KR102074591B1 - PI3P binding probe derived from RavZ protein and endosome detection method using the same - Google Patents

Abstract

The present invention relates to: a probe for detecting endosomes, comprising a membrane targeting (MT) sequence of RavZ protein which forms a bond with PI3P, and a gene of a reporter fluorescent protein, in order to confirm the formation and distribution of endosomes; and a detection method for confirming the formation and distribution of endosomes using the probe.

Description

PI3P binding probe derived from RavZ protein and endosome detection method using the same}

In order to confirm the formation and distribution of intracellular vesicles, an intracellular vesicle detection probe comprising a Membrane targeting (MT) site and a reporter fluorescent protein of RavZ protein forming a bond with PI3P and intracellular vesicles using the same To a method of detection of formation and distribution.

Phosphoinositide is one of the phospholipids present in small amounts in cells and plays a variety of important roles, such as intracellular signal transduction. Phosphoinositides exist in various phosphorylated forms by phosphatase, which act as phospholipid markers of specific organelles. Among them, phosphatidylinositol-3-phosphate (PI3P) is known as a marker for early endosomes (vesicles) in cells and is known to play an important role in the formation of early autovesicles. (It plays an important role in mediating autophagy. (Levine, B, and Klionsky, DJ (2004) Development by self-digestion: molecular mechanisms and biological functions of autophagy Dev Cell 6, 463-477). In addition, the PI3P-positive endosomal probe developed in the past can be targeted to the PI3P-positive endosomes, thereby inhibiting endogenous PI3P function. There was a possibility.

RavZ protein is derived from Legionella pneumophila and is known to play a role in interfering with autophagy of host cells, and the domain present in RavZ contains a cell membrane targeting site that binds to PI3P. . In this study, we developed a probe to probe PI3P-positive vesicles by drug treatment using PI3P binding site of RavZ protein.

1.Patent Application No. 10-2016-7032300 2. Patent Application No. 10-2012-7004431

Kwon, et al. The 1: 2 complex between RavZ and LC3 reveals a mechanism for deconjugation of LC3 on the phagophore membrane

Disclosure of Invention An object of the present invention is to provide a probe for detecting intracellular vesicles comprising a gene of a reporter fluorescent protein and a Membrane targeting (MT) sequence of RavZ protein that forms a bond with PI3P to confirm the formation and distribution of intracellular vesicles. It is.

Another object of the present invention is to provide a method for detecting the formation and distribution of intracellular vesicles, the method using the probe.

In order to solve the above problems of the present invention, the present invention includes a membrane binding (Membrane targeting (MT)) site and a reporter fluorescent protein of RavZ protein forming a bond with PI3P to confirm the formation and distribution of intracellular vesicles It provides a probe for detecting intracellular vesicles.

The probe of the present invention includes a membrane binding (MT) site (RavZ (MT)) and a reporter fluorescent protein that bind to PI3P present in the cytoplasm among domains present in the RavZ protein, wherein RavZ (MT) is a monomer. Or a dimer (RavZ (2xMT)). RavZ (MT), which is a monomer, does not have targeting characteristics for intracellular vesicles, but when RavZ (MT) is a dimer (RavZ (2xMT)), it is targeted to intracellular vesicles, which can be confirmed by fluorescent labeling.

In one embodiment of the present invention, examples of the reporter protein are green fluorescent protein (GFP), modified green fluorescent protein (mGFP), enhanced green fluorescent protein (enhanced green fluorescent protein). EGFP), red fluorescent protein (RFP), modified red fluorescent protein (mRFP) and the like, preferably enhanced green fluorescent protein (EGFP) or modified red fluorescent protein ( mRFP) can be used. Probe of the present invention can be confirmed by the formation of the intracellular vesicle formation and distribution directly in the state of maintaining the cell by including the reporter protein.

In addition, when RavZ (MT) is present as a monomer, additionally, FRB or FKBP may be combined with each other. That is, in one embodiment of the present invention, such a probe was manufactured as RavZ (MT) -EGFP-FRB or mRFP-FKBP-RavZ (MT), respectively. The FRB / rapamycin / FKBP system is used, and the FRB / rapamycin / FKBP system is a complex using the property that FRB (FKBP12-rapamycin binding) and FKBP (FK506 binding protein) are bound by rapamycin. In the absence of mycin, the complex is not formed and exists as a separate probe (RavZ (MT) -EGFP-FRB / mRFP-FKBP-RavZ (MT)), and when treated with rapamycin, the complex forms a MT of RavZ. The site forms a dimer, and as described above, RavZ (2xMT) targets intracellular vesicles. Through this process, the probe of the present invention can label the vesicles formed in the cells (including endosome, autoendoplasmic reticulum or autophagy) so that the appearance and distribution of vesicles in the cell can be confirmed.

In order to achieve another object of the present invention, the present invention provides a method for detecting the formation and distribution of intracellular vesicles, using the probe.

More specifically, the method for detecting intracellular vesicles of the present invention utilizes a feature that targets vesicles (endosomes) in cells when EGFP-RavZ (2xMT), which is a RavZ (MT) dimer, is expressed in cells. That is, as a result of expressing a probe including an early endosomal marker (Rab5) and a late endosomal marker (Rab7) together to identify a portion of the probe that is labeled in the cell, the initial endo at the site targeted by the probe It was confirmed that the probe including some markers is also located.

In addition, a detection method using a probe (RavZ (MT) -EGFP-FRB / mRFP-FKBP-RavZ (MT)) in which a FRB or FKBP is bound to a RavZ (MT) monomer, respectively, may be performed using the two types of probes. After each expression in the cells, the rapamycin treatment step, FRB, FKBP of the two probes are combined with each other to form RavZ (2xMT), so it is targeted to the endosomes in the cell. Through this process, the formation and distribution of intracellular vesicles (endosomes) can be confirmed by treating rapamycin as needed without affecting other functions of the cells.

In particular, the reporter protein that binds to the FRB or FKBP-coupled probe may be manufactured to have a different color, and the reporter protein may be manufactured to include the reporter protein in at least one of the two probes. Due to the change, the formation and distribution of endosomes can be confirmed.

A probe for detecting autophagy comprising a Membrane targeting (MT) site of RavZ protein and a reporter fluorescent protein that forms a bond with PI3P of the present invention is an intracellular vesicle containing an initial autophagy using the interaction of the protein. Can be effectively detected.

Figure 1 shows the results of confirming the binding to RavZ (MT) and RavZ (2xMT) and PI3P of the present invention.
Figure 2 shows the results of targeting the intracellular vesicles RavZ (MT) probe using the FRB / rapamycin / FKBP system.

In the previous studies, the inventors found that when there is only one PI3P binding site of Ravz protein (GFP-RavZ (PI3P)), it is not located in the endosomes in the cell but located in the cytoplasm, and when two PI3P binding sites exist (GFP-RavZ (2xPI3P)) It was confirmed that the cells are located in Rab5-positive endosomes.

Therefore, in the present invention, RavZ (PI3P) -FKBP and GFP-RavZ (PI3P) -FRB were produced by incorporating the above technique and further combining the above technique with the FRB-rapamycin-FKBP system, which is usually present as a monomer. When expressed in cells, they are distributed in the cytoplasm. Treatment of rapamycin in the cells forms a dimer of RavZ (PI3P) as the FRB-rapamycin-FKBP complex is formed, and it can be confirmed through the distribution of fluorescent proteins that they are located in the endosome. As described above, the probe of the present invention is normally located in the cytoplasm and can detect the formation and distribution of PI3P-positive endosomes when necessary without affecting the function of the PI3P-positive endosomes. In the present invention, since the probe contains a membrane targeting site of PI3P, it is described as RavZ (MT) for monomers and RavZ (2xMT) for dimers.

In the present invention, "intracellular vesicle" includes "endosome", which means vesicles surrounding molecules introduced from the cell membrane, and autophagy generated as a result of endocytosis such as autophagy. ). In particular, the endosome in the present invention refers to the initial endosome associated with the synthesis of PI3P, the probe of the present invention includes a site that specifically binds to the PI3P, by binding to the PI3P expressed in the early endosome It includes a detectable function.

In the present invention, "probe" (probe) is a substance that can be diagnosed by distinguishing the intracellular vesicles from other cells, polypeptides showing an increase or decrease by the formation and distribution of intracellular vesicles (endosome) Or organic biomolecules such as nucleic acids (eg mRNA), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, polysaccharides, etc.) and the like.

"Probe" refers to a nucleic acid fragment, such as RNA or DNA, that corresponds to a few bases to hundreds of bases that can achieve specific binding with mRNA. Probes can be made in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes and the like. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

"Nucleic acid" is meant to include any DNA or RNA, such as chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in tissue samples. One or both strands of a double-stranded nucleic acid molecule and any fragment or portion of an intact nucleic acid molecule.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role in protein coding or transcription or in the regulation of other gene expression. The gene may consist of any nucleic acid encoding a functional protein or only a portion of a nucleic acid encoding or expressing a protein. Nucleic acid sequences can include gene abnormalities in exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences, or unique sequences adjacent to genes.

A "protein" is also intended to include fragments, analogs and derivatives of a protein that retain essentially the same biological activity or function as the reference protein.

"Transformation, transfection or transfection" refers to the process by which extracellular DNA enters a host cell in the presence or absence of an accompanying substance. A “transfected cell” refers to a cell in which extracellular DNA is introduced into the cell and has extracellular DNA. DNA can be introduced into cells so that nucleic acids can be inserted into chromosomes or replicated into extrachromosomal material. Cells into which external DNA is introduced are called "transformers."

The term "vector" refers to any nucleic acid comprising a competent nucleotide sequence that is inserted into a host cell, recombined with and inserted into the host cell genome, or spontaneously replicates as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and the like.

By "label" or "label" is meant a compound or composition that directly or indirectly facilitates detection of a reagent conjugated to, or fused to, conjugated to, or fused to a reagent, such as a nucleic acid probe or antibody. The label may itself be detected (eg, a radioisotope label or fluorescent label) or, in the case of an enzyme label, may catalyze the chemical modification of the detectable substrate compound or composition.

Throughout this specification, the terms “comprises” and “comprising”, unless otherwise indicated in the context, include any given step or element, or group of steps or elements, but any other step or element, or step or element It should be understood that this group is not excluded.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated concretely.

The probe of the present invention includes a membrane binding (MT) site (RavZ (MT)) and a reporter fluorescent protein that bind to PI3P present in the cytoplasm among domains present in the RavZ protein. The RavZ (MT) may be a monomer or a dimer (RavZ (2xMT)), but in the case of the monomer, RavZ (MT) does not have specific binding to PI3P.

The dimer RavZ (2xMT) binds to PI3P, a lipid expressed in intracellular vesicles. In order to confirm what the target is GFP-RavZ (2xMT) prepared in one embodiment of the present invention, Rab5-RFP and early endosomal marker Rab7-RFP was expressed together. As a result, it was confirmed that EGFP-RavZ (2xMT) and Rab5-RFP were targeted at almost the same position. Therefore, it was confirmed that EGFP-RavZ (2xMT) can be utilized as a probe that binds to PI3P expressed in early endosomes.

In addition, RavZ (MT) of the present invention can be produced and used as a monomer, RavZ (PI3P) -FKBP and GFP-RavZ (PI3P) -FRB in combination with the FRB-rapamycin-FKBP system. As described above, FRB-rapamycin-FKBP utilizes the properties of two proteins that bind in the presence of rapamycin. In the absence of rapamycin, the probe is present as a monomer, and when treated with rapamycin, it forms a dimer. Finally, it binds to PI3P. This property does not normally affect the function of other organs in the cell and, if necessary, has the advantage of being able to probe the distribution of endosomes in the cell by treating the drug.

The RavZ protein is derived from Legionella pneumophila, and is known to play a role in inhibiting autophagy by directly binding to and removing autophagosome membrane proteins. The PI3P membrane binding (MT) site derived from the RavZ protein may include, but is not limited to, the amino acid sequence of SEQ ID NO: 1 or a nucleotide sequence encoding the same. Such genes or proteins include variants by wild type as well as deletions, insertions, non-conservative or conservative substitutions or combinations thereof.

The gene encoding the protein, etc., by referring to the nucleotide sequence known in the art, deletes the oligonucleotide complementary to its terminal portion, and then uses the primer as a primer and genomic DNA or cDNA of a specific cell as a template. It can be obtained by performing.

The present invention includes both functional equivalents of the proteins. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence with the original amino acid sequence as a result of the addition, substitution, or deletion of the amino acid It has the same identity and refers to the protein which shows substantially homogeneous physiological activity. 'Substantially homogeneous physiological activity' refers to the activity of each protein involved in the mechanism of child interaction.

In addition, the present invention can be expressed by fusing a fluorescent protein as a reporter gene in order to detect the intracellular level of the endosomes. The reporter fluorescent protein gene may be expressed in accordance with the promoter activity of the upstream (upstream) of the gene can be confirmed whether the expression in the cell. Examples of reporter fluorescent proteins include green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), modified red fluorescence Protein (mRFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), indigo fluorescent protein (CFP), and enhanced indigo fluorescent protein (ECFP) and Renila luciferase, red fluorescent protein (DsRed), and the like, but are not limited to the examples above. Preferably, green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), modified red fluorescent protein (mRFP) ), Most preferably enhanced green fluorescent protein (EGFP) and modified red fluorescent protein (mRFP) are used as reporter proteins.

In another aspect, the present invention relates to a recombinant vector for detecting intracellular vesicles, comprising the probe sequence for detecting intracellular vesicles.

The vector may include, for example, each of the genes contained in the probe or may include a sequence encoding a recombinant protein to which they are fused.

The "vector" refers to a gene construct which is an expression vector capable of expressing a protein of interest in a suitable host cell, and which contains essential regulatory elements operably linked to express the gene insert. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. Plasmid vectors include (a) an initiation point for replication to efficiently replicate hundreds of plasmid vectors per host cell, (b) antibiotic resistance genes to allow selection of host cells transformed with the plasmid vector, and (c It has a structure that includes restriction enzyme cleavage site where foreign DNA fragments can be inserted. Although no suitable restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods can facilitate ligation of the vector and foreign DNA.

The "operably linked" means that when the gene is linked downstream of the promoter sequence of the present invention, it is linked in a form capable of expression of the gene, and further include any sequence to achieve the above object. Can be. Examples of the arbitrary sequences include an operator sequence, a sequence for in frame, and the like, as well as a cis element such as an enhancer and a splicing signal in addition to the gene of the present invention. (splicing signal), poly A addition signal (poly A addition signal), a selection marker (selection marker), a ribosome binding sequence (ribosome binding sequence, SD sequence) and the like may be further included. As an example of a selection marker, chloramphenicol resistance gene, ampicillin resistance gene, dihydrofolate reductase, neomycin resistance gene, etc. may be used, but additional components for operably linked by the above examples are limited. It is not.

In another aspect, the present invention relates to a composition for detecting intracellular vesicles for determining the intracellular vesicle formation and distribution, comprising the probe or the vector.

In one embodiment of the present invention, the detection may be performed by measuring the expression level of the reporter fluorescent protein. The expression level measurement includes all of the mRNA or protein expression levels thereof. Analytical methods for measuring mRNA expression levels include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real time reverse transcriptase (Realtime RT-PCR), and RNase protection assay (RPA; RNase protection assay, Northern blotting, DNA chip, and the like, but are not limited thereto.

At this time, the primers used may initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures. The primers of the present invention are sense and antisense nucleic acids having 7 to 50 nucleotide sequences as primers specific for each marker gene. Primers can incorporate additional features that do not change the basic properties of the primers that serve as a starting point for DNA synthesis. The primers can be chemically synthesized using other well known methods and can be modified using many means known in the art.

The protein expression level can be determined by using an antibody that specifically binds to the protein of the gene. "Antibody" means a specific protein molecule directed against an antigenic site. Polyclonal antibodies, monoclonal antibodies and recombinant antibodies. Analytical methods for this purpose include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, and rockets. Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorter (FACS), protein chip, etc. .

In another aspect, the present invention provides a cell transformed with the recombinant vector. The transformation method is a method known in the art, such as, but not limited to, heat shock method (heat shock method, calcium phosphate precipitation method, electroporation, gene gun, silicone Although there are carbide carbide whiskers, sonication, and precipitation using polyethylene (polyethylenglycol), a natural introduction method, etc., the method of introducing the vector of the present invention is not limited by the above examples.

In one embodiment of the present invention, the cell line used for transfection (transformation) is preferably an animal cell, more preferably a mammalian cell line. The medium that can be used in the present invention may be any medium conventionally used for culturing animal cells, for example, Eagles' MEM (Eagle's minimum essential medium, Eagle, H. Science 130: 432 (1959)), α -MEM (Stanner, CP et al., Nat. New Biol. 230: 52 (1971)), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147: 923 (1978)), 199 medium (Morgan et al., Proc. Soc. Exp. Bio. Med., 73: 1 (1950)), CMRL 1066, RPMI 1640 (Moore et al., J. Amer. Med. Assoc. 199: 519 (1967) ), F12 (Ham, Proc. Natl. Acad. Sci. USA 53: 288 (1965)), F10 (Ham, RG Exp. Cell Res. 29: 515 (1963)), DMEM (Dulbecco's modification of Eagle's medium, Dulbecco , R. et al., Virology 8: 396 (1959)), mixtures of DMEM and F12 (Barnes, D. et al., Anal. Biochem. 102: 255 (1980)), Way-mouth's MB752 / 1 (Waymouth , CJ Natl. Cancer Inst. 22: 1003 (1959)), McCoy's 5A (McCoy, TA, et al., Proc. Soc.Exp. Biol. Med. 100: 115 (1959)) and the MCDB series (Ham, RG et al., In Vitro 14:11 (1978)) and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing the invention, the preferred materials and methods are described herein.

1. Cell Culture, Transfection, Drug Treatment and Cells Imaging

HEK293T cells or MEFs were moistened at 37 ° C. with 5% (v / v) CO ₂ in cell culture medium (Dulbecco's modified Eagle's medium [DMEM]: 10% [v / v] FBS and penicillin / streptomycin supplement). Grown in. Primary neuronal cultures were performed.

For serum starvation, HeLa cells or MEFs were incubated for 24 h in cell culture medium without FBS. The cells were transfected with DNA constructs using either calcium phosphate method or Lipofectamine 2000 (Life Technologies, Carlsbad, Calif., USA) and incubated for 24 hours.

Fluorescence images were obtained by confocal laser-scanning microscope (Radiance 2000, Zeiss) and analyzed by NIH Image J software (National Institutes of Health, Bethesda, MD, USA).

To induce autophagy, HeLa cells, WT MEFs, atg5-/-MEFs, or cultured neurons with 10 nM rapamycin for 4 hours in the presence or absence of 10 mM NH ₄ Cl depending on cell type and experiment Incubated for

2. RavZ  of mine PI3P  Identify membrane targeting

Among the domains present in the RavZ protein, in order to confirm the binding property of the membrane binding site that binds to the cell membrane phospholipid, PI3P, the sequence (SEQ ID NO: 1) and the GFP, which is a fluorescent protein, are fused to EGFP-RavZ (MT) ( SEQ ID NO: 6) was prepared. (FIG. 1A)

In the EGFP-RavZ (MT), a dimer, EGFP-RavZ (2xMT) (SEQ ID NO: 7) was prepared to further include an MT site, and each probe was expressed in cells. (Figure 1A)

As a result, as shown in Figure 1A, it was confirmed that the monomer probe is located mostly in the cytoplasm, in the case of the dimer probe, it was confirmed that it can be targeted to the endoplasmic reticulum distribution thereof.

In addition, as a result of comparing the early endosomal marker (Rab5) and the late endosomal marker (Rab7) together, as shown in Figure 1B, it can be confirmed that the expression position of the initial endosomal marker and the dimer probe of the present invention are the same. there was. That is, it was confirmed from these results that the dimer probe of the present invention had the property of binding to the initial endosomes.

In addition, antimycin A (Antimycin A) is a drug that inhibits intracellular ATP synthesis. When processed into cells, PI3P, PI4P, and PI (4,5) P ₂ are lipids generated by phosphorylation of PI in cells. The back is not made. GFP-PLCδ1-PH, which binds to the plasma membrane and binds to PI (4,5) P ₂ as a control, disappears from plasma membrane targeting by treatment with antimycin A. In the case of EGFP-RavZ (2xMT), intracellular endosomes We noticed that most of the targeting for is gone. (Figure 1C)

3. FRB / rapamycin Of FKBP  System Probe  making

The monomer probe prepared in Example 2 was prepared using a FRB / rapamycin / FKBP system operable probe by rapamycin. The amino acid sequence of the FRB protein (SEQ ID NO: 2) and the amino acid sequence of the FKBP protein (SEQ ID NO: 3) are respectively bound to the monomer probe (SEQ ID NO: 6), and the fluorescent protein is attached to each probe to RavZ (MT) -EGFP-. FRB (SEQ ID NO: 8) and mRFP-FKBP-RavZ (MT) (SEQ ID NO: 9) were produced, respectively. Since the produced probe RavZ (MT) is present as a monomer, it can be confirmed that all are located in the cytoplasm. (Figure 2A phase)

After treatment with 1 μM of rapamycin in the cells, as a result of observation, it can be seen that each probe is targeted to the endosomes as shown in the lower part of FIG. 2A. This shows that the FRB / rapamycin / FKBP complex is formed so that RavZ (MT) -EGFP-FRB and mRFP-FKBP-RavZ (MT) exist as dimer probes to target intracellular endosomes.

In addition, the same result can be confirmed by labeling only one of the probes with a fluorescent substance. As shown in FIG. 2B, RavZ (MT) -EGFP-FRB and fluorescently unlabeled FKBP-RavZ (MT) were expressed and treated with rapamycin in the same manner. .

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

<110> Kyungpook National University Industry- Academic Cooperation Foundation          Hannam University Institute for Industry-Academia Cooperation <120> PI3P binding probe derived from RavZ protein and endosome          detection method using the same <130> PN1807-233 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 105 <212> PRT <213> legionella pneumophila <400> 1 Pro Val Gln Leu Ser Glu Phe Ile Val Ala Leu Glu Asp Tyr Gly Lys   1 5 10 15 Leu Arg Ser Gln Gln Ser Glu Lys Ser Met Leu Asn Phe Ile Gly Tyr              20 25 30 Ser Lys Thr Ala Lys Leu Thr Ala Val Glu Leu Leu Ile Gly Ile Leu          35 40 45 Asn Asp Ile Lys Gly Lys Asn Glu Ile Ser Glu Ser Gln Tyr Asp Lys      50 55 60 Leu Val Lys Glu Val Asp Cys Leu Met Asp Ser Ser Leu Gly Lys Leu  65 70 75 80 Val Gln Phe His Leu Lys Asn Leu Gly Ala Glu Ser Leu Gln Lys Leu                  85 90 95 Val Leu Pro Cys Val Lys Phe Asp Asp             100 105 <210> 2 <211> 93 <212> PRT <213> Artificial Sequence <220> <223> FKBP12-rapamycin binding sequence <400> 2 Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu Glu Ala Ser Arg   1 5 10 15 Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu              20 25 30 Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr          35 40 45 Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp      50 55 60 Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala  65 70 75 80 Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys                  85 90 <210> 3 <211> 107 <212> PRT <213> Unknown <220> <223> Human FKBP12, FK506 binding protein <400> 3 Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro   1 5 10 15 Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp              20 25 30 Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe          35 40 45 Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala      50 55 60 Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr  65 70 75 80 Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr                  85 90 95 Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu             100 105 <210> 4 <211> 239 <212> PRT <213> Unknown <220> 223 EGFP sequence <400> 4 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu   1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly              20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile          35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr      50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys  65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu                  85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu             100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly         115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr     130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser                 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly             180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu         195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe     210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235 <210> 5 <211> 225 <212> PRT <213> Unknown <220> <223> mRFP sequence <400> 5 Met Ala Ser Ser Glu Asp Val Ile Lys Glu Phe Met Arg Phe Lys Val   1 5 10 15 Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu              20 25 30 Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys Val          35 40 45 Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln      50 55 60 Phe Gln Tyr Gly Ser Lys Ala Tyr Val Lys His Pro Ala Asp Ile Pro  65 70 75 80 Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val                  85 90 95 Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser Ser             100 105 110 Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys Leu Arg Gly Thr Asn         115 120 125 Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu     130 135 140 Ala Ser Thr Glu Arg Met Tyr Pro Glu Asp Gly Ala Leu Lys Gly Glu 145 150 155 160 Ile Lys Met Arg Leu Lys Leu Lys Asp Gly Gly His Tyr Asp Ala Glu                 165 170 175 Val Lys Thr Thr Tyr Met Ala Lys Lys Pro Val Gln Leu Pro Gly Ala             180 185 190 Tyr Lys Thr Asp Ile Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr         195 200 205 Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg His Ser Thr Gly     210 215 220 Ala 225 <210> 6 <211> 346 <212> PRT <213> Unknown <220> <223> EGFP-RavZ (MT) sequence <400> 6 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu   1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly              20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile          35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr      50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys  65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu                  85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu             100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly         115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr     130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser                 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly             180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu         195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe     210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Leu 225 230 235 240 Glu Pro Val Gln Leu Ser Glu Phe Ile Val Ala Leu Glu Asp Tyr Gly                 245 250 255 Lys Leu Arg Ser Gln Gln Ser Glu Lys Ser Met Leu Asn Phe Ile Gly             260 265 270 Tyr Ser Lys Thr Ala Lys Leu Thr Ala Val Glu Leu Leu Ile Gly Ile         275 280 285 Leu Asn Asp Ile Lys Gly Lys Asn Glu Ile Ser Glu Ser Gln Tyr Asp     290 295 300 Lys Leu Val Lys Glu Val Asp Cys Leu Met Asp Ser Ser Leu Gly Lys 305 310 315 320 Leu Val Gln Phe His Leu Lys Asn Leu Gly Ala Glu Ser Leu Gln Lys                 325 330 335 Leu Val Leu Pro Cys Val Lys Phe Asp Asp             340 345 <210> 7 <211> 453 <212> PRT <213> Unknown <220> 223 EGFP-RavZ (2xMT) sequence <400> 7 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu   1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly              20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile          35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr      50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys  65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu                  85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu             100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly         115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr     130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser                 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly             180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu         195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe     210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Leu 225 230 235 240 Glu Pro Val Gln Leu Ser Glu Phe Ile Val Ala Leu Glu Asp Tyr Gly                 245 250 255 Lys Leu Arg Ser Gln Gln Ser Glu Lys Ser Met Leu Asn Phe Ile Gly             260 265 270 Tyr Ser Lys Thr Ala Lys Leu Thr Ala Val Glu Leu Leu Ile Gly Ile         275 280 285 Leu Asn Asp Ile Lys Gly Lys Asn Glu Ile Ser Glu Ser Gln Tyr Asp     290 295 300 Lys Leu Val Lys Glu Val Asp Cys Leu Met Asp Ser Ser Leu Gly Lys 305 310 315 320 Leu Val Gln Phe His Leu Lys Asn Leu Gly Ala Glu Ser Leu Gln Lys                 325 330 335 Leu Val Leu Pro Cys Val Lys Phe Asp Asp Val Glu Pro Val Gln Leu             340 345 350 Ser Glu Phe Ile Val Ala Leu Glu Asp Tyr Gly Lys Leu Arg Ser Gln         355 360 365 Gln Ser Glu Lys Ser Met Leu Asn Phe Ile Gly Tyr Ser Lys Thr Ala     370 375 380 Lys Leu Thr Ala Val Glu Leu Leu Ile Gly Ile Leu Asn Asp Ile Lys 385 390 395 400 Gly Lys Asn Glu Ile Ser Glu Ser Gln Tyr Asp Lys Leu Val Lys Glu                 405 410 415 Val Asp Cys Leu Met Asp Ser Ser Leu Gly Lys Leu Val Gln Phe His             420 425 430 Leu Lys Asn Leu Gly Ala Glu Ser Leu Gln Lys Leu Val Leu Pro Cys         435 440 445 Val Lys Phe Asp Asp     450 <210> 8 <211> 346 <212> PRT <213> Unknown <220> RavZ (MT) -EGFP-FRB probe sequence <400> 8 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu   1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly              20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile          35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr      50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys  65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu                  85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu             100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly         115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr     130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser                 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly             180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu         195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe     210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Leu 225 230 235 240 Glu Pro Val Gln Leu Ser Glu Phe Ile Val Ala Leu Glu Asp Tyr Gly                 245 250 255 Lys Leu Arg Ser Gln Gln Ser Glu Lys Ser Met Leu Asn Phe Ile Gly             260 265 270 Tyr Ser Lys Thr Ala Lys Leu Thr Ala Val Glu Leu Leu Ile Gly Ile         275 280 285 Leu Asn Asp Ile Lys Gly Lys Asn Glu Ile Ser Glu Ser Gln Tyr Asp     290 295 300 Lys Leu Val Lys Glu Val Asp Cys Leu Met Asp Ser Ser Leu Gly Lys 305 310 315 320 Leu Val Gln Phe His Leu Lys Asn Leu Gly Ala Glu Ser Leu Gln Lys                 325 330 335 Leu Val Leu Pro Cys Val Lys Phe Asp Asp             340 345 <210> 9 <211> 487 <212> PRT <213> Unknown <220> <223> mRFP-FKBP-RavZ (MT) probe sequence <400> 9 Met Ala Ser Ser Glu Asp Val Ile Lys Glu Phe Met Arg Phe Lys Val   1 5 10 15 Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu              20 25 30 Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys Val          35 40 45 Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln      50 55 60 Phe Gln Tyr Gly Ser Lys Ala Tyr Val Lys His Pro Ala Asp Ile Pro  65 70 75 80 Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val                  85 90 95 Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser Ser             100 105 110 Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys Leu Arg Gly Thr Asn         115 120 125 Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu     130 135 140 Ala Ser Thr Glu Arg Met Tyr Pro Glu Asp Gly Ala Leu Lys Gly Glu 145 150 155 160 Ile Lys Met Arg Leu Lys Leu Lys Asp Gly Gly His Tyr Asp Ala Glu                 165 170 175 Val Lys Thr Thr Tyr Met Ala Lys Lys Pro Val Gln Leu Pro Gly Ala             180 185 190 Tyr Lys Thr Asp Ile Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr         195 200 205 Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg His Ser Thr Gly     210 215 220 Ala Ser Gly Leu Arg Ser Arg Ser Ala Ala Ala Gly Ala Gly Gly Ala 225 230 235 240 Ala Arg Ala Ala Leu Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp                 245 250 255 Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr             260 265 270 Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn         275 280 285 Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp     290 295 300 Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr 305 310 315 320 Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile                 325 330 335 Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu             340 345 350 Ser Ala Gly Gly Ser Ala Gly Gly Ser Ala Gly Gly Ser Ala Gly Gly         355 360 365 Ser Ala Gly Gly Ser Ala Gly Gly Pro Arg Ala Gln Val Glu Pro Val     370 375 380 Gln Leu Ser Glu Phe Ile Val Ala Leu Glu Asp Tyr Gly Lys Leu Arg 385 390 395 400 Ser Gln Gln Ser Glu Lys Ser Met Leu Asn Phe Ile Gly Tyr Ser Lys                 405 410 415 Thr Ala Lys Leu Thr Ala Val Glu Leu Leu Ile Gly Ile Leu Asn Asp             420 425 430 Ile Lys Gly Lys Asn Glu Ile Ser Glu Ser Gln Tyr Asp Lys Leu Val         435 440 445 Lys Glu Val Asp Cys Leu Met Asp Ser Ser Leu Gly Lys Leu Val Gln     450 455 460 Phe His Leu Lys Asn Leu Gly Ala Glu Ser Leu Gln Lys Leu Val Leu 465 470 475 480 Pro Cys Val Lys Phe Asp Asp                 485

Claims (10)

A Membrane targeting (MT) sequence consisting of the amino acid of SEQ ID NO: 1 of the RavZ protein forming a bond with PI3P;
And Endosome detection probe comprising a reporter fluorescent protein sequence consisting of amino acids of SEQ ID NO: 4 or 5. delete The method of claim 1,
The probe is a probe for detecting intracellular vesicles, characterized in that it comprises one or more repeating the Membrane targeting (MT) sequence of RavZ protein. The method of claim 1,
The probe for detecting vesicles in cells comprising the amino acid sequence of SEQ ID NO: 6 or 7. The method of claim 1,
The probe for detecting the intracellular vesicles, characterized in that coupled with the FKBP protein consisting of the amino acid of SEQ ID NO: 3 or FRB consisting of the amino acid of SEQ ID NO: 2. delete The method of claim 1,
The reporter fluorescent protein may be a green fluorescent protein (GFP), a modified green fluorescent protein, an enhanced green fluorescent protein (EGFP), a red fluorescent protein (RFP), or a modified red fluorescent protein. (mRFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), indigo fluorescent protein (CFP), and enhanced indigo fluorescent protein ( ECFP) and Renilla luciferase (Renila luciferase) and red fluorescent protein (DsRed) probes for detecting intracellular vesicles, characterized in that at least one selected from the group comprising. The method of claim 1,
The probe is for detecting the formation and distribution of intracellular vesicles, probes for detecting intracellular vesicles. An intracellular vesicle detection composition comprising the probe for detecting intracellular vesicles of claim 1 or a vector for expressing the same. Preparing a first probe to which a Membrane targeting (MT) sequence consisting of amino acids of SEQ ID NO: 1 and a FRB protein consisting of amino acids of SEQ ID NO: 2 are bound to the RavZ protein forming a bond with PI3P;
Preparing a second probe to which a Membrane targeting (MT) sequence consisting of amino acids of SEQ ID NO: 1 and a FKBP protein consisting of amino acids of SEQ ID NO: 3 are bound to the RavZ protein;
Preparing a probe by further binding a fluorescent protein sequence consisting of amino acids of SEQ ID NO: 4 or SEQ ID NO: 5 to the first or second probe;
Simultaneously expressing the first probe and the second probe in a cell; And
Comprising the step of treating the cells with rapamycin, intracellular vesicle probe method.

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