT ACA CCA C;

Primer Ur2, sequence: CAC CAT CAC CAA ATA GCA AAG C;

Primer Uf3, sequence: AAT GTT AAG GAA GTG GAA GAA TGG;

Primer Ur3, sequence: ATG ATG AAG GTG CTT GAA GTG ATG.

6, a kind of streptococcus-salivarius urease, its aminoacid sequence is as described in the SEQ ID NO.3.

7, the method for preparing the streptococcus-salivarius urease comprises with the described 57.I urease gene of claim 1 plasmid U ₁₊₂₊₃The transformed competence colibacillus intestinal bacteria obtain the streptococcus-salivarius urease.

8, the application of 57.I urease gene according to claim 1 in preparation treatment or the sick medicine of prevention oral cavity dental caries.

9, the application of streptococcus-salivarius urease according to claim 6 in preparation treatment or the sick medicine of prevention oral cavity dental caries.

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