CN111378050B

CN111378050B - Fusion protein and application thereof

Info

Publication number: CN111378050B
Application number: CN201811639168.1A
Authority: CN
Inventors: 储军; 王亮
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2018-12-29
Filing date: 2018-12-29
Publication date: 2023-04-07
Anticipated expiration: 2038-12-29
Also published as: CN111378050A

Abstract

The invention relates to the technical field of biological medicines, and particularly relates to a fusion protein and application thereof. The fusion protein fuses a plasma membrane positioning sequence, dab1 protein, photosensitive protein and fluorescent protein, positions the fusion protein to a lipid raft or non-lipid raft area of a cell plasma membrane, and can realize the rapid activation and regulation of a PI3K/Akt signal channel locally with high space-time resolution by illumination. The rapid, effective and specific switch control of the signal channel is helpful to understand the cascade transmission rule of the signal, and has very important significance for researching diseases closely related to the signal channel.

Description

Fusion protein and application thereof

Technical Field

The invention relates to the technical field of biological medicines, and particularly relates to a fusion protein and application thereof.

Background

Phosphoinositide 3-kinases (PI 3K) are intracellular phosphatidylinositol kinases that, upon receiving signals from tyrosine kinases and G protein-coupled receptors, catalyze the formation of Ptd Ins (3, 4, 5) P2, a small substrate molecule, ptd Ins (4, 5) P3 (hereinafter PI 3P), at the plasma membrane. PI3P activates Akt and other downstream proteins. The signal channel is widely existed in various cells, is an important growth-promoting and storage signal channel in the cells, and regulates a plurality of activities such as cell proliferation, differentiation, growth, survival, metabolism, migration and the like. Research shows that the PI3K/Akt pathway abnormality is closely related to the occurrence and development of human tumors, neurodegenerative diseases and insulin resistance related diseases such as II type diabetes, cardiovascular and cerebrovascular diseases and the like. Through the rapid, effective and specific switch control of the signal path, the method is helpful for understanding the cascade transmission rule of signals, and has very important significance for researching diseases closely related to the signal path.

The traditional method adopts chemical substances (such as IGF-1) to activate a PI3K/Akt pathway for researching the migration, the cell morphological change and the like of normal cells and tumor cells, and has lower space-time resolution and more complex induction method.

Optogenetics is widely applied in many fields mainly involving neuroscience as an emerging discipline, and the optogenetics technology can realize the rapid activation and regulation of specific signal channel molecules in cells by combining genetic expression of protein and light control and imaging of laser. Optical modulation of the PI3K/Akt pathway has been achieved, for example Idevall-Hagren O et al use photocontrol tools to control PI3K production at the plasma membrane, see FIG. 1. The method uses the action of light control protein to pull the catalytic subunit P110 alpha of PI3K to the vicinity of a plasma membrane of CIBN/Cry2, thereby catalyzing the formation of PI 3P. Compared with the traditional chemical small molecule induction method, the method is simple and convenient, the space-time resolution is higher, but 2 fusion proteins need to be expressed in cells, namely the expression quantity of each protein required by the best effect of CIBN-CAAX and mCH-CRY2-iSH2 needs to be wasted for searching, and the iSH2 is derived from PI3K subunits, so that excessive expression can influence the induction result and is not beneficial to the control of a PI3K/Akt channel.

Disclosure of Invention

In view of this, the present invention provides a fusion protein and its application. The fusion protein provided by the invention can quickly, effectively and locally activate a PI3K/Akt signal channel with high space-time resolution.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

the invention provides a fusion protein, which comprises a plasma membrane positioning sequence, dab1 protein, light-sensitive protein and fluorescent protein; the plasma membrane localization sequence is a lyn protein localization sequence or a KRas protein localization sequence.

In some embodiments, the lyn protein localization sequence is:

(1) SEQ ID NO: 1;

or (2) SEQ ID NO:1 by deletion, substitution or addition of one or more amino acid residues to obtain an amino acid sequence with equivalent functions.

In some embodiments, the KRas protein localization sequence is:

(1) SEQ ID NO: 2;

or (2) SEQ ID NO:2 by deletion, substitution or addition of one or more amino acid residues to obtain an amino acid sequence with equivalent functions.

In some embodiments, the light sensitive protein is an cryptochrome Cry2olig protein.

In some embodiments, the fluorescent protein is the red fluorescent protein mCherry or the yellow fluorescent protein mCit.

In some embodiments, the fusion protein has an amino acid sequence as shown in SEQ ID NO. 3-5 or an amino acid sequence with equivalent functions obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 3-5.

In one embodiment, the fusion protein has the amino acid sequence shown as SEQ ID NO. 3. The fusion protein consists of a lyn protein positioning sequence, a Dab1 protein, a cryptochrome Cry2olig protein and a red fluorescent protein mCherry, namely lyn-Dab1-Cry2olig-mCH.

In one embodiment, the fusion protein has an amino acid sequence as set forth in SEQ ID NO. 4. The fusion protein consists of a lyn protein positioning sequence, a Dab1 protein, an cryptochrome Cry2olig protein and a yellow fluorescent protein mCit, namely lyn-Dab1-Cry2olig-mCit.

In one embodiment, the fusion protein has the amino acid sequence shown as SEQ ID NO. 5. The fusion protein consists of a Dab1 protein, a cryptochrome Cry2olig protein, a yellow fluorescent protein mCit and a KRas protein positioning sequence, namely Dab1-Cry2olig-mCit-KRas.

The invention also provides a DNA molecule encoding the fusion protein of the invention.

In some embodiments, the present invention provides DNA molecules encoding the amino acid sequence shown in SEQ ID NO. 3, having the nucleotide sequence shown in SEQ ID NO.6.

In some embodiments, the present invention provides a DNA molecule encoding the amino acid sequence shown in SEQ ID NO. 4, the nucleotide sequence of which is shown in SEQ ID NO.7.

In some embodiments, the present invention provides a DNA molecule encoding the amino acid sequence shown in SEQ ID NO. 5, the nucleotide sequence of which is shown in SEQ ID NO.8.

Due to the degeneracy of the codons, there are a wide variety of DNA molecules which, in addition to the nucleotide sequences shown in SEQ ID Nos. 6 to 8, are capable of encoding the fusion proteins according to the invention.

The invention also provides a recombinant DNA vector comprising the DNA molecule of the invention.

The recombinant DNA vector is an expression vector, and a person skilled in the art clones the DNA molecule of the fusion protein into the expression vector, transforms a host cell, and obtains the fusion protein by induction expression. The expression vector comprises pCAG series vectors. In some embodiments, the expression vector is pCAG-DsRed2 in the pCAG series.

The invention also provides a host cell comprising the recombinant DNA vector.

Furthermore, the invention also provides application of the fusion protein in preparing a PI3K signal pathway activator. The diseases related to the PI3K/Akt signal pathway comprise neurodegenerative diseases, cardiovascular and cerebrovascular diseases and diabetes

Research shows that the fusion protein provided by the invention can be respectively positioned in lyn-opto-Dab1/opto-Dab1-KRas of a plasma membrane lipid raft (lipid raft)/non-lipid raft (non-lipid raft) region, and the activation effect of PI3K kinase in different proton membrane regions can be more finely researched. According to research, the lyn-opto-Dab1/opto-Dab1-KRas protein fused with a specific plasma membrane localization sequence only has a diffusion process on a plasma membrane and does not contain a rapid cytoplasm diffusion process, and the spatial resolution is higher compared with the opto-Dab1 fusion protein.

The fusion protein provided by the invention fuses a plasma membrane positioning sequence of lyn, dab1 protein, photosensitive protein and fluorescent protein, and can realize the rapid activation and regulation of a PI3K/Akt signal channel locally and at high spatial and temporal resolution by illumination. The rapid, effective and specific switch control of the signal path is helpful for understanding the cascade transmission rule of the signal, and has very important significance for researching diseases closely related to the signal path.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

Fig. 1 is a schematic structural diagram of a prior art optically-activated PI3K pathway;

FIG. 2 is a schematic structural diagram of the fusion protein lyn-Dab1-Cry2olig-mCH of the present invention;

FIG. 3 is a fluorescent analysis of COS7 cells co-transfected with the fusion protein lyn-Dab1-cry2olig-mCit and Akt-PH-mCH;

FIG. 4 is a fluorescence analysis diagram of COS7 cell co-transfected by the fusion protein Dab1-Cry2olig-mCit-KRas and Akt-PH-mCH;

FIG. 5 is a fluorescent analysis of the light activation of the fusion protein lyn-opto-Dab1-mCH in COS7 cells and the resulting change in cell morphology.

Detailed Description

The invention discloses a fusion protein and application thereof, and can be realized by appropriately improving process parameters by referring to the content in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1 preparation of lyn-Dab1-Cry2olig-mCH fusion protein

By utilizing a molecular cloning basic technology, a DNA sequence for coding a lyn protein membrane positioning sequence, dab1 protein, a photosensitive protein Cry2olig and a red fluorescent protein mCH (mCherry) is connected, and is introduced into an expression vector pCAG-DsRed2 to obtain a recombinant expression vector, and the recombinant expression vector is transformed into host bacteria Escherichia coli to obtain a fusion protein (a structural schematic diagram is shown in figure 2), and the fusion protein has an amino acid sequence shown as SEQ ID NO. 3, and a nucleotide sequence shown as SEQ ID NO.6.

Example 2 preparation of lyn-Dab1-Cry2olig-mCit fusion protein

By using a molecular cloning basic technology, a DNA sequence for coding a lyn protein membrane positioning sequence, dab1 protein, light sensitive protein Cry2olig and yellow fluorescent protein mCit (mCit) is connected, and the DNA sequence is introduced into an expression vector pCAG-DsRed2 to obtain a recombinant expression vector, and the recombinant expression vector is transformed into host bacterium escherichia coli to obtain a fusion protein through induced expression, wherein the fusion protein has an amino acid sequence shown as SEQ ID NO. 4, and a nucleotide sequence is shown as SEQ ID NO.7.

Example 3 preparation of Dab1-Cry2olig-mCit-KRas fusion protein

By utilizing a molecular cloning basic technology, DNA sequences for coding Dab1 protein, light-sensitive protein Cry2olig, yellow fluorescent protein mCit (mCit) and KRas protein membrane positioning sequences are connected and are introduced into an expression vector pCAG-DsRed2 to obtain a recombinant expression vector, the recombinant expression vector is transformed into host bacterium escherichia coli, and a fusion protein is obtained by induced expression, and has an amino acid sequence shown as SEQ ID NO. 5 and a nucleotide sequence shown as SEQ ID NO.8.

Example 4

Firstly, cloning Akt-PH-mCH gene fragment, transferring the Akt-PH-mCH gene fragment into an expression vector pCAG-DsRed2 to obtain an eukaryotic expression vector containing Akt-PH-mCH.

COS7 cells (purchased from ATCC) cultured in a glass-bottomed dish were transfected with the recombinant expression vector containing lyn-Opto-Dab1-mCit and the expression vector containing Akt-PH-mCh of example 2, respectively, by a Lipofectamine 2000 kit, and after overnight culture, the cells were starved for 16 hours with a serum-free, phenol red-free medium (purchased from GIBCO). Cells expressing Akt-PH-mCH probes were found using a commercial confocal microscope (e.g., zeiss LSM780 confocal microscope) using red fluorescence (fluorescence can be excited at 561nm, and received at 580-620 nm). In the experiment, a 488nm/561nm scanning area, fluorescence confocal imaging results are shown in figure 3.

The above experiment was carried out on the fusion protein Dab1-Cry2olig-mCit-KRas in the same manner, and the results are shown in fig. 4.

As can be seen from the results of FIGS. 3 to 4, the phenomenon of Akt-PH-mCH transferring to the plasma membrane occurs from 0s to 200s, and the results show that both lyn-Dab1-Cry2olig-mCit and Dab1-Cry2olig-mCit-KRas can successfully activate the PI3K pathway and generate a large amount of PI3P small molecules at the plasma membrane.

With time from 0s to 200s, akt-PH-mCh undergoes a phenomenon of translocation to the plasma membrane, and the results indicate that the PI3K pathway is activated, producing a large number of small PI3P molecules at the plasma membrane, as shown in fig. 3.

Example 5

COS7 cells (purchased from ATCC) cultured in a glass-bottomed dish were transfected with the recombinant expression vector containing lyn-Opto-Dab1-mCH of example 1 using Lipofectamine 2000 kit, and after overnight culture, the cells were starved for 16 hours using a serum-free, phenol red-free medium (purchased from GIBCO). Cells expressing Opto-Dab1-mCH were found using a commercial confocal microscope (such as Zeiss LSM780 confocal microscope) using red fluorescence (fluorescence can be excited at 561nm, fluorescence received at 580-620 nm). A small area (e.g., a circular area with a diameter of 6.6 μm) at the cell edge is selected, followed by 10 scans with 488nm excitation lines at 0.2% power (sized as required) with 0.79 μ s dwell time per pixel, followed by activation, and then the fluorescence and bright field morphologies of the cells are observed using the red fluorescence channel and the bright field channel, respectively. Imaging was performed every 5 seconds. It can be seen that at 200s, cell membrane movement was evident in the circular area and nearby, with folds and extension into the extracellular space, see fig. 5.

From the results of fig. 5, it can be seen that the white schematic circle region (i.e., activation region) is rapidly scanned by 488nm excitation light, and then the enhancement of the red signal of the activation region can be observed, and the deformation of the cytoplasmic membrane can be observed in the bright field image, and the results show that the fusion protein provided by the invention can realize the rapid activation of the PI3K/Akt signal pathway locally and with high spatial and temporal resolution, and can realize the observation of the morphological change of the cell with high spatial and temporal resolution.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

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Phe Leu Leu Leu Pro Trp Lys Trp Gly Met Lys Tyr Phe Trp Asp Thr

945 950 955 960

Leu Leu Asp Ala Asp Leu Glu Cys Asp Ile Leu Gly Trp Gln Tyr Ile

965 970 975

Ser Gly Ser Ile Pro Asp Gly His Glu Leu Asp Arg Leu Asp Asn Pro

980 985 990

Ala Leu Gln Gly Ala Lys Tyr Asp Pro Glu Gly Glu Tyr Ile Arg Gln

995 1000 1005

Trp Leu Pro Glu Leu Ala Arg Leu Pro Thr Glu Trp Ile His His Pro

1010 1015 1020

Trp Asp Ala Pro Leu Thr Val Leu Lys Ala Ser Gly Val Glu Leu Gly

1025 1030 1035 1040

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

1045 1050 1055

Leu Ala Lys Ala Ile Ser Arg Thr Arg Gly Ala Gln Ile Met Ile Gly

1060 1065 1070

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

1075 1080 1085

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

1090 1095 1100

Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala

1105 1110 1115 1120

Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu

1125 1130 1135

Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe Gly Tyr Gly Leu Met

1140 1145 1150

Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys

1155 1160 1165

Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys

1170 1175 1180

Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp

1185 1190 1195 1200

Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp

1205 1210 1215

Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn

1220 1225 1230

Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe

1235 1240 1245

Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His

1250 1255 1260

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

1265 1270 1275 1280

Asn His Tyr Leu Ser Tyr Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu

1285 1290 1295

Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile

1300 1305 1310

Thr Leu Gly Met Asp Glu Leu Tyr Lys Ser Gly Arg Asp Ser Arg Ser

1315 1320 1325

<210> 5

<211> 1320

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Ser Thr Glu Thr Glu Leu Gln Val Ala Val Lys Thr Ser Ala Lys

1 5 10 15

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

20 25 30

Lys Arg Phe Lys Gly Glu Gly Val Arg Tyr Lys Ala Lys Leu Ile Gly

35 40 45

Ile Asp Glu Val Ser Ala Ala Arg Gly Asp Lys Leu Cys Gln Asp Ser

50 55 60

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

65 70 75 80

His Lys Gln Lys Ile Phe Leu Thr Ile Ser Phe Gly Gly Ile Lys Ile

85 90 95

Phe Asp Glu Lys Thr Gly Ala Leu Gln His His His Ala Val His Glu

100 105 110

Ile Ser Tyr Ile Ala Lys Asp Ile Thr Asp His Arg Ala Phe Gly Tyr

115 120 125

Val Cys Gly Lys Glu Gly Asn His Arg Phe Val Ala Ile Lys Thr Ala

130 135 140

Gln Ala Ala Glu Pro Val Ile Leu Asp Leu Arg Asp Leu Phe Gln Leu

145 150 155 160

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

165 170 175

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

180 185 190

Val Glu Asp Pro Val Tyr Gln Tyr Ile Val Phe Glu Ala Gly His Glu

195 200 205

Pro Ile Arg Asp Pro Glu Thr Glu Glu Asn Ile Tyr Gln Val Pro Thr

210 215 220

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

225 230 235 240

Ser Ala Val Thr Gln Leu Glu Leu Phe Gly Asp Met Ser Thr Pro Pro

245 250 255

Asp Ile Thr Ser Pro Pro Thr Pro Ala Thr Pro Gly Asp Ala Phe Leu

260 265 270

Pro Ser Ser Ser Gln Thr Leu Pro Gly Ser Ala Asp Val Phe Gly Ser

275 280 285

Met Ser Phe Gly Thr Ala Ala Val Pro Ser Gly Tyr Val Ala Met Gly

290 295 300

Ala Val Leu Pro Ser Phe Trp Gly Gln Gln Pro Leu Val Gln Gln Gln

305 310 315 320

Ile Ala Met Gly Ala Gln Pro Pro Val Ala Gln Val Ile Pro Gly Ala

325 330 335

Gln Pro Ile Ala Trp Gly Gln Pro Gly Leu Phe Pro Ala Thr Gln Gln

340 345 350

Ala Trp Pro Thr Val Ala Gly Gln Phe Pro Pro Ala Ala Phe Met Pro

355 360 365

Thr Gln Thr Val Met Pro Leu Ala Ala Ala Met Phe Gln Gly Pro Leu

370 375 380

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

385 390 395 400

Pro Gln Ser Asp Lys Pro Arg Gln Lys Met Gly Lys Glu Ser Phe Lys

405 410 415

Asp Phe Gln Met Val Gln Pro Pro Pro Val Pro Ser Arg Lys Pro Asp

420 425 430

Gln Pro Ser Leu Thr Cys Thr Ser Glu Ala Phe Ser Ser Tyr Phe Asn

435 440 445

Lys Val Gly Val Ala Gln Asp Thr Asp Asp Cys Asp Asp Phe Asp Ile

450 455 460

Ser Gln Leu Asn Leu Thr Pro Val Thr Ser Thr Thr Pro Ser Thr Asn

465 470 475 480

Ser Pro Pro Thr Pro Ala Pro Arg Gln Ser Ser Pro Ser Lys Ser Ser

485 490 495

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

500 505 510

Phe Glu Ser Pro Ser Lys Ser Glu Glu Gln Glu Ala Pro Asp Gly Ser

515 520 525

Gln Ala Ser Ser Thr Ser Asp Pro Phe Gly Glu Pro Ser Gly Glu Pro

530 535 540

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

545 550 555 560

Thr Met Lys Met Asp Lys Lys Thr Ile Val Trp Phe Arg Arg Asp Leu

565 570 575

Arg Ile Glu Asp Asn Pro Ala Leu Ala Ala Ala Ala His Glu Gly Ser

580 585 590

Val Phe Pro Val Phe Ile Trp Cys Pro Glu Glu Glu Gly Gln Phe Tyr

595 600 605

Pro Gly Arg Ala Ser Arg Trp Trp Met Lys Gln Ser Leu Ala His Leu

610 615 620

Ser Gln Ser Leu Lys Ala Leu Gly Ser Asp Leu Thr Leu Ile Lys Thr

625 630 635 640

His Asn Thr Ile Ser Ala Ile Leu Asp Cys Ile Arg Val Thr Gly Ala

645 650 655

Thr Lys Val Val Phe Asn His Leu Tyr Asp Pro Val Ser Leu Val Arg

660 665 670

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

675 680 685

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

690 695 700

Lys Gly Lys Pro Phe Thr Ser Phe Asn Ser Tyr Trp Lys Lys Cys Leu

705 710 715 720

Asp Met Ser Ile Glu Ser Val Met Leu Pro Pro Pro Trp Arg Leu Met

725 730 735

Pro Ile Thr Ala Ala Ala Glu Ala Ile Trp Ala Cys Ser Ile Glu Glu

740 745 750

Leu Gly Leu Glu Asn Glu Ala Glu Lys Pro Ser Asn Ala Leu Leu Thr

755 760 765

Arg Ala Trp Ser Pro Gly Trp Ser Asn Ala Asp Lys Leu Leu Asn Glu

770 775 780

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

785 790 795 800

Val Gly Asn Ser Thr Ser Leu Leu Ser Pro Tyr Leu His Phe Gly Glu

805 810 815

Ile Ser Val Arg His Val Phe Gln Cys Ala Arg Met Lys Gln Ile Ile

820 825 830

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

835 840 845

Leu Arg Gly Ile Gly Leu Arg Glu Tyr Ser Arg Tyr Ile Cys Phe Asn

850 855 860

Phe Pro Phe Thr His Glu Gln Ser Leu Leu Ser His Leu Arg Phe Phe

865 870 875 880

Pro Trp Asp Ala Asp Val Asp Lys Phe Lys Ala Trp Arg Gln Gly Arg

885 890 895

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

900 905 910

Gly Trp Met His Asn Arg Ile Arg Val Ile Val Ser Ser Phe Ala Val

915 920 925

Lys Phe Leu Leu Leu Pro Trp Lys Trp Gly Met Lys Tyr Phe Trp Asp

930 935 940

Thr Leu Leu Asp Ala Asp Leu Glu Cys Asp Ile Leu Gly Trp Gln Tyr

945 950 955 960

Ile Ser Gly Ser Ile Pro Asp Gly His Glu Leu Asp Arg Leu Asp Asn

965 970 975

Pro Ala Leu Gln Gly Ala Lys Tyr Asp Pro Glu Gly Glu Tyr Ile Arg

980 985 990

Gln Trp Leu Pro Glu Leu Ala Arg Leu Pro Thr Glu Trp Ile His His

995 1000 1005

Pro Trp Asp Ala Pro Leu Thr Val Leu Lys Ala Ser Gly Val Glu Leu

1010 1015 1020

Gly Thr Asn Tyr Ala Lys Pro Ile Val Asp Ile Asp Thr Ala Arg Glu

1025 1030 1035 1040

Leu Leu Ala Lys Ala Ile Ser Arg Thr Arg Gly Ala Gln Ile Met Ile

1045 1050 1055

Gly Ala Ala Ala Arg Asp Pro Pro Val Ala Thr Met Val Ser Lys Gly

1060 1065 1070

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

1075 1080 1085

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

1090 1095 1100

Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys

1105 1110 1115 1120

Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe Gly Tyr Gly Leu

1125 1130 1135

Met Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe

1140 1145 1150

Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe

1155 1160 1165

Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly

1170 1175 1180

Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu

1185 1190 1195 1200

Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His

1205 1210 1215

Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn

1220 1225 1230

Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp

1235 1240 1245

His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro

1250 1255 1260

Asp Asn His Tyr Leu Ser Tyr Gln Ser Lys Leu Ser Lys Asp Pro Asn

1265 1270 1275 1280

Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly

1285 1290 1295

Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Lys Lys Lys Lys Lys Lys

1300 1305 1310

Ser Lys Thr Lys Cys Val Ile Met

1315 1320

<210> 6

<211> 3957

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgggatgta ttaaatcaaa gaggaaggac aacctcaatg acgatgaatc aactgagaca 60

gaacttcaag tagctgtgaa aaccagcgcc aagaaagact ccaggaagaa aggtcaggat 120

cgcagcgaag ccactttgat aaagaggttt aaaggcgaag gggtccggta caaagccaag 180

ctgattggga ttgatgaagt gtccgcagct cggggagaca agttatgtca agattccatg 240

atgaagctca agggtgttgt tgctggcgca cgttccaagg gagaacacaa acagaaaatc 300

tttttaacca tctcctttgg aggaatcaaa atctttgatg agaagacggg ggcccttcag 360

catcaccatg ctgttcatga aatttcctac attgcgaagg acatcacaga tcatcgggct 420

ttcggatacg tttgcgggaa ggaagggaat cacagatttg tggccatcaa aacagcccag 480

gcggctgaac ctgttatcct ggacttgaga gatctctttc aactcatcta tgagctgaag 540

caaagagaag aattggaaaa aaaggcacaa aaggataagc agtgtgaaca agctgtgtac 600

cagaccattt tggaagagga tgtggaagat cccgtgtacc agtacattgt gtttgaggct 660

ggacatgagc caatccgtga tcctgaaaca gaagagaaca tttaccaggt tcccaccagc 720

caaaagaagg aaggtgttta tgatgtgcca aaaagtcaac ctgtaagtgc tgtgacccaa 780

ttagaacttt ttggagacat gtccacccct cctgatataa cctctccccc tactcctgca 840

accccaggtg atgcctttct cccgtcgtcg tcccagacgc ttccggggag tgcagatgtg 900

tttggctcta tgtctttcgg cactgctgct gtaccctcag gttatgtcgc tatgggcgcc 960

gtcctcccat ccttctgggg ccagcagccc cttgttcaac agcagatcgc catgggtgct 1020

cagccacccg tcgctcaggt gataccagga gctcagccca tcgcatgggg ccagccaggt 1080

ctctttcctg ccacccagca agcctggccc actgtggccg ggcagttccc gccagccgcc 1140

ttcatgccca cacaaactgt tatgccttta gcagccgcca tgttccaagg tcccctcacc 1200

ccccttgcaa ccgtcccagg cacgaatgac tctgccaggt caagtccaca gagtgacaag 1260

cccaggcaga aaatggggaa ggagtctttc aaggatttcc agatggtcca gcctccaccc 1320

gtaccctccc ggaagcctga ccagccctcc ctgacctgta cctcagaggc cttctccagt 1380

tacttcaaca aagtcggggt ggcacaggat acagacgact gtgatgactt tgacatctcc 1440

caactgaact tgacccctgt gacttctacc acaccatcta ccaactcacc tccaacccca 1500

gcccctaggc agagctctcc atccaaatca tcagcatccc acgtcagtga cccgaccgca 1560

gatgacatct tcgaagaagg ctttgaaagt cccagcaaaa gtgaagaaca agaagcacct 1620

gatggatcac aggcctcctc caccagtgat ccatttgggg agcccagtgg tgagcccagt 1680

ggtgataata taagtccaca agacggtagc ggttccctcg aggccaccat gaagatggac 1740

aaaaagacta tagtttggtt tagaagagac ctaaggattg aggataatcc tgcattagca 1800

gcagctgctc acgaaggatc tgtttttcct gtcttcattt ggtgtcctga agaagaagga 1860

cagttttatc ctggaagagc ttcaagatgg tggatgaaac aatcacttgc tcacttatct 1920

caatccttga aggctcttgg atctgacctc actttaatca aaacccacaa cacgatttca 1980

gcgatcttgg attgtatccg cgttaccggt gctacaaaag tcgtctttaa ccacctctat 2040

gatcctgttt cgttagttcg ggaccatacc gtaaaggaga agctggtgga acgtgggatc 2100

tctgtgcaaa gctacaatgg agatctattg tatgaaccgt gggagatata ctgcgaaaag 2160

ggcaaacctt ttacgagttt caattcttac tggaagaaat gcttagatat gtcgattgaa 2220

tccgttatgc ttcctcctcc ttggcggttg atgccaataa ctgcagcggc tgaagcgatt 2280

tgggcgtgtt cgattgaaga actagggctg gagaatgagg ccgagaaacc gagcaatgcg 2340

ttgttaacta gagcttggtc tccaggatgg agcaatgctg ataagttact aaatgagttc 2400

atcgagaagc agttgataga ttatgcaaag aacagcaaga aagttgttgg gaattctact 2460

tcactacttt ctccgtatct ccatttcggg gaaataagcg tcagacacgt tttccagtgt 2520

gcccggatga aacaaattat atgggcaaga gataagaaca gtgaaggaga agaaagtgca 2580

gatctttttc ttaggggaat cggtttaaga gagtattctc ggtatatatg tttcaacttc 2640

ccgtttactc acgagcaatc gttgttgagt catcttcggt ttttcccttg ggatgctgat 2700

gttgataagt tcaaggcctg gagacaaggc aggaccggtt atccgttggt ggatgccgga 2760

atgagagagc tttgggctac cggatggatg cataacagaa taagagtgat tgtttcaagc 2820

tttgctgtga agtttcttct ccttccatgg aaatggggaa tgaagtattt ctgggataca 2880

cttttggatg ctgatttgga atgtgacatc cttggctggc agtatatctc tgggagtatc 2940

cccgatggcc acgagcttga tcgcttggac aatcccgcgt tacaaggcgc caaatatgac 3000

ccagaaggtg agtacataag gcaatggctt cccgagcttg cgagattgcc aactgaatgg 3060

atccatcatc catgggacgc tcctttaacc gtactcaaag cttctggtgt ggaactcgga 3120

acaaactatg cgaaacccat tgtagacatc gacacagctc gtgagctact agctaaagct 3180

atttcaagaa cccgtggagc acagatcatg atcggagcag cagcccggga tccaccggtc 3240

gccaccatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc 3300

ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc 3360

gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc 3420

ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg 3480

aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg 3540

gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg 3600

caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc 3660

cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag 3720

gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac 3780

gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac 3840

aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag 3900

tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaa 3957

<210> 7

<211> 3987

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgggatgta ttaaatcaaa gaggaaggac aacctcaatg acgatgaatc aactgagaca 60

gaacttcaag tagctgtgaa aaccagcgcc aagaaagact ccaggaagaa aggtcaggat 120

cgcagcgaag ccactttgat aaagaggttt aaaggcgaag gggtccggta caaagccaag 180

ctgattggga ttgatgaagt gtccgcagct cggggagaca agttatgtca agattccatg 240

atgaagctca agggtgttgt tgctggcgca cgttccaagg gagaacacaa acagaaaatc 300

tttttaacca tctcctttgg aggaatcaaa atctttgatg agaagacggg ggcccttcag 360

catcaccatg ctgttcatga aatttcctac attgcgaagg acatcacaga tcatcgggct 420

ttcggatacg tttgcgggaa ggaagggaat cacagatttg tggccatcaa aacagcccag 480

gcggctgaac ctgttatcct ggacttgaga gatctctttc aactcatcta tgagctgaag 540

caaagagaag aattggaaaa aaaggcacaa aaggataagc agtgtgaaca agctgtgtac 600

cagaccattt tggaagagga tgtggaagat cccgtgtacc agtacattgt gtttgaggct 660

ggacatgagc caatccgtga tcctgaaaca gaagagaaca tttaccaggt tcccaccagc 720

caaaagaagg aaggtgttta tgatgtgcca aaaagtcaac ctgtaagtgc tgtgacccaa 780

ttagaacttt ttggagacat gtccacccct cctgatataa cctctccccc tactcctgca 840

accccaggtg atgcctttct cccgtcgtcg tcccagacgc ttccggggag tgcagatgtg 900

tttggctcta tgtctttcgg cactgctgct gtaccctcag gttatgtcgc tatgggcgcc 960

gtcctcccat ccttctgggg ccagcagccc cttgttcaac agcagatcgc catgggtgct 1020

cagccacccg tcgctcaggt gataccagga gctcagccca tcgcatgggg ccagccaggt 1080

ctctttcctg ccacccagca agcctggccc actgtggccg ggcagttccc gccagccgcc 1140

ttcatgccca cacaaactgt tatgccttta gcagccgcca tgttccaagg tcccctcacc 1200

ccccttgcaa ccgtcccagg cacgaatgac tctgccaggt caagtccaca gagtgacaag 1260

cccaggcaga aaatggggaa ggagtctttc aaggatttcc agatggtcca gcctccaccc 1320

gtaccctccc ggaagcctga ccagccctcc ctgacctgta cctcagaggc cttctccagt 1380

tacttcaaca aagtcggggt ggcacaggat acagacgact gtgatgactt tgacatctcc 1440

caactgaact tgacccctgt gacttctacc acaccatcta ccaactcacc tccaacccca 1500

gcccctaggc agagctctcc atccaaatca tcagcatccc acgtcagtga cccgaccgca 1560

gatgacatct tcgaagaagg ctttgaaagt cccagcaaaa gtgaagaaca agaagcacct 1620

gatggatcac aggcctcctc caccagtgat ccatttgggg agcccagtgg tgagcccagt 1680

ggtgataata taagtccaca agacggtagc ggttccctcg aggccaccat gaagatggac 1740

aaaaagacta tagtttggtt tagaagagac ctaaggattg aggataatcc tgcattagca 1800

gcagctgctc acgaaggatc tgtttttcct gtcttcattt ggtgtcctga agaagaagga 1860

cagttttatc ctggaagagc ttcaagatgg tggatgaaac aatcacttgc tcacttatct 1920

caatccttga aggctcttgg atctgacctc actttaatca aaacccacaa cacgatttca 1980

gcgatcttgg attgtatccg cgttaccggt gctacaaaag tcgtctttaa ccacctctat 2040

gatcctgttt cgttagttcg ggaccatacc gtaaaggaga agctggtgga acgtgggatc 2100

tctgtgcaaa gctacaatgg agatctattg tatgaaccgt gggagatata ctgcgaaaag 2160

ggcaaacctt ttacgagttt caattcttac tggaagaaat gcttagatat gtcgattgaa 2220

tccgttatgc ttcctcctcc ttggcggttg atgccaataa ctgcagcggc tgaagcgatt 2280

tgggcgtgtt cgattgaaga actagggctg gagaatgagg ccgagaaacc gagcaatgcg 2340

ttgttaacta gagcttggtc tccaggatgg agcaatgctg ataagttact aaatgagttc 2400

atcgagaagc agttgataga ttatgcaaag aacagcaaga aagttgttgg gaattctact 2460

tcactacttt ctccgtatct ccatttcggg gaaataagcg tcagacacgt tttccagtgt 2520

gcccggatga aacaaattat atgggcaaga gataagaaca gtgaaggaga agaaagtgca 2580

gatctttttc ttaggggaat cggtttaaga gagtattctc ggtatatatg tttcaacttc 2640

ccgtttactc acgagcaatc gttgttgagt catcttcggt ttttcccttg ggatgctgat 2700

gttgataagt tcaaggcctg gagacaaggc aggaccggtt atccgttggt ggatgccgga 2760

atgagagagc tttgggctac cggatggatg cataacagaa taagagtgat tgtttcaagc 2820

tttgctgtga agtttcttct ccttccatgg aaatggggaa tgaagtattt ctgggataca 2880

cttttggatg ctgatttgga atgtgacatc cttggctggc agtatatctc tgggagtatc 2940

cccgatggcc acgagcttga tcgcttggac aatcccgcgt tacaaggcgc caaatatgac 3000

ccagaaggtg agtacataag gcaatggctt cccgagcttg cgagattgcc aactgaatgg 3060

atccatcatc catgggacgc tcctttaacc gtactcaaag cttctggtgt ggaactcgga 3120

acaaactatg cgaaacccat tgtagacatc gacacagctc gtgagctact agctaaagct 3180

atttcaagaa cccgtggagc acagatcatg atcggagcag cagcccggga tccaccggtc 3240

gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 3300

ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 3360

acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg 3420

cccaccctcg tgaccacctt cggctacggc ctgatgtgct tcgcccgcta ccccgaccac 3480

atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 3540

atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 3600

accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 3660

gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag 3720

aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag 3780

ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac 3840

aaccactacc tgagctacca gtccaaactg agcaaagacc ccaacgagaa gcgcgatcac 3900

atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac 3960

aagagcggcc gcgactctag atcataa 3987

<210> 8

<211> 3963

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgtcaactg agacagaact tcaagtagct gtgaaaacca gcgccaagaa agactccagg 60

aagaaaggtc aggatcgcag cgaagccact ttgataaaga ggtttaaagg cgaaggggtc 120

cggtacaaag ccaagctgat tgggattgat gaagtgtccg cagctcgggg agacaagtta 180

tgtcaagatt ccatgatgaa gctcaagggt gttgttgctg gcgcacgttc caagggagaa 240

cacaaacaga aaatcttttt aaccatctcc tttggaggaa tcaaaatctt tgatgagaag 300

acgggggccc ttcagcatca ccatgctgtt catgaaattt cctacattgc gaaggacatc 360

acagatcatc gggctttcgg atacgtttgc gggaaggaag ggaatcacag atttgtggcc 420

atcaaaacag cccaggcggc tgaacctgtt atcctggact tgagagatct ctttcaactc 480

atctatgagc tgaagcaaag agaagaattg gaaaaaaagg cacaaaagga taagcagtgt 540

gaacaagctg tgtaccagac cattttggaa gaggatgtgg aagatcccgt gtaccagtac 600

attgtgtttg aggctggaca tgagccaatc cgtgatcctg aaacagaaga gaacatttac 660

caggttccca ccagccaaaa gaaggaaggt gtttatgatg tgccaaaaag tcaacctgta 720

agtgctgtga cccaattaga actttttgga gacatgtcca cccctcctga tataacctct 780

ccccctactc ctgcaacccc aggtgatgcc tttctcccgt cgtcgtccca gacgcttccg 840

gggagtgcag atgtgtttgg ctctatgtct ttcggcactg ctgctgtacc ctcaggttat 900

gtcgctatgg gcgccgtcct cccatccttc tggggccagc agccccttgt tcaacagcag 960

atcgccatgg gtgctcagcc acccgtcgct caggtgatac caggagctca gcccatcgca 1020

tggggccagc caggtctctt tcctgccacc cagcaagcct ggcccactgt ggccgggcag 1080

ttcccgccag ccgccttcat gcccacacaa actgttatgc ctttagcagc cgccatgttc 1140

caaggtcccc tcacccccct tgcaaccgtc ccaggcacga atgactctgc caggtcaagt 1200

ccacagagtg acaagcccag gcagaaaatg gggaaggagt ctttcaagga tttccagatg 1260

gtccagcctc cacccgtacc ctcccggaag cctgaccagc cctccctgac ctgtacctca 1320

gaggccttct ccagttactt caacaaagtc ggggtggcac aggatacaga cgactgtgat 1380

gactttgaca tctcccaact gaacttgacc cctgtgactt ctaccacacc atctaccaac 1440

tcacctccaa ccccagcccc taggcagagc tctccatcca aatcatcagc atcccacgtc 1500

agtgacccga ccgcagatga catcttcgaa gaaggctttg aaagtcccag caaaagtgaa 1560

gaacaagaag cacctgatgg atcacaggcc tcctccacca gtgatccatt tggggagccc 1620

agtggtgagc ccagtggtga taatataagt ccacaagacg gtagcggttc cctcgaggcc 1680

accatgaaga tggacaaaaa gactatagtt tggtttagaa gagacctaag gattgaggat 1740

aatcctgcat tagcagcagc tgctcacgaa ggatctgttt ttcctgtctt catttggtgt 1800

cctgaagaag aaggacagtt ttatcctgga agagcttcaa gatggtggat gaaacaatca 1860

cttgctcact tatctcaatc cttgaaggct cttggatctg acctcacttt aatcaaaacc 1920

cacaacacga tttcagcgat cttggattgt atccgcgtta ccggtgctac aaaagtcgtc 1980

tttaaccacc tctatgatcc tgtttcgtta gttcgggacc ataccgtaaa ggagaagctg 2040

gtggaacgtg ggatctctgt gcaaagctac aatggagatc tattgtatga accgtgggag 2100

atatactgcg aaaagggcaa accttttacg agtttcaatt cttactggaa gaaatgctta 2160

gatatgtcga ttgaatccgt tatgcttcct cctccttggc ggttgatgcc aataactgca 2220

gcggctgaag cgatttgggc gtgttcgatt gaagaactag ggctggagaa tgaggccgag 2280

aaaccgagca atgcgttgtt aactagagct tggtctccag gatggagcaa tgctgataag 2340

ttactaaatg agttcatcga gaagcagttg atagattatg caaagaacag caagaaagtt 2400

gttgggaatt ctacttcact actttctccg tatctccatt tcggggaaat aagcgtcaga 2460

cacgttttcc agtgtgcccg gatgaaacaa attatatggg caagagataa gaacagtgaa 2520

ggagaagaaa gtgcagatct ttttcttagg ggaatcggtt taagagagta ttctcggtat 2580

atatgtttca acttcccgtt tactcacgag caatcgttgt tgagtcatct tcggtttttc 2640

ccttgggatg ctgatgttga taagttcaag gcctggagac aaggcaggac cggttatccg 2700

ttggtggatg ccggaatgag agagctttgg gctaccggat ggatgcataa cagaataaga 2760

gtgattgttt caagctttgc tgtgaagttt cttctccttc catggaaatg gggaatgaag 2820

tatttctggg atacactttt ggatgctgat ttggaatgtg acatccttgg ctggcagtat 2880

atctctggga gtatccccga tggccacgag cttgatcgct tggacaatcc cgcgttacaa 2940

ggcgccaaat atgacccaga aggtgagtac ataaggcaat ggcttcccga gcttgcgaga 3000

ttgccaactg aatggatcca tcatccatgg gacgctcctt taaccgtact caaagcttct 3060

ggtgtggaac tcggaacaaa ctatgcgaaa cccattgtag acatcgacac agctcgtgag 3120

ctactagcta aagctatttc aagaacccgt ggagcacaga tcatgatcgg agcagcagcc 3180

cgggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg 3240

cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag 3300

ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag 3360

ctgcccgtgc cctggcccac cctcgtgacc accttcggct acggcctgat gtgcttcgcc 3420

cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac 3480

gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg 3540

aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag 3600

gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc 3660

atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag 3720

gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc 3780

gtgctgctgc ccgacaacca ctacctgagc taccagtcca aactgagcaa agaccccaac 3840

gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc 3900

atggacgagc tgtacaagaa gaagaaaaag aagaagtcca agaccaagtg cgtgatcatg 3960

taa 3963

Claims

1. A fusion protein, comprising a plasma membrane localization sequence, a Dab1 protein, a light sensitive protein and a fluorescent protein;

the amino acid sequence of the fusion protein is shown in any one of SEQ ID NO 3-5.

2. A DNA molecule encoding the fusion protein of claim 1.

3. A recombinant DNA vector comprising the DNA molecule of claim 2.

4. A host cell comprising the recombinant DNA vector of claim 3.

5. Use of the fusion protein of claim 1 in the preparation of a medicament for a disease associated with the PI3K/Akt signaling pathway, including neurodegenerative diseases, cardiovascular and cerebrovascular diseases, and diabetes.