CN104844696A - Design, synthesis and application of transcription activator like effector function protein - Google Patents
Design, synthesis and application of transcription activator like effector function protein Download PDFInfo
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- CN104844696A CN104844696A CN201410056380.0A CN201410056380A CN104844696A CN 104844696 A CN104844696 A CN 104844696A CN 201410056380 A CN201410056380 A CN 201410056380A CN 104844696 A CN104844696 A CN 104844696A
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Abstract
The invention relates to a transcription activator like effector (TALE) function protein. The repeat variable amino acid di-residues of the TALE function protein are arginine R and valine respectively, or valine V and isoleucine I respectively, or glycine G and aspartic acid D respectively, or arginine R and tryptophan W respectively, or threonine T and glycine G respectively, or cysteine C and glycine G respectively, or glutamic acid E and cysteine C respectively.
Description
Technical field
The present invention relates to the design of one " transcriptional activation increment effector (TranscriptionActivator Like Effectors, TALE) " functional protein, synthesis and the application of above-mentioned functions albumen, particularly above-mentioned functions albumen.
Background technology
In the genome times afterwards comprehensively, we are in the urgent need to the novel biotechnology such as efficient genetically manipulated, synthesis.Such as, we usually need the expression of suppression or a reticent gene.Traditional gene knockout (Gene Knockout) technology depends on abiogenous homologous recombination in cell, and its efficiency is very low, is generally 10
-6level; Although RNAi technology is simple, be difficult to again obtain absolutely inhibition.Except suppressing or reticent specific gene, we usually also need for specific gene, carry out certain several base, the amendment of even a certain section sequence.Equally only depend on homologous recombination technique, be difficult to obtain desirable effect.
TALE (Transcription Activator Like Effectors) is first the upper discovery of phytopathogen Xanthomonas campestris (Xanthomonas), be attached to DNA specifically, in this pathogenic bacterial infection process, plant gene regulated and controled.TALE albumen can enter in nucleus through nuclear membrane and be combined with specific DNA binding domain, with the expression of disease and resistibility genes involved in regulating plant genome.Briefly, TALE is made up of 4 or more the series connection " module " of specific recognition DNA and the N-end of both sides and C-end sequence, and each " module " comprises 34 amino acid, wherein the 12nd and 13 amino acids are critical sites of targets identification, be referred to as and repeat variable bis-amino acid residue (repeat variable diresidue, or RVDs).TALE identifies that the mechanism of DNA is that a Nucleotide on DNA target spot is identified by the RVD on a tumor-necrosis factor glycoproteins.
In theory, for any one base of A, T, G, C, the RVDs of particular combination with it can be found.Therefore, to any section of DNA sequence, we can design easily, synthesize the TALE of corresponding module composition.The problem that it may be noted that is, 1) although corresponding RVD can be designed for A, T, G, C, and, the corresponding relation between them needs to optimize further; 2) can the TALE of design and synthesis in conjunction with the desired location on genome, also be decided by a lot of other factors by target efficiently; 3) composition of module also has the space optimized further.
Solve although TALE also has a lot of problem to await further investigation, this does not hinder its application.A maximum application prospect is TALEN.TALEN is a fusion rotein, forms by merging with the endonuclease (Nuclease) that can produce double-strand break (double strand break, DSB) on DNA sequence dna with certain segment DNA sequence-specific identification TALE.TALEN is heterodimer molecule (the TALE-Nuclease acting in conjunction of Ji Liang unit), can at two cutting DNAs between nearer specific recognition sequence of being separated by.
The DSB that TALEN produces can be repaired by following two kinds of approach: 1) non-homologous end joining (Non Homologous EndJoining, NHEJ): NHEJ is with natural repair mechanism, can be used to introduce nucleotide deletion so that inactivation or knock out a specific target gene; 2) homologous recombination (Homologous Recombination, HR): DSB promotes homologous recombination, under a DNA masterplate exists, can produce specific DNA sequences and change, also can by integrated transgene on DNA sequence dna.NHEJ approach may be used for gene silencing, and HR can be used for amendment gene (Gene Editing) or gene knock-in (Gene Knock-in).No matter be which kind of approach, the reparation that TALEN produces with depend on merely compared with homologous recombination, the efficiency that restructuring occurs gene improves greatly, this, for we are engaged in the technique means that genome customizedization (genome customization) provide convenience, brings new hope for developing more easy novel gene group targeting modification technology.
The application prospect that TALE another one is large is TALEA (transcription activator-like (TAL) effector activator).TALEA is a fusion rotein, the TALE and transcription factor activation region VP64 (VP64Activation Domain) that identify specific DNA sequence is merged, can be built into the activating transcription factor TALEA identifying specific DNA sequence in promotor.This fusion rotein by conjunction with the specific DNA sequence near gene promoter, and is combined with Polymerase II by VP64 active region, thus transcribing of activated gene, improve the expression of endogenous targets gene.In actually operating, target sequence (a general 12-18 base) need be chosen in the promotor upstream of target gene, build TALE identification module.
TALE technology has started to show up prominently at life science.2011, France and the U.S.'s two group cooperations, utilize TALEN technology, knock out inactivation IgM function in rats, efficiency was up to 60%.2011, comprise several groups of China, utilize TALEN technology, in zebra fish, knock out the genes such as inactivation hey2, efficiency also reached more than 30%.
TALE has special constitutional features, comprises nitrogen end (N end) secretion signal, the DNA binding domain of central authorities and the activation domain of nuclear localization sequence (Nuclear localization signal, NLS) and carbon teminal (C end).In DNA binding domain, nearly all TALE albumen had been found that is all have the repeating unit of different amts (12 ~ 30), high conservative to form, in these repeating units (generally containing 33 ~ 35 amino acid), except the 12nd and 13 amino acids are not quite similar, other integral parts are all very conservative.Wherein the 12nd and 13 variable amino acid be called as the variable bis-amino acid residue RVD of tumor-necrosis factor glycoproteins (repeat variable di-residues).TALE mainly identifies DNA sequence dna by the RVD in repeating unit.The RVD reported at present has 14 kinds.NI (l-asparagine and Isoleucine) specific recognition A, HD (Histidine and aspartic acid) specific recognition C, NN (l-asparagine and l-asparagine) can identify G and A, NK (l-asparagine and Methionin) specific recognition G, NS (l-asparagine and Serine) can identify G, A, C and T, NG (l-asparagine and glycine) specific recognition T, NH (l-asparagine and Histidine) specific recognition G, N* (l-asparagine, 13rd vacancy) can T be identified, C, G and A, NP (l-asparagine and proline(Pro)) can identify T, A and C, HN (Histidine and l-asparagine) can identify G and A, NT (l-asparagine and Threonine) can identify G and A, SN (Serine and l-asparagine) specific recognition G, SH (Serine and Histidine) specific recognition G.
Prior art Problems existing: the TALE specificity had is strong not, and some TALE recognition efficiencies are not high enough.Therefore, develop new efficient TA LE sequence (namely new RVD), just become the technical problem that this area is in the urgent need to address.
Summary of the invention
Object of the present invention is just in order to solve the problems of the technologies described above, wish that carrying out some by design to TALE albumen improves, realize the optimization in the RVD sequence of TALE albumen, to reach the RVD sequence having particular combination with it for any one base of A, T, G, C.
RV specific recognition A base; VI specific recognition C base; GD specific recognition C base; RW specific recognition C base; TG identifies T base, can also more weak identification C and G base; CG can identify G base, can also more weak identification T and C base; EC can identify more by force A, G and C base, can also more weak identification T base.
The technical solution used in the present invention is as follows.
Design for HHB gene order design and synthesis TALEN in the present invention, the experimental technique adopted is luciferase strand restructuring annealing experiment (the luciferase single-strand annealing recombination assay relatively commonly used in the world, SSA), and be used for detecting native gene non-homogeneous restructuring detection efficiency method---SURVEYOR tests.Experimental principle is as shown in accompanying drawing 1,2.
Although there is general demand in the new TALE better for identification specificity, recognition efficiency is higher, also method (SSA, SURVEYOR of test TALE recognition efficiency is there is in prior art, refer to hereafter), but since TALE is found, the TALE rarely found report of new texture, in many reasons, screening operation amount is too large to such an extent as to exceed the scope of standing, and is one of topmost reason.
Total institute is known, because namely primary amino acid has 20 kinds, therefore has 20 containing two amino acid whose RVD
2i.e. 400 kinds of permutation and combination situations, screening experiment amount is quite large.Utilize this character of the plasmid incompatibility of plasmid in intestinal bacteria in the present invention, in permutation and combination a large amount of in intestinal bacteria, filter out useful RVD.Concrete steps as shown in Figure 1.First the method sets up screening system, then TALEN storehouse is set up according to 400 kinds of situations of 20 kinds of primary amino acids permutation and combination between two, TALEN in TALEN storehouse cotransformation together with reporter plasmid is entered intestinal bacteria, select positive colony amplification wherein, last and reporter plasmid again cotransformation is verified.We make the experimental amount of screening experiment greatly reduce at the said new method of design, thus make from combination possible in a large number, pick out effective novel TALE and become feasible.
Use aforesaid method, we have picked out effective TALE from the combination of hundreds of seed amino acid, as follows respectively: the variable bis-amino acid residue RVD (repeat variable di-residues) that repeats in " transcriptional activation increment effector (TALE) " functional protein is respectively arginine R and α-amino-isovaleric acid V, or be respectively α-amino-isovaleric acid V and Isoleucine I, or be respectively glycine G and aspartic acid D, or be respectively arginine R and tryptophane W, or be respectively Threonine T and glycine G, or be respectively halfcystine C and glycine G, or be respectively L-glutamic acid E and halfcystine C.Further, we also confirm the base of above-mentioned novel TALE specific recognition, can be applied in the specific recognition of these bases by these novel TALE, as follows respectively:
The variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of described " transcriptional activation increment effector " functional protein is respectively arginine R and α-amino-isovaleric acid V, for the specific recognition to VITAMIN B4 (A); The variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of described " transcriptional activation increment effector " functional protein is respectively α-amino-isovaleric acid V and Isoleucine I, for the specific recognition to cytosine(Cyt) (C); The variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of described " transcriptional activation increment effector " functional protein is respectively glycine G and aspartic acid D, for the specific recognition to cytosine(Cyt) (C); The variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of described " transcriptional activation increment effector " functional protein is respectively arginine R and tryptophane W, for the specific recognition to cytosine(Cyt) (C); The variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of described " transcriptional activation increment effector " functional protein is respectively Threonine T and glycine G, for identifying more by force the carrying out of thymus pyrimidine (T), can also to cytosine(Cyt) (C) and the more weak identification of guanine (G) base; The variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of described " transcriptional activation increment effector " functional protein is respectively halfcystine C and glycine G, for identifying more by force the carrying out of guanine (G), more weak identification can also be carried out to thymus pyrimidine (T) and cytosine(Cyt) (C); The variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of described " transcriptional activation increment effector " functional protein is respectively L-glutamic acid E and halfcystine C, for identifying more by force the carrying out of VITAMIN B4 (A), guanine (G) and cytosine(Cyt) (C), more weak identification can also be carried out to thymus pyrimidine (T).
Accompanying drawing explanation
Fig. 1 screening experiment schematic flow sheet
Fig. 2 luciferase strand restructuring annealing experiment (luciferase single-strand annealing recombination assay, SSA) schematic diagram: first, luciferase gene is transformed.Terminator codon and target-gene sequence is added in the middle of luciferase gene; Immediately go up other one section of luciferase gene sequence again by molecular biology method subsequently, in way, the sequence (about 800bp) of light gray areas is identical.When TALEN is effective, TALEN can shear the DNA break of a formation double-strand at target sequence location double-stranded DNA, because two light gray areas sequences (i.e. a fragment gene in " terminator codon " left side and a fragment gene on " target sequence " right side in Fig. 1) are identical, now homologous recombination can be there is, just define an activated luciferase reporter gene, the expression of luciferase can be measured in experiment to detect the power of the effect of TALEN.
Fig. 3 SURVEYOR schematic diagram: first by the method for PCR, goal gene fragment is increased out from genome.Comprising sequence disappearance occurring and do not change.Then amplification fragment is out annealed in vitro again, the fragment that disappearance now occurs and the sequence do not changed can be annealed and be formed with the unpaired duplex structure of part.Following SURVEYOR enzyme carries out enzyme and conscientiously tests, and this enzyme can identify that unpaired region is carried out enzyme and cut.We can detect by the content running a, b, c in glue detection figure the efficiency that disappearance occurs, thus draw the action effect of TALEN.
The SSA experimental result of Fig. 4 RVD-RV
The SSA experimental result of Fig. 5 RVD-VI
The SSA experimental result of Fig. 6 RVD-GD
The SSA experimental result of Fig. 7 RVD-RW
The SSA experimental result of Fig. 8 RVD-TG
The SSA experimental result of Fig. 9 RVD-CG
The SSA experimental result of Figure 10 RVD-EC
Figure 11 SURVEYOR experimental result
Embodiment
Embodiment 1
The variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of " transcriptional activation increment effector " functional protein is respectively arginine R and α-amino-isovaleric acid V, is called for short RVD-RV.Corresponding SSA experimental result is shown in Fig. 4, wherein positive control identifies A according to the TALE:NI (l-asparagine and Isoleucine) of Design with Rule synthesis existing in field, HD (Histidine and aspartic acid) identifies C, NN (l-asparagine and l-asparagine) identifies G, and NG (l-asparagine and glycine) identifies T.Compared with positive control, RVD-RV is higher for the recognition rate of VITAMIN B4 (A), and compared with positive control, relative efficiency reaches 150%.Therefore this TALE can be used for the specific recognition to VITAMIN B4 (A).
Embodiment 2
The variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of " transcriptional activation increment effector " functional protein is respectively α-amino-isovaleric acid V and Isoleucine I, is called for short RVD-VI.Corresponding SSA experimental result is shown in Fig. 5.Compared with positive control, RVD-VI is very high for the recognition rate of cytosine(Cyt) (C), and compared with positive control, relative efficiency reaches 198%.Therefore this TALE can be used for the specific recognition to cytosine(Cyt) (C).
Embodiment 3
The variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of " transcriptional activation increment effector " functional protein is respectively glycine G and aspartic acid D, is called for short RVD-GD.Corresponding SSA experimental result is shown in Fig. 6.Compared with positive control, RVD-GD is very high for the recognition rate of cytosine(Cyt) (C), and compared with positive control, relative efficiency reaches 103%.Therefore this TALE can be used for the specific recognition to cytosine(Cyt) (C).
Embodiment 4
The variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of " transcriptional activation increment effector " functional protein is respectively arginine R and tryptophane W, is called for short RVD-RW.Corresponding SSA experimental result is shown in Fig. 7.Compared with positive control, RVD-RW is higher for the recognition rate of cytosine(Cyt) (C), and compared with positive control, relative efficiency reaches 68.5%.Therefore this TALE can be used for the specific recognition to cytosine(Cyt) (C).
Embodiment 5
The variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of " transcriptional activation increment effector " functional protein is respectively Threonine T and glycine G, is called for short RVD-TG.Corresponding SSA experimental result is shown in Fig. 8.Compared with positive control, RVD-TG is higher for the recognition rate of thymus pyrimidine (T), and compared with positive control, relative efficiency reaches 132%; Carry out more weak identification for cytosine(Cyt) (C) and guanine (G) base, compared with positive control, relative efficiency is respectively 61% and 36%.Therefore this TALE can be used for identifying more by force the carrying out of thymus pyrimidine (T), can also to cytosine(Cyt) (C) and the more weak identification of guanine (G) base.
Embodiment 6
The variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of " transcriptional activation increment effector " functional protein is respectively halfcystine C and glycine G, is called for short RVD-CG.Corresponding SSA experimental result is shown in Fig. 9.Compared with positive control, RVD-CG is higher for the recognition rate of guanine (G), and compared with positive control, relative efficiency reaches 88.8%; Carry out more weak identification for thymus pyrimidine (T) and cytosine(Cyt) (C), compared with positive control, relative efficiency is respectively 1% and 34%.Therefore this TALE can be used for identifying more by force the carrying out of guanine (G), more weak identification can also be carried out to thymus pyrimidine (T) and cytosine(Cyt) (C).
Embodiment 7
The variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of " transcriptional activation increment effector " functional protein is respectively L-glutamic acid E and halfcystine C, is called for short RVD-EC.Corresponding SSA experimental result is shown in Figure 10.Compared with positive control, RVD-EC is higher for the recognition rate of VITAMIN B4 (A), guanine (G) and cytosine(Cyt) (C), and compared with positive control, relative efficiency is respectively 211%, 421 and 200%; Carry out more weak identification to thymus pyrimidine (T), compared with positive control, relative efficiency is 13%.Therefore this TALE can be used for identifying more by force the carrying out of VITAMIN B4 (A), guanine (G) and cytosine(Cyt) (C), more weak identification can also be carried out to thymus pyrimidine (T).
Annex:
(1)
In embodiment 1, RVD-RV aminoacid sequence:
MAPKKKRKVYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHGTVDLRTLGYSQQQQEKI
KPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVG
KQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNL
TPEQVVAIASRVGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLP
VLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASRVGGKQAL
ETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASH
DGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPD
QVVAIASRVGGKQALETVQRLLPVLCQAHGLTPDQVVAIASRVGGKQALETVQRLLPVLC
QAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETV
QRLLPVLCQAHGLTPAQVVAIASRVGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDG
GKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQV
VAIASRVGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQA
HGLTPAQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGL
PHAPALIKRTNRRIPERTSHRVA
(2)
In embodiment 2, RVD-VI aminoacid sequence:
MAPKKKRKVYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHGTVDLRTLGYSQQQQEKI
KPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVG
KQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNL
TPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPV
LCQAHGLTPAQVVAIASVIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETV
QRLLPVLCQAHGLTPDQVVAIASVIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASVIGGK
QALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAI
ASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLT
PDQVVAIASVIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVL
CQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASVIGGKQALETV
QRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGG
KQALETVQRLLPVLCQAHGLTPDQVVAIASVIGGKQALETVQRLLPVLCQAHGLTPAQVVA
IASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRT
NRRIPERTSHRVA
(3)
In embodiment 3, RVD-GD aminoacid sequence:
MAPKKKRKVYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHGTVDLRTLGYSQQQQEK
IKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGV
GKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPL
NLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRL
LPVLCQAHGLTPDQVVAIASGDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGK
QALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVA
IASGDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAH
GLTPAQVVAIASGDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASGDGGKQALETVQRL
LPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASGDGGK
QALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVA
IASGDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHG
LTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGRPALESIVAQLS
RPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVA
(4)
In embodiment 4, RVD-RW aminoacid sequence:
MAPKKKRKVYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHGTVDLRTLGYSQQQQEK
IKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGV
GKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPL
NLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASRWGGKQALETVQRL
LPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASRWGGK
QALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVA
IASRWGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAH
GLTPAQVVAIASRWGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRL
LPVLCQAHGLTPDQVVAIASRWGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQ
ALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAI
ASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHG
LTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLP
VLCQAHGLTPDQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPAL
DAVKKGLPHAPALIKRTNRRIPERTSHRVA
(5)
In embodiment 5, RVD-TG aminoacid sequence:
MAPKKKRKVYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHGTVDLRTLGYSQQQQEKI
KPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVG
KQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNL
TPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASTGGGKQALETVQRLLPV
LCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALET
VQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHD
GGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQ
VVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQA
HGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASTGGGKQALETVQR
LLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQ
ALETVQRLLPVLCQAHGLTPAQVVAIASTGGGKQALETVQRLLPVLCQAHGLTPAQVVAIA
SNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLT
PAQVVAIASTGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAP
ALIKRTNRRIPERTSHRVA
(6)
In embodiment 6, RVD-CG aminoacid sequence:
MAPKKKRKVYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHGTVDLRTLGYSQQQQEK
IKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGV
GKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPL
NLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLL
PVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQ
ALETVQRLLPVLCQAHGLTPAQVVAIASCGGGKQALETVQRLLPVLCQAHGLTPDQVVAI
ASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHG
LTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLP
VLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQAL
ETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIAS
NGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLT
PAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVL
CQAHGLTPDQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDA
VKKGLPHAPALIKRTNRRIPERTSHRVA
(7)
In embodiment 7, RVD-EC aminoacid sequence:
MAPKKKRKVYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHGTVDLRTLGYSQQQQEK
IKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGV
GKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPL
NLTPEQVVAIASECGGKQALETVQRLLPVLCQAHGLTPDQVVAIASECGGKQALETVQRL
LPVLCQAHGLTPDQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGK
QALETVQRLLPVLCQAHGLTPAQVVAIASECGGKQALETVQRLLPVLCQAHGLTPDQVVA
IASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHG
LTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLL
PVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQ
ALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAI
ASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGL
TPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGRPALESIVAQLSR
PDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVA
Claims (8)
1. one kind " transcriptional activation increment effector (Transcription Activator Like Effectors, TALE) " functional protein, it is characterized in that the variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of described " transcriptional activation increment effector " functional protein is respectively arginine R and α-amino-isovaleric acid V, or be respectively α-amino-isovaleric acid V and Isoleucine I, or be respectively glycine G and aspartic acid D, or be respectively arginine R and tryptophane W, or be respectively Threonine T and glycine G, or be respectively halfcystine C and glycine G, or be respectively L-glutamic acid E and halfcystine C.
2. the application of functional protein according to claim 1, it is characterized in that the variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of described " transcriptional activation increment effector " functional protein is respectively arginine R and α-amino-isovaleric acid V, for the specific recognition to VITAMIN B4 in DNA (A).
3. the application of functional protein according to claim 1, it is characterized in that the variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of described " transcriptional activation increment effector " functional protein is respectively α-amino-isovaleric acid V and Isoleucine I, for the specific recognition to cytosine(Cyt) in DNA (C).
4. the application of functional protein according to claim 1, it is characterized in that the variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of described " transcriptional activation increment effector " functional protein is respectively glycine G and aspartic acid D, for the specific recognition to cytosine(Cyt) in DNA (C).
5. the application of functional protein according to claim 1, it is characterized in that the variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of described " transcriptional activation increment effector " functional protein is respectively arginine R and tryptophane W, for the specific recognition to cytosine(Cyt) in DNA (C).
6. the application of functional protein according to claim 1, it is characterized in that the variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of described " transcriptional activation increment effector " functional protein is respectively Threonine T and glycine G, for identifying more by force thymus pyrimidine in DNA (T), can also to cytosine(Cyt) in DNA (C) and the more weak identification of guanine (G) base.
7. the application of functional protein according to claim 1, it is characterized in that the variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of described " transcriptional activation increment effector " functional protein is respectively halfcystine C and glycine G, for identifying more by force the carrying out of guanine in DNA (G), more weak identification can also be carried out to thymus pyrimidine in DNA (T) and cytosine(Cyt) (C).
8. the application of functional protein according to claim 1, it is characterized in that the variable bis-amino acid residue RVD (repeat variable di-residues) that repeats of described " transcriptional activation increment effector " functional protein is respectively L-glutamic acid E and halfcystine C, for identifying more by force the carrying out of VITAMIN B4 in DNA (A), guanine (G) and cytosine(Cyt) (C), more weak identification can also be carried out to thymus pyrimidine in DNA (T).
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