CN113699160B - Mutation method of rat mitochondrial gene G14098A and application thereof - Google Patents
Mutation method of rat mitochondrial gene G14098A and application thereof Download PDFInfo
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Abstract
A mutation method of rat mitochondrial gene G14098A and application thereof. The invention designs two TALE target point recognition sequences containing mutation sites by utilizing DdCBE base editing technology according to target mutation sites, and positions DdCBE to a corresponding target sequence by screening DdCBE combinations so as to mutate target bases from G to A. The mutation method of the present invention has high accuracy and low off-target effect. According to the invention, the G at 14098 th site of mitochondrial gene of a rat can be mutated into A by microinjection of rat fertilized eggs. Hybridizing a female mutant rat and a wild rat for mutation passage analysis, and indicating that the mutation can be stably passed; behavioral and cardiac function evaluations confirmed that the rat model has a human mitochondrial mutation disease phenotype.
Description
Technical Field
The invention relates to a mitochondrial gene mutation method and an animal modeling application thereof.
Background
As life science and medical research step into new fields, the demand for animal models is increasing. The animal model is an insurmountable link in the research and development process of vaccines and medicines, and particularly, the research on major infectious diseases and complex human diseases needs a stable and effective disease animal model as a support.
Mitochondrial DNA (mtDNA) mutations are mainly manifested as base changes, which can lead to a variety of systemic diseases in humans. To date, the pathogenic variation of human mtDNA has exceeded 270 and the number is increasing. Currently, there is no effective treatment regimen for pathogenic mutations in mtDNA. Therefore, there is an urgent need for animal models comprising precise human mtDNA variations to reveal the pathophysiological processes of such diseases and to develop therapeutic approaches for these diseases. Previously, mammalian animal models with pathogenic mtDNA mutations can be obtained by mitochondrial transplantation or using PolG D257A/WT Pedigree screening of novel mtDNA mutations [ Stewart, J.B.J. Inherit MetabDis,2020]. However, neither of these strategies can yield animal models with precise mtDNA mutations on demand.
Disclosure of Invention
In humans, the G14710A mutation in mtDNA TRNE gene is associated with mitochondrial myopathy, which the inventors found to correspond to at the site G14098 in rats, and thus a gene mutation method was designed to be applied to G14098A mutant rat modeling to aid in the study of the associated disease.
The invention aims to provide a precise and effective mutation method of rat mitochondrial gene G14098A and related application thereof.
According to a first aspect of the present invention, there is provided a method for mutating a rat mitochondrial gene G14098A, comprising:
respectively selecting one TALE recognition target sequence at the upper and lower streams of the position of the target mutation base G, wherein the interval sequence between the two TALE recognition target sequences is 7-18bp and does not contain the base G except the target mutation base G, and the two TALE recognition target sequences and the interval sequence define the target mutation sequence;
screening a pair of DdCBE combinations according to the selected TALE recognition target sequence; and
the screened combinations of DdCBE were co-injected to localize them to the target mutant sequence, thereby editing the target mutant base G into A to introduce mutations.
The invention adopts a DddA-derived cytosine base editor (DdCBE) to successfully realize the accurate conversion from C.G to T.A in mtDNA (high accuracy and low off-target effect), thereby opening up a way for establishing an animal model with specific mtDNA mutation and related research and application.
According to a preferred embodiment of the invention, the two TALE recognition target sequences selected may each be:
left recognition of target sequence: ttaactgtgactaat (SEQ ID NO. 1); and
right recognition target sequence: agttgaattacggcgat (SEQ ID NO. 2).
According to a further preferred embodiment of the present invention, the screened combination of dcbe may be:
rat G14098A Left TALE-G1333C (L1333C) and Rat G14098A Right TALE-G1333N (R1333N);
rat G14098A Left TALE-G1397C (L1397C) and Rat G14098A Right TALE-G1397N (L1397N);
rat G14098A Left TALE-G1333N (L1333N) and Rat G14098A Right TALE-G1397C (R1333C); or
Rat G14098A Left TALE-G1397N (L1397N) or Rat G14098A Right TALE-G1333C (L1397C).
The DdCBE combination is more preferably: rat G14098A Left TALE-G1397C (L1397C) and Rat G14098A Right TALE-G1397N (L1397N).
According to another aspect of the invention, the application of the method in constructing a rat model with mitochondrial gene target site G14098A mutation is also provided.
Female mutant rats of the mitochondrial gene G14098A of the F0 generation established by the method of the present invention were mated with wild type rats, and the mutation of the mitochondrial gene G14098A could be detected in the offspring rats. In addition, the behavioral and cardiac function analysis of the mitochondrial gene G14098A mutant rat can simulate and research the clinical phenotype of the human mitochondrial gene G14710A mutation.
According to other aspects of the invention, the application of the method in simulating and researching mitochondrial encephalomyopathy caused by human mitochondrial G14710A mutation is also provided.
Drawings
FIG. 1 homology alignment of pathogenic mutation sites of human and rat mitochondria;
FIG. 2 transfection of different combinations of DdCBE in C6 cells results in a comparison of mitochondrial G14098A mutations;
FIG. 3 shows the identification result of mitochondrial gene G14098A mutant rat;
FIG. 4 analysis of the mutation of each tissue of mitochondrial gene G14098A mutant rat;
FIG. 5 mitochondrial Gene G14098A mutant rat-related behavioral analysis;
FIG. 6 mitochondrial gene G14098A mutant rat cardiac function assay.
Detailed Description
First, according to the mitochondrial gene mutation G14710A of human, the corresponding rat mitochondrial gene mutation G14098A was determined (see fig. 1). Based on the rat mutation site, the recognition site (target or target sequence) of TALE was designed. The invention adopts DdCBE fixed-point mitochondrial editing: and (3) positioning the fused base editing tool to the target gene position by utilizing a pair of TALE recognition sites, thereby realizing the mutation of the target base. The invention selects and designs TALE recognition targets as follows:
according to the target design principle of TALE, one TALE recognition site is respectively selected at the upstream and the downstream of the target, the middle spacing sequence of the two TALE recognition sites is 7-18bp, the target mutation base G is contained, and the base G outside the target base is avoided.
Mutations were introduced in the rat mitochondrial gene using two dcbe fused TALE recognition sites to target the corresponding mutation sequences and editing the target mutation G to a.
Aiming at rat mitochondrial gene G14098 mutation, the invention selects the following two TALE left and right recognition targets:
left side identification of target:
ttaactgtgactaat(SEQ ID NO.1)
and identifying a target spot on the right side:
agttgaattacggcgat(SEQ ID NO.2)
aiming at the selected TALE target sequences, different DdCBE is constructed, and the corresponding different DdCBE is as follows:
Rat G14098A Left TALE-G1333C(SEQ ID NO.3)；
Rat G14098A Right TALE-G1333N(SEQ ID NO.4)；
Rat G14098A Left TALE-G1397C(SEQ ID NO.5)；
Rat G14098A Right TALE-G1397N(SEQ ID NO.6)；
Rat G14098A Left TALE-G1333N(SEQ ID NO.7)；
Rat G14098A Right TALE-G1397C(SEQ ID NO.8)；
Rat G14098A Left TALE-G1397N(SEQ ID NO.9)；
Rat G14098A Right TALE-G1333C(SEQ ID NO.10)。
DdCBE was introduced into PB transposon expression vectors for mutation of rat mitochondrial gene G14098A, respectively.
Example 1
DdCBE-mediated mutation of mitochondrial gene G14098A was performed on a rat cell line. Gene editing of cell lines (by electroporation or lipofection) is performed by routine procedures, as exemplified by lipofection.
(1) Taking C6 cells as an example, the invention carries out the culture and transfection of eukaryotic cells: c6 cells were inoculated and cultured in DMEM high-glucose medium supplemented with 10% FBS (Gemini) containing penicillin (100U/ml) and streptomycin (100. Mu.g/ml).
(2) The cells were divided into 6-well plates before transfection, and transfection was performed when the density reached 70% -80%.
The DdCBE system plasmid combination is as follows:
A:Rat G14098A Left TALE-G1333C/Rat G14098A Right TALE-G1333N(L1333C+R1333N)；
B:Rat G14098A Left TALE-G1397C/Rat G14098A Right TALE-G1397N(L1397C+R1397N)；
C:Rat G14098A Left TALE-G1333N/Rat G14098A Right TALE-G1397C(L1333N+R L1333C)；
D:Rat G14098A Left TALE-G1397N/Rat G14098A Right TALE-G1333C(L1397N+R L1397C)；
(3) Transfection is exemplified by lipofection. According to Lipofectamine TM In the manual of 2000Transfection Reagent (Invitrogen, 11668-019), for example, combination A, 1. Mu.g of Rat G14098A Left TALE-G1333C (L1333C) plasmid and 1. Mu.g of Rat G14098A Right TALE-G1333N (R1333N) plasmid were mixed and co-transfected into each well of cells, and the solution was changed after 6 to 8 hours, and the cells were harvested after 72 hours.
(4) Genotyping analysis
A. A portion of the cells was lysed in a lysis solution (10. Mu.M Tris-HCl,0.4M NaCl, 2. Mu.M EDTA,1% SDS) using 100. Mu.g/ml proteinase K, and then phenol-chloroform extracted and dissolved in 50. Mu.l of deionized water.
B. PCR amplification is carried out by using a pair of primers G14098-For (TCAAGTCTCCGGGTACTCTC) and G14098-Rev (AAATTGAGGCGCCCGTTGG), and PCR recovery products are obtained by purifying with AxyPrep PCR clean kit (AXYGEN, AP-PCR-250G) according to a PCR reaction system:
300-400ng genomic DNA
25μl 2×Buffer
1μl dNTP
2μl G14098--For(20μm)
2μl G14098--Rev(20μm)
1μl DNA Polymerase(Vazyme,P505-d3)
Water was added to the reaction system to 50. Mu.l.
C. The obtained PCR-recovered product was ligated to a cloning vector
-Blunt Cloning Kit (TransGen, CB 101) ligation reaction system:
1 μ l PCR product
1μl
-Blunt Cloning vector
Gently mixed, and reacted at room temperature (20-37 ℃) for 5 minutes. After the reaction was completed, the centrifuge tube was placed on ice.
The ligation product transformed DH5 competent cells (TransGen, CD 201).
D. Clones were picked and the target gene mutation was sequenced using the universal primer M13-F, the sequencing results are as follows (mutated bases are in italics, bold, underlined):
target point mutation conditions:
Wt：gacatgaaaaa
Mut：gacataaaaaa
the results show that: the mitochondrial gene 14098 site was mutated from G to a, and dcbe combined with B: G14098A Left TALE-G1397C/Rat G14098A Right TALE-G1397N mediated mitochondrial G14098A mutation efficiency is highest (figure 2).
Example 2
Carrying out DdCBE-mediated mitochondrial gene G14098A mutation on rat fertilized eggs to realize target base mutation, establishing a mitochondrial gene G14098 mutant rat model, carrying out behavioral and cardiac function evaluation, and identifying whether the model accords with clinical phenotype caused by human mitochondrial mutation.
The embryo collection, microinjection, embryo culture and embryo transplantation of rats are carried out according to the conventional operation.
(1) Microinjection: fertilized eggs were injected with G14098A Left TALE-G1397C/Rat G14098A Right TALE-G1397N plasmid. Performing embryo transplantation conventionally;
(2) Genotype analysis:
A. extracting genome DNA by shearing tail of conventional mice: after lysing digestion with 100. Mu.g/ml proteinase K in lysis buffer (10. Mu.M Tris-HCl,0.4M NaCl, 2. Mu.M EDTA,1% SDS), phenol-chloroform extraction was followed by dissolution in 50. Mu.l deionized water.
B. PCR amplification is carried out by using a pair of primers G14098-For (TCAAGTCTCCGGGTACTCTC) and G14098-Rev (AAATTGAGGCGCCCGTTGG), and PCR recovery products are obtained by purifying with AxyPrep PCR clean kit (AXYGEN, AP-PCR-250G) according to a PCR reaction system:
300-400ng genomic DNA
25μl 2×Buffer
1μl dNTP
2μl G14098--For(20μm)
2μl G14098--Rev(20μm)
1μl DNA Polymerase(Vazyme,P505-d3)
Water was added to the reaction system to 50. Mu.l.
C. The obtained PCR-recovered product was ligated to a cloning vector
-Blunt Cloning Kit (TransGen, CB 101) in the ligation reaction system:
2 μ l PCR product
1μl
-Blunt Cloning vector
Gently mixed, and reacted at room temperature (20-37 ℃) for 5 minutes. After the reaction was completed, the centrifuge tube was placed on ice.
The ligation product transformed DH5 competent cells (TransGen, CD 201).
D. Clones were picked and the target gene mutation was sequenced with the universal primer M13-F, the sequencing results are as follows (bold italics with lower dash for mutated bases):
target point mutation conditions:
Wt：gacatgaaaaa
Mut：gacataaaaaa
the results show that: the site 14098 of the mitochondrial gene is mutated from G to A (shown in figure 3), and the mutation condition analysis of each tissue is carried out on one mutant Rat, and the result shows that the mutation can be detected in each tissue (shown in figure 3), and the result proves that the mutation of the mitochondrial gene G14098A can be realized by using the G14098A Left TALE-G1397C/Rat G14098A Right TALE-G1397N plasmid in a microinjection mode.
(4) By crossing the obtained G1408A male and female mutant rats with wild-type rats, and performing genomic DNA extraction, PCR and sequencing analysis on the offspring rats, the mutation was found to be stably inherited into the offspring rats (see FIG. 4). It shows that we obtain mitochondrial gene G14098A mutant rats capable of being inherited stably.
(5) Mitochondrial gene G14098A point mutation behavioral analysis of rat
The obtained mitochondrial gene G14098A point mutation rat can cause mitochondrial function defect, further influences the function of muscle tissues, and judges the muscle function index of the rat through the detection of a behavioral method.
Open field experiment: the experiment was carried out in a black box of 80cm by 50 cm. Spontaneous activity of rats was recorded for 5 minutes using the SuperMaze digital tracking system. The movement trajectories of the rats were analyzed by SuperMaze software.
Rotating rod experiment: the locomotion and balance ability of rats was evaluated using a rotarod apparatus (ZH-600B, anhui Zhenghua bioinstrumentation, inc.) with an automatic timer and a drop sensor. All rats were left on a stationary rotarod for 3 minutes prior to training. Training was performed at 20rpm and the rats were dropped and immediately placed back on the rotating bar for a maximum of 5 times. The test was performed once daily for three consecutive days. The incubation period of the rats falling off the rotarod was recorded.
And (3) a grip force experiment: the muscle strength of the rat limbs was measured using a grip dynamometer (ZH-YLS-13A, anhui Zhenghua Biofacilities). Three measurements were recorded and the average was calculated.
The experimental results of the open field showed that the mutations resulted in a decrease in the distance and mean velocity of movement, a decrease in the ability to move and balance, and a significant decrease in the grasping power of the limbs, as shown in fig. 5.
(6) Mitochondrial gene G14098A mutant rat cardiac function evaluation
The obtained mitochondrial gene G14098A mutant rat is subjected to echocardiography analysis to detect parameters related to cardiac function.
The cardiac ultrasound method comprises the following steps: rats were anesthetized with isoflurane and echocardiographic observations were performed using a micro-ultrasound imaging system (Vevo 3100). At least three consecutive cardiac cycles are recorded.
The heart function evaluation results showed that the mutant rats exhibited an dilated cardiomyopathy phenotype, larger ventricles, thinner walls, and decreased contractile function, as compared to the wild rats, as shown in fig. 6.
Sequence listing
<110> institute of animal research for medical experiments of Chinese academy of medical sciences
Mutation method of <120> rat mitochondrial gene G14098A and application thereof
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Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
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Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu
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Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
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Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
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His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
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Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln
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Glu Ser Ile Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala
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Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro
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Gln Thr Ser Glu Ser Gly Gly Gly Gly Ser Pro Gly Ala Ala Ala Asp
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Tyr Lys Asp Asp Asp Asp Lys Gly Ser Val Asp Leu Arg Thr Leu Gly
65 70 75 80
Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val Arg Ser Thr
85 90 95
Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe Thr His Ala
100 105 110
His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly Thr Val Ala
115 120 125
Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu Ala Thr His Glu
130 135 140
Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala Arg Ala Leu Glu
145 150 155 160
Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro Pro Leu Gln Leu
165 170 175
Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly Gly Val Thr Ala
180 185 190
Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr Gly Ala Pro Leu
195 200 205
Asn Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
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Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
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Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
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Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
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Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser
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Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
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Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile
305 310 315 320
Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
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Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val
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Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
355 360 365
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln
370 375 380
Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr
385 390 395 400
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
405 410 415
Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu
420 425 430
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
435 440 445
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
450 455 460
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
465 470 475 480
Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly
485 490 495
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
500 505 510
Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile
515 520 525
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
530 535 540
Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
545 550 555 560
His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
565 570 575
Val Leu Cys Gln Ala His Gly Leu Thr Pro Ala Gln Val Val Ala Ile
580 585 590
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
595 600 605
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val
610 615 620
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
625 630 635 640
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln
645 650 655
Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr
660 665 670
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
675 680 685
Ala Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu
690 695 700
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
705 710 715 720
Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln
725 730 735
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
740 745 750
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly
755 760 765
Arg Pro Ala Leu Glu Ser Ile Val Ala Gln Leu Ser Arg Pro Asp Pro
770 775 780
Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu
785 790 795 800
Gly Gly Arg Pro Ala Leu Asp Ala Val Lys Lys Gly Leu Gly Gly Ser
805 810 815
Gly Ser Tyr Ala Leu Gly Pro Tyr Gln Ile Ser Ala Pro Gln Leu Pro
820 825 830
Ala Tyr Asn Gly Gln Thr Val Gly Thr Phe Tyr Tyr Val Asn Asp Ala
835 840 845
Gly Gly Leu Glu Ser Lys Val Phe Ser Ser Gly Gly Ser Gly Gly Ser
850 855 860
Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val
865 870 875 880
Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile
885 890 895
Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu
900 905 910
Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr
915 920 925
Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile
930 935 940
Lys Met Leu
945
<210> 5
<211> 865
<212> PRT
<213> Artificial Sequence
<400> 5
Met Leu Gly Phe Val Gly Arg Val Ala Ala Ala Pro Ala Ser Gly Ala
1 5 10 15
Leu Arg Arg Leu Thr Pro Ser Ala Ser Leu Pro Pro Ala Gln Leu Leu
20 25 30
Leu Arg Ala Ala Pro Thr Ala Val His Pro Val Arg Asp Tyr Ala Ala
35 40 45
Gln Thr Ser Glu Ser Gly Gly Gly Gly Ser Pro Gly Ala Ala Ala Asp
50 55 60
Tyr Lys Asp Asp Asp Asp Lys Gly Ser Val Asp Leu Arg Thr Leu Gly
65 70 75 80
Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val Arg Ser Thr
85 90 95
Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe Thr His Ala
100 105 110
His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly Thr Val Ala
115 120 125
Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu Ala Thr His Glu
130 135 140
Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala Arg Ala Leu Glu
145 150 155 160
Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro Pro Leu Gln Leu
165 170 175
Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly Gly Val Thr Ala
180 185 190
Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr Gly Ala Pro Leu
195 200 205
Asn Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly
210 215 220
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
225 230 235 240
Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly
245 250 255
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
260 265 270
Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser
275 280 285
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
290 295 300
Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
305 310 315 320
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
325 330 335
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val
340 345 350
Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
355 360 365
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Ala Gln
370 375 380
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
385 390 395 400
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
405 410 415
Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu
420 425 430
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
435 440 445
Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
450 455 460
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
465 470 475 480
Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
485 490 495
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
500 505 510
Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile
515 520 525
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
530 535 540
Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
545 550 555 560
His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
565 570 575
Val Leu Cys Gln Ala His Gly Leu Thr Pro Ala Gln Val Val Ala Ile
580 585 590
Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
595 600 605
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val
610 615 620
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
625 630 635 640
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln
645 650 655
Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr
660 665 670
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
675 680 685
Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala Leu
690 695 700
Glu Ser Ile Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala
705 710 715 720
Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro
725 730 735
Ala Leu Asp Ala Val Lys Lys Gly Leu Gly Gly Ser Ala Ile Pro Val
740 745 750
Lys Arg Gly Ala Thr Gly Glu Thr Lys Val Phe Thr Gly Asn Ser Asn
755 760 765
Ser Pro Lys Ser Pro Thr Lys Gly Gly Cys Ser Gly Gly Ser Thr Asn
770 775 780
Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln
785 790 795 800
Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn
805 810 815
Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser Thr
820 825 830
Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro
835 840 845
Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met
850 855 860
Leu
865
<210> 6
<211> 1011
<212> PRT
<213> Artificial Sequence
<400> 6
Met Leu Gly Phe Val Gly Arg Val Ala Ala Ala Pro Ala Ser Gly Ala
1 5 10 15
Leu Arg Arg Leu Thr Pro Ser Ala Ser Leu Pro Pro Ala Gln Leu Leu
20 25 30
Leu Arg Ala Ala Pro Thr Ala Val His Pro Val Arg Asp Tyr Ala Ala
35 40 45
Gln Thr Ser Glu Ser Gly Gly Gly Gly Ser Pro Gly Ala Ala Ala Asp
50 55 60
Tyr Lys Asp Asp Asp Asp Lys Gly Ser Val Asp Leu Arg Thr Leu Gly
65 70 75 80
Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val Arg Ser Thr
85 90 95
Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe Thr His Ala
100 105 110
His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly Thr Val Ala
115 120 125
Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu Ala Thr His Glu
130 135 140
Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala Arg Ala Leu Glu
145 150 155 160
Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro Pro Leu Gln Leu
165 170 175
Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly Gly Val Thr Ala
180 185 190
Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr Gly Ala Pro Leu
195 200 205
Asn Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
210 215 220
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
225 230 235 240
Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
245 250 255
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
260 265 270
Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser
275 280 285
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
290 295 300
Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile
305 310 315 320
Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
325 330 335
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val
340 345 350
Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
355 360 365
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln
370 375 380
Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr
385 390 395 400
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
405 410 415
Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu
420 425 430
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
435 440 445
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
450 455 460
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
465 470 475 480
Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly
485 490 495
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
500 505 510
Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile
515 520 525
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
530 535 540
Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
545 550 555 560
His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
565 570 575
Val Leu Cys Gln Ala His Gly Leu Thr Pro Ala Gln Val Val Ala Ile
580 585 590
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
595 600 605
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val
610 615 620
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
625 630 635 640
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln
645 650 655
Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr
660 665 670
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
675 680 685
Ala Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu
690 695 700
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
705 710 715 720
Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln
725 730 735
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
740 745 750
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly
755 760 765
Arg Pro Ala Leu Glu Ser Ile Val Ala Gln Leu Ser Arg Pro Asp Pro
770 775 780
Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu
785 790 795 800
Gly Gly Arg Pro Ala Leu Asp Ala Val Lys Lys Gly Leu Gly Gly Ser
805 810 815
Gly Ser Tyr Ala Leu Gly Pro Tyr Gln Ile Ser Ala Pro Gln Leu Pro
820 825 830
Ala Tyr Asn Gly Gln Thr Val Gly Thr Phe Tyr Tyr Val Asn Asp Ala
835 840 845
Gly Gly Leu Glu Ser Lys Val Phe Ser Ser Gly Gly Pro Thr Pro Tyr
850 855 860
Pro Asn Tyr Ala Asn Ala Gly His Val Glu Gly Gln Ser Ala Leu Phe
865 870 875 880
Met Arg Asp Asn Gly Ile Ser Glu Gly Leu Val Phe His Asn Asn Pro
885 890 895
Glu Gly Thr Cys Gly Phe Cys Val Asn Met Thr Glu Thr Leu Leu Pro
900 905 910
Glu Asn Ala Lys Met Thr Val Val Pro Pro Glu Gly Ser Gly Gly Ser
915 920 925
Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val
930 935 940
Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile
945 950 955 960
Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu
965 970 975
Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr
980 985 990
Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile
995 1000 1005
Lys Met Leu
1010
<210> 7
<211> 879
<212> PRT
<213> Artificial Sequence
<400> 7
Met Leu Gly Phe Val Gly Arg Val Ala Ala Ala Pro Ala Ser Gly Ala
1 5 10 15
Leu Arg Arg Leu Thr Pro Ser Ala Ser Leu Pro Pro Ala Gln Leu Leu
20 25 30
Leu Arg Ala Ala Pro Thr Ala Val His Pro Val Arg Asp Tyr Ala Ala
35 40 45
Gln Thr Ser Glu Ser Gly Gly Gly Gly Ser Pro Gly Ala Ala Ala Asp
50 55 60
Tyr Lys Asp Asp Asp Asp Lys Gly Ser Val Asp Leu Arg Thr Leu Gly
65 70 75 80
Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val Arg Ser Thr
85 90 95
Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe Thr His Ala
100 105 110
His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly Thr Val Ala
115 120 125
Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu Ala Thr His Glu
130 135 140
Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala Arg Ala Leu Glu
145 150 155 160
Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro Pro Leu Gln Leu
165 170 175
Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly Gly Val Thr Ala
180 185 190
Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr Gly Ala Pro Leu
195 200 205
Asn Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly
210 215 220
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
225 230 235 240
Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly
245 250 255
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
260 265 270
Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser
275 280 285
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
290 295 300
Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
305 310 315 320
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
325 330 335
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val
340 345 350
Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
355 360 365
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Ala Gln
370 375 380
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
385 390 395 400
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
405 410 415
Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu
420 425 430
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
435 440 445
Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
450 455 460
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
465 470 475 480
Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
485 490 495
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
500 505 510
Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile
515 520 525
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
530 535 540
Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
545 550 555 560
His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
565 570 575
Val Leu Cys Gln Ala His Gly Leu Thr Pro Ala Gln Val Val Ala Ile
580 585 590
Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
595 600 605
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val
610 615 620
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
625 630 635 640
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln
645 650 655
Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr
660 665 670
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
675 680 685
Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala Leu
690 695 700
Glu Ser Ile Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala
705 710 715 720
Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro
725 730 735
Ala Leu Asp Ala Val Lys Lys Gly Leu Gly Gly Ser Gly Ser Tyr Ala
740 745 750
Leu Gly Pro Tyr Gln Ile Ser Ala Pro Gln Leu Pro Ala Tyr Asn Gly
755 760 765
Gln Thr Val Gly Thr Phe Tyr Tyr Val Asn Asp Ala Gly Gly Leu Glu
770 775 780
Ser Lys Val Phe Ser Ser Gly Gly Ser Gly Gly Ser Thr Asn Leu Ser
785 790 795 800
Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu Ser
805 810 815
Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn Lys Pro
820 825 830
Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu
835 840 845
Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp Ala
850 855 860
Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met Leu
865 870 875
<210> 8
<211> 933
<212> PRT
<213> Artificial Sequence
<400> 8
Met Leu Gly Phe Val Gly Arg Val Ala Ala Ala Pro Ala Ser Gly Ala
1 5 10 15
Leu Arg Arg Leu Thr Pro Ser Ala Ser Leu Pro Pro Ala Gln Leu Leu
20 25 30
Leu Arg Ala Ala Pro Thr Ala Val His Pro Val Arg Asp Tyr Ala Ala
35 40 45
Gln Thr Ser Glu Ser Gly Gly Gly Gly Ser Pro Gly Ala Ala Ala Asp
50 55 60
Tyr Lys Asp Asp Asp Asp Lys Gly Ser Val Asp Leu Arg Thr Leu Gly
65 70 75 80
Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val Arg Ser Thr
85 90 95
Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe Thr His Ala
100 105 110
His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly Thr Val Ala
115 120 125
Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu Ala Thr His Glu
130 135 140
Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala Arg Ala Leu Glu
145 150 155 160
Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro Pro Leu Gln Leu
165 170 175
Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly Gly Val Thr Ala
180 185 190
Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr Gly Ala Pro Leu
195 200 205
Asn Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
210 215 220
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
225 230 235 240
Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
245 250 255
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
260 265 270
Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser
275 280 285
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
290 295 300
Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile
305 310 315 320
Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
325 330 335
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val
340 345 350
Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
355 360 365
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln
370 375 380
Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr
385 390 395 400
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
405 410 415
Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu
420 425 430
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
435 440 445
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
450 455 460
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
465 470 475 480
Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly
485 490 495
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
500 505 510
Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile
515 520 525
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
530 535 540
Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
545 550 555 560
His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
565 570 575
Val Leu Cys Gln Ala His Gly Leu Thr Pro Ala Gln Val Val Ala Ile
580 585 590
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
595 600 605
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val
610 615 620
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
625 630 635 640
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln
645 650 655
Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr
660 665 670
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
675 680 685
Ala Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu
690 695 700
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
705 710 715 720
Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln
725 730 735
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
740 745 750
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly
755 760 765
Arg Pro Ala Leu Glu Ser Ile Val Ala Gln Leu Ser Arg Pro Asp Pro
770 775 780
Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu
785 790 795 800
Gly Gly Arg Pro Ala Leu Asp Ala Val Lys Lys Gly Leu Gly Gly Ser
805 810 815
Ala Ile Pro Val Lys Arg Gly Ala Thr Gly Glu Thr Lys Val Phe Thr
820 825 830
Gly Asn Ser Asn Ser Pro Lys Ser Pro Thr Lys Gly Gly Cys Ser Gly
835 840 845
Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln
850 855 860
Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu
865 870 875 880
Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr
885 890 895
Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro
900 905 910
Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn
915 920 925
Lys Ile Lys Met Leu
930
<210> 9
<211> 943
<212> PRT
<213> Artificial Sequence
<400> 9
Met Leu Gly Phe Val Gly Arg Val Ala Ala Ala Pro Ala Ser Gly Ala
1 5 10 15
Leu Arg Arg Leu Thr Pro Ser Ala Ser Leu Pro Pro Ala Gln Leu Leu
20 25 30
Leu Arg Ala Ala Pro Thr Ala Val His Pro Val Arg Asp Tyr Ala Ala
35 40 45
Gln Thr Ser Glu Ser Gly Gly Gly Gly Ser Pro Gly Ala Ala Ala Asp
50 55 60
Tyr Lys Asp Asp Asp Asp Lys Gly Ser Val Asp Leu Arg Thr Leu Gly
65 70 75 80
Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val Arg Ser Thr
85 90 95
Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe Thr His Ala
100 105 110
His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly Thr Val Ala
115 120 125
Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu Ala Thr His Glu
130 135 140
Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala Arg Ala Leu Glu
145 150 155 160
Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro Pro Leu Gln Leu
165 170 175
Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly Gly Val Thr Ala
180 185 190
Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr Gly Ala Pro Leu
195 200 205
Asn Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly
210 215 220
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
225 230 235 240
Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly
245 250 255
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
260 265 270
Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser
275 280 285
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
290 295 300
Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
305 310 315 320
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
325 330 335
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val
340 345 350
Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
355 360 365
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Ala Gln
370 375 380
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
385 390 395 400
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
405 410 415
Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu
420 425 430
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
435 440 445
Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
450 455 460
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
465 470 475 480
Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
485 490 495
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
500 505 510
Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile
515 520 525
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
530 535 540
Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
545 550 555 560
His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
565 570 575
Val Leu Cys Gln Ala His Gly Leu Thr Pro Ala Gln Val Val Ala Ile
580 585 590
Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
595 600 605
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val
610 615 620
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
625 630 635 640
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln
645 650 655
Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr
660 665 670
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
675 680 685
Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala Leu
690 695 700
Glu Ser Ile Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala
705 710 715 720
Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro
725 730 735
Ala Leu Asp Ala Val Lys Lys Gly Leu Gly Gly Ser Gly Ser Tyr Ala
740 745 750
Leu Gly Pro Tyr Gln Ile Ser Ala Pro Gln Leu Pro Ala Tyr Asn Gly
755 760 765
Gln Thr Val Gly Thr Phe Tyr Tyr Val Asn Asp Ala Gly Gly Leu Glu
770 775 780
Ser Lys Val Phe Ser Ser Gly Gly Pro Thr Pro Tyr Pro Asn Tyr Ala
785 790 795 800
Asn Ala Gly His Val Glu Gly Gln Ser Ala Leu Phe Met Arg Asp Asn
805 810 815
Gly Ile Ser Glu Gly Leu Val Phe His Asn Asn Pro Glu Gly Thr Cys
820 825 830
Gly Phe Cys Val Asn Met Thr Glu Thr Leu Leu Pro Glu Asn Ala Lys
835 840 845
Met Thr Val Val Pro Pro Glu Gly Ser Gly Gly Ser Thr Asn Leu Ser
850 855 860
Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu Ser
865 870 875 880
Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn Lys Pro
885 890 895
Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu
900 905 910
Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp Ala
915 920 925
Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met Leu
930 935 940
<210> 10
<211> 997
<212> PRT
<213> Artificial Sequence
<400> 10
Met Leu Gly Phe Val Gly Arg Val Ala Ala Ala Pro Ala Ser Gly Ala
1 5 10 15
Leu Arg Arg Leu Thr Pro Ser Ala Ser Leu Pro Pro Ala Gln Leu Leu
20 25 30
Leu Arg Ala Ala Pro Thr Ala Val His Pro Val Arg Asp Tyr Ala Ala
35 40 45
Gln Thr Ser Glu Ser Gly Gly Gly Gly Ser Pro Gly Ala Ala Ala Asp
50 55 60
Tyr Lys Asp Asp Asp Asp Lys Gly Ser Val Asp Leu Arg Thr Leu Gly
65 70 75 80
Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val Arg Ser Thr
85 90 95
Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe Thr His Ala
100 105 110
His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly Thr Val Ala
115 120 125
Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu Ala Thr His Glu
130 135 140
Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala Arg Ala Leu Glu
145 150 155 160
Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro Pro Leu Gln Leu
165 170 175
Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly Gly Val Thr Ala
180 185 190
Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr Gly Ala Pro Leu
195 200 205
Asn Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
210 215 220
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
225 230 235 240
Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
245 250 255
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
260 265 270
Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser
275 280 285
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
290 295 300
Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile
305 310 315 320
Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
325 330 335
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val
340 345 350
Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
355 360 365
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln
370 375 380
Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr
385 390 395 400
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
405 410 415
Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu
420 425 430
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
435 440 445
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
450 455 460
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
465 470 475 480
Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly
485 490 495
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
500 505 510
Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile
515 520 525
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
530 535 540
Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
545 550 555 560
His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
565 570 575
Val Leu Cys Gln Ala His Gly Leu Thr Pro Ala Gln Val Val Ala Ile
580 585 590
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
595 600 605
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val
610 615 620
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
625 630 635 640
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln
645 650 655
Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr
660 665 670
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
675 680 685
Ala Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu
690 695 700
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
705 710 715 720
Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln
725 730 735
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
740 745 750
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly
755 760 765
Arg Pro Ala Leu Glu Ser Ile Val Ala Gln Leu Ser Arg Pro Asp Pro
770 775 780
Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu
785 790 795 800
Gly Gly Arg Pro Ala Leu Asp Ala Val Lys Lys Gly Leu Gly Gly Ser
805 810 815
Pro Thr Pro Tyr Pro Asn Tyr Ala Asn Ala Gly His Val Glu Gly Gln
820 825 830
Ser Ala Leu Phe Met Arg Asp Asn Gly Ile Ser Glu Gly Leu Val Phe
835 840 845
His Asn Asn Pro Glu Gly Thr Cys Gly Phe Cys Val Asn Met Thr Glu
850 855 860
Thr Leu Leu Pro Glu Asn Ala Lys Met Thr Val Val Pro Pro Glu Gly
865 870 875 880
Ala Ile Pro Val Lys Arg Gly Ala Thr Gly Glu Thr Lys Val Phe Thr
885 890 895
Gly Asn Ser Asn Ser Pro Lys Ser Pro Thr Lys Gly Gly Cys Ser Gly
900 905 910
Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln
915 920 925
Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu
930 935 940
Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr
945 950 955 960
Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro
965 970 975
Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn
980 985 990
Lys Ile Lys Met Leu
995
Claims (5)
1. A method of mutating a rat mitochondrial gene G14098A comprising:
respectively selecting one TALE recognition target sequence at the upper and lower streams of the position of the target mutation base G, wherein the interval sequence between the two TALE recognition target sequences is 7-18bp and does not contain the base G except the target mutation base G, and the two TALE recognition target sequences and the interval sequence define the target mutation sequence;
screening a pair of DdCBE combinations according to the selected TALE recognition target sequence; and
co-injecting the screened DdCBE combination to target mutant sequence, thereby editing the target mutant base G into A to introduce mutation,
wherein the two selected TALE recognition target sequences are:
left recognition of target sequence: ttaactgtgactaat; and
right recognition target sequence: the agttgaattacggcgat,
wherein the screened DdCBE combination is as follows:
rat G14098A Left TALE-G1333C as shown in sequence 3 and Rat G14098A Right TALE-G1333N as shown in sequence 4;
rat G14098A Left TALE-G1397C as shown in sequence 5 and Rat G14098A Right TALE-G1397N as shown in sequence 6;
rat G14098A Left TALE-G1333N as shown in sequence 7 and Rat G14098A Right TALE-G1397C as shown in sequence 8; or
Rat G14098A Left TALE-G1397N as shown in sequence 9 and Rat G14098A Right TALE-G1333C as shown in sequence 10.
2. A mutation method according to claim 1, wherein the DdCBE selected is a combination of:
rat G14098A Left TALE-G1397C and Rat G14098A Right TALE-G1397N.
3. Use of the method according to claim 1 or 2 for constructing a rat model of mitochondrial gene target site G14098A mutation.
4. Use according to claim 3, wherein the mutation is carried out in the rat cell line C6.
5. Use according to claim 3, wherein the mutation is carried out in rat zygotes.
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