CN1950505A - Process for producing polypeptide - Google Patents

Abstract

A process for producing a target protein at low temperature, comprises inducing expression of not only a vector having introduced therein a gene coding for the target protein but also a vector having a chaperone gene introduced therein.

Description

Produce the method for polypeptide

Technical field

The present invention relates under cold condition by expression have the coding target protein that is integrated gene carrier and have the method that the carrier of the chaperone gene that is integrated is produced target protein.

Background field

Recently, the genomic analysis of various biologies just is being tending towards finishing, and thinks that these are analyzed in the proteic comprehensive functional analysis that will turn in the future as the expression of gene product.Just constantly increase apace by illustrating interaction between single proteic character and the detailed analysis albumen help the to explain research of biological phenomena.On the other hand, in determining the three-dimensional structure of receptor protein,intracellular, great interest is arranged, described protein-specific ground in conjunction with the material of various physiologically actives with transfer function.This is because the proteic active substance of bind receptor can be the candidate substances of new drug.Therefore, the screening new drug has been invested in concern.Be to determine so proteic characteristic, general method comprises goes into vector gene with the gene integration of correspondence, transforms host for example bacterium, yeast or insect cell and check by expressing the characteristic of the recombinant protein that obtains.

When estimating proteic exact nature, whether described albumen is folded into correct tertiary structure is very important.Yet, if use above-mentioned host expression system to prepare the albumen that derives from the allos biology according to the protein expression method, then people may often run into such situation, in this condition, because described proteic unusual folding paraprotein that acquisition has different tertiary structures.Such albumen has formed the aggregate that is called inclusion body in the host.The formation of inclusion body is favourable, avoids the proteolysis enzyme liberating in the host cell because expressed proteins is protected, and described albumen can easily separate from cell by centrifugal.Yet, in order to obtain the target protein of biologic activity, must dissolve inclusion body, then with its regeneration (refolding) by sex change.When each albumen was repeated trial and error, mainly dependence experience of dissolving/regenerated method was carried out.Can not obtain satisfied output in many cases, and in addition, it is always not possible to regenerate.In addition, concerning quite a lot of heterologous protein, can not obtain high expression level, because described albumen is by the proteasome degradation in the intestinal bacteria (Escherichia coli).Never talkative, fully established the method for problem that solves the insoluble of such expression product or degraded.At present, use the albumen of above-mentioned host expression system mass production biologic activity must be not successful.For addressing this problem, attempted and methods such as chaperone coexpression, and carried out some reports (for example, referring to patent documentation 1 to 3).

DnaK, DnaJ and GrpE are the collaborative chaperones that plays a role in proteic folding.This is following considers.At first, when DnaJ in conjunction with as the folding albumen of substrate the time, the ATP on the DnaK is hydrolyzed, to form folding albumen/DnaJ/DnaK mixture (ADP bond type).Then, GrpE exchanges ADP/ATP, to discharge substrate protein (for example, referring to non-patent literature 1) from mixture.Triggering the factor is one of molecular chaperoning that participates in protein folding, and has such catalytic activity, the cis-trans isomerization reaction (peptidyl cis-trans propyl isomerism enzyme (PPIase) activity) of the peptide bond of the N-end side of the proline residue during promptly in cell, folding in the catalysis target protein amino acid.

Known in many cases in intestinal bacteria the coexpression of undissolved foreign protein and GroEL and GroES cause the successful dissolving of this foreign protein.The example of described foreign protein comprises Tyrosylprotein kinase (for example, referring to non-patent literature 2 and 3), glutamate racemase (for example, referring to non-patent literature 4) and Tetrahydrofolate dehydrogenase (for example, referring to non-patent literature 5).In addition, know because and the solvability of the increase of the human growth hormone that causes of the coexpression of DnaK (for example, non-patent literature 6), because and the solvability of the increase of the trans-glutaminases (transglutaminase) that causes of the coexpression of DnaJ (for example, non-patent literature 4) and because and the solvability (for example, referring to non-patent literature 2) of the increase of the Tyrosylprotein kinase that causes of the coexpression of DnaK, DnaJ and GrpE.

Carried out by will be in intestinal bacteria the fusion rotein of insoluble albumen conduct and chaperone etc. express its dissolved attempted.For example, known by with mouse anti lysozyme of chicken Fab antibody fragment as and the fusion rotein of TcFKBP18 (from the chaperone of archeobacteria) express and make its dissolving obtain success (referring to patent documentation 7).

Yet, by also unresolved relevant all proteic expression of aforesaid method or folding problem.Therefore, need establishment to be used for producing effectively proteic method consumingly all the time.

If the colibacillary culture temperature that will be in logarithmic phase drops to 10-20 ℃ from 37 ℃, the growth of Bacillus coli cells is stagnated temporarily so, has induced one group of proteic expression that is called cold shock protein during this period.According to the level of inducing described albumen is divided into two groups: I group (10 times or more) and II group (being lower than 10 times).Albumen in the I group comprises CspA, CspB, CspG and CsdA (for example, referring to non-patent literature 7 and 8).In these albumen, the expression level of CspA (for example, referring to patent documentation 5) after temperature is converted to 10 ℃ from 37 ℃ 1.5 hours reaches 13% (for example, referring to non-patent literature 9) of total cell protein.So, the trial of having carried out using the promotor of cspA gene to produce recombinant protein at low temperatures.

For the system that under cold condition, uses cspA genetic expression recombinant protein, except the above-mentioned efficient transcription initiation that the promotor of passing through this gene at low temperatures produces, also shown following effectiveness.

(1) if encoding function CspA albumen is not (specifically from the cspA genetic transcription and mRNA that can be translated, if the part of the proteic N-end sequence of its coding CspA), the long-time section of this mRNA ground suppresses the expression that other e. coli proteins comprise cold shock protein so.In this time period, preferably translate described mRNA (for example, referring to non-patent literature 7 and patent documentation 6).This phenomenon is called LACE (the cryogenic dependence microbiotic effect that the cspA of brachymemma expresses) effect.

(2) sequence of being made up of 15 Nucleotide that is called the downstream frame is positioned at 12 Nucleotide in downstream position afterwards of the initiator codon of cspA gene.This sequence makes it possible to produce high translation efficiency under cold condition.

(3) 5 '-non-translational region of being made up of 159 Nucleotide is between the transcription initiation site and initiator codon of the mRNA of cspA gene.This zone has negative effect and has positive-effect under the condition under the end temperature the expression of 37 ℃ of following CspA.

Particularly, the phenomenon described in (1) is hinting the specific expressed feasibility of having only target protein of using the cspA gene to carry out above.Therefore, expection can be used for this system the production of high purity recombinant protein or isotope-labeled proteic preparation to carry out structural analysis.

Yet the expression of recombinant proteins system under the above-mentioned cold condition still can not be used for all recombinant proteins.Each albumen has inherence (intrinsic) molecular weight, iso-electric point and amino acid to be formed, and needs to form unique senior orderly structure with the performance function.Even use above-mentioned expression system, also there is such albumen, for described albumen, can not obtain enough expression levels and maybe can not obtain active albumen.

Patent documentation 1:JP-A 11-9274

Patent documentation 2:JP-A 2000-255702

Patent documentation 3:JP-A 2000-189163

Patent documentation 4:JP-A 8-308564

Patent documentation 5:WO 90/09447

Patent documentation 6:WO 98/27220

Patent documentation 7:WO 2004/001041

Non-patent literature 1:Proc.Natl.Acad.Sci.USA, 91:10345-10349 (1994)

Non-patent literature 2:Cell Mol.Biol., 40:635-644 (1994)

Non-patent literature 3:Proc.Natl.Acad.Sci.USA, 92:1048-1052 (1995)

Non-patent literature 4:J.Biochem., 117:495-498 (1995)

Non-patent literature 5:Protein.Eng., 7:925-931 (1994)

Non-patent literature 6:Biotechnol., 10:301-304 (1992)

Non-patent literature 7:J.Bacteriol., 178:4919-4925 (1996)

Non-patent literature 8:J.Bacteriol., 178:2994-2997 (1996)

Non-patent literature 9:Proc.Natl.Acad.Sci.USA, 87:283-287 (1990)

Summary of the invention

Problem by the present invention's solution

Consider above-mentioned prior art, main purpose of the present invention provides the method that keeps its active desired polypeptides of producing effectively.

The method of dealing with problems

Result as further investigation, after the inventor has found to use under cold condition the expression of recombinant proteins system to make coding desired polypeptides or proteic genetic expression, by in used host, increasing the chaperone expression of gene, can express as active soluble proteins as the soluble proteins expressed proteins being difficult to according to ordinary method.

In addition, the inventor found according to the method described above with desired polypeptides or albumen as and the fusion rotein of chaperone be expressed in the solubilising of target protein effective especially.With albumen is compared as ordinary method of expressing with the fusion rotein of chaperone or the effect of using the expression of recombinant proteins system to express the method for target protein under cold condition, the effect of such method in the solubilising of target protein is astonishing and unexpected.

For example, the invention provides the method that is used for expressing effectively desired polypeptides, this method changes identical host over to the carrier with the chaperone gene that is integrated and induces two expression of gene to realize by the carrier that will have the goal gene that is integrated.

A first aspect of the present invention relates to the method for producing polypeptide, this method comprises such host is exposed under the cold condition to induce polypeptide expression, described host has the gene of the polypeptide that the coding that changed over to this host wants, wherein in this host, the expression of gene of coding chaperone is enhanced.

According to this first aspect, can strengthen the expression of gene of coding chaperone by for example such method, described method is: induce the chaperone expression of gene among the host; Chaperone gene on modification host's the karyomit(e); Change the chaperone gene over to host; Or use such host, in this host, the chaperone expression of gene is enhanced.

According to this first aspect, can preferably use coding to be selected from the gene of the chaperone of DnaK, DnaJ, GrpE, GroEL, GroES and the triggering factor.

According to this first aspect, the gene of the polypeptide that the coding that is changed over to the host can be wanted is connected to the downstream of such DNA, and described dna encoding derives from 5 '-non-translational region of the mRNA of cold shock protein gene.With intestinal bacteria cspA gene is that example illustrates described cold shock protein gene.

According to this first aspect, can use the DNA of the polypeptide that carrier wants coding and the gene of coding chaperone to change the host over to.The gene of the DNA of the polypeptide that coding can be wanted and coding chaperone interconnects, thus with regard to codified the fusion rotein of this polypeptide of wanting and this chaperone.With RAV-2 (RAV-2) reversed transcriptive enzyme α subunit, RAV-2 β subunit, DNA enzyme or people Dicer PAZ structural domain polypeptide is the polypeptide that the example explanation is wanted.

With intestinal bacteria is the host that the example explanation is used according to this first aspect.

A second aspect of the present invention relates to a cover plasmid vector that is used to produce the polypeptide of wanting, and it comprises:

(1) first carrier, its downstream in promotor has:

(a) coding derives from the DNA of 5 '-non-translational region of the mRNA of cold shock protein gene; With

(b) can be used for inserting the gene of the polypeptide that coding wants and be positioned at the restriction endonuclease recognition sequence in downstream of the DNA of (a); With

(2) have second carrier of gene of coding chaperone,

Wherein select (1) and (2) thus the feasible uncompatibility that do not produce of replication orgin of carrier.

For example, according to this second aspect, first carrier can comprise such DNA, and this dna encoding derives from 5 '-non-translational region of the mRNA of intestinal bacteria cspA gene, and second carrier can comprise the gene that coding is selected from the chaperone of DnaK, DnaJ, GrpE, GroEL, GroES and the triggering factor.The plasmid that can duplicate in intestinal bacteria can be used as described carrier.

A third aspect of the present invention relates to such expression vector, and this carrier has in the downstream of promotor:

(a) coding derives from the DNA of 5 '-non-translational region of mRNA of the gene of cold shock protein; With

(b) has the gene that can be used for inserting the polypeptide that coding wants and be positioned at the DNA of restriction endonuclease recognition sequence in downstream of the DNA of (a); With

(c) gene of coding chaperone.

With such carrier is the expression vector that example illustrates this third aspect, described such carrier contains the DNA of coding from 5 '-non-translational region of the mRNA of intestinal bacteria cspA gene, or described such carrier contains the gene that coding is selected from the chaperone of DnaK, DnaJ, GrpE, GroEL, GroES and the triggering factor.

According to this third aspect, the restriction endonuclease recognition sequence that can be used for inserting the gene of the polypeptide that coding wants can be positioned at such position, the gene of the polypeptide that described coding wants be can insert in this position, thereby the polypeptide conduct wanted and the expressing fusion protein of chaperone made.

Described expression vector can be the plasmid that can duplicate in intestinal bacteria.

The invention effect

According to method of the present invention, may obtain quite a large amount of pass through ordinary method beyond expression of words, keep its active polypeptide simultaneously.

The accompanying drawing summary

Fig. 1 shows the check result of the expression of the hDi-ASI that is undertaken by coexpression." A " and " B " shows the result of CBB dyeing and antibody staining respectively.In the drawings, " T " represents cell extract fraction and " S " expression soluble component.

Fig. 2 shows reversed transcriptive enzyme (RTase) α or reversed transcriptive enzyme β and triggers the check result of the Expression of Fusion Protein of the factor." A " and " B " shows the result of single expression and coexpression respectively, and " C " and " D " shows the result of amalgamation and expression in the result of the amalgamation and expression in the T7 promoter expression system and the cold shock expression system respectively.In the drawings, " α " expression RAV-2 reversed transcriptive enzyme α, " β " expression RAV-2 reversed transcriptive enzyme β, " T " expression cell extract fraction, " S " represents soluble fraction, and " P " represents insoluble fraction.

Fig. 3 shows the DNA enzyme and triggers the check result of the Expression of Fusion Protein of the factor." A ", " B " and " C " show the result of single expression system, coexpression system and amalgamation and expression system respectively.In the drawings, " S " represents soluble fraction, and " P " represents insoluble fraction.

Fig. 4 shows the result that the DNA enzymic activity is measured.In the drawings, the independent substrate of " M " expression, " 1 " represents the result of the DNA enzymic activity measurement that the soluble fraction of use supersound process is in contrast carried out, and " 2 " represent the result of the DNA enzymic activity measurement that the soluble fraction of the supersound process of use amalgamation and expression system is carried out.

Implement optimization model of the present invention

Hereinafter, the present invention will be described.

(1) cold shock carrier

The carrier that contains the gene of the polypeptide that coding wants used according to the invention is so a kind of carrier, in this carrier, be converted to the temperature lower (that is, by cold shock) by culture temperature and induce described polypeptide expression than normal growth temperature with the host.It is used as according to expression vector of the present invention.As used herein, term " low temperature " is meant the temperature of the normal growth temperature that is lower than the host.Hereinafter, above-mentioned carrier can be called the cold shock carrier.Be used to use the system of the polypeptide that such vector expression wants to be called the cold shock expression system.The carrier that has such DNA and be connected to the gene of the polypeptide that the coding in described DNA downstream wants can be used as such carrier, and described dna encoding derives from 5 '-non-translational region of the mRNA of cold shock protein gene.As used herein, term " downstream " is meant the downstream position with respect to transcriptional orientation.

Although do not want to limit the present invention, in preferred embodiments, the carrier of the gene of the polypeptide that comprising encodes wants comprises following elements:

(A) promotor that in employed host, works;

(B) control region of the effect of the promotor of adjusting (A); With

(C) coding derives from the part of 5 '-non-translational region of mRNA of cold shock protein gene or the part in such zone of encoding, and in this zone, has carried out the substituting of at least one Nucleotide, disappearance, inserts or has added at described non-translational region.

Be described in detail below.

Promotor about (A) does not have concrete qualification, as long as it has the activity of initial rna transcription in used host.Particularly, it is can be by itself and such part being used in combination any promotor as cold reactive promotor, and described part coding derives from the 5 '-non-translational region of mRNA of the cold shock protein gene of (C).

Regulatory gene about (B) does not have concrete qualification, as long as it can be used for regulating the expression of gene in the promotor downstream that is positioned at (A).For example, can suppress gene translation target protein by such Regional Integration being gone into carrier from the promotor downstream, with from the mRNA complementary RNA (sense-rna) of promoter transcription from this regional transcription.By under the control of the suitable promotor of the promotor that is different from (A), transcribing the expression of the adjustable desired polypeptides of described sense-rna.Can use the operator gene in the expression regulation district that is present in range gene.For example, can use the lac operator gene that derives from the intestinal bacteria lactose operon according to the present invention.Can by use suitable inductor for example the function of lactose or its analog (preferably, isopropyl-(IPTG)) cancellation lac operator gene promotor is played a role.Usually such operator gene sequence is placed the downstream of promotor and transcription initiation site near.

The part of 5 '-non-translational region of mRNA that coding derives from the cold shock protein of (C) is the part in the mRNA zone of coding initiator codon 5 '.At intestinal bacteria cold shock protein gene (cspA, cspB, cspG and csdA) (J.Bacteriol., 178:4919-4925 (1996); J.Bacteriol., found described part characteristically 178:2994-2997 (1996)).Come in the mRNA of such genetic transcription 5 ' terminal 100 Nucleotide or more the part of polynucleotide be not translated into albumen.This part is extremely important for the cold reaction of genetic expression.If this 5 '-non-translational region is attached to the mRNA (adhering at its 5 ' end) of arbitrary protein, under cold condition, take place so from mRNA to proteic translation.As long as keep function, can in the nucleotide sequence of 5 '-non-translational region of the mRNA that derives from cold shock protein, substitute, lack, insert or add one or more Nucleotide.

As used herein, " zone " is meant the scope of nucleic acid (DNA or RNA).As used herein, " 5 ' of mRNA-non-translational region " is meant proteins encoded not and is positioned at zone as the 5 ' side of the mRNA of synthetic as a result that transcribes from DNA.Hereinafter, described zone is called " 5 '-UTR (5 '-non-translational region) ".Unless otherwise noted, 5 '-UTR is meant 5 '-non-translational region or its modifier of the mRNA of intestinal bacteria cspA gene.

The part that coding derives from 5 '-UTR of top listed cold shock protein gene can be used for carrier of the present invention.Particularly, preferably can use the part that derives from intestinal bacteria cspA gene.In addition, can use such part,, in this part, nucleotide sequence carried out partly modifying as long as it has contribution to polypeptide cold specific expressed.For example, can use such part, in this part, modify the nucleotide sequence in described zone by relevant (B) described operator gene above integrating.Selectively, the part of 5 '-UTR of coding cold shock protein gene can be placed between the initiator codon of the promotor of (A) and the gene that coding is treated polypeptide expressed.Operator gene can be integrated into this part.

Except said elements, comprise anti-downstream frame sequence (anti-downstream box sequence) complementary nucleotide sequence in the ribosome-RNA(rRNA) with used host by downstream at 5 '-non-translational region, may increase expression efficiency.For example, under colibacillary situation, anti-downstream frame sequence is present in position 1467 to the position 1481 in the 16S ribosome-RNA(rRNA).May use the zone of the N-terminal peptide of coding cold shock protein, this zone is contained and this sequence height complementary nucleotide sequence.Wherein transcription termination sequence (terminator) carrier that is placed in the gene downstream of target protein helps the high expression level of target protein, and this is because the stability of described carrier has obtained increase.

Carrier of the present invention can be any carrier in the common carrier (for example, plasmid, phage or virus vector), as long as it can be used for reaching the purpose as carrier.For zone contained except said elements, carrier of the present invention can have replication orgin for example, as the drug resistance gene or operator gene (for example, the lacI of lac operon of selective marker ^qGene) the necessary regulatory gene of function.If carrier of the present invention is integrated in this host's the genomic dna, then can not produce inconvenience after it is changed over to the host.

The structure of plasmid vector is described below particularly.Unless otherwise noted, the zone of the gene of intestinal bacteria CspA albumen, the described protein expression of participation and the promoter region in this gene are called " CspA ", " cspA gene " and " cspA promotor " herein.In the nucleotide sequence (SEQ ID NO:1) of natural cspA gene, respectively, main transcription initiation site (+1) is positioned at Nucleotide 426, SD sequence (ribosome binding sequence) is positioned at Nucleotide 609 to Nucleotide 611, the initiator codon of CspA is positioned at Nucleotide 621 to Nucleotide 623, and the terminator codon of CspA is positioned at Nucleotide 832 to Nucleotide 834, in the GeneBank gene database, under catalog number (Cat.No.) M30139, registered described natural CspA gene, and this gene can obtain openly.In addition, the coding of 620 the part from Nucleotide 462 to the nucleosides 5 ' UTR in the sequence.With a 5 ' UTR that example comes illustration partly to modify who describes among the WO 99/27117.Be used for that the carrier pCold08NC2 of embodiment comprises 5 '-the UTR district is the example.This nucleotides sequence is shown in SEQ ID NO:2.

Can will be integrated into carrier to increase the expression efficiency of goal gene with the anti-downstream frame sequence height complementary nucleotide sequence (downstream frame sequence) that is present in the 16S ribosome-RNA(rRNA).The downstream frame sequence and the above-mentioned anti-downstream frame sequence in zone that is present in the N-terminal portions of coding intestinal bacteria CspA has only 67% complementarity.The nucleotide sequence that use has than the complementarity of top nucleotide sequence higher (preferably 80% or higher, more preferably 100% (DB sequence ideally)) can make the expression of gene that is connected the downstream have higher efficient.

In addition, the nucleotide sequence of coded markings sequence or the nucleotide sequence of proteins encoded enzyme identification aminoacid sequence can be integrated into carrier, described flag sequence is the peptide that is used to promote the purifying of the goal gene product of expressing, and described proteolytic enzyme identification aminoacid sequence is used to remove the extra peptide (for example, flag sequence) in the goal gene product.

Usefulness such as the histidine mark (His mark) be made up of several histidine residues, maltose binding protein, glutathione-S-transferase can be acted on the flag sequence of purifying.Use chelate column easily purifying be attached with the polypeptide of histidine mark.For other flags sequence, also can carry out purifying easily by the part that use has specificity avidity.Factor Xa, zymoplasm, enteropeptidase etc. can be used as the proteolytic enzyme that is used to remove extra peptide.The nucleotide sequence of the aminoacid sequence that coding can be cut with these proteolytic enzyme specificitys is integrated into carrier.

(2) enhancing of chaperone expression

The method that is used to produce polypeptide of the present invention is characterised in that and strengthens the expression of gene of coding chaperone after the genetic expression of coding desired polypeptides.

According to the present invention, can be with any albumen as chaperone, as long as it is to participate in proteic folding albumen.The example comprises and derives from colibacillary DnaK, DnaJ, GrpE, GroEL, GroES and the triggering factor.

Although do not want to limit the present invention, carry out polypeptide expression for using the cold shock carrier, preferably use to participate in isomerized albumen on the proline(Pro) in polypeptide, for example trigger the factor (being also referred to as peptidyl cis-trans propyl isomerism enzyme (PPI enzyme)).Chaperone is not limited to and derives from colibacillary chaperone.For example, can use the chaperone that derives from archeobacteria, yeast, microorganism or psychrophilic organism.

Can be by the genetic expression of the coding chaperone on the induced chromosome, or pass through known technology, the for example modification of chaperone gene on the karyomit(e) (increase of copy number or the insertion of promotor), by changing the chaperone gene over to host, or realize the enhancing of chaperone genetic expression by the strain that host's mutagenic obtained height is expressed the chaperone gene.

For example, if the condition of inducing chaperone to express among the known host can be used the chaperone expression of gene on the described condition induced chromosome so.Can use site-directed mutagenesis or the gene insertion technology of utilizing homologous recombination host chromosome to be carried out the modification of the chaperone gene on the karyomit(e).For example, can use such inducible promoter to come abduction delivering, described promotor has been inserted into the upstream of the gene of derivative coding chaperone on host chromosome.

In another embodiment, obtain such host's who is used for express polypeptide mutant strain and used as the host, in described mutant strain, the chaperone expression of gene is enhanced.According to known method, for example by with mutagenic compound for example reagent or treatment with ultraviolet light host microorganism, the strain of selecting chaperone expression of gene wherein to be enhanced then obtains mutant strain.

The enhancing of the expression of gene of coding chaperone is meant with normal amount to be compared, and the proteic amount of the chaperone among the host increases.Confirm that it is possible whether having strengthened the chaperone expression of gene according to the method described above, for example, measure chaperone albumen by the antibody that uses the identification chaperone, or by using known method (for example, RT-PCR method, Northern hybridization or use the hybrid method of DNA array) to measure from the amount of the mRNA of the genetic transcription of coding chaperone.

It is favourable can controlling the expression of chaperone and the expression of target protein independently, thus but the selection of time of amount that the optimization chaperone is expressed and expression and do not reduce the expression level of target protein.For realizing this purpose, preferably the chaperone gene is placed the downstream of controllable promotor.In addition, be preferably used for controllable initiating that chaperone expresses and to be used for the target protein expression promoter different.

Can be by with the chaperone gene insertion vector and change it over to host and strengthen the chaperone expression of gene.Described carrier can be any in the common carrier (for example, plasmid, phage or virus vector), as long as it can be used for reaching the purpose as carrier.

Usually, two kinds of plasmids that are closely related can not stably coexist as among the single host.This phenomenon is called uncompatibility.According to the present invention, if plasmid is used as the carrier (being also referred to as the chaperone plasmid hereinafter) that comprises the chaperone gene, so preferably use the carrier with such replicon, this carrier does not show and is used for the uncompatibility of the expression vector of target protein.For example, (for example, the pCold07 that describes among the WO 99/27117) carrier is as the expression vector of target protein, the p15A replicon among the carrier pACYC etc. can be used for the chaperone plasmid so if having the ColE1 replicon with one.

According to the present invention, can further randomly comprise selectable marker gene, thereby can after transforming, easily select with the carrier that contains the chaperone gene.These selectable marker genes comprise amicillin resistance (Amp ^r) gene, kalamycin resistance (Km ^r) gene and chlorampenicol resistant (Cm ^r) gene.Be not both with the selectable marker gene that comprises in the expression vector of foreign protein and want.

The specific examples of chaperone plasmid used according to the invention comprises plasmid pG-KJE8, the plasmid Gro7 that expresses GroEL/GroES, the plasmid pKJE7 that expresses DnaK/DnaJ/GrpE that expresses DnaK/DnaJ/GrpE and GroEL/GroES, the plasmid pG-Tf2 that expresses the GroEL/GroES and the triggering factor, and expresses the plasmid pTf16 (all plasmids are all from Takara Bio) that triggers the factor.

Can use will the encode gene of chaperone of above-mentioned carrier to change the host over to, maybe can be integrated on the host chromosome and use.

According to the present invention, the foreign protein that express can be any albumen, as long as it is unsettled and/or insoluble in the host.These foreign proteins comprise Interferon, rabbit, interleukin-, interleukin-1 receptor, the antagonist of interleukin-1 receptor, granulocyte colony-stimulating factor, rHuGM-CSF, macrophage colony stimulating factor, erythropoietin, thrombopoietin, leukaemia inhibitory factor, stem cell factor, tumour necrosis factor, tethelin, proinsulin, rhIGF-1, fibroblast growth factor, Thr6 PDGF BB, transforming growth factor, pHGF, the bone morphogenetic factor, nerve growth factor, ciliary neurotrophic factor, brain derived neurotrophic factor, the neurogliocyte derived neurotrophic factor, neurotrophin, uPA, tissue plasminogen activator, thrombin, C albumen, glucocerebrosidase, hemocuprein, feritin, N,O-Diacetylmuramidase, P450, renninogen, trypsin inhibitor, elastase inhibitor, lipocortin, RMETHU LEPTIN, immunoglobulin (Ig), single-chain antibody, complement component, the blood white protein, the cdear pollen-antigen, the stress protein of hypoxia inducible, protein kinase, the proto-oncogene product, the albumen of transcription regulaton factor and formation virus.

Expression vector of the present invention is changed over to the host and carries out the expression of target protein.Host's example includes, but not limited to prokaryotic organism (for example, bacterium), yeast, fungi, plant, insect cell and mammalian cell.The feature of expression vector must be complementary with used host.For example, if expressing protein in the mammal cell line system, then expression vector preferably uses from the mammalian genes group isolating promotor (for example, the mouse metallothionein promoter) or separate the promotor (for example, bacilliform virus promoter, vaccinia virus 7.5K promotor) of the virus of breeding in the comfortable for example cell.

Among other things, preferably use prokaryotic organism for example intestinal bacteria as the host.If gram negative bacterium is used as the host, so can be at tenuigenin or periplasmic space expressing protein.

Method for chaperone carrier and expression vector being changed over to the host according to the present invention does not have concrete qualification, and can use various known methods.The example comprises according to the transfection of calcium phosphate precipitation method, electroporation, liposome fusion, nuclear injection, uses virus or phage-infect.The present invention also comprises the host of containing expression vector of the present invention.The method that changes the host over to can be a step form, wherein shifts chaperone carrier and expression vector simultaneously, or is two step forms, wherein changes expression vector after changing the chaperone carrier over to again over to, or changes the chaperone carrier after changing expression vector over to again over to.Use can be screened the strain of cotransformation corresponding to the medicine of selectable marker gene.Can be by confirmation expression of exogenous gene such as for example Western blots.

In one aspect, the present invention relates to the carrier that a cover is used for express polypeptide, the combination that it comprises above-mentioned cold shock carrier and contains the carrier of chaperone gene.Aspect this, preferably the carrier that uses the gene that can insert coded polypeptide is as the cold shock carrier, and this expression of gene is wanted for the realization of purpose.The example comprises having (a) the such DNA and (b) carrier of such restriction endonuclease recognition sequence, described dna encoding derives from 5 '-non-translational region of the mRNA of cold shock protein gene, and described recognition sequence can be used for inserting the gene of the polypeptide that coding wants and be positioned at the downstream of the DNA of (a).

In yet another aspect, the present invention relates to such carrier, in this carrier, the gene of coding chaperone is inserted into the cold shock carrier.In this case, can prepare the transformant that can be used for expressing desired polypeptides by transforming the host with single carrier.

Aspect this, can on such position, arrange to can be used for to insert the restriction endonuclease recognition sequence of the gene of coding desired polypeptides, in described position, to be connected to the gene of coding desired polypeptides in the gene frame of coding chaperone, thereby just can express the fusion rotein of chaperone and desired polypeptides.In the fusion rotein of chaperone and desired polypeptides, chaperone can be connected N-end side or the C-end side or the both sides of desired polypeptides.Fusion rotein can have amino acid or the peptide as joint between chaperone and desired polypeptides.The chain length of joint is preferably 1 to 50 amino acid, more preferably 3 to 40 amino acid, most preferably 5 to 30 amino acid.The aminoacid sequence of joint can be protease recognition sequence or such sequence, has inserted a plurality of protease recognition sequences of arranging in this sequence.The example of these protease recognition sequences comprises for example recognition sequence of factor Xa, zymoplasm, enteropeptidase (all enzymes all can obtain from Takara Bio) and PreScission proteolytic enzyme (Amersham Biosciences) of various proteolytic enzyme.For example, protease recognition sequence is preferably by 4 to 8 sequences that amino acid is formed.

Can insert above-mentioned carrier by the gene of the desired polypeptides of will encoding and change it over to fusion polypeptide that appropriate host obtains desired polypeptides and chaperone.If fusion polypeptide has the joint of the recognition sequence that comprises proteolytic enzyme, so by obtaining with the described polypeptide of protease digestion and the isolating desired polypeptides of chaperone.

(3) method of production polypeptide

Produce the method for polypeptide of the present invention according to for example the following step.

The downstream of the DNA that the gene insertion of coding desired polypeptides is such, described dna encoding derives from 5 '-non-translational region of the mRNA of the cold shock protein gene in the cold shock carrier.Change the recombinant expression vector that makes up as mentioned above over to appropriate host with the preparation transformant.

If use the carrier of the gene that comprises above-mentioned coding chaperone in the mode of combination, change described carrier over to transformant so again.If the cold shock carrier has the gene of coding chaperone, can transform the host with carrier individually so.

Cultivate transformant under normal operation.For example, under colibacillary situation, can under about 37 ℃, cultivate.Can in all culturing steps, in the host, express chaperone, or can be when desired polypeptides be expressed abduction delivering.Depend on the state that the chaperone gene exists among the host, can induce the chaperone expression of gene.For example, if induce inherent chaperone expression of gene in the host, then the host can be placed under the appropriate condition.If the gene of coding chaperone (for example, changes under host's the situation at a molecular gene of accompanying that will be inserted into carrier) under the control of allogeneic promoter, then use the method that is suitable for controlling the promotor of transcribing to carry out induced expression.If use such host, so above-mentioned induction method is unessential, and in described host, the chaperone expression of gene is strengthened consistently.

Usually after increasing cell number under the above-mentioned general culture temperature, induce the expression of desired polypeptides.Among the hosts, induce the cold shock reaction by reduce culture temperature from above-mentioned state, thereby cause the preferred expression of desired polypeptides.Although do not want to limit the present invention, by culture temperature being adjusted downward to the temperature that is lower than general culture temperature, for example reduce 5 ℃ or more, preferably 10 ℃ or more, induce the cold shock reaction.If use the cold shock carrier of the operator gene in downstream, can come evoked promoter by the function of using suitable method to eliminate operator gene so with the promotor of placing.

After cold shock, continue further to cultivate transformant at low temperatures with express polypeptide.Desired polypeptides can be produced by collect described polypeptide from the culture that is obtained.Can be from the polypeptide the transformant cell purification culture, described transformant cell is collected from culture or culture supernatants or both.Can use the combination of known protein purification technique (for example ammonium sulfate fractional separation, ultrafiltration and various chromatography) to carry out the purifying of polypeptide.

Can promote purifying by the express polypeptide that design exists with such form, the polypeptide of this form is bonded to carrier by suitable part.For example, design vector like this, thus make the N-end side of the mark of about 6 histidine residues attached to polypeptide.Then, the fusion polypeptide of gained can be bonded to metal (for example, nickel) inner complex carrier by histidine residues.Use such carrier can be easily with polypeptide expressed with derive from host's albumen sepn.Can be by cutting the polypeptide expressed that is bonded to carrier with proteolytic enzyme in the joint and easily only desired polypeptides being discharged from carrier.In the time of need not cutting when using the imidazoles wash-out, may discharge polypeptide expressed from carrier very naturally.Except above-mentioned histidine mark, may use such method, in described method, use glutathione-S-transferase or its part serves as a mark and use gsh resin (glutathione resin) to carry out affinity chromatography, may use such method, use maltose binding protein or its part to serve as a mark in the method and use the maltose resin to carry out purifying etc.

In addition, can use the avidity of antagonist.Can be designed for the mark of purifying, to make it to be positioned at the N-end side or the C-end side of expressed proteins.General aps gene engineering of those skilled in the art and affinity purification.

Because the polypeptide expression except desired polypeptides is suppressed in such system (in this system, using above-mentioned cold shock vector expression polypeptide), so method of the present invention is favourable for producing highly purified polypeptide.

Embodiment

The following example illustrates the present invention in more detail, but is not interpreted as limiting its scope.

In aforesaid method, as people such as J.Sambrook (writing .), Molecular Cloning:A Laboratory Manual the 3rd edition comprises the preparation of plasmid and the basic skills of digestion with restriction enzyme described in the Cold Spring Harbor Laboratory (2001).

Embodiment 1: by with the inspection of the expression of the hDi-ASI of chaperone coexpression

(1) structure of expression vector

For express by the PAZ+RNA enzyme III structural domain of people Dicer (from the N-terminal of the aminoacid sequence of people Dicer the 679th to the 1924th) polypeptide formed, following construction of expression vector.

At first, use dna synthesizer, and carry out purifying according to ordinary method based on the synthetic synthetic primer 5 and 6 (SEQ ID NOS:4 and 5) of the nucleotide sequence under the Genbank catalog number (Cat.No.) AB028449 that can openly obtain.Synthetic primer 5 is such synthetic DNA, this DNA has the recognition sequence of restriction enzyme KpnI to the Nucleotide 14 at Nucleotide 9, and has amino acid 679 in the aminoacid sequence (SEQ ID NO:3) corresponding to people Dicer to the Nucleotide 36 to the nucleotide sequence of amino acid 685 at Nucleotide 16.Synthetic primer 6 has the recognition sequence of restriction enzyme HindIII to the Nucleotide 14 at Nucleotide 9, and has amino acid/11 919 in the aminoacid sequence (SEQ ID NO:3) corresponding to people Dicer to the Nucleotide 35 to the nucleotide sequence of amino acid/11 924 at Nucleotide 18.

Use the synthetic primer to carry out PCR.The reaction conditions of PCR is as follows.

In brief, prepare the reaction mixture of 50 μ l cumulative volumes by adding 2 μ l template DNAs (human cDNA library, human pancreas, Takara Bio), 5 μ l, 10 * LA PCR damping fluid (Takara Bio), 5 μ ldNTP mixtures (Takara Bio), 10pmol synthetic primer 5,10pmol synthetic primer 6,0.5U Takara LA Taq (Takara Bio) and sterilized water.Reaction mixture is placed TaKaRa PCR thermal cycler (Thermal Cycler) SP (Takara Bio) and carries out following reaction: 30 circulations: 94 ℃ were carried out 1 minute, and 55 ℃ are carried out carrying out 3 minutes in 1 minute and 72 ℃.

After the reaction, 5 μ l reaction mixtures are carried out electrophoresis on 1.0% sepharose.And from running gel, reclaim and the target DNA fragment of the observed about 2.7-kbp of purifying, and this fragment is carried out ethanol sedimentation.Behind ethanol sedimentation, the DNA that reclaims is resuspended in the 5 μ l sterilized waters, and it is carried out double digested with restriction enzyme KpnI (Takara Bio) and restriction enzyme HindIII (Takara Bio).Extraction and purifying KpnI-HindIII digest are to obtain the dna fragmentation of KpnI-HindIII digestion behind electrophoresis on 1.0% sepharose.

Then, description according to WO99/27117, use e. coli jm109/pMM047 (FERM BP-6523) (to be preserved in Independent Administrative Leged Industrial Technology Complex Inst on October 31st, 1997 (original preservation date) and to specially permit biological sustenance center (International PatentOrganism Depositary, National Institute of Advanced Science andTechnology), AIST Tsukuba Central 6,1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki 305-8566, Japan) the plasmid pMM047 that carries makes up pCold08NC2 as starting material.Plasmid pCold08NC2 is to have following from the upstream to the order in downstream: cspA promotor, lac operator gene, modified derive from 5 of intestinal bacteria cspA gene '-UTR and multiple clone site.In addition, described plasmid have the lacI gene, the nucleotide sequence of the aminoacid sequence discerned by factor Xa with the complete complementary of anti-downstream frame sequence downstream frame sequence, the histidine mark of forming by 6 histidine residues and coding in the intestinal bacteria 16S ribosome-RNA(rRNA).The nucleotides sequence in 5 among the carrier pCold08NC2 '-UTR district is shown in SEQ ID NO:2.

With with the identical restriction enzyme cut vector pCold08NC2 of restriction enzyme that uses when the dna fragmentation of preparation KpnI-HindIII digestion, and to terminal dephosphorylation.Mix and use dna ligation kit (Takara Bio) that it is interconnected the dna fragmentation of prepared carrier and KpnI-HindIII digestion.Use 20 μ l to connect mixture transformed into escherichia coli JM109.Transformant is grown on the LB substratum that contains the penbritin that agar that concentration is 1.5% (w/v) and concentration is 50 μ g/ml.

The plasmid that has the target DNA fragment of insertion by the order-checking confirmation.This recombinant plasmid is called pCold08 hDi-ASI.This plasmid is called as and is expressed as plasmid pCold08 hDi-ASI, and since on September 26th, 2003 (original preservation date), it is deposited in Independent Administrative Leged Industrial Technology Complex Inst with preserving number FERMBP-10076 always and speciallys permit biological sustenance center, AIST Tsukuba Central 6,1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki 305-8566, Japan.PCold08 hDi-ASI is the plasmid that comprises such nucleotide sequence, amino acid 679 in the aminoacid sequence of this sequence encoding people Dicer (SEQ ID NO:3) is to the aminoacid sequence (nucleotide sequence of SEQ ID NO:6, the aminoacid sequence of SEQ ID NO:7) of amino acid/11 924.Have perfectly (Perfect) DB sequence, His flag sequence and factor Xa sequence from the albumen of plasmid expression.Described proteic aminoacid sequence is shown in SEQ ID NO:8, and nucleotides sequence is shown in SEQ ID NO:9.

(2) preparation of cotransformation body (co-transformant)

According to the Calcium Chloride Method above-mentioned plasmid pCold08/hDi-ASI of 1ng and 1ng chaperone plasmid pGro7 (it expresses GroEL and GroES), pKJE7 (it expresses DnaK, DnaJ and GrpE), pG-Tf2 (it expresses GroEL, GroES and the triggering factor) or pTF16 (it expresses the triggering factor) (all from Takara Bio) transformed into escherichia coli BL21.

Containing the flat board that concentration is respectively the paraxin of 20 μ g/ml and 100 μ g/ml and penbritin by use screens to obtain to contain the cotransformation body of pCold08/hDi-ASI and pGro7, pKJE7, pG-Tf2 or pTF16.The coexpression hDi-ASI that is obtained and wherein a kind of clone of chaperone be called as T1, T2, T3 and T4.

By with the independent transformed into escherichia coli BL21 of pCold08/hDi-ASI (Novagen) with use and contain concentration and screen the transformant for preparing in contrast as the flat board of the penbritin of 100ug/ml.The clone of the conventional expression system of no chaperone transfer is called C1.

(3) expression of hDi-ASI

Use the transformant separately that obtains in (1) to check the expression of hDi-ASI.Use 5ml LB liquid nutrient medium (comprising 1% bacto-tryptone, 0.5% yeast extract, 0.5%NaCl, 20 μ g/ml paraxin, 50 μ g/ml penbritins) to cultivate.Use the culture medium culturing C1 in contrast of no paraxin.

Under 37 ℃, cultivate transformant separately.After cultivating beginning, by adding L-arabinose or add the expression that chaperone is usually induced at the Fourth Ring with the final concentration of 0.5mg/ml (for T1, T2 or T4) with the final concentration of 5ng/ml (for T3) to substratum.When turbidity (OD600) reaches about 0.4 the time, cultivated 15 minutes down at 15 ℃, in culture, add IPTG with the final concentration of 0.5mM, and further cultivate 24 hours down to induce the expression of hDi-ASI at 15 ℃.

In the expression of inducing hDi-ASI after 24 hours, collecting cell.By the supersound process ruptured cell with preparation cell extract fraction.Then, by centrifugal under 15,000 * g soluble fraction is separated with insoluble fraction.With each corresponding to about 3.75 * 10 ⁶The part of the fraction separately of individual cell is carried out SDS-PAGE.By CBB dyeing (A) with use and resist-the results are shown among Fig. 1 of (B) analyzing of the Western blot (Qiagen) of His traget antibody.

As shown in fig. 1, in cell extract fraction, observe target protein, but in soluble fraction, almost do not observe this albumen with molecular weight of 144000 as the C1 of conventional expression system contrast.On the other hand, seen at T4, arrive, at hDi-ASI with trigger under the situation of factor coexpression,, observe the increase of hDi-ASI in the cell extract fraction when when comparing.Described albumen mainly is detected in soluble fraction.(wherein hDi-ASI and another kind of chaperone coexpression) almost do not observe this albumen in soluble fraction under the situation of T1, T2 or T3.

As mentioned above, show and do not have the conventional expression system of chaperone transfer, perhaps with expression GroEL and GroES; Dnak, DnaJ and GrpE; Or the coexpression that the factor was compared and triggered to GroEL, GroES and the coexpression of the chaperone of the triggering factor is effective on expression level that increases hDi-ASI and solvability.

(4) expression and purifying

With the pTf16 and the pCold08 hDi-ASI transformed into escherichia coli BL21 of preparation in top (2), and transformant is grown in the LB substratum that contains the penbritin that agar that concentration is 1.5% (w/v) and concentration is 50 μ g/ml.The colony inoculation to two of growth is contained 500ml TB liquid nutrient medium fully, and (6g bacto-tryptone, 12g bacterium are with yeast extract, 2ml glycerine, 17mM KH ₂PO ₄, 72mM K ₂HPO ₄, the 25mg penbritin) container in.After the inoculation, with the final concentration of 0.5mg/ml to wherein adding pectinose.Cell is cultivated until logarithmic phase under 37 ℃, 130rpm, be cooled to 15 ℃ then.After the cooling, with the final concentration of 1.0mM to wherein adding IPTG, and under 15 ℃, 130rpm culturing cell 24 hours to carry out induced expression.Then by centrifugal collecting cell, to obtain the 3.3g wet cell.The 3.3g wet cell is resuspended in the 13.16ml binding buffer liquid (50mMTris hydrochloride buffer (pH8.5), 100mM sodium-chlor, 1mM magnesium chloride, proteinase inhibitor (completely, not containing EDTA, Boehringer Mannheim)).To carry out centrifugal (12,000rpm, 20 minutes) with supernatant liquor extract and precipitate and separate by supersound process disruptive cell.

Following use nickel post is further purified about 13ml supernatant liquor extract.

In brief, will inject the post of  50-mm corresponding to the Ni-NTA agar (Qiagen) of 10ml resin volume and use the 30ml distilled water wash.Then with 100ml binding buffer liquid washing resin and collect.Under 4 ℃, mixed gently about 1 hour to wherein adding the supernatant liquor and the use rotation shaker of about 13ml from the cell rupture formulations prepared from solutions.The resin that has wherein adsorbed target protein is injected the pillar of  50-mm and uses 50ml binding buffer liquid washing 2 times.Buffer A (20mM tris-hydrochloride buffer (pH8.5), 100mM sodium-chlor, 1mM magnesium chloride, 10% glycerine, 20mM imidazoles), 50ml buffer B (20mM tris-hydrochloride buffer (pH8.5), 800mM sodium-chlor, 1mM magnesium chloride, 10% glycerine, 20mM imidazoles) and the 50ml buffer A washing resin of using 50ml then are to remove the unnecessary proteins except target protein.

After the washing, carry out wash-out with 30ml damping fluid C (20mM tris-hydrochloride buffer (pH8.5), 100mM sodium-chlor, 1mM magnesium chloride, 10% glycerine, 100mM imidazoles).Use Centricon YM-10 (Amicon) to concentrate the sample of wash-out, to wherein adding 10ml damping fluid D (50mM tris-hydrochloride buffer (pH8.5), 250mM sodium-chlor, 1mM magnesium chloride, 0.1mM DTT, 0.1%Triton X-100,10% glycerine), and concentrate this mixture.Repeat twice of this program.Enriched material is dialysed to obtain about 220 μ l protein samples to 500ml damping fluid E (50mM tris-hydrochloride buffer (pH8.5), 250mM sodium-chlor, 1mM magnesium chloride, 0.1mM DTT, 0.1%Triton X-100,50% glycerine).When its part is carried out electrophoresis on the 10%SDS polyacrylamide, be approximately the band of observing target protein on 144,000 the position corresponding to molecular weight.This sample is used for following activity to be determined.

(5) measurement of dsRNA degrading activity

The dsRNA degrading activity of the protein sample of preparation in the following measurement above-mentioned (4).

At first, use TurboScript T7 transcript reagent box (GTS) synthetic as the dsRNA that is used for the substrate of activity measurement according to incidental scheme.

Particularly, make up pDON-rsGFP by plasmid pDON-AI (Takara Bio) will be inserted from the gene (SEQ ID NO:10) of the coding red shift green fluorescent protein (being called GFP hereinafter) of plasmid pQBI1125 (Wako Pure ChemicalIndustries).Use pDON-rsGFP to carry out PCR to obtain amplified production as template and use synthetic primer 3 (SEQ IDNO:11) (it has the T7 promoter sequence) and synthetic primer 4 (SEQ ID NO:12).The double-stranded DNA of use gained is as template and use t7 rna polymerase to prepare the dsRNA of about 700bp by the RNA building-up reactions.By add dsRNA that 1 μ g prepares above, above the 1 μ l in (3) protein sample, 2 μ l 5x reaction buffers (100mM tris hydrochloride buffer (pH8.5), 750mM sodium-chlor, 12.5mM magnesium chloride) of preparation and the water that do not contain nuclease prepare the reaction mixture of cumulative volume 10 μ l.After 18 hours, the described reaction mixture of 10 μ l is carried out electrophoresis 37 ℃ of following reaction mixture reactions on 15% polyacrylamide gel.Behind the electrophoresis, with ethidium bromide to gel-colored to observe the product of cutting.As a result, observe the degraded product of about 21 base pairs, thereby show its dsRNA degrading activity.

As mentioned above, show that the albumen with dsRNA degrading activity expresses according to method of the present invention.

Embodiment 2: the inspection of the expression of reversed transcriptive enzyme α and reversed transcriptive enzyme β

The coexpression of expression, target protein and the triggering factor of following mutual more independent target protein and the Expression of Fusion Protein of the target protein and the triggering factor.Use two kinds of expression systems (that is, wherein use system's (cold shock expression system) of cold shock carrier and wherein use the expression system (T7 promoter expression system) of the combination of T7 promotor and t7 rna polymerase) to carry out Expression of Fusion Protein.

(1) structure of plasmid vector

Use dna synthesizer to trigger gene order (the Genbank catalog number (Cat.No.) NC_000913 of the factor based on intestinal bacteria, position 454357 to position 455655) synthesizes synthetic primer TFN and TFCP (SEQ ID NOS:13 and 14), and it is carried out purifying according to ordinary method.

Synthetic primer TFN is such synthetic DNA, this DNA have coding trigger from intestinal bacteria the factor aminoacid sequence N-terminal the 1st to the 9th amino acid whose nucleotide sequence and at the recognition sequence of the restriction enzyme NdeI of Nucleotide 4 to the Nucleotide 9.Synthetic primer TFCP is such synthetic DNA, and this DNA has and encodes and trigger nucleotide sequence complementary nucleotide sequence, the recognition sequence of restriction enzyme EcoRI, the recognition sequence of restriction enzyme BamHI and the recognition sequence of restriction enzyme HindIII of the recognition sequence of the 1st to the 9th amino acid whose nucleotide sequence complementary nucleotide sequence of C-terminal of aminoacid sequence of the factor and proteins encoded enzyme factor Xa from intestinal bacteria.From the genomic dna of intestinal bacteria HB101 (Takara Bio) extraction as the template of PCR.

Use synthetic primer and genomic dna to carry out PCR.The reaction conditions of PCR is as follows.In brief, prepare the reaction mixture that cumulative volume is 100 μ l by adding template DNA, 10 μ l 10xPyrobest damping fluid II (Takara Bio), 8 μ l dNTP mixtures (Takara Bio), 100pmol synthetic primer TFN, 100pmol synthetic primer TFCP, 2.5U PyrobestDNA polysaccharase (Takara Bio) and the sterilized water that 1 μ l prepares as mentioned above.Reaction mixture is placed PCR thermal cycler SP (Takara Bio) and carries out following reaction: 30 circulations: carried out under 94 ℃ 30 seconds, and carried out carrying out under 30 seconds and 72 ℃ 2 minutes under 59 ℃.

After the reaction, the reaction mixture of 100 μ l is carried out electrophoresis on 1% sepharose.Target DNA fragment from running gel recovery and the observed about 1.5kbp of purifying carries out ethanol sedimentation with it.Behind the ethanol sedimentation, the DNA that reclaims is suspended in the 15 μ l sterilized waters, and carries out double digested with restriction enzyme NdeI (Takara Bio) and restriction enzyme HindIII (Takara Bio).Extraction and purifying NdeI-HindIII digest are to obtain the dna fragmentation of NdeI-HindIII digestion after carrying out electrophoresis on 1% sepharose.

Then, with restriction enzyme NdeI and HindIII double digested plasmid vector pColdII (Takara Bio), and to terminal dephosphorylation.Mix and use dna ligation kit (Takara Bio) that it is interconnected the dna fragmentation of prepared carrier and NdeI-HindIII digestion.Use the connection mixture transformed into escherichia coli JM109 of 10 μ l.Transformant is grown on the LB substratum that contains the penbritin that agar that concentration is 1.5% (w/v) and concentration is 100 μ g/ml.The plasmid that has the target DNA fragment of insertion by the order-checking confirmation.Subsequently, introduce silent mutation to eliminate the recognition sequence (Genbank catalog number (Cat.No.) NC_000913, position 455107 to position 455112) of the restriction enzyme EcoRI in the triggering factor gene sequence in the plasmid.The cold shock expression system that has that is obtained is called pColdTF with the recombinant plasmid that contains intestinal bacteria triggering factor gene sequence.

Following structure uses the T7 promoter expression system to express the plasmid vector of the fusion rotein of the target protein and the intestinal bacteria triggering factor.

At first, with restriction enzyme EcoRI (Takara Bio) and restriction enzyme EcoO109I (Takara Bio) double digested pColdTF, and to the dna fragmentation of terminal dephosphorylation with acquisition EcoRI-EcoO109I digestion.Then, use restriction enzyme EcoRI and restriction enzyme EcoO109I double digested pCold08NC2, and it is carried out electrophoresis on 1% sepharose.Extract and purifying EcoRI-EcoO109I digest, and dna fragmentation mixing and the use dna ligation kit (Takara Bio) of itself and EcoRI-EcoO109I digestion is connected.Use 10 μ l to connect mixture transformed into escherichia coli JM109.Transformant is grown on the LB substratum that contains the penbritin that agar that concentration is 1.5% (w/v) and concentration is 100 μ g/ml.The recombinant plasmid (wherein the multiple clone site among the pColdTF being modified) that is obtained is called pColdTF-II.

With restriction enzyme XbaI (Takara Bio) digestion pColdTF-II, it is flat terminal with T4DNA polysaccharase (Takara Bio) it to be become, and degrades to obtain to contain the flat terminal fragment of NdeI that intestinal bacteria trigger the gene of the factor with restriction enzyme NdeI then.

With restriction enzyme BamHI (Takara Bio) dissimilation plasmid carrier pET16b (Novagen), it is flat terminal with the T4DNA polysaccharase it to be become, then with restriction enzyme NdeI digestion, and to terminal dephosphorylation.Prepared carrier and the flat terminal dna fragmentation of NdeI that contains intestinal bacteria triggering factor gene are mixed and with dna ligation kit it interconnected.Use 10 μ l to connect mixture transformed into escherichia coli JM109.Transformant is grown on the LB substratum that contains the penbritin that agar that concentration is 1.5% (w/v) and concentration is 100 μ g/ml.The T7 promoter expression system that has that is obtained is called pETTF with the recombinant plasmid that contains intestinal bacteria triggering factor gene sequence.

(2) structure of the carrier of expression reversed transcriptive enzyme α and reversed transcriptive enzyme β

Based on the synthetic double-stranded DNA of the aminoacid sequence of RAV-2 (RAV-2) reversed transcriptive enzyme α subunit (being called RAV-2 reversed transcriptive enzyme α hereinafter) (Genbank catalog number (Cat.No.) BAA22090, the 1st to the 572nd amino acid that begins from N-terminal) with sequence of SEQ ID NO:15.Select modified nucleotide sequence and do not change amino acid sequence coded according to colibacillary codon, and design this sequence to make it the having recognition sequence of restriction enzyme EcoRI and the recognition sequence of restriction enzyme XbaI at two ends.

Based on the synthetic double-stranded DNA of the aminoacid sequence (Genbank catalog number (Cat.No.) BAA22090) of RAV-2 (RAV-2) reversed transcriptive enzyme β subunit (being called RAV-2 reversed transcriptive enzyme β hereinafter) with sequence of SEQ ID NO:16.Select this nucleotide sequence of modification and do not change amino acid sequence coded according to colibacillary codon, and design this sequence to make it the having recognition sequence of restriction enzyme EcoRI and the recognition sequence of restriction enzyme XbaI at two ends.

With restriction enzyme EcoRI (Takara Bio) and two synthetic double-stranded DNAs of XbaI (Takara Bio) double digested, and it is carried out electrophoresis on 1% sepharose.From running gel reclaim and the dna fragmentation of the observed purpose size of purifying with the gene that obtains to contain coding RAV-2 reversed transcriptive enzyme α through the dna fragmentation of EcoRI-XbaI digestion and contain the dna fragmentation that digests through EcoRI-XbaI of the gene of coding RAV-2 reversed transcriptive enzyme β.

With the pColdTF of preparation in restriction enzyme EcoRI and the XbaI double digested (1), and to terminal dephosphorylation.Mix and use dna ligation kit (Takara Bio) that it is interconnected a kind of in the dna fragmentation of prepared carrier and two kinds of EcoRI-XbaI digestion.Use 10 μ l to connect mixture transformed into escherichia coli JM109.Transformant is grown on the LB substratum that contains the penbritin that agar that concentration is 1.5% (w/v) and concentration is 100 μ g/ml.The plasmid of expressing fusion rotein that triggers the factor and RAV-2 reversed transcriptive enzyme α and the fusion rotein that triggers the factor and RAV-2 reversed transcriptive enzyme β in the cold shock expression system is called pColdTF-α and pColdTF-β.

In addition, according to described method (except the pCold08Nc2 that uses embodiment 1 to make up replaces the pColdTF) the preparation single expression RAV-2 reversed transcriptive enzyme α of pColdTF-α and pColdTF-β and the plasmid of single expression RAV-2 reversed transcriptive enzyme β of being used for.Prepared plasmid is called pCold08-α and pCold08-β.

Following being structured in expressed fusion rotein that triggers the factor and RAV-2 reversed transcriptive enzyme α and the plasmid that triggers the fusion rotein of the factor and RAV-2 reversed transcriptive enzyme β in the T7 promoter expression system.

With restriction enzyme XbaI (Takara Bio) digestion pColdTF-α and pColdTF-β, it is flat terminal with T4 archaeal dna polymerase (Takara Bio) it to be become, digest with restriction enzyme EcoRI then, and it is carried out electrophoresis on 1% sepharose.Reclaim and the dna fragmentation of the observed purpose size of purifying from running gel, put down terminal dna fragmentation with the flat terminal dna fragmentation of EcoRI of the gene that obtains to contain coding RAV-2 reversed transcriptive enzyme α and the EcoRI that contains the gene of coding RAV-2 reversed transcriptive enzyme β.

With the pETTF of preparation in restriction enzyme SalI (Takara Bio) digestion (1), it is flat terminal with the T4DNA polysaccharase it to be become, digest with restriction enzyme EcoRI then, and to terminal dephosphorylation.With a kind of the mixing in prepared carrier and the flat terminal dna fragmentation of two kinds of EcoRI, and use dna ligation kit that it is interconnected.Use 10 μ l to connect mixture transformed into escherichia coli JM109.Transformant is grown on the LB substratum that contains the penbritin that agar that concentration is 1.5% (w/v) and concentration is 100 μ g/ml.Be used for being called pETTF-α and pETTF-β at the fusion rotein of the T7 promoter expression system expression triggering factor and RAV-2 reversed transcriptive enzyme α and the plasmid of the fusion rotein that triggers the factor and RAV-2 reversed transcriptive enzyme β.

(3) preparation of transformant

Use the pColdTF-α transformed into escherichia coli BL21 that in the cold shock expression system, expresses the fusion rotein that triggers the factor and RAV-2 reversed transcriptive enzyme α according to Calcium Chloride Method.Containing concentration by use is that the flat board of the penbritin of 100 μ g/ml screens and obtains transformant.

The transformant that also prepares the e. coli bl21 that transforms with pColdTF-β in a similar manner, described pColdTF-β is used for expressing the fusion rotein that triggers the factor and RAV-2 reversed transcriptive enzyme β at the cold shock expression system.

Also preparation is used for the following transformant that compares with above-mentioned amalgamation and expression system: the transformant of expressing RAV-2 reversed transcriptive enzyme α or RAV-2 reversed transcriptive enzyme β at single expression system; In coexpression system, express the transformant of the RAV-2 reversed transcriptive enzyme α and the triggering factor; In coexpression system, express the transformant of the RAV-2 reversed transcriptive enzyme β and the triggering factor; In the T7 promoter expression system, express the transformant of the fusion rotein that triggers the factor and RAV-2 reversed transcriptive enzyme α; With the transformant of in the T7 promoter expression system, expressing the fusion rotein that triggers the factor and RAV-2 reversed transcriptive enzyme β.

Obtain to be used for the transformant expressed at single expression system according to such preparation method, this method is except transforming the BL21 with plasmid pCold08-α or pCold08-β, is used in the method for the transformant of cold shock expression system expressed fusion protein similar with above-mentioned about preparation.

Be prepared as follows and be used for the transformant expressed at coexpression system.At first, use plasmid pTf16 and plasmid pCold08-α or pCold08-β transformed into escherichia coli BL21 according to the calcium chloride method.Contain by use that concentration is respectively the paraxin of 100 μ g/ml and 20 μ g/ml and the flat board of penbritin screens the cotransformation body that obtains to contain plasmid pTf16 and pCold08-α or pCold08-β.

Obtain to be used for transformant according to such preparation method at T7 promoter expression system expressed fusion protein, this method is except using plasmid pETTF-α or pETTF-β, with with BL21 (DE3) (Novagen) transform outside, be used in the method for the transformant of cold shock expression system expressed fusion protein similar with above-mentioned relevant preparation.

(4) expression of reversed transcriptive enzyme α and reversed transcriptive enzyme β

Use the transformant that obtains in (3) to check the expression of reversed transcriptive enzyme α and reversed transcriptive enzyme β.Using 5ml to contain concentration is that the LB liquid nutrient medium of the penbritin of 50 μ g/ml is cultivated and is used in the transformant of cold shock expression system expressed fusion protein and is used for the transformant of single expression system.Use 5ml to contain the LB liquid nutrient medium that concentration is respectively penbritin, paraxin and the pectinose of 50 μ g/ml, 20 μ g/ml and 0.5mg/ml and cultivate the transformant that is used for coexpression system.37 ℃ of transformant of cultivating down separately.When turbidity (OD600) reaches about 0.4 the time, under 15 ℃, cultivated 15 minutes, in culture, add IPTG with the final concentration of 1mM, and cultivate 24 hours to carry out induced expression at 15 ℃.At induced expression after 24 hours, collecting cell.

Using 5ml to contain concentration is that the LB liquid nutrient medium of the penbritin of 50 μ g/ml is cultivated the transformant that is used at T7 promoter expression system expressed fusion protein.Cultivate transformant down at 37 ℃.When turbidity (OD600) reaches about 0.4 the time, in culture, add IPTG with the final concentration of 1mM, and cultivate 3 hours to carry out induced expression at 37 ℃.At induced expression after 3 hours, collecting cell.

Suspension cell in PBS, and pass through the supersound process ruptured cell with preparation cell extract fraction.Then, by centrifugal under 15,000 * g soluble fraction and insoluble fraction are separated.With each corresponding to about 3.75 * 10 ⁶The part of the fraction separately of individual cell is carried out SDS-PAGE.The Fig. 2 that the results are shown in by the CBB staining analysis.

As shown in Figure 2, the single expression (A) of the target protein that uses pCold08-α or pCold08-β and use the target protein of pCold08-α and pTf16 or pCold08-β and pTf16 and the situation (B) of the coexpression of the triggering factor under, in the cell extract fraction, observe the molecular weight 63 that corresponds respectively to RAV-2 reversed transcriptive enzyme α and RAV-2 reversed transcriptive enzyme β, 000Da and 98, the expression product of 000Da, but in soluble fraction, almost do not observe this expression product.Under the situation (C) that the fusion rotein of the target protein that uses pETTF-α or pETTF-β and the triggering factor is expressed, in insoluble fraction, detect major part detected target protein in the cell extract fraction in the T7 promoter expression system.On the other hand, under the situation (D) of target protein that uses pColdTF-α or pColdTF-β and the amalgamation and expression that triggers the factor, in soluble fraction, detect major part detected target protein in the cell extract fraction.

The expression of embodiment 3:DNA enzyme

(1) structure of the carrier of expressible dna enzyme

(be preserved in Independent Administrative Leged Industrial Technology Complex Inst and specially permit biological sustenance center on February 16th, 2005 (original preservation date) to use pCold08-End1 (FERM BP-10313), AIST Tsukuba Central 6,1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki305-8566, Japan) as the plasmid of single expression DNA enzyme.This plasmid comprises the nucleotide sequence of the DNA enzyme that coding is made up of 254 amino-acid residues, and make up described plasmid like this, thereby make it express the fusion rotein of 271 such amino-acid residues, in this fusion rotein, added recognition sequence and the joint sequence of His mark, factor Xa for the DNA enzyme.

Following structure is used to express the plasmid of the fusion rotein that triggers the factor and DNA enzyme.

At first, use dna synthesizer based on synthetic synthetic primer NUCN of the nucleotide sequence of pCold08-End1 and NUCC (SEQ ID NOS:17 and 18), and according to the described primer of ordinary method purifying.Synthetic primer NUCN is such synthetic DNA, and this DNA has coding from the 1st to the 7th amino acid whose nucleotide sequence of DNA enzyme N-terminal with at the recognition sequence of the restriction enzyme EcoRI of Nucleotide 4 to the Nucleotide 9.Synthetic primer NUCC is such synthetic DNA, and this DNA has and encodes from the 247th to the 254th amino acid whose nucleotide sequence complementary nucleotide sequence of the N-terminal of DNA enzyme and at the recognition sequence of the restriction enzyme BamHI of Nucleotide 4 to the Nucleotide 9.

Use the synthetic primer to carry out PCR.The reaction conditions of PCR is as follows.In brief, prepare the reaction mixture of cumulative volume 100 μ l by adding 1 μ l template DNA (pCold08-End1), 10 μ l 10x Pyrobest damping fluid II (Takara Bio), 8 μ l dNTP mixtures (Takara Bio), 100pmol synthetic primer NUCN, 100pmol synthetic primer NUCC, 2.5U Pyrobest archaeal dna polymerase (Takara Bio) and sterilized water.Reaction mixture is placed PCR thermal cycler SP (Takara Bio) and following the reaction: 30 circulations: 94 ℃ were carried out 30 seconds, and 58 ℃ are carried out carrying out 1 minute in 30 seconds and 72 ℃.

After the reaction, 100 μ l reaction mixtures are carried out electrophoresis on 1% sepharose.Carry out ethanol sedimentation from the target DNA fragment of running gel recovery and the observed about 0.8kbp of purifying and with it.Behind ethanol sedimentation, the DNA that reclaims is suspended in the 15 μ l sterilized waters, and carries out double digested with restriction enzyme EcoRI (Takara Bio) and restriction enzyme BamHI (Takara Bio).After carrying out electrophoresis on 1% the sepharose, extraction and purifying EcoRI-BamHI digest are to obtain the dna fragmentation of EcoRI-BamHI digestion.

Then, with the pColdTF of preparation among restriction enzyme EcoRI and the BamHI double digested embodiment 2 (2), and to terminal dephosphorylation.Mix and use dna ligation kit (Takara Bio) that it is interconnected a kind of in the dna fragmentation of prepared carrier and two kinds of EcoRI-BamHI digestion.Use 10 μ l to connect mixture transformed into escherichia coli JM109.Transformant is grown on the LB substratum that contains the penbritin that agar that concentration is 1.5% (w/v) and concentration is 100 μ g/ml.Preparation has the plasmid of the target DNA fragment of insertion.The plasmid that will be used to express the fusion rotein that triggers the factor and DNA enzyme is called pColdTF-End1.

(2) preparation of transformant

Use the pColdTF-End1 transformed into escherichia coli BL21 that expresses the fusion rotein that triggers the factor and DNA enzyme according to Calcium Chloride Method.Containing concentration by use is that the flat board of the penbritin of 100 μ g/ml screens and obtains transformant.

Also preparation be used for amalgamation and expression system being used for of comparing in the transformant of single expression system expressible dna enzyme be used at coexpression system expressible dna enzyme and trigger the transformant of the factor.

According to the transformant that such preparation method's acquisition is expressed in single expression system, this method is except transforming the BL21 with plasmid pCold08-End1, and is similar with the preparation method of above-mentioned transformant.

Be prepared as follows the transformant of in coexpression system, expressing.At first, according to Calcium Chloride Method, use plasmid pCold08-End1 and plasmid pTf16 transformed into escherichia coli BL21.Contain by use that concentration is respectively the paraxin of 100 μ g/ml and 20 μ g/ml and the flat board of penbritin screens the cotransformation body that obtains to contain plasmid pCold08-End1 and pTf16.

(3) expression of DNA enzyme

Use the transformant that obtains in (2) to check the expression of DNA enzyme.Using 5ml to contain concentration is that the LB liquid nutrient medium of the penbritin of 50 μ g/ml is cultivated transformant that is used for the amalgamation and expression system and the transformant that is used for single expression system.Use 5ml to contain the LB liquid nutrient medium that concentration is respectively penbritin, paraxin and the pectinose of 50 μ g/ml, 20 μ g/ml and 0.5mg/ml and cultivate the transformant that is used for coexpression system.37 ℃ of transformant of cultivating down separately.When turbidity (OD600) reaches about 0.8 the time, cultivated 15 minutes down at 15 ℃, in culture, add IPTG with the final concentration of 1mM, and cultivate 24 hours down to carry out induced expression at 15 ℃.Carrying out induced expression after 24 hours, collecting cell.With cell suspension in PBS, and by the supersound process ruptured cell with preparation cell extract fraction.Then, by centrifugal under 15,000 * g soluble fraction is separated with insoluble fraction.Each is carried out SDS-PAGE (gel of 5-20%) corresponding to the part of the fraction separately of 0.05OD (OD600).Be shown among Fig. 3 by the painted analytical results of CBB.

As shown in Figure 3, the situation (A) of the single expression of the target protein that uses pCold08-End1 and use the target protein of pCold08-End1 and pTf16 and the situation (B) of the coexpression of the triggering factor under, after soluble fraction or insoluble fraction being carried out CBB dyeing, do not observe molecular weight 31, the obvious band of the expression product of 000Da corresponding to the DNA enzyme.Under the situation (C) of DNA enzyme that uses pColdTF-End1 and the amalgamation and expression that triggers the factor,, detect band corresponding to target protein for soluble fraction.

(4) measurement of DNA enzymic activity

Measure the DNA enzymic activity of the soluble fraction of the supersound process of the amalgamation and expression system of preparation in (3).Also obtain soluble fractions in contrast from the independent colibacillary supersound process that transforms with carrier pColdTF (not having the insertion fragment of integrating), and in identical time measurement activity.To obtain the soluble fraction of supersound process in contrast, just except using pColdTF with top (2) and the similar mode of mode in (3).

Use the substrate of λ-HindIII digest (Takara Bio) as activity measurement.By adding 1 μ g λ-HindIII digest, preparing the reaction mixture of cumulative volume 50 μ l corresponding to soluble fraction, the 5 μ l 10x reaction buffers (400mM tris-hydrochloride buffer (pH7.5), 100mM sodium-chlor, 60mM magnesium chloride, 10mM calcium chloride) of the supersound process of 0.025OD (OD600) and the water that do not contain nuclease.Reaction mixture was reacted 2 hours down at 20 ℃.The reaction mixture of 10 μ l is carried out electrophoresis to analyze the product of cutting on 1% sepharose.The results are shown among Fig. 4.

As shown in Figure 4, when the soluble fraction of using supersound process in contrast when (swimming lane 1), almost do not observe the degraded of substrate.On the other hand, when using the DNA enzyme and trigger the soluble fraction (swimming lane 2) of supersound process of amalgamation and expression system of the factor, substrate is degraded and observes the activity of DNA enzyme.

The inspection of the expression of embodiment 4:hDi-PAZ

(1) structure of expression vector

For expression comprises the polypeptide of the PAZ structural domain (from the 895th to the 1064th amino acid of the N-terminal of the aminoacid sequence of people Dicer) of people Dicer (from the 892nd to the 1064th amino acid of the N-terminal of the aminoacid sequence of people Dicer; Be also referred to as PAZ), following construction of expression vector.

At first, use dna synthesizer based on the synthetic synthetic primer 1 and 2 (SEQ ID NOS:19 and 20) of the nucleotide sequence under Genbank catalog number (Cat.No.) AB028449 that can openly obtain, and according to the described primer of method purifying of routine.Synthetic primer 1 is such synthetic DNA, this DNA have the recognition sequence of the restriction enzyme KpnI of Nucleotide 9 to the Nucleotide 14 and Nucleotide 16 to the Nucleotide 36 corresponding to the nucleotide sequence of the amino acid 892 in the aminoacid sequence (SEQ ID NO:3) of people Dicer to amino acid 898.The synthetic primer 2 have the recognition sequence of the restriction enzyme HindIII of Nucleotide 9 to the Nucleotide 14 and Nucleotide 15 to the Nucleotide 36 corresponding to the nucleotide sequence of the amino acid/11 058 in the aminoacid sequence (SEQ ID NO:3) of people Dicer to amino acid/11 064.

Use described synthetic primer to carry out PCR.The pCold08/hDi-ASI that uses preparation among the embodiment 1-(2) is as template DNA.The reaction conditions of PCR is as follows.

In brief, by adding 1ng template DNA, 10 μ l 10x LA PCR damping fluids (Takara Bio), 8 μ l dNTP mixtures (Takara Bio), 10pmol synthetic primer 5,10pmol synthetic primer 6, the Takara Ex Taq (Takara Bio) of 0.5U and the reaction mixture that sterilized water prepares cumulative volume 100 μ l.Reaction mixture is placed TaKaRa PCR thermal cycler MP (Takara Bio) and carries out following reaction: 30 circulations: 94 ℃ are carried out 30 seconds, 55 ℃ and carry out carrying out 2 minutes in 30 seconds and 72 ℃.

After the reaction, 95 μ l reaction mixtures are carried out electrophoresis on 1.0% sepharose.Reclaim and the target DNA fragment of the observed about 530bp of purifying from running gel, and it is carried out ethanol sedimentation.Behind the ethanol sedimentation, the DNA that reclaims is suspended in the 5 μ l sterilized waters, and carries out double digested with restriction enzyme KpnI (Takara Bio) and restriction enzyme HindIII (Takara Bio).Behind electrophoresis on 1.0% sepharose, extract and the dna fragmentation of purifying KpnI-HindIII digest to obtain to digest through KpnI-HindIII.

With with the identical restriction enzyme cut vector pCold08NC2 of enzyme used when the dna fragmentation of preparation KpnI-HindIII digestion, and to terminal dephosphorylation.The dna fragmentation of prepared carrier and KpnI-HindIII digestion is mixed and with dna ligation kit (Takara Bio) it interconnected.Use 6 μ l to connect mixture transformed into escherichia coli JM109.Transformant is grown on the LB substratum that contains the penbritin that agar that concentration is 1.5% (w/v) and concentration is 100 μ g/ml.

Plasmid with target DNA fragment of insertion is called pCold08/hDi-PAZ.PCold08/hDi-PAZ is the plasmid that contains such nucleotide sequence, and the amino acid 892 in this nucleotide sequence coded aminoacid sequence from people Dicer (SEQ ID NO:3) is to the aminoacid sequence of amino acid/11 064.Have perfect DB sequence, His flag sequence and factor Xa sequence from the albumen of described plasmid expression.

Be used to express the plasmid of the fusion rotein that triggers the factor and PAZ according to the preparation of such method, this method except with the pColdTF-II of preparation among the embodiment 2-(1) as the carrier, similar with the method for the preparation of above-mentioned relevant pCold08/hDi-PAZ.Described plasmid is called pColdTF/hDi-PAZ.

(2) preparation of transformant

The pColdTF/hDi-PAZ transformed into escherichia coli BL21 that is used to express the fusion rotein that triggers the factor and hDi-PAZ according to Calcium Chloride Method.Containing concentration by use is that the flat board of the penbritin of 100 μ g/ml screens and obtains transformant.

Also preparation is used for expressing the transformant of hDi-PAZ and being used for expressing hDi-PAZ and triggering the transformant of the factor at coexpression system at single expression system with amalgamation and expression system being used for of comparing.

According to the transformant that such preparation method's acquisition is expressed in single expression system, this preparation method is except transforming the BL21 with plasmid pCold08/hDi-PAZ, and is similar with the preparation method of the transformant that is used for expressing in the amalgamation and expression system.Be prepared as follows and be used for the transformant expressed at coexpression system.At first, use plasmid pCold08/hDi-PAZ and plasmid pTf16 transformed into escherichia coli A19 according to Calcium Chloride Method.Contain by use that concentration is respectively the paraxin of 50 μ g/ml and 100 μ g/ml and the flat board of penbritin screens the cotransformation body that obtains to contain plasmid pCold08/hDi-PAZ and pTf16.

(3) expression of hDi-PAZ

Use the transformant that obtains in (2) to check the expression of hDi-PAZ.Using 3ml to contain concentration is that the LB liquid nutrient medium of the penbritin of 50 μ g/ml is cultivated transformant that is used for the amalgamation and expression system and the transformant that is used for single expression system.Use 3ml to contain the LB liquid nutrient medium that concentration is respectively penbritin, paraxin and the pectinose of 50 μ g/ml, 20 μ g/ml and 0.5mg/ml and cultivate the transformant that is used for coexpression system.37 ℃ of transformant of cultivating down separately.When turbidity (OD600) reaches about 0.4 the time, under 15 ℃, cultivated 15 minutes, in culture, add IPTG with the final concentration of 0.5mM, and under 15 ℃, cultivate 24 hours to carry out induced expression.Carrying out induced expression after 24 hours, collecting cell.With cell suspension in cell rupture solution (50mM tris-HCl (pH8.5), 100mM NaCl, 1mMMgCl ₂, proteinase inhibitor (not containing EDTA fully)) in and break with preparation cell extract fraction by supersound process.Then, by centrifugal under 15,000 * g soluble fraction is separated with insoluble fraction.With each corresponding to about 2.5 * 10 ⁶The part of the fraction separately of individual cell is carried out SDS-PAGE.Analyze by CBB dyeing.

The result, in the single expression of the target protein that uses pCold08/hDi-PAZ with use the target protein of pCold08/hDi-PAZ and pTf16 and trigger under the situation of coexpression of the factor, in the cell extract fraction, observe molecular weight 24 corresponding to target protein, the expression product of 000Da, but in soluble fraction, almost do not detect this expression product.Under the situation of target protein that uses pColdTF/hDi-PAZ to carry out and the amalgamation and expression that triggers the factor, to compare with contrast, the amount of the target protein in the cell extract fraction increases.Major part detects in soluble fraction.

Industrial usability

The method according to this invention may produce quite a large amount of high-purities that has and keep simultaneously it Active purpose polypeptide.

Sequence table independence literal

SEQ ID NO:2; 5 of the sudden change of coding intestinal bacteria cspA gene '-gene of UTR

SEQ ID NO:4; The synthetic primer 5 of the gene of amplification coding people dicer mutant

SEQ ID NO:5; The synthetic primer 6 of the gene of amplification coding people dicer mutant

SEQ ID NO:6; The gene of coding people dicer mutant

SEQ ID NO:7; The aminoacid sequence of people dicer mutant

SEQ ID NO:8; The aminoacid sequence of people dicer mutant

SEQ ID NO:9; The gene of coding people dicer mutant

SEQ ID NO:10; The gene of coding red shift green fluorescent protein

SEQ ID NO:11; The synthetic primer 3 of the gene of amplification coding red shift green fluorescent protein

SEQ ID NO:12; The synthetic primer 4 of the gene of amplification coding red shift green fluorescent protein

SEQ ID NO:13; Amplification coding triggers the synthetic primer TFN of the gene of the factor

SEQ ID NO:14; Amplification coding triggers the synthetic primer TFCP of the gene of the factor

SEQ ID NO:15; The gene of coding RAV-2 reversed transcriptive enzyme α subunit

SEQ ID NO:16; The gene of coding RAV-2 reversed transcriptive enzyme β subunit

SEQ ID NO:17; The synthetic primer NUCN of the gene of amplification coding DNA enzyme

SEQ ID NO:18; The synthetic primer NUCC of the gene of amplification coding DNA enzyme

SEQ ID NO:19; The synthetic primer 1 of the gene of amplification coding people dicer PAZ structural domain

SEQ ID NO:20; The synthetic primer 2 of the gene of amplification coding people dicer PAZ structural domain

Sequence table

<110>TAKARA?BIO?INC.

＜120〉method of production polypeptide

<130>665240

<150>JP?2004-152598

<151>2004-05-21

<150>JP?2005-067984

<151>2005-03-14

<160>20

<170>PatentIn?version?3.3

<210>1

<211>1209

<212>DNA

＜213〉intestinal bacteria (Escherichia coli)

<400>1

aagcttcgat?gcaattcacg?atcccgcagt?gtgatttgag?gagttttcaa?tggaatataa 60

agatccaatg?catgagctgt?tgagcagcct?ggaacagatt?gtttttaaag?atgaaacgca 120

gaaaattacc?ctgacgcaca?gaacaacgtc?ctgtaccgaa?attgagcagt?tacgaaaagg 180

gacaggatta?aaaatcgatg?atttcgcccg?ggttttgggc?gtatcagtcg?ccatggtaaa 240

ggaatgggaa?tccagacgcg?tgaagccttc?aagtgccgaa?ctaaaattga?tgcgtttgat 300

tcaagccaac?ccggcattaa?gtaagcagtt?gatggaatag?acttttatcc?actttattgc 360

tgtttacggt?cctgatgaca?ggaccgtttt?ccaaccgatt?aatcataaat?atgaaaaata 420

attgttgcat?cacccgccaa?tgcgtggctt?aatgcacatc?aacggtttga?cgtacagacc 480

attaaagcag?tgtagtaagg?caagtccctt?caagagttat?cgttgatacc?cctcgtagtg 540

cacattcctt?taacgcttca?aaatctgtaa?agcacgccat?atcgccgaaa?ggcacactta 600

attattaaag?gtaatacact?atgtccggta?aaatgactgg?tatcgtaaaa?tggttcaacg 660

ctgacaaagg?cttcggcttc?atcactcctg?acgatggctc?taaagatgtg?ttcgtacact 720

tctctgctat?ccagaacgat?ggttacaaat?ctctggacga?aggtcagaaa?gtgtccttca 780

ccatcgaaag?cggcgctaaa?ggcccggcag?ctggtaacgt?aaccagcctg?taatctctgc 840

ttaaaagcac?agaatctaag?atccctgcca?tttggcgggg?atttttttat?ttgttttcag 900

gaaataaata?atcgatcgcg?taataaaatc?tattattatt?tttgtgaaga?ataaatttgg 960

gtgcaatgag?aatgcgcaac?gccgtaagta?aggcgggaat?aatttcccgc?cgaagactct 1020

tactctttca?atttgcaggc?taaaaacgcc?gccagctcat?aactctcctg?tttaatatgc 1080

aattcacaca?gtgaatctct?tatcatccag?gtgaaaaata?aaagcgtgaa?acaaatcact 1140

attaaagaaa?gtaatctata?tttctgcgca?ttccagctct?gtgttgattt?cacgagtatg 1200

tactgcacc 1209

<210>2

<211>143

<212>RNA

＜213〉artificial sequence

<220>

＜223〉gene of 5 '-UTR of the sudden change of coding intestinal bacteria (Escherichia coli) cspA gene

<400>2

aauugugagc?ggauaacaau?uugaugugcu?agcgcauauc?caguguagua?aggcaagucc 60

cuucaagagc?cuuuaacgcu?ucaaaaucug?uaaagcacgc?cauaucgccg?aaaggcacac 120

uuaauuauua?aagguaauac?acu 143

<210>3

<211>1924

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>3

Met?Lys?Ser?Pro?Ala?Leu?Gln?Pro?Leu?Ser?Met?Ala?Gly?Leu?Gln?Leu

1 5 10 15

Met?Thr?Pro?Ala?Ser?Ser?Pro?Met?Gly?Pro?Phe?Phe?Gly?Leu?Pro?Trp

20 25 30

Gln?Gln?Glu?Ala?Ile?His?Asp?Asn?Ile?Tyr?Thr?Pro?Arg?Lys?Tyr?Gln

35 40 45

Val?Glu?Leu?Leu?Glu?Ala?Ala?Leu?Asp?His?Asn?Thr?Ile?Val?Cys?Leu

50 55 60

Asn?Thr?Gly?Ser?Gly?Lys?Thr?Phe?Ile?Ala?Ser?Thr?Thr?Leu?Leu?Lys

65 70 75 80

Ser?Cys?Leu?Tyr?Leu?Asp?Leu?Gly?Glu?Thr?Ser?Ala?Arg?Asn?Gly?Lys

85 90 95

Arg?Thr?Val?Phe?Leu?Val?Asn?Ser?Ala?Asn?Gln?Val?Ala?Gln?Gln?Val

100 105 110

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

115 120 125

Leu?Glu?Val?Asn?Ala?Ser?Trp?Thr?Lys?Glu?Arg?Trp?Asn?Gln?Glu?Phe

130 135 140

Thr?Lys?His?Gln?Val?Leu?Ile?Met?Thr?Cys?Tyr?Val?Ala?Leu?Asn?Val

145 150 155 160

Leu?Lys?Asn?Gly?Tyr?Leu?Ser?Leu?Ser?Asp?Ile?Asn?Leu?Leu?Val?Phe

165 170 175

Asp?Glu?Cys?His?Leu?Ala?Ile?Leu?Asp?His?Pro?Tyr?Arg?Glu?Phe?Met

180 185 190

Lys?Leu?Cys?Glu?Ile?Cys?Pro?Ser?Cys?Pro?Arg?Ile?Leu?Gly?Leu?Thr

195 200 205

Ala?Ser?Ile?Leu?Asn?Gly?Lys?Trp?Asp?Pro?Glu?Asp?Leu?Glu?Glu?Lys

210 215 220

Phe?Gln?Lys?Leu?Glu?Lys?Ile?Leu?Lys?Ser?Asn?Ala?Glu?Thr?Ala?Thr

225 230 235 240

Asp?Leu?Val?Val?Leu?Asp?Arg?Tyr?Thr?Ser?Gln?Pro?Cys?Glu?Ile?Val

245 250 255

Val?Asp?Cys?Gly?Pro?Phe?Thr?Asp?Arg?Ser?Gly?Leu?Tyr?Glu?Arg?Leu

260 265 270

Leu?Met?Glu?Leu?Glu?Glu?Ala?Leu?Asn?Phe?Ile?Asn?Asp?Cys?Asn?Ile

275 280 285

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

290 295 300

Leu?Ser?Asp?Cys?Arg?Ala?Val?Leu?Val?Val?Leu?Gly?Pro?Trp?Cys?Ala

305 310 315 320

Asp?Lys?Val?Ala?Gly?Met?Met?Val?Arg?Glu?Leu?Gln?Lys?Tyr?Ile?Lys

325 330 335

His?Glu?Gln?Glu?Glu?Leu?His?Arg?Lys?Phe?Leu?Leu?Phe?Thr?Asp?Thr

340 345 350

Phe?Leu?Arg?Lys?Ile?His?Ala?Leu?Cys?Glu?Glu?His?Phe?Ser?Pro?Ala

355 360 365

Ser?Leu?Asp?Leu?Lys?Phe?Val?Thr?Pro?Lys?Val?Ile?Lys?Leu?Leu?Glu

370 375 380

Ile?Leu?Arg?Lys?Tyr?Lys?Pro?Tyr?Glu?Arg?His?Ser?Phe?Glu?Ser?Val

385 390 395 400

Glu?Trp?Tyr?Asn?Asn?Arg?Asn?Gln?Asp?Asn?Tyr?Val?Ser?Trp?Ser?Asp

405 410 415

Ser?Glu?Asp?Asp?Asp?Glu?Asp?Glu?Glu?Ile?Glu?Glu?Lys?Glu?Lys?Pro

420 425 430

Glu?Thr?Asn?Phe?Pro?Ser?Pro?Phe?Thr?Asn?Ile?Leu?Cys?Gly?Ile?Ile

435 440 445

Phe?Val?Glu?Arg?Arg?Tyr?Thr?Ala?Val?Val?Leu?Asn?Arg?Leu?Ile?Lys

450 455 460

Glu?Ala?Gly?Lys?Gln?Asp?Pro?Glu?Leu?Ala?Tyr?Ile?Ser?Ser?Asn?Phe

465 470 475 480

Ile?Thr?Gly?His?Gly?Ile?Gly?Lys?Asn?Gln?Pro?Arg?Asn?Asn?Thr?Met

485 490 495

Glu?Ala?Glu?Phe?Arg?Lys?Gln?Glu?Glu?Val?Leu?Arg?Lys?Phe?Arg?Ala

500 505 510

His?Glu?Thr?Asn?Leu?Leu?Ile?Ala?Thr?Ser?Ile?Val?Glu?Glu?Gly?Val

515 520 525

Asp?Ile?Pro?Lys?Cys?Asn?Leu?Val?Val?Arg?Phe?Asp?Leu?Pro?Thr?Glu

530 535 540

Tyr?Arg?Ser?Tyr?Val?Gln?Ser?Lys?Gly?Arg?Ala?Arg?Ala?Pro?Ile?Ser

545 550 555 560

Asn?Tyr?Ile?Met?Leu?Ala?Asp?Thr?Asp?Lys?Ile?Lys?Ser?Phe?Glu?Glu

565 570 575

Asp?Leu?Lys?Thr?Tyr?Lys?Ala?Ile?Glu?Lys?Ile?Leu?Arg?Asn?Lys?Cys

580 585 590

Ser?Lys?Ser?Val?Asp?Thr?Gly?Glu?Thr?Asp?Ile?Asp?Pro?Val?Met?Asp

595 600 605

Asp?Asp?His?Val?Phe?Pro?Pro?Tyr?Val?Leu?Arg?Pro?Asp?Asp?Gly?Gly

610 615 620

Pro?Arg?Val?Thr?Ile?Asn?Thr?Ala?Ile?Gly?His?Ile?Asn?Arg?Tyr?Cys

625 630 635 640

Ala?Arg?Leu?Pro?Ser?Asp?Pro?Phe?Thr?His?Leu?Ala?Pro?Lys?Cys?Arg

645 650 655

Thr?Arg?Glu?Leu?Pro?Asp?Gly?Thr?Phe?Tyr?Ser?Thr?Leu?Tyr?Leu?Pro

660 665 670

Ile?Asn?Ser?Pro?Leu?Arg?Ala?Ser?Ile?Val?Gly?Pro?Pro?Met?Ser?Cys

675 680 685

Val?Arg?Leu?Ala?Glu?Arg?Val?Val?Ala?Leu?Ile?Cys?Cys?Glu?Lys?Leu

690 695 700

His?Lys?Ile?Gly?Glu?Leu?Asp?Asp?His?Leu?Met?Pro?Val?Gly?Lys?Glu

705 710 715 720

Thr?Val?Lys?Tyr?Glu?Glu?Glu?Leu?Asp?Leu?His?Asp?Glu?Glu?Glu?Thr

725 730 735

Ser?Val?Pro?Gly?Arg?Pro?Gly?Ser?Thr?Lys?Arg?Arg?Gln?Cys?Tyr?Pro

740 745 750

Lys?Ala?Ile?Pro?Glu?Cys?Leu?Arg?Asp?Ser?Tyr?Pro?Arg?Pro?Asp?Gln

755 760 765

Pro?Cys?Tyr?Leu?Tyr?Val?Ile?Gly?Met?Val?Leu?Thr?Thr?Pro?Leu?Pro

770 775 780

Asp?Glu?Leu?Asn?Phe?Arg?Arg?Arg?Lys?Leu?Tyr?Pro?Pro?Glu?Asp?Thr

785 790 795 800

Thr?Arg?Cys?Phe?Gly?Ile?Leu?Thr?Ala?Lys?Pro?Ile?Pro?Gln?Ile?Pro

805 810 815

His?Phe?Pro?Val?Tyr?Thr?Arg?Ser?Gly?Glu?Val?Thr?Ile?Ser?Ile?Glu

820 825 830

Leu?Lys?Lys?Ser?Gly?Phe?Met?Leu?Ser?Leu?Gln?Met?Leu?Glu?Leu?Ile

835 840 845

Thr?Arg?Leu?His?Gln?Tyr?Ile?Phe?Ser?His?Ile?Leu?Arg?Leu?Glu?Lys

850 855 860

Pro?Ala?Leu?Glu?Phe?Lys?Pro?Thr?Asp?Ala?Asp?Ser?Ala?Tyr?Cys?Val

865 870 875 880

Leu?Pro?Leu?Asn?Val?Val?Asn?Asp?Ser?Ser?Thr?Leu?Asp?Ile?Asp?Phe

885 890 895

Lys?Phe?Met?Glu?Asp?Ile?Glu?Lys?Ser?Glu?Ala?Arg?Ile?Gly?Ile?Pro

900 905 910

Ser?Thr?Lys?Tyr?Thr?Lys?Glu?Thr?Pro?Phe?Val?Phe?Lys?Leu?Glu?Asp

915 920 925

Tyr?Gln?Asp?Ala?Val?Ile?Ile?Pro?Arg?Tyr?Arg?Asn?Phe?Asp?Gln?Pro

930 935 940

His?Arg?Phe?Tyr?Val?Ala?Asp?Val?Tyr?Thr?Asp?Leu?Thr?Pro?Leu?Ser

945 950 955 960

Lys?Phe?Pro?Ser?Pro?Glu?Tyr?Glu?Thr?Phe?Ala?Glu?Tyr?Tyr?Lys?Thr

965 970 975

Lys?Tyr?Asn?Leu?Asp?Leu?Thr?Asn?Leu?Asn?Gln?Pro?Leu?Leu?Asp?Val

980 985 990

Asp?His?Thr?Ser?Ser?Arg?Leu?Asn?Leu?Leu?Thr?Pro?Arg?His?Leu?Asn

995 1000 1005

Gln?Lys?Gly?Lys?Ala?Leu?Pro?Leu?Ser?Ser?Ala?Glu?Lys?Arg?Lys

1010 1015 1020

Ala?Lys?Trp?Glu?Ser?Leu?Gln?Asn?Lys?Gln?Ile?Leu?Val?Pro?Glu

1025 1030 1035

Leu?Cys?Ala?Ile?His?Pro?Ile?Pro?Ala?Ser?Leu?Trp?Arg?Lys?Ala

1040 1045 1050

Val?Cys?Leu?Pro?Ser?Ile?Leu?Tyr?Arg?Leu?His?Cys?Leu?Leu?Thr

1055 1060 1065

Ala?Glu?Glu?Leu?Arg?Ala?Gln?Thr?Ala?Ser?Asp?Ala?Gly?Val?Gly

1070 1075 1080

Val?Arg?Ser?Leu?Pro?Ala?Asp?Phe?Arg?Tyr?Pro?Asn?Leu?Asp?Phe

1085 1090 1095

Gly?Trp?Lys?Lys?Ser?Ile?Asp?Ser?Lys?Ser?Phe?Ile?Ser?Ile?Ser

1100 1105 1110

Asn?Ser?Ser?Ser?Ala?Glu?Asn?Asp?Asn?Tyr?Cys?Lys?His?Ser?Thr

1115 1120 1125

Ile?Val?Pro?Glu?Asn?Ala?Ala?His?Gln?Gly?Ala?Asn?Arg?Thr?Ser

1130 1135 1140

Ser?Leu?Glu?Asn?His?Asp?Gln?Met?Ser?Val?Asn?Cys?Arg?Thr?Leu

1145 1150 1155

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

1160 1165 1170

Leu?Thr?Ala?Ile?Asn?Gly?Leu?Ser?Tyr?Asn?Gln?Asn?Leu?Ala?Asn

1175 1180 1185

Gly?Ser?Tyr?Asp?Leu?Ala?Asn?Arg?Asp?Phe?Cys?Gln?Gly?Asn?Gln

1190 1195 1200

Leu?Asn?Tyr?Tyr?Lys?Gln?Glu?Ile?Pro?Val?Gln?Pro?Thr?Thr?Ser

1205 1210 1215

Tyr?Ser?Ile?Gln?Asn?Leu?Tyr?Ser?Tyr?Glu?Asn?Gln?Pro?Gln?Pro

1220 1225 1230

Ser?Asp?Glu?Cys?Thr?Leu?Leu?Ser?Asn?Lys?Tyr?Leu?Asp?Gly?Asn

1235 1240 1245

Ala?Asn?Lys?Ser?Thr?Ser?Asp?Gly?Ser?Pro?Val?Met?Ala?Val?Met

1250 1255 1260

Pro?Gly?Thr?Thr?Asp?Thr?Ile?Gln?Val?Leu?Lys?Gly?Arg?Met?Asp

1265 1270 1275

Ser?Glu?Gln?Ser?Pro?Ser?Ile?Gly?Tyr?Ser?Ser?Arg?Thr?Leu?Gly

1280 1285 1290

Pro?Asn?Pro?Gly?Leu?Ile?Leu?Gln?Ala?Leu?Thr?Leu?Ser?Asn?Ala

1295 1300 1305

Ser?Asp?Gly?Phe?Asn?Leu?Glu?Arg?Leu?Glu?Met?Leu?Gly?Asp?Ser

1310 1315 1320

Phe?Leu?Lys?His?Ala?Ile?Thr?Thr?Tyr?Leu?Phe?Cys?Thr?Tyr?Pro

1325 1330 1335

Asp?Ala?His?Glu?Gly?Arg?Leu?Ser?Tyr?Met?Arg?Ser?Lys?Lys?Val

1340 1345 1350

Ser?Asn?Cys?Asn?Leu?Tyr?Arg?Leu?Gly?Lys?Lys?Lys?Gly?Leu?Pro

1355 1360 1365

Ser?Arg?Met?Val?Val?Ser?Ile?Phe?Asp?Pro?Pro?Val?Asn?Trp?Leu

1370 1375 1380

Pro?Pro?Gly?Tyr?Val?Val?Asn?Gln?Asp?Lys?Ser?Asn?Thr?Asp?Lys

1385 1390 1395

Trp?Glu?Lys?Asp?Glu?Met?Thr?Lys?Asp?Cys?Met?Leu?Ala?Asn?Gly

1400 1405 1410

Lys?Leu?Asp?Glu?Asp?Tyr?Glu?Glu?Glu?Asp?Glu?Glu?Glu?Glu?Ser

1415 1420 1425

Leu?Met?Trp?Arg?Ala?Pro?Lys?Glu?Glu?Ala?Asp?Tyr?Glu?Asp?Asp

1430 1435 1440

Phe?Leu?Glu?Tyr?Asp?Gln?Glu?His?Ile?Arg?Phe?Ile?Asp?Asn?Met

1445 1450 1455

Leu?Met?Gly?Ser?Gly?Ala?Phe?Val?Lys?Lys?Ile?Ser?Leu?Ser?Pro

1460 1465 1470

Phe?Ser?Thr?Thr?Asp?Ser?Ala?Tyr?Glu?Trp?Lys?Met?Pro?Lys?Lys

1475 1480 1485

Ser?Ser?Leu?Gly?Ser?Met?Pro?Phe?Ser?Ser?Asp?Phe?Glu?Asp?Phe

1490 1495 1500

Asp?Tyr?Ser?Ser?Trp?Asp?Ala?Met?Cys?Tyr?Leu?Asp?Pro?Ser?Lys

1505 1510 1515

Ala?Val?Glu?Glu?Asp?Asp?Phe?Val?Val?Gly?Phe?Trp?Asn?Pro?Ser

1520 1525 1530

Glu?Glu?Asn?Cys?Gly?Val?Asp?Thr?Gly?Lys?Gln?Ser?Ile?Ser?Tyr

1535 1540 1545

Asp?Leu?His?Thr?Glu?Gln?Cys?Ile?Ala?Asp?Lys?Ser?Ile?Ala?Asp

1550 1555 1560

Cys?Val?Glu?Ala?Leu?Leu?Gly?Cys?Tyr?Leu?Thr?Ser?Cys?Gly?Glu

1565 1570 1575

Arg?Ala?Ala?Gln?Leu?Phe?Leu?Cys?Ser?Leu?Gly?Leu?Lys?Val?Leu

1580 1585 1590

Pro?Val?Ile?Lys?Arg?Thr?Asp?Arg?Glu?Lys?Ala?Leu?Cys?Pro?Thr

1595 1600 1605

Arg?Glu?Asn?Phe?Asn?Ser?Gln?Gln?Lys?Asn?Leu?Ser?Val?Ser?Cys

1610 1615 1620

Ala?Ala?Ala?Ser?Val?Ala?Ser?Ser?Arg?Ser?Ser?Val?Leu?Lys?Asp

1625 1630 1635

Ser?Glu?Tyr?Gly?Cys?Leu?Lys?Ile?Pro?Pro?Arg?Cys?Met?Phe?Asp

1640 1645 1650

His?Pro?Asp?Ala?Asp?Lys?Thr?Leu?Asn?His?Leu?Ile?Ser?Gly?Phe

1655 1660 1665

Glu?Asn?Phe?Glu?Lys?Lys?Ile?Asn?Tyr?Arg?Phe?Lys?Asn?Lys?Ala

1670 1675 1680

Tyr?Leu?Leu?Gln?Ala?Phe?Thr?His?Ala?Ser?Tyr?His?Tyr?Asn?Thr

1685 1690 1695

Ile?Thr?Asp?Cys?Tyr?Gln?Arg?Leu?Glu?Phe?Leu?Gly?Asp?Ala?Ile

1700 1705 1710

Leu?Asp?Tyr?Leu?Ile?Thr?Lys?His?Leu?Tyr?Glu?Asp?Pro?Arg?Gln

1715 1720 1725

His?Ser?Pro?Gly?Val?Leu?Thr?Asp?Leu?Arg?Ser?Ala?Leu?Val?Asn

1730 1735 1740

Asn?Thr?Ile?Phe?Ala?Ser?Leu?Ala?Val?Lys?Tyr?Asp?Tyr?His?Lys

1745 1750 1755

Tyr?Phe?Lys?Ala?Val?Ser?Pro?Glu?Leu?Phe?His?Val?Ile?Asp?Asp

1760 1765 1770

Phe?Val?Gln?Phe?Gln?Leu?Glu?Lys?Asn?Glu?Met?Gln?Gly?Met?Asp

1775 1780 1785

Ser?Glu?Leu?Arg?Arg?Ser?Glu?Glu?Asp?Glu?Glu?Lys?Glu?Glu?Asp

1790 1795 1800

Ile?Glu?Val?Pro?Lys?Ala?Met?Gly?Asp?Ile?Phe?Glu?Ser?Leu?Ala

1805 1810 1815

Gly?Ala?Ile?Tyr?Met?Asp?Ser?Gly?Met?Ser?Leu?Glu?Thr?Val?Trp

1820 1825 1830

Gln?Val?Tyr?Tyr?Pro?Met?Met?Arg?Pro?Leu?Ile?Glu?Lys?Phe?Ser

1835 1840 1845

Ala?Asn?Val?Pro?Arg?Ser?Pro?Val?Arg?Glu?Leu?Leu?Glu?Met?Glu

1850 1855 1860

Pro?Glu?Thr?Ala?Lys?Phe?Ser?Pro?Ala?Glu?Arg?Thr?Tyr?Asp?Gly

1865 1870 1875

Lys?Val?Arg?Val?Thr?Val?Glu?Val?Val?Gly?Lys?Gly?Lys?Phe?Lys

1880 1885 1890

Gly?Val?Gly?Arg?Ser?Tyr?Arg?Ile?Ala?Lys?Ser?Ala?Ala?Ala?Arg

1895 1900 1905

Arg?Ala?Leu?Arg?Ser?Leu?Lys?Ala?Asn?Gln?Pro?Gln?Val?Pro?Asn

1910 1915 1920

Ser

<210>4

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the synthetic primer 5 of the gene of amplification coding people dicer mutant

<400>4

tcgagctcgg?tacccgcctc?cattgttggt?ccacca 36

<210>5

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the synthetic primer 6 of the gene of amplification coding people dicer mutant

<400>5

tatctagaaa?gcttttagct?attgggaacc?tgaggt 36

<210>6

<211>3741

<212>DNA

＜213〉artificial sequence

<220>

＜223〉gene of coding people dicer mutant

<400>6

gcctccattg?ttggtccacc?aatgagctgt?gtacgattgg?ctgaaagagt?tgtcgctctc 60

atttgctgtg?agaaactgca?caaaattggc?gaactggatg?accatttgat?gccagttggg 120

aaagagactg?ttaaatatga?agaggagctt?gatttgcatg?atgaagaaga?gaccagtgtt 180

ccaggaagac?caggttccac?gaaacgaagg?cagtgctacc?caaaagcaat?tccagagtgt 240

ttgagggata?gttatcccag?acctgatcag?ccctgttacc?tgtatgtgat?aggaatggtt 300

ttaactacac?ctttacctga?tgaactcaac?tttagaaggc?ggaagctcta?tcctcctgaa 360

gataccacaa?gatgctttgg?aatactgacg?gccaaaccca?tacctcagat?tccacacttt 420

cctgtgtaca?cacgctctgg?agaggttacc?atatccattg?agttgaagaa?gtctggtttc 480

atgttgtctc?tacaaatgct?tgagttgatt?acaagacttc?accagtatat?attctcacat 540

attcttcggc?ttgaaaaacc?tgcactagaa?tttaaaccta?cagacgctga?ttcagcatac 600

tgtgttctac?ctcttaatgt?tgttaatgac?tccagcactt?tggatattga?ctttaaattc 660

atggaagata?ttgagaagtc?tgaagctcgc?ataggcattc?ccagtacaaa?gtatacaaaa 720

gaaacaccct?ttgtttttaa?attagaagat?taccaagatg?ccgttatcat?tccaagatat 780

cgcaattttg?atcagcctca?tcgattttat?gtagctgatg?tgtacactga?tcttacccca 840

ctcagtaaat?ttccttcccc?tgagtatgaa?acttttgcag?aatattataa?aacaaagtac 900

aaccttgacc?taaccaatct?caaccagcca?ctgctggatg?tggaccacac?atcttcaaga 960

cttaatcttt?tgacacctcg?acatttgaat?cagaagggga?aagcgcttcc?tttaagcagt 1020

gctgagaaga?ggaaagccaa?atgggaaagt?ctgcagaata?aacagatact?ggttccagaa 1080

ctctgtgcta?tacatccaat?tccagcatca?ctgtggagaa?aagctgtttg?tctccccagc 1140

atactttatc?gccttcactg?ccttttgact?gcagaggagc?taagagccca?gactgccagc 1200

gatgctggcg?tgggagtcag?atcacttcct?gcggatttta?gataccctaa?cttagacttc 1260

gggtggaaaa?aatctattga?cagcaaatct?ttcatctcaa?tttctaactc?ctcttcagct 1320

gaaaatgata?attactgtaa?gcacagcaca?attgtccctg?aaaatgctgc?acatcaaggt 1380

gctaatagaa?cctcctctct?agaaaatcat?gaccaaatgt?ctgtgaactg?cagaacgttg 1440

ctcagcgagt?cccctggtaa?gctccacgtt?gaagtttcag?cagatcttac?agcaattaat 1500

ggtctttctt?acaatcaaaa?tctcgccaat?ggcagttatg?atttagctaa?cagagacttt 1560

tgccaaggaa?atcagctaaa?ttactacaag?caggaaatac?ccgtgcaacc?aactacctca 1620

tattccattc?agaatttata?cagttacgag?aaccagcccc?agcccagcga?tgaatgtact 1680

ctcctgagta?ataaatacct?tgatggaaat?gctaacaaat?ctacctcaga?tggaagtcct 1740

gtgatggccg?taatgcctgg?tacgacagac?actattcaag?tgctcaaggg?caggatggat 1800

tctgagcaga?gcccttctat?tgggtactcc?tcaaggactc?ttggccccaa?tcctggactt 1860

attcttcagg?ctttgactct?gtcaaacgct?agtgatggat?ttaacctgga?gcggcttgaa 1920

atgcttggcg?actccttttt?aaagcatgcc?atcaccacat?atctattttg?cacttaccct 1980

gatgcgcatg?agggccgcct?ttcatatatg?agaagcaaaa?aggtcagcaa?ctgtaatctg 2040

tatcgccttg?gaaaaaagaa?gggactaccc?agccgcatgg?tggtgtcaat?atttgatccc 2100

cctgtgaatt?ggcttcctcc?tggttatgta?gtaaatcaag?acaaaagcaa?cacagataaa 2160

tgggaaaaag?atgaaatgac?aaaagactgc?atgctggcga?atggcaaact?ggatgaggat 2220

tacgaggagg?aggatgagga?ggaggagagc?ctgatgtgga?gggctccgaa?ggaagaggct 2280

gactatgaag?atgatttcct?ggagtatgat?caggaacata?tcagatttat?agataatatg 2340

ttaatggggt?caggagcttt?tgtaaagaaa?atctctcttt?ctcctttttc?aaccactgat 2400

tctgcatatg?aatggaaaat?gcccaaaaaa?tcctccttag?gtagtatgcc?attttcatca 2460

gattttgagg?attttgacta?cagctcttgg?gatgcaatgt?gctatctgga?tcctagcaaa 2520

gctgttgaag?aagatgactt?tgtggtgggg?ttctggaatc?catcagaaga?aaactgtggt 2580

gttgacacgg?gaaagcagtc?catttcttac?gacttgcaca?ctgagcagtg?tattgctgac 2640

aaaagcatag?cggactgtgt?ggaagccctg?ctgggctgct?atttaaccag?ctgtggggag 2700

agggctgctc?agcttttcct?ctgttcactg?gggctgaagg?tgctcccggt?aattaaaagg 2760

actgatcggg?aaaaggccct?gtgccctact?cgggagaatt?tcaacagcca?acaaaagaac 2820

ctttcagtga?gctgtgctgc?tgcttctgtg?gccagttcac?gctcttctgt?attgaaagac 2880

tcggaatatg?gttgtttgaa?gattccacca?agatgtatgt?ttgatcatcc?agatgcagat 2940

aaaacactga?atcaccttat?atcggggttt?gaaaattttg?aaaagaaaat?caactacaga 3000

ttcaagaata?aggcttacct?tctccaggct?tttacacatg?cctcctacca?ctacaatact 3060

atcactgatt?gttaccagcg?cttagaattc?ctgggagatg?cgattttgga?ctacctcata 3120

accaagcacc?tttatgaaga?cccgcggcag?cactccccgg?gggtcctgac?agacctgcgg 3180

tctgccctgg?tcaacaacac?catctttgca?tcgctggctg?taaagtacga?ctaccacaag 3240

tacttcaaag?ctgtctctcc?tgagctcttc?catgtcattg?atgactttgt?gcagtttcag 3300

cttgagaaga?atgaaatgca?aggaatggat?tctgagctta?ggagatctga?ggaggatgaa 3360

gagaaagaag?aggatattga?agttccaaag?gccatggggg?atatttttga?gtcgcttgct 3420

ggtgccattt?acatggatag?tgggatgtca?ctggagacag?tctggcaggt?gtactatccc 3480

atgatgcggc?cactaataga?aaagttttct?gcaaatgtac?cccgttcccc?tgtgcgagaa 3540

ttgcttgaaa?tggaaccaga?aactgccaaa?tttagcccgg?ctgagagaac?ttacgacggg 3600

aaggtcagag?tcactgtgga?agtagtagga?aaggggaaat?ttaaaggtgt?tggtcgaagt 3660

tacaggattg?ccaaatctgc?agcagcaaga?agagccctcc?gaagcctcaa?agctaatcaa 3720

cctcaggttc?ccaatagcta?a 3741

<210>7

<211>1246

<212>PRT

＜213〉artificial sequence

<220>

＜223〉aminoacid sequence of people dicer mutant

<400>7

Ala?Ser?Ile?Val?Gly?Pro?Pro?Met?Ser?Cys?Val?Arg?Leu?Ala?Glu?Arg

1 5 10 15

Val?Val?Ala?Leu?Ile?Cys?Cys?Glu?Lys?Leu?His?Lys?Ile?Gly?Glu?Leu

20 25 30

Asp?Asp?His?Leu?Met?Pro?Val?Gly?Lys?Glu?Thr?Val?Lys?Tyr?Glu?Glu

35 40 45

Glu?Leu?Asp?Leu?His?Asp?Glu?Glu?Glu?Thr?Ser?Val?Pro?Gly?Arg?Pro

50 55 60

Gly?Ser?Thr?Lys?Arg?Arg?Gln?Cys?Tyr?Pro?Lys?Ala?Ile?Pro?Glu?Cys

65 70 75 80

Leu?Arg?Asp?Ser?Tyr?Pro?Arg?Pro?Asp?Gln?Pro?Cys?Tyr?Leu?Tyr?Val

85 90 95

Ile?Gly?Met?Val?Leu?Thr?Thr?Pro?Leu?Pro?Asp?Glu?Leu?Asn?Phe?Arg

100 105 110

Arg?Arg?Lys?Leu?Tyr?Pro?Pro?Glu?Asp?Thr?Thr?Arg?Cys?Phe?Gly?Ile

115 120 125

Leu?Thr?Ala?Lys?Pro?Ile?Pro?Gln?Ile?Pro?His?The?Pro?Val?Tyr?Thr

130 135 140

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

145 150 155 160

Met?Leu?Ser?Leu?Gln?Met?Leu?Glu?Leu?Ile?Thr?Arg?Leu?His?Gln?Tyr

165 170 175

Ile?Phe?Ser?His?Ile?Leu?Arg?Leu?Glu?Lys?Pro?Ala?Leu?Glu?Phe?Lys

180 185 190

Pro?Thr?Asp?Ala?Asp?Ser?Ala?Tyr?Cys?Val?Leu?Pro?Leu?Asn?Val?Val

195 200 205

Asn?Asp?Ser?Ser?Thr?Leu?Asp?Ile?Asp?Phe?Lys?Phe?Met?Glu?Asp?Ile

210 215 220

Glu?Lys?Ser?Glu?Ala?Arg?Ile?Gly?Ile?Pro?Ser?Thr?Lys?Tyr?Thr?Lys

225 230 235 240

Glu?Thr?Pro?Phe?Val?Phe?Lys?Leu?Glu?Asp?Tyr?Gln?Asp?Ala?Val?Ile

245 250 255

Ile?Pro?Arg?Tyr?Arg?Asn?Phe?Asp?Gln?Pro?His?Arg?Phe?Tyr?Val?Ala

260 265 270

Asp?Val?Tyr?Thr?Asp?Leu?Thr?Pro?Leu?Ser?Lys?Phe?Pro?Ser?Pro?Glu

275 280 285

Tyr?Glu?Thr?Phe?Ala?Glu?Tyr?Tyr?Lys?Thr?Lys?Tyr?Asn?Leu?Asp?Leu

290 295 300

Thr?Asn?Leu?Asn?Gln?Pro?Leu?Leu?Asp?Val?Asp?His?Thr?Ser?Ser?Arg

305 310 315 320

Leu?Asn?Leu?Leu?Thr?Pro?Arg?His?Leu?Asn?Gln?Lys?Gly?Lys?Ala?Leu

325 330 335

Pro?Leu?Ser?Ser?Ala?Glu?Lys?Arg?Lys?Ala?Lys?Trp?Glu?Ser?Leu?Gln

340 345 350

Asn?Lys?Gln?Ile?Leu?Val?Pro?Glu?Leu?Cys?Ala?Ile?His?Pro?Ile?Pro

355 360 365

Ala?Ser?Leu?Trp?Arg?Lys?Ala?Val?Cys?Leu?Pro?Ser?Ile?Leu?Tyr?Arg

370 375 380

Leu?His?Cys?Leu?Leu?Thr?Ala?Glu?Glu?Leu?Arg?Ala?Gln?Thr?Ala?Ser

385 390 395 400

Asp?Ala?Gly?Val?Gly?Val?Arg?Ser?Leu?Pro?Ala?Asp?Phe?Arg?Tyr?Pro

405 410 415

Asn?Leu?Asp?Phe?Gly?Trp?Lys?Lys?Ser?Ile?Asp?Ser?Lys?Ser?Phe?Ile

420 425 430

Ser?Ile?Ser?Asn?Ser?Ser?Ser?Ala?Glu?Asn?Asp?Asn?Tyr?Cys?Lys?His

435 440 445

Ser?Thr?Ile?Val?Pro?Glu?Asn?Ala?Ala?His?Gln?Gly?Ala?Asn?Arg?Thr

450 455 460

Ser?Ser?Leu?Glu?Asn?His?Asp?Gln?Met?Ser?Val?Asn?Cys?Arg?Thr?Leu

465 470 475 480

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

485 490 495

Thr?Ala?Ile?Asn?Gly?Leu?Ser?Tyr?Asn?Gln?Asn?Leu?Ala?Asn?Gly?Ser

500 505 510

Tyr?Asp?Leu?Ala?Asn?Arg?Asp?Phe?Cys?Gln?Gly?Asn?Gln?Leu?Asn?Tyr

515 520 525

Tyr?Lys?Gln?Glu?Ile?Pro?Val?Gln?Pro?Thr?Thr?Ser?Tyr?Ser?Ile?Gln

530 535 540

Asn?Leu?Tyr?Ser?Tyr?Glu?Asn?Gln?Pro?Gln?Pro?Ser?Asp?Glu?Cys?Thr

545 550 555 560

Leu?Leu?Ser?Asn?Lys?Tyr?Leu?Asp?Gly?Asn?Ala?Asn?Lys?Ser?Thr?Ser

565 570 575

Asp?Gly?Ser?Pro?Val?Met?Ala?Val?Met?Pro?Gly?Thr?Thr?Asp?Thr?Ile

580 585 590

Gln?Val?Leu?Lys?Gly?Arg?Met?Asp?Ser?Glu?Gln?Ser?Pro?Ser?Ile?Gly

595 600 605

Tyr?Ser?Ser?Arg?Thr?Leu?Gly?Pro?Asn?Pro?Gly?Leu?Ile?Leu?Gln?Ala

610 615 620

Leu?Thr?Leu?Ser?Asn?Ala?Ser?Asp?Gly?Phe?Asn?Leu?Glu?Arg?Leu?Glu

625 630 635 640

Met?Leu?Gly?Asp?Ser?Phe?Leu?Lys?His?Ala?Ile?Thr?Thr?Tyr?Leu?Phe

645 650 655

Cys?Thr?Tyr?Pro?Asp?Ala?His?Glu?Gly?Arg?Leu?Ser?Tyr?Met?Arg?Ser

660 665 670

Lys?Lys?Val?Ser?Asn?Cys?Asn?Leu?Tyr?Arg?Leu?Gly?Lys?Lys?Lys?Gly

675 680 685

Leu?Pro?Ser?Arg?Met?Val?Val?Ser?Ile?Phe?Asp?Pro?Pro?Val?Asn?Trp

690 695 700

Leu?Pro?Pro?Gly?Tyr?Val?Val?Asn?Gln?Asp?Lys?Ser?Asn?Thr?Asp?Lys

705 710 715 720

Trp?Glu?Lys?Asp?Glu?Met?Thr?Lys?Asp?Cys?Met?Leu?Ala?Asn?Gly?Lys

725 730 735

Leu?Asp?Glu?Asp?Tyr?Glu?Glu?Glu?Asp?Glu?Glu?Glu?Glu?Ser?Leu?Met

740 745 750

Trp?Arg?Ala?Pro?Lys?Glu?Glu?Ala?Asp?Tyr?Glu?Asp?Asp?Phe?Leu?Glu

755 760 765

Tyr?Asp?Gln?Glu?His?Ile?Arg?Phe?Ile?Asp?Asn?Met?Leu?Met?Gly?Ser

770 775 780

Gly?Ala?Phe?Val?Lys?Lys?Ile?Ser?Leu?Ser?Pro?Phe?Ser?Thr?Thr?Asp

785 790 795 800

Ser?Ala?Tyr?Glu?Trp?Lys?Met?Pro?Lys?Lys?Ser?Ser?Leu?Gly?Ser?Met

805 810 815

Pro?Phe?Ser?Ser?Asp?Phe?Glu?Asp?Phe?Asp?Tyr?Ser?Ser?Trp?Asp?Ala

820 825 830

Met?Cys?Tyr?Leu?Asp?Pro?Ser?Lys?Ala?Val?Glu?Glu?Asp?Asp?Phe?Val

835 840 845

Val?Gly?Phe?Trp?Asn?Pro?Ser?Glu?Glu?Asn?Cys?Gly?Val?Asp?Thr?Gly

850 855 860

Lys?Gln?Ser?Ile?Ser?Tyr?Asp?Leu?His?Thr?Glu?Gln?Cys?Ile?Ala?Asp

865 870 875 880

Lys?Ser?Ile?Ala?Asp?Cys?Val?Glu?Ala?Leu?Leu?Gly?Cys?Tyr?Leu?Thr

885 890 895

Ser?Cys?Gly?Glu?Arg?Ala?Ala?Gln?Leu?Phe?Leu?Cys?Ser?Leu?Gly?Leu

900 905 910

Lys?Val?Leu?Pro?Val?Ile?Lys?Arg?Thr?Asp?Arg?Glu?Lys?Ala?Leu?Cys

915 920 925

Pro?Thr?Arg?Glu?Asn?Phe?Asn?Ser?Gln?Gln?Lys?Asn?Leu?Ser?Val?Ser

930 935 940

Cys?Ala?Ala?Ala?Ser?Val?Ala?Ser?Ser?Arg?Ser?Ser?Val?Leu?Lys?Asp

945 950 955 960

Ser?Glu?Tyr?Gly?Cys?Leu?Lys?Ile?Pro?Pro?Arg?Cys?Met?Phe?Asp?His

965 970 975

Pro?Asp?Ala?Asp?Lys?Thr?Leu?Asn?His?Leu?Ile?Ser?Gly?Phe?Glu?Asn

980 985 990

Phe?Glu?Lys?Lys?Ile?Asn?Tyr?Arg?Phe?Lys?Asn?Lys?Ala?Tyr?Leu?Leu

995 1000 1005

Gln?Ala?Phe?Thr?His?Ala?Ser?Tyr?His?Tyr?Asn?Thr?Ile?Thr?Asp

1010 1015 1020

Cys?Tyr?Gln?Arg?Leu?Glu?Phe?Leu?Gly?Asp?Ala?Ile?Leu?Asp?Tyr

1025 1030 1035

Leu?Ile?Thr?Lys?His?Leu?Tyr?Glu?Asp?Pro?Arg?Gln?His?Ser?Pro

1040 1045 1050

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

1055 1060 1065

Phe?Ala?Ser?Leu?Ala?Val?Lys?Tyr?Asp?Tyr?His?Lys?Tyr?Phe?Lys

1070 1075 1080

Ala?Val?Ser?Pro?Glu?Leu?Phe?His?Val?Ile?Asp?Asp?Phe?Val?Gln

1085 1090 1095

Phe?Gln?Leu?Glu?Lys?Asn?Glu?Met?Gln?Gly?Met?Asp?Ser?Glu?Leu

1100 1105 1110

Arg?Arg?Ser?Glu?Glu?Asp?Glu?Glu?Lys?Glu?Glu?Asp?Ile?Glu?Val

1115 1120 1125

Pro?Lys?Ala?Met?Gly?Asp?Ile?Phe?Glu?Ser?Leu?Ala?Gly?Ala?Ile

1130 1135 1140

Tyr?Met?Asp?Ser?Gly?Met?Ser?Leu?Glu?Thr?Val?Trp?Gln?Val?Tyr

1145 1150 1155

Tyr?Pro?Met?Met?Arg?Pro?Leu?Ile?Glu?Lys?Phe?Ser?Ala?Asn?Val

1160 1165 1170

Pro?Arg?Ser?Pro?Val?Arg?Glu?Leu?Leu?Glu?Met?Glu?Pro?Glu?Thr

1175 1180 1185

Ala?Lys?Phe?Ser?Pro?Ala?Glu?Arg?Thr?Tyr?Asp?Gly?Lys?Val?Arg

1190 1195 1200

Val?Thr?Val?Glu?Val?Val?Gly?Lys?Gly?Lys?Phe?Lys?Gly?Val?Gly

1205 1210 1215

Arg?Ser?Tyr?Arg?Ile?Ala?Lys?Ser?Ala?Ala?Ala?Arg?Arg?Ala?Leu

1220 1225 1230

Arg?Ser?Leu?Lys?Ala?Asn?Gln?Pro?Gln?Val?Pro?Asn?Ser

1235 1240 1245

<210>8

<211>1267

<212>PRT

＜213〉artificial sequence

<220>

＜223〉aminoacid sequence of people dicer mutant

<400>8

Met?Asn?His?Lys?Val?His?His?His?His?His?His?Ile?Glu?Gly?Arg?Asn

1 5 10 15

Ser?Ser?Ser?Val?Pro?Ala?Ser?Ile?Val?Gly?Pro?Pro?Met?Ser?Cys?Val

20 25 30

Arg?Leu?Ala?Glu?Arg?Val?Val?Ala?Leu?Ile?Cys?Cys?Glu?Lys?Leu?His

35 40 45

Lys?Ile?Gly?Glu?Leu?Asp?Asp?His?Leu?Met?Pro?Val?Gly?Lys?Glu?Thr

50 55 60

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

65 70 75 80

Val?Pro?Gly?Arg?Pro?Gly?Ser?Thr?Lys?Arg?Arg?Gln?Cys?Tyr?Pro?Lys

85 90 95

Ala?Ile?Pro?Glu?Cys?Leu?Arg?Asp?Ser?Tyr?Pro?Arg?Pro?Asp?Gln?Pro

100 105 110

Cys?Tyr?Leu?Tyr?Val?Ile?Gly?Met?Val?Leu?Thr?Thr?Pro?Leu?Pro?Asp

115 120 125

Glu?Leu?Asn?Phe?Arg?Arg?Arg?Lys?Leu?Tyr?Pro?Pro?Glu?Asp?Thr?Thr

130 135 140

Arg?Cys?Phe?Gly?Ile?Leu?Thr?Ala?Lys?Pro?Ile?Pro?Gln?Ile?Pro?His

145 150 155 160

Phe?Pro?Val?Tyr?Thr?Arg?Ser?Gly?Glu?Val?Thr?Ile?Ser?Ile?Glu?Leu

165 170 175

Lys?Lys?Ser?Gly?Phe?Met?Leu?Ser?Leu?Gln?Met?Leu?Glu?Leu?Ile?Thr

180 185 190

Arg?Leu?His?Gln?Tyr?Ile?Phe?Ser?His?Ile?Leu?Arg?Leu?Glu?Lys?Pro

195 200 205

Ala?Leu?Glu?Phe?Lys?Pro?Thr?Asp?Ala?Asp?Ser?Ala?Tyr?Cys?Val?Leu

210 215 220

Pro?Leu?Asn?Val?Val?Asn?Asp?Ser?Ser?Thr?Leu?Asp?Ile?Asp?Phe?Lys

225 230 235 240

Phe?Met?Glu?Asp?Ile?Glu?Lys?Ser?Glu?Ala?Arg?Ile?Gly?Ile?Pro?Ser

245 250 255

Thr?Lys?Tyr?Thr?Lys?Glu?Thr?Pro?Phe?Val?Phe?Lys?Leu?Glu?Asp?Tyr

260 265 270

Gln?Asp?Ala?Val?Ile?Ile?Pro?Arg?Tyr?Arg?Asn?Phe?Asp?Gln?Pro?His

275 280 285

Arg?Phe?Tyr?Val?Ala?Asp?Val?Tyr?Thr?Asp?Leu?Thr?Pro?Leu?Ser?Lys

290 295 300

Phe?Pro?Ser?Pro?Glu?Tyr?Glu?Thr?Phe?Ala?Glu?Tyr?Tyr?Lys?Thr?Lys

305 310 315 320

Tyr?Asn?Leu?Asp?Leu?Thr?Asn?Leu?Asn?Gln?Pro?Leu?Leu?Asp?Val?Asp

325 330 335

His?Thr?Ser?Ser?Arg?Leu?Asn?Leu?Leu?Thr?Pro?Arg?His?Leu?Asn?Gln

340 345 350

Lys?Gly?Lys?Ala?Leu?Pro?Leu?Ser?Ser?Ala?Glu?Lys?Arg?Lys?Ala?Lys

355 360 365

Trp?Glu?Ser?Leu?Gln?Asn?Lys?Gln?Ile?Leu?Val?Pro?Glu?Leu?Cys?Ala

370 375 380

Ile?His?Pro?Ile?Pro?Ala?Ser?Leu?Trp?Arg?Lys?Ala?Val?Cys?Leu?Pro

385 390 395 400

Ser?Ile?Leu?Tyr?Arg?Leu?His?Cys?Leu?Leu?Thr?Ala?Glu?Glu?Leu?Arg

405 410 415

Ala?Gln?Thr?Ala?Ser?Asp?Ala?Gly?Val?Gly?Val?Arg?Ser?Leu?Pro?Ala

420 425 430

Asp?Phe?Arg?Tyr?Pro?Asn?Leu?Asp?Phe?Gly?Trp?Lys?Lys?Ser?Ile?Asp

435 440 445

Ser?Lys?Ser?Phe?Ile?Ser?Ile?Ser?Asn?Ser?Ser?Ser?Ala?Glu?Asn?Asp

450 455 460

Asn?Tyr?Cys?Lys?His?Ser?Thr?Ile?Val?Pro?Glu?Asn?Ala?Ala?His?Gln

465 470 475 480

Gly?Ala?Asn?Arg?Thr?Ser?Ser?Leu?Glu?Asn?His?Asp?Gln?Met?Ser?Val

485 490 495

Asn?Cys?Arg?Thr?Leu?Leu?Ser?Glu?Ser?Pro?Gly?Lys?Leu?His?Val?Glu

500 505 510

Val?Ser?Ala?Asp?Leu?Thr?Ala?Ile?Asn?Gly?Leu?Ser?Tyr?Asn?Gln?Asn

515 520 525

Leu?Ala?Asn?Gly?Ser?Tyr?Asp?Leu?Ala?Asn?Arg?Asp?Phe?Cys?Gln?Gly

530 535 540

Asn?Gln?Leu?Asn?Tyr?Tyr?Lys?Gln?Glu?Ile?Pro?Val?Gln?Pro?Thr?Thr

545 550 555 560

Ser?Tyr?Ser?Ile?Gln?Asn?Leu?Tyr?Ser?Tyr?Glu?Asn?Gln?Pro?Gln?Pro

565 570 575

Ser?Asp?Glu?Cys?Thr?Leu?Leu?Ser?Asn?Lys?Tyr?Leu?Asp?Gly?Asn?Ala

580 585 590

Asn?Lys?Ser?Thr?Ser?Asp?Gly?Ser?Pro?Val?Met?Ala?Val?Met?Pro?Gly

595 600 605

Thr?Thr?Asp?Thr?Ile?Gln?Val?Leu?Lys?Gly?Arg?Met?Asp?Ser?Glu?Gln

610 615 620

Ser?Pro?Ser?Ile?Gly?Tyr?Ser?Ser?Arg?Thr?Leu?Gly?Pro?Asn?Pro?Gly

625 630 635 640

Leu?Ile?Leu?Gln?Ala?Leu?Thr?Leu?Ser?Asn?Ala?Ser?Asp?Gly?Phe?Asn

645 650 655

Leu?Glu?Arg?Leu?Glu?Met?Leu?Gly?Asp?Ser?Phe?Leu?Lys?His?Ala?Ile

660 665 670

Thr?Thr?Tyr?Leu?Phe?Cys?Thr?Tyr?Pro?Asp?Ala?His?Glu?Gly?Arg?Leu

675 680 685

Ser?Tyr?Met?Arg?Ser?Lys?Lys?Val?Ser?Asn?Cys?Asn?Leu?Tyr?Arg?Leu

690 695 700

Gly?Lys?Lys?Lys?Gly?Leu?Pro?Ser?Arg?Met?Val?Val?Ser?Ile?Phe?Asp

705 710 715 720

Pro?Pro?Val?Asn?Trp?Leu?Pro?Pro?Gly?Tyr?Val?Val?Asn?Gln?Asp?Lys

725 730 735

Ser?Asn?Thr?Asp?Lys?Trp?Glu?Lys?Asp?Glu?Met?Thr?Lys?Asp?Cys?Met

740 745 750

Leu?Ala?Asn?Gly?Lys?Leu?Asp?Glu?Asp?Tyr?Glu?Glu?Glu?Asp?Glu?Glu

755 760 765

Glu?Glu?Ser?Leu?Met?Trp?Arg?Ala?Pro?Lys?Glu?Glu?Ala?Asp?Tyr?Glu

770 775 780

Asp?Asp?Phe?Leu?Glu?Tyr?Asp?Gln?Glu?His?Ile?Arg?Phe?Ile?Asp?Asn

785 790 795 800

Met?Leu?Met?Gly?Ser?Gly?Ala?Phe?Val?Lys?Lys?Ile?Ser?Leu?Ser?Pro

805 810 815

Phe?Ser?Thr?Thr?Asp?Ser?Ala?Tyr?Glu?Trp?Lys?Met?Pro?Lys?Lys?Ser

820 825 830

Ser?Leu?Gly?Ser?Met?Pro?Phe?Ser?Ser?Asp?Phe?Glu?Asp?Phe?Asp?Tyr

835 840 845

Ser?Ser?Trp?Asp?Ala?Met?Cys?Tyr?Leu?Asp?Pro?Ser?Lys?Ala?Val?Glu

850 855 860

Glu?Asp?Asp?Phe?Val?Val?Gly?Phe?Trp?Asn?Pro?Ser?Glu?Glu?Asn?Cys

865 870 875 880

Gly?Val?Asp?Thr?Gly?Lys?Gln?Ser?Ile?Ser?Tyr?Asp?Leu?His?Thr?Glu

885 890 895

Gln?Cys?Ile?Ala?Asp?Lys?Ser?Ile?Ala?Asp?Cys?Val?Glu?Ala?Leu?Leu

900 905 910

Gly?Cys?Tyr?Leu?Thr?Ser?Cys?Gly?Glu?Arg?Ala?Ala?Gln?Leu?Phe?Leu

915 920 925

Cys?Ser?Leu?Gly?Leu?Lys?Val?Leu?Pro?Val?Ile?Lys?Arg?Thr?Asp?Arg

930 935 940

Glu?Lys?Ala?Leu?Cys?Pro?Thr?Arg?Glu?Asn?Phe?Asn?Ser?Gln?Gln?Lys

945 950 955 960

Asn?Leu?Ser?Val?Ser?Cys?Ala?Ala?Ala?Ser?Val?Ala?Ser?Ser?Arg?Ser

965 970 975

Ser?Val?Leu?Lys?Asp?Ser?Glu?Tyr?Gly?Cys?Leu?Lys?Ile?Pro?Pro?Arg

980 985 990

Cys?Met?Phe?Asp?His?Pro?Asp?Ala?Asp?Lys?Thr?Leu?Asn?His?Leu?Ile

995 1000 1005

Ser?Gly?Phe?Glu?Asn?Phe?Glu?Lys?Lys?Ile?Asn?Tyr?Arg?Phe?Lys

1010 1015 1020

Asn?Lys?Ala?Tyr?Leu?Leu?Gln?Ala?Phe?Thr?His?Ala?Ser?Tyr?His

1025 1030 1035

Tyr?Asn?Thr?Ile?Thr?Asp?Cys?Tyr?Gln?Arg?Leu?Glu?Phe?Leu?Gly

1040 1045 1050

Asp?Ala?Ile?Leu?Asp?Tyr?Leu?Ile?Thr?Lys?His?Leu?Tyr?Glu?Asp

1055 1060 1065

Pro?Arg?Gln?His?Ser?Pro?Gly?Val?Leu?Thr?Asp?Leu?Arg?Ser?Ala

1070 1075 1080

Leu?Val?Asn?Asn?Thr?Ile?Phe?Ala?Ser?Leu?Ala?Val?Lys?Tyr?Asp

1085 1090 1095

Tyr?His?Lys?Tyr?Phe?Lys?Ala?Val?Ser?Pro?Glu?Leu?Phe?His?Val

1100 1105 1110

Ile?Asp?Asp?Phe?Val?Gln?Phe?Gln?Leu?Glu?Lys?Asn?Glu?Met?Gln

1115 1120 1125

Gly?Met?Asp?Ser?Glu?Leu?Arg?Arg?Ser?Glu?Glu?Asp?Glu?Glu?Lys

1130 1135 1140

Glu?Glu?Asp?Ile?Glu?Val?Pro?Lys?Ala?Met?Gly?Asp?Ile?Phe?Glu

1145 1150 1155

Ser?Leu?Ala?Gly?Ala?Ile?Tyr?Met?Asp?Ser?Gly?Met?Ser?Leu?Glu

1160 1165 1170

Thr?Val?Trp?Gln?Val?Tyr?Tyr?Pro?Met?Met?Arg?Pro?Leu?Ile?Glu

1175 1180 1185

Lys?Phe?Ser?Ala?Asn?Val?Pro?Arg?Ser?Pro?Val?Arg?Glu?Leu?Leu

1190 1195 1200

Glu?Met?Glu?Pro?Glu?Thr?Ala?Lys?Phe?Ser?Pro?Ala?Glu?Arg?Thr

1205 1210 1215

Tyr?Asp?Gly?Lys?Val?Arg?Val?Thr?Val?Glu?Val?Val?Gly?Lys?Gly

1220 1225 1230

Lys?Phe?Lys?Gly?Val?Gly?Arg?Ser?Tyr?Arg?Ile?Ala?Lys?Ser?Ala

1235 1240 1245

Ala?Ala?Arg?Arg?Ala?Leu?Arg?Ser?Leu?Lys?Ala?Asn?Gln?Pro?Gln

1250 1255 1260

Val?Pro?Asn?Ser

1265

<210>9

<211>3804

<212>DNA

＜213〉artificial sequence

<220>

＜223〉gene of coding people dicer mutant

<400>9

atgaatcaca?aagtgcatca?tcatcatcat?catatcgaag?gtaggaattc?gagctcggta 60

cccgcctcca?ttgttggtcc?accaatgagc?tgtgtacgat?tggctgaaag?agttgtcgct 120

ctcatttgct?gtgagaaact?gcacaaaatt?ggcgaactgg?atgaccattt?gatgccagtt 180

gggaaagaga?ctgttaaata?tgaagaggag?cttgatttgc?atgatgaaga?agagaccagt 240

gttccaggaa?gaccaggttc?cacgaaacga?aggcagtgct?acccaaaagc?aattccagag 300

tgtttgaggg?atagttatcc?cagacctgat?cagccctgtt?acctgtatgt?gataggaatg 360

gttttaacta?cacctttacc?tgatgaactc?aactttagaa?ggcggaagct?ctatcctcct 420

gaagatacca?caagatgctt?tggaatactg?acggccaaac?ccatacctca?gattccacac 480

tttcctgtgt?acacacgctc?tggagaggtt?accatatcca?ttgagttgaa?gaagtctggt 540

ttcatgttgt?ctctacaaat?gcttgagttg?attacaagac?ttcaccagta?tatattctca 600

catattcttc?ggcttgaaaa?acctgcacta?gaatttaaac?ctacagacgc?tgattcagca 660

tactgtgttc?tacctcttaa?tgttgttaat?gactccagca?ctttggatat?tgactttaaa 720

ttcatggaag?atattgagaa?gtctgaagct?cgcataggca?ttcccagtac?aaagtataca 780

aaagaaacac?cctttgtttt?taaattagaa?gattaccaag?atgccgttat?cattccaaga 840

tatcgcaatt?ttgatcagcc?tcatcgattt?tatgtagctg?atgtgtacac?tgatcttacc 900

ccactcagta?aatttccttc?ccctgagtat?gaaacttttg?cagaatatta?taaaacaaag 960

tacaaccttg?acctaaccaa?tctcaaccag?ccactgctgg?atgtggacca?cacatcttca 1020

agacttaatc?ttttgacacc?tcgacatttg?aatcagaagg?ggaaagcgct?tcctttaagc 1080

agtgctgaga?agaggaaagc?caaatgggaa?agtctgcaga?ataaacagat?actggttcca 1140

gaactctgtg?ctatacatcc?aattccagca?tcactgtgga?gaaaagctgt?ttgtctcccc 1200

agcatacttt?atcgccttca?ctgccttttg?actgcagagg?agctaagagc?ccagactgcc 1260

agcgatgctg?gcgtgggagt?cagatcactt?cctgcggatt?ttagataccc?taacttagac 1320

ttcgggtgga?aaaaatctat?tgacagcaaa?tctttcatct?caatttctaa?ctcctcttca 1380

gctgaaaatg?ataattactg?taagcacagc?acaattgtcc?ctgaaaatgc?tgcacatcaa 1440

ggtgctaata?gaacctcctc?tctagaaaat?catgaccaaa?tgtctgtgaa?ctgcagaacg 1500

ttgctcagcg?agtcccctgg?taagctccac?gttgaagttt?cagcagatct?tacagcaatt 1560

aatggtcttt?cttacaatca?aaatctcgcc?aatggcagtt?atgatttagc?taacagagac 1620

ttttgccaag?gaaatcagct?aaattactac?aagcaggaaa?tacccgtgca?accaactacc 1680

tcatattcca?ttcagaattt?atacagttac?gagaaccagc?cccagcccag?cgatgaatgt 1740

actctcctga?gtaataaata?ccttgatgga?aatgctaaca?aatctacctc?agatggaagt 1800

cctgtgatgg?ccgtaatgcc?tggtacgaca?gacactattc?aagtgctcaa?gggcaggatg 1860

gattctgagc?agagcccttc?tattgggtac?tcctcaagga?ctcttggccc?caatcctgga 1920

cttattcttc?aggctttgac?tctgtcaaac?gctagtgatg?gatttaacct?ggagcggctt 1980

gaaatgcttg?gcgactcctt?tttaaagcat?gccatcacca?catatctatt?ttgcacttac 2040

cctgatgcgc?atgagggccg?cctttcatat?atgagaagca?aaaaggtcag?caactgtaat 2100

ctgtatcgcc?ttggaaaaaa?gaagggacta?cccagccgca?tggtggtgtc?aatatttgat 2160

ccccctgtga?attggcttcc?tcctggttat?gtagtaaatc?aagacaaaag?caacacagat 2220

aaatgggaaa?aagatgaaat?gacaaaagac?tgcatgctgg?cgaatggcaa?actggatgag 2280

gattacgagg?aggaggatga?ggaggaggag?agcctgatgt?ggagggctcc?gaaggaagag 2340

gctgactatg?aagatgattt?cctggagtat?gatcaggaac?atatcagatt?tatagataat 2400

atgttaatgg?ggtcaggagc?ttttgtaaag?aaaatctctc?tttctccttt?ttcaaccact 2460

gattctgcat?atgaatggaa?aatgcccaaa?aaatcctcct?taggtagtat?gccattttca 2520

tcagattttg?aggattttga?ctacagctct?tgggatgcaa?tgtgctatct?ggatcctagc 2580

aaagctgttg?aagaagatga?ctttgtggtg?gggttctgga?atccatcaga?agaaaactgt 2640

ggtgttgaca?cgggaaagca?gtccatttct?tacgacttgc?acactgagca?gtgtattgct 2700

gacaaaagca?tagcggactg?tgtggaagcc?ctgctgggct?gctatttaac?cagctgtggg 2760

gagagggctg?ctcagctttt?cctctgttca?ctggggctga?aggtgctccc?ggtaattaaa 2820

aggactgatc?gggaaaaggc?cctgtgccct?actcgggaga?atttcaacag?ccaacaaaag 2880

aacctttcag?tgagctgtgc?tgctgcttct?gtggccagtt?cacgctcttc?tgtattgaaa 2940

gactcggaat?atggttgttt?gaagattcca?ccaagatgta?tgtttgatca?tccagatgca 3000

gataaaacac?tgaatcacct?tatatcgggg?tttgaaaatt?ttgaaaagaa?aatcaactac 3060

agattcaaga?ataaggctta?ccttctccag?gcttttacac?atgcctccta?ccactacaat 3120

actatcactg?attgttacca?gcgcttagaa?ttcctgggag?atgcgatttt?ggactacctc 3180

ataaccaagc?acctttatga?agacccgcgg?cagcactccc?cgggggtcct?gacagacctg 3240

cggtctgccc?tggtcaacaa?caccatcttt?gcatcgctgg?ctgtaaagta?cgactaccac 3300

aagtacttca?aagctgtctc?tcctgagctc?ttccatgtca?ttgatgactt?tgtgcagttt 3360

cagcttgaga?agaatgaaat?gcaaggaatg?gattctgagc?ttaggagatc?tgaggaggat 3420

gaagagaaag?aagaggatat?tgaagttcca?aaggccatgg?gggatatttt?tgagtcgctt 3480

gctggtgcca?tttacatgga?tagtgggatg?tcactggaga?cagtctggca?ggtgtactat 3540

cccatgatgc?ggccactaat?agaaaagttt?tctgcaaatg?taccccgttc?ccctgtgcga 3600

gaattgcttg?aaatggaacc?agaaactgcc?aaatttagcc?cggctgagag?aacttacgac 3660

gggaaggtca?gagtcactgt?ggaagtagta?ggaaagggga?aatttaaagg?tgttggtcga 3720

agttacagga?ttgccaaatc?tgcagcagca?agaagagccc?tccgaagcct?caaagctaat 3780

caacctcagg?ttcccaatag?ctaa 3804

<210>10

<211>720

<212>DNA

＜213〉artificial sequence

<220>

＜223〉gene of coding red shift green fluorescent protein

<400>10

atggctagca?aaggagaaga?actcttcact?ggagttgtcc?caattcttgt?tgaattagat 60

ggtgatgtta?acggccacaa?gttctctgtc?agtggagagg?gtgaaggtga?tgcaacatac 120

ggaaaactta?ccctgaagtt?catctgcact?actggcaaac?tgcctgttcc?atggccaaca 180

ctagtcacta?ctctgtgcta?tggtgttcaa?tgcttttcaa?gatacccgga?tcatatgaaa 240

cggcatgact?ttttcaagag?tgccatgccc?gaaggttatg?tacaggaaag?gaccatcttc 300

ttcaaagatg?acggcaacta?caagacacgt?gctgaagtca?agtttgaagg?tgataccctt 360

gttaatagaa?tcgagttaaa?aggtattgac?ttcaaggaag?atggaaacat?tctgggacac 420

aaattggaat?acaactataa?ctcacacaat?gtatacatca?tggcagacaa?acaaaagaat 480

ggaatcaaag?tgaacttcaa?gacccgccac?aacattgaag?atggaagcgt?tcaactagca 540

gaccattatc?aacaaaatac?tccaattggc?gatggccctg?tccttttacc?agacaaccat 600

tacctgtcca?cacaatctgc?cctttcgaaa?gatcccaacg?aaaagagaga?ccacatggtc 660

cttcttgagt?ttgtaacagc?tgctgggatt?acacatggca?tggatgaact?gtacaactga 720

<210>11

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the synthetic primer 3 of the gene of amplification coding red shift green fluorescent protein

<400>11

gggtaatacg?actcactata?gggagaatgg?ctagcaaagg?ag 42

<210>12

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the synthetic primer 4 of the gene of amplification coding red shift green fluorescent protein

<400>12

gggtaatacg?actcactata?gggagatcag?ttgtacagtt?ca 42

<210>13

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉amplification coding triggers the synthetic primer TFN of the gene of the factor

<400>13

ggccatatgc?aagtttcagt?tgaaaccact?c 31

<210>14

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉amplification coding triggers the synthetic primer TFCP of the gene of the factor

<400>14

gcaagcttgg?atccgaattc?tccctacctt?cgatcgcctg?ctggttcatc?agctcgttga 60

<210>15

<211>1732

<212>DNA

＜213〉artificial sequence

<220>

＜223〉gene of coding RAV-2 reversed transcriptive enzyme a subunit

<400>15

gaattcgacc?gttgctctgc?acctggctat?cccgctgaaa?tggaaaccgg?accacacccc 60

ggtttggatc?gaccagtggc?cgctgccgga?aggtaaactg?gttgctgtta?cccagctggt 120

tgaaaaagaa?ctgcagctgg?gtcacatcga?accgtctctg?tcttgctgga?acaccccggt 180

gttcgttatc?cgtaaagctt?ctggttctta?ccgtctgctg?cacgacctgc?gtgctgttaa 240

cgctaaactg?gttccgttcg?gtgctgttca?gcagggtgct?ccggttctgt?ctgctctgcc 300

gcgtggttgg?ccgctgatgg?ttctggacct?gaaagactgc?ttcttctcta?tcccgctggc 360

tgaacaggac?cgtgaagctt?tcgctttcac?cctgccgtct?gttaacaacc?aggctccggc 420

tcgtcgtttc?cagtggaaag?ttctgccgca?gggtatgacc?tgctctccga?ccatctgcca 480

gctggttgtt?ggtcaggttc?tggaaccgct?gcgtctgaaa?cacccggctc?tgcgtatgct 540

gcactacatg?gacgacctgc?tgctggctgc?ttcttctcac?gacggtctgg?aagctgctgg 600

taaagaagtt?atcggtaccc?tggaacgtgc?tggtttcacc?atctctccgg?acaaaatcca 660

gcgtgaaccg?ggtgttcagt?acctgggtta?caaactgggt?tctacctacg?ttgctccggt 720

tggtctggtt?gctgaaccgc?gtatcgctac?cctgtgggac?gttcagaaac?tggttggttc 780

tctgcagtgg?ctgcgtccgg?ctctgggtat?cccgccgcgt?ctgatgggtc?cgttctacga 840

acagctgcgt?ggttctgacc?cgaacgaagc?tcgtgaatgg?aacctggaca?tgaaaatggc 900

ttggcgtgaa?atcgttcagc?tgtctaccac?cgctgctctg?gaacgttggg?acccggctca 960

gccgctggaa?ggtgctgttg?ctcgttgcga?acagggtgct?atcggtgttc?tgggtcaggg 1020

tctgtctacc?cacccgcgtc?cgtgcctgtg?gctgttctct?acccagccga?ccaaggcttt 1080

caccgcttgg?ctggaagttc?tgaccctgct?gatcaccaaa?ctgcgtgctt?ctgctgttcg 1140

taccttcggt?aaagaagttg?acatcctgct?gctgccggct?tgcttccgtg?aagacctgcc 1200

gctgccggaa?ggtatcctgc?tggctctgcg?tggtttcgct?ggtaaaatcc?gttcttctga 1260

caccccgtct?atcttcgaca?tcgctcgtcc?gctgcacgtt?tctctgaaag?ttcgtgttac 1320

cgaccacccg?gttccgggtc?cgaccgtttt?caccgacgct?tcttcttcta?cccacaaagg 1380

tgttgttgtt?tggcgtgaag?gtccgcgttg?ggaaatcaaa?gaaatcgttg?acctgggtgc 1440

ttctgttcag?cagctagaag?ctcgtgctgt?tgctatggct?ctgctgctgt?ggccgaccac 1500

cccgaccaac?gttgttaccg?actctgcttt?cgttgctaaa?atgctgctga?aaatgggtca 1560

ggaaggtgtt?ccgtctaccg?ctgctgcttt?catcctggaa?gacgctctgt?ctcagcgttc 1620

tgctatggct?gctgttctgc?acgttcgttc?tcactctgaa?gttccgggtt?tcttcaccga 1680

aggtaacgac?gttgctgact?ctcaggctac?cttccaggct?tactaatcta?ga 1732

<210>16

<211>2709

<212>DNA

＜213〉artificial sequence

<220>

＜223〉gene of coding RAV-2 reversed transcriptive enzyme β subunit

<400>16

gaattcgacc?gttgctctgc?acctggctat?cccgctgaaa?tggaaaccgg?accacacccc 60

ggtttggatc?gaccagtggc?cgctgccgga?aggtaaactg?gttgctgtta?cccagctggt 120

tgaaaaagaa?ctgcagctgg?gtcacatcga?accgtctctg?tcttgctgga?acaccccggt 180

gttcgttatc?cgtaaagctt?ctggttctta?ccgtctgctg?cacgacctgc?gtgctgttaa 240

cgctaaactg?gttccgttcg?gtgctgttca?gcagggtgct?ccggttctgt?ctgctctgcc 300

gcgtggttgg?ccgctgatgg?ttctggacct?gaaagactgc?ttcttctcta?tcccgctggc 360

tgaacaggac?cgtgaagctt?tcgctttcac?cctgccgtct?gttaacaacc?aggctccggc 420

tcgtcgtttc?cagtggaaag?ttctgccgca?gggtatgacc?tgctctccga?ccatctgcca 480

gctggttgtt?ggtcaggttc?tggaaccgct?gcgtctgaaa?cacccggctc?tgcgtatgct 540

gcactacatg?gacgacctgc?tgctggctgc?ttcttctcac?gacggtctgg?aagctgctgg 600

taaagaagtt?atcggtaccc?tggaacgtgc?tggtttcacc?atctctccgg?acaaaatcca 660

gcgtgaaccg?ggtgttcagt?acctgggtta?caaactgggt?tctacctacg?ttgctccggt 720

tggtctggtt?gctgaaccgc?gtatcgctac?cctgtgggac?gttcagaaac?tggttggttc 780

tctgcagtgg?ctgcgtccgg?ctctgggtat?cccgccgcgt?ctgatgggtc?cgttctacga 840

acagctgcgt?ggttctgacc?cgaacgaagc?tcgtgaatgg?aacctggaca?tgaaaatggc 900

ttggcgtgaa?atcgttcagc?tgtctaccac?cgctgctctg?gaacgttggg?acccggctca 960

gccgctggaa?ggtgctgttg?ctcgttgcga?acagggtgct?atcggtgttc?tgggtcaggg 1020

tctgtctacc?cacccgcgtc?cgtgcctgtg?gctgttctct?acccagccga?ccaaggcttt 1080

caccgcttgg?ctggaagttc?tgaccctgct?gatcaccaaa?ctgcgtgctt?ctgctgttcg 1140

taccttcggt?aaagaagttg?acatcctgct?gctgccggct?tgcttccgtg?aagacctgcc 1200

gctgccggaa?ggtatcctgc?tggctctgcg?tggtttcgct?ggtaaaatcc?gttcttctga 1260

caccccgtct?atcttcgaca?tcgctcgtcc?gctgcacgtt?tctctgaaag?ttcgtgttac 1320

cgaccacccg?gttccgggtc?cgaccgtttt?caccgacgct?tcttcttcta?cccacaaagg 1380

tgttgttgtt?tggcgtgaag?gtccgcgttg?ggaaatcaaa?gaaatcgttg?acctgggtgc 1440

ttctgttcag?cagctagaag?ctcgtgctgt?tgctatggct?ctgctgctgt?ggccgaccac 1500

cccgaccaac?gttgttaccg?actctgcttt?cgttgctaaa?atgctgctga?aaatgggtca 1560

ggaaggtgtt?ccgtctaccg?ctgctgcttt?catcctggaa?gacgctctgt?ctcagcgttc 1620

tgctatggct?gctgttctgc?acgttcgttc?tcactctgaa?gttccgggtt?tcttcaccga 1680

aggtaacgac?gttgctgact?ctcaggctac?cttccaggct?tacccgctgc?gtgaagctaa 1740

agacctgcac?accgctctgc?acatcggtcc?gcgtgctctg?tctaaagctt?gcaacatctc 1800

tatgcagcag?gctcgtgaag?ttgttcagac?ctgcccgcac?tgcaactctg?ctccggctct 1860

ggaagctggt?gttaacccgc?gtggtctggg?tccgctgcag?atctggcaga?ccgacttcac 1920

cctggaaccg?cgtatggctc?cgcgttcttg?gctggctgtt?accgttgaca?ccgcttcttc 1980

tgctatcgtt?gttacccagc?acggtcgtgt?tacctctgtt?gctgctcagc?accactgggc 2040

taccgctatc?gctgttctgg?gtcgtccgaa?agctatcaaa?accgacaacg?gttcttgctt 2100

cacctctaaa?tctacccgtg?aatggctggc?tcgttggggt?atcgctcaca?ccaccggtat 2160

cccgggtaac?tctcagggtc?aggctatggt?tgaacgtgct?aaccgtctgc?tgaaagacaa 2220

aatccgtgtt?ctggctgaag?gtgacggttt?catgaaacgt?atcccggctt?ctaaacaggg 2280

tgaactgctg?gctaaagcta?tgtacgctct?gaaccacttc?gaacgtggtg?aaaacaccaa 2340

aaccccggtt?cagaaacact?ggcgtccgac?cgttctgacc?gaaggtccgc?cggttaaaat 2400

ccgtatcgaa?accggtgaat?gggaaaaagg?ttggaacgtt?ctggtttggg?gtcgtggtta 2460

cgctgctgtt?aaaaaccgtg?acaccgacaa?agttatctgg?gttccgtctc?gtaaagttaa 2520

accggacatc?acccagaaag?acgaagttac?caaaaaagac?gaagcttctc?cgctgttcgc 2580

tggttcttct?gactggatcc?cgtggggtga?cgaacaggaa?ggtctgcagg?aagaagctgc 2640

ttctaacaaa?caggaaggtc?cgggtgaaga?caccctggct?gctaacgaat?cttgaattag 2700

ctttctaga 2709

<210>17

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the synthetic primer NUCN of the gene of amplification coding DNA enzyme

<400>17

ggcgaattcg?atgtttttgt?taattttagg?g 31

<210>18

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the synthetic primer NUCC of the gene of amplification coding DNA enzyme

<400>18

gcgggatcct?taacgaacta?agccgttatt?ttggc 35

<210>19

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the synthetic primer 1 of the gene of amplification coding people dicer PAZ structural domain

<400>19

tcgagctcgg?tacccattga?ctttaaattc?atggaa 36

<210>20

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the synthetic primer 2 of the gene of amplification coding people dicer PAZ structural domain

<400>20

tatctagaaa?gcttaaaggc?agtgaaggcg?ataaag 36

Claims (18)

1. be used to produce the method for polypeptide, this method comprises such host is exposed under the cold condition to induce described polypeptide expression, this host has the gene of the polypeptide that the coding that changed over to this host wants, and wherein in this host, the expression of gene of coding chaperone is enhanced.

2. the process of claim 1 wherein that described method is: induce the chaperone genetic expression among the host by being selected from the expression of gene of a kind of method enhancing coding chaperone in such method; Modify the chaperone gene on the host chromosome; Change the chaperone gene over to host; With the such host of use, in this host, the chaperone expression of gene is enhanced.

3. the process of claim 1 wherein that described chaperone is selected from DnaK, DnaJ, GrpE, GroEL, GroES and the triggering factor.

4. the process of claim 1 wherein that the gene of polypeptide that the coding that is changed over to the host is wanted is connected to the downstream of such DNA, this dna encoding derives from 5 '-non-translational region of the mRNA of cold shock protein gene.

5. the method for claim 4, the gene that wherein will be changed over to the polypeptide that host's coding wants is connected to the downstream of such DNA, and this dna encoding derives from 5 '-non-translational region of the mRNA of intestinal bacteria cspA gene.

6. the process of claim 1 wherein that the gene that uses the polypeptide that carrier wants coding and the gene of coding chaperone change the host over to.

7. the method for claim 6, wherein the gene of the gene of the polypeptide that coding is wanted and coding chaperone interconnects, thus the fusion rotein of polypeptide that codified is wanted and chaperone.

8. the method for claim 7, the wherein said polypeptide of wanting is selected from RAV-2 reversed transcriptive enzyme α subunit, RAV-2 reversed transcriptive enzyme β subunit, DNA enzyme and Dicer PAZ structural domain polypeptide.

9. the process of claim 1 wherein that described host is intestinal bacteria.

10. be used to produce a cover plasmid vector of the polypeptide of wanting, it comprises:

(1) first carrier, its downstream in promotor has:

(a) coding derives from the DNA of 5 '-non-translational region of the mRNA of cold shock protein gene; With

(b) can be used for inserting the gene of the polypeptide that coding wants and be positioned at the restriction endonuclease recognition sequence in downstream of the DNA of (a); With

(2) have second carrier of gene of coding chaperone,

Wherein select the replication orgin of the carrier of (1) and (2) like this, thus the feasible uncompatibility that do not produce.

11. a cover carrier of claim 10, wherein said first carrier comprises such DNA, and this dna encoding derives from 5 '-non-translational region of the mRNA of intestinal bacteria cspA gene.

12. a cover carrier of claim 10, wherein said second carrier comprises the gene that coding is selected from the chaperone of DnaK, DnaJ, GrpE, GroEL, GroES and the triggering factor.

13. a cover carrier of claim 10, it is made up of the plasmid that can duplicate in intestinal bacteria.

14. an expression vector, its downstream at promoter has:

(a) coding derives from the DNA of 5 '-non-translational region of the mRNA of cold shock protein gene;

(b) has the gene that can be used for inserting the polypeptide that coding wants and be positioned at the DNA of restriction endonuclease recognition sequence in downstream of the DNA of (a); With

(c) gene of coding chaperone.

15. the expression vector of claim 14, it comprises the DNA that coding derives from 5 '-non-translational region of intestinal bacteria cspA gene.

16. the expression vector of claim 14, it is to comprise the plasmid of gene that coding is selected from the chaperone of DnaK, DnaJ, GrpE, GroEL, GroES and the triggering factor.

17. the expression vector of claim 14, the restriction endonuclease recognition sequence that wherein can be used for inserting the gene of the polypeptide that coding wants is positioned at such position, can insert the gene of the polypeptide that coding wants in this position, thus can with the polypeptide wanted as and the fusion rotein of chaperone express.

18. the expression vector of claim 14, it is the plasmid that can duplicate in intestinal bacteria.

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