CN1950505A - Process for producing polypeptide - Google Patents
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- CN1950505A CN1950505A CN 200580014919 CN200580014919A CN1950505A CN 1950505 A CN1950505 A CN 1950505A CN 200580014919 CN200580014919 CN 200580014919 CN 200580014919 A CN200580014919 A CN 200580014919A CN 1950505 A CN1950505 A CN 1950505A
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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
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.
Applications Claiming Priority (3)
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JP152598/2004 | 2004-05-21 | ||
JP2004152598 | 2004-05-21 | ||
JP067984/2005 | 2005-03-10 |
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Publication Number | Publication Date |
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CN1950505A true CN1950505A (en) | 2007-04-18 |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102191207A (en) * | 2010-03-01 | 2011-09-21 | 华东师范大学 | Gene engineering bacterium of expression of soluble FGF-21 and construction method and application thereof |
CN103880962A (en) * | 2014-03-19 | 2014-06-25 | 国家纳米科学中心 | Fusion protein and encoding gene and preparation method of fusion protein as well as pharmaceutical composition and preparation method of pharmaceutical composition |
CN108794637A (en) * | 2017-08-09 | 2018-11-13 | 芜湖英特菲尔生物制品产业研究院有限公司 | A kind of canine recombinant long-acting interferon α and the fusion protein and preparation method thereof for preparing this long-acting interferon |
CN108864293A (en) * | 2017-08-09 | 2018-11-23 | 芜湖英特菲尔生物制品产业研究院有限公司 | A kind of fusion protein being made of dog albumin and dog interferon γ and preparation method thereof and a kind of canine recombinant long-acting interferon γ |
-
2005
- 2005-05-19 CN CN 200580014919 patent/CN1950505A/en active Pending
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102191207A (en) * | 2010-03-01 | 2011-09-21 | 华东师范大学 | Gene engineering bacterium of expression of soluble FGF-21 and construction method and application thereof |
CN103880962A (en) * | 2014-03-19 | 2014-06-25 | 国家纳米科学中心 | Fusion protein and encoding gene and preparation method of fusion protein as well as pharmaceutical composition and preparation method of pharmaceutical composition |
CN103880962B (en) * | 2014-03-19 | 2016-01-06 | 国家纳米科学中心 | Fusion rotein and encoding gene thereof and preparation method and a kind of pharmaceutical composition and preparation method thereof |
CN108794637A (en) * | 2017-08-09 | 2018-11-13 | 芜湖英特菲尔生物制品产业研究院有限公司 | A kind of canine recombinant long-acting interferon α and the fusion protein and preparation method thereof for preparing this long-acting interferon |
CN108864293A (en) * | 2017-08-09 | 2018-11-23 | 芜湖英特菲尔生物制品产业研究院有限公司 | A kind of fusion protein being made of dog albumin and dog interferon γ and preparation method thereof and a kind of canine recombinant long-acting interferon γ |
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Open date: 20070418 |