CN109666068A

CN109666068A - Proline optical probe and its preparation method and application

Info

Publication number: CN109666068A
Application number: CN201910149337.1A
Authority: CN
Inventors: 杨弋; 赵玉政; 李写; 徐磊; 王巧慧
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2019-02-28
Filing date: 2019-02-28
Publication date: 2019-04-23
Anticipated expiration: 2039-02-28
Also published as: CN109666068B

Abstract

The present invention relates to a kind of proline optical probes and its preparation method and application.On the one hand, the present invention relates to a kind of optical probes, and comprising proline sensitivity polypeptide or its functional variety and optical activity polypeptide or its functional variety, wherein optical activity polypeptide or its functional variety are located in the sequence of proline sensitivity polypeptide or its functional variety.The present invention also relates to the preparation method of above-mentioned probe and its applications in detection proline.

Description

Proline optical probe and its preparation method and application

Technical field

The present invention relates to optical probe technical field more particularly to a kind of proline optical probe and preparation method thereof and answer With.

Background technique

Proline is widely distributed in various animals and plants as one of 20 kinds of natural amino acids.In plant, dried meat ammonia Acid accumulation is a very common and important phenomenon.When harm of the plant by external environment, the intracorporal dried meat ammonia of plant Acid content can occur acutely to increase, and the mechanism and its physiological action in plant that this phenomenon generates still are not ground Study carefully clear.And in mammalian cells, proline is as its physiological action of nonessential amino acid at the beginning not It is widely recognized, is merely possible to the raw material of protein synthesis and since the special of its structure makes it to protein The maintenance of skeleton structure has important role.But in recent years, the regulation of intracellular ROS is made with Proline Metabolism access It was found that people it is gradual again by sight again focused on grinding for proline in mammalian cell and its metabolic pathway On studying carefully.

Proline Metabolism is mainly to carry out in cell cytoplasm and mitochondria in mammal, and mainly proline passes through Transport protein matter on mitochondria enters in mitochondria, passes through a proline oxidase (POX/ on mitochondrial inner membrane PRODH) Δ 1- pyrrolin -5- carboxylic acid (P5C) will be generated after proline oxidative dehydrogenation, and P5C shuttles back and cytoplasm and passes through born of the same parents PYCR (P5C reductase) in matter transforms back into proline.Material is thus formed the circulations of a Proline Metabolism.Δ 1- pyrroles Quinoline -5- carboxylic acid (P5C) can form a tautomeric equilibrium, last GSA mono- with GSA (glutamic acid-γ-semialdehyde) in cytoplasm It is then that can pass through bird that aspect, which can generate glutamic acid to enter tricarboxylic acid cycle be metabolized on the other hand by P5CDH dehydrogenation, Propylhomoserin transaminase generation ornithine enters urea cycle and is metabolized.Generally speaking be exactly POX and PYCR (P5CDH) to P5C and We term it proline circulations for the metabolic pathway that the adjusting that mutually converts of the proline between cytoplasm and mitochondria is formed (Liu W etc., Biofactors.2012,38 (6): 398-406；The Autophagy.2012,8 such as Liu W (9): 1407-1409； Liu W etc., Cancer Res.2012,72 (14): 3677-3686).

Recent study finds that proline oxidase (POX) gene can be by promoting cell cycle arrest, inducing cell point Change and the approach negativity such as the apoptosis of promotion cell adjust the growth of tumour cell, and finds the different of its expression in kinds of tumors Often.Proline also consumes ADP and generates ATP and free electron while oxidative dehydrogenation generates P5C under the action of POX, Along with the generation of ROS (reactivity keto) during the electronics transmits in Intramitochondrial electronics respiratory chain, and ROS is then The apoptosis to cell, proliferation and cell cycle have vital regulation (Liu Y etc., Cancer Res.2009,69 (16):6414-6422；Phang J M etc., Szabados L etc., Trends Plant Sci.2010,15 (2): 89-97； Pandhare J etc., J Biol Chem.2006,281 (4): 2044-2052；Liu Y etc., Oncogene.2006,25 (41): 5640-5647)。

There is above-mentioned important role just because of proline, therefore the detection of proline content is also particularly important.Dried meat ammonia The common detection method of acid includes acid ninhydrine development process (Chen etc., Applie Environmental Microbiology 2006,72:4001-4006), spectrophotometer (Hortala MA etc., J Am Chem So2003,125 (1): 20-21；Pu F Deng Anal Chem 2010,82 (19): 8211-8216), high performance liquid chromatography (Wadud S etc., Journal of chromatography B,Analytical technologies in the biomedical and life sciences 2002,767 (2): 369-374) etc..

But these methods are not particularly suited for living cells research, there are many defects: needing by time-consuming sample treatment Process, such as clasmatosis, separation and Extraction purifying etc.；Cannot be carried out in living cells and subcellular organelle it is in situ, in real time, dynamic, High-throughput and high-spatial and temporal resolution detection.This field there is still a need for can in the cell, outer real-time positioning, quantitative, high-throughput detection The method of proline.

Summary of the invention

The purpose of the present invention is to provide in the cell, outer real-time positioning, high-throughput, quantitative detection proline probe and Method.

In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:

The present invention provides a kind of proline optical probe, includes proline sensitivity polypeptide or its functional variety and optical activity Polypeptide or its functional variety, wherein optical activity polypeptide or its functional variety are located at proline sensitivity polypeptide or its functional variety In sequence.Proline sensitivity polypeptide or its functional variety are divided into first part and second by optical activity polypeptide or its functional variety Part.In one embodiment, optical probe of the present invention is sensitive to proline.

The present invention provides a kind of proline optical probe, including proline sensitivity polypeptide B and optical activity polypeptide A, Middle optical activity polypeptide A is located in the sequence of proline sensitivity polypeptide B, proline sensitivity polypeptide B is divided for first part B1 and Second part B2 forms the probe structure of B1-A-B2 formula.

In one embodiment, proline sensitivity polypeptide includes proline binding protein or its functional variety.At one In embodiment, proline binding protein is originated from Agrobacterium, such as Agrobacterium fabrum.In an embodiment In, proline binding protein is originated from Atu2422-GABA receptor protein or its analog.In one embodiment, proline knot Hop protein includes Atu2422-GABA receptor protein or its functional variety.In a specific embodiment, proline combination egg It is white that there is sequence or its functional variety shown in SEQ ID NO:1, or with it have 35%, 40%, 50%, 60%, 70%, 80%, the sequence of 85%, 90%, 95%, 99% sequence identity.

In one embodiment, optical activity polypeptide is fluorescin or its functional variety.In one embodiment, Fluorescin is selected from yellow fluorescence protein (cpYFP as shown in SEQ ID NO:2), orange fluorescence albumen (such as SEQ ID CpmOrange shown in NO:3), the red fluorescent protein (mKate as shown in SEQ ID NO:4 or 8, such as SEQ ID NO:5 institute The mcherry shown), green fluorescent protein (cpGFP as shown in SEQ ID NO:6), blue fluorescent protein (such as SEQ ID NO: CpBFP shown in 7), apple DsRed (cpmApple as shown in SEQ ID NO:9).Preferably, optical activity polypeptide It is cpYFP.In one embodiment, fluorescin have SEQ ID NO:2-9 in it is any shown in sequence.

In one embodiment, optical probe also includes the one or more connectors for flanking the optical activity polypeptide. Connector of the present invention can be any amino acid sequence of any length.In one embodiment, optical activity polypeptide side The wing includes no more than the connector of 5 amino acid, such as the connector of 0,1,2,3,4 amino acid.In one embodiment, connector Positioned at the N-terminal and/or C-terminal of optical activity polypeptide.In one embodiment, optical probe of the present invention does not include connector.One In a embodiment, optical probe is as follows: first part's B1- optical activity polypeptide A-proline of proline sensitivity polypeptide The second part B2 of sensitive polypeptide.

In one embodiment, optical probe of the present invention also includes positioning sequence, for example thin for navigating to probe The specific cells device of born of the same parents.

Optical activity polypeptide of the present invention can be located at or be fused to any position of proline sensitivity polypeptide.At one In embodiment, optical activity polypeptide is located in the section selected from the following of proline sensitivity polypeptide: residue 117-123, residue 249-259 and residue 323-330, number correspond to the overall length of proline sensitivity polypeptide.In one embodiment, optical activity One or more amino acid in the section selected from the following of polypeptide displacement proline sensitivity polypeptide: residue 117-123, residue 249-259 and residue 323-330, number correspond to the overall length of proline sensitivity polypeptide.

In one embodiment, optical activity polypeptide is located at the site selected from the following of proline sensitivity polypeptide: 117/ 118,117/119,117/120,117/121,118/119,118/120,118/121,119/120,119/121,120/121, 120/122,120/123,121/122,121/123,122/123,249/250,249/251,249/252,249/253,249/ 254,249/255,249/256,249/257,249/258,249/259,250/251,250/252,250/253,250/254, 250/255,250/256,250/257,250/258,250/259,251/252,251/253,251/254,251/255,251/ 256,251/257,251/258,251/259,252/253,252/254,252/255,252/256,252/257,252/258, 252/259,253/254,253/255,253/256,253/257,253/258,253/259,254/255,254/256,254/ 257,254/258,254/259,255/256,255/257,255/258,255/259,256/257,256/258,256/259, 257/258,257/259,258/259,323/330,324/330,325/330,326/327,326/328,326/329,326/ 330,327/328,327/329,327/330,328/329,328/330 and 329/330.Herein, if in the form of " X/Y " table Two numbers in the site shown are continuous integers, then it represents that optical activity polypeptide be located at amino acid described in the number it Between.Such as indicate that optical activity polypeptide is located between the amino acid 1 17 and 118 of proline sensitivity polypeptide positioned at site 117/118. If two numbers in the site indicated in the form of " X/Y " are not continuous integers, then it represents that the displacement of optical activity polypeptide should Amino acid between amino acid shown in number.Such as indicate that optical activity polypeptide displacement proline is sensitive positioned at site 249/259 The amino acid 250-258 of polypeptide.Preferably, optical activity polypeptide is located at following sites of proline sensitivity polypeptide: 120/121, 121/122,121/123,324/330,325/330 and 326/330.

In the exemplary embodiment, B1-A-B2 formula optical probe of the present invention can be when cpYFP is located at Atu2422's The probe formed when 120/121,121/122,121/123,324/330,325/330 and 326/330, such as SEQ ID NO:10- Shown in 15.In one embodiment, optical probe of the present invention has sequence shown in SEQ ID NO:10-15 or is made from it.

The present invention also provides the proline sensitivity polypeptides with one or more mutation, including have one or more mutation Proline binding protein.The amino acid mutation includes the modification, substitution, the truncation of missing or sequence of amino acid.At one In embodiment, described be mutated can be selected from the F77, A100, T102 of proline sensitivity polypeptide, D121, Y150, D226, G227 and The mutation in the sites such as Y275.Illustratively, the mutation is selected from F77S, F77Y, F77C, the F77L of proline sensitivity polypeptide, F77P, F77H, F77Q, F77W, F77I, F77T, F77N, F77K, F77R, F77V, F77A, F77D, F77E, F77M, F77A, A100S, A100Y, A100C, A100L, A100P, A100H, A100Q, A100W, A100R, A100I, A100T, A100N, A100K, A100M, A100V, A100F, A100D, A100E, A100G, T102S, T102Y, T102C, T102L, T102P, T102H, T102Q, T102W, T102R, T102I, T102A, T102N, T102K, T102M, T102V, T102F, T102D, T102E, T102G, D121S, D121Y, D121C, D121L, D121P, D121H, D121Q, D121W, D121I, D121T, D121N, D121K, D121R, D121V, D121A, D121F, D121E, D121M, D121A, Y150S, Y150T, Y150C, Y150L, Y150P, Y150H, Y150Q, Y150W, Y150R, Y150I, Y150A, Y150N, Y150K, Y150M, Y150V, Y150F, Y150D, Y150E, Y150G, D226S, D226T, D226C, D226L, D226P, D226H, D226Q, D226W, D226R, D226I, D226A, D226N, D226K, D226M, D226V, D226F, D226Y, D226E, D226G, G227S, G227T, G227C, G227L, G227P, G227H, G227Q, G227W, G227R, G227I, G227A, G227N, G227K, G227M, G227V, G227F, G227Y, G227E, G227D, Y275S, Y275T, Y275C, Y275L, Y275P, Y275H, Y275Q, Y275W, Y275R, Y275I, Y275A, Y275N, Y275K, Y275M, Y275V, Y275F, Y275G, Y275E and Y275D.In one embodiment, above-mentioned mutation is selected from F77L, A100G, D121E, D121S, D121T, D121V, D226E, D226N and Y275F.In one embodiment, proline sensitivity polypeptide described herein sequence shown in SEQ ID NO:1, Or has in the sequence of 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity with it and wrap Containing the mutation at following site: F77, A100, T102, D121, Y150, D226 and Y275.

Proline sensitivity polypeptide (such as proline binding protein) in optical probe of the present invention may include one or more Amino acid mutation.In some embodiments, the proline sensitivity polypeptide in optical probe include it is described herein have one or The proline binding protein of multiple mutation.The mutation includes modification, substitution, missing or the truncation of amino acid.Implement at one In mode, the mutation is selected from F77, A100, T102, D121, Y150, D226, G227 and the Y275 etc. of proline sensitivity polypeptide The mutation in site.Illustratively, the mutation is selected from F77S, F77Y, F77C, F77L, the F77P of proline sensitivity polypeptide, F77H, F77Q, F77W, F77I, F77T, F77N, F77K, F77R, F77V, F77A, F77D, F77E, F77M, F77A, A100S, A100Y, A100C, A100L, A100P, A100H, A100Q, A100W, A100R, A100I, A100T, A100N, A100K, A100M, A100V, A100F, A100D, A100E, A100G, T102S, T102Y, T102C, T102L, T102P, T102H, T102Q, T102W, T102R, T102I, T102A, T102N, T102K, T102M, T102V, T102F, T102D, T102E, T102G, D121S, D121Y, D121C, D121L, D121P, D121H, D121Q, D121W, D121I, D121T, D121N, D121K, D121R, D121V, D121A, D121F, D121E, D121M, D121A, Y150S, Y150T, Y150C, Y150L, Y150P, Y150H, Y150Q, Y150W, Y150R, Y150I, Y150A, Y150N, Y150K, Y150M, Y150V, Y150F, Y150D, Y150E, Y150G, D226S, D226T, D226C, D226L, D226P, D226H, D226Q, D226W, D226R, D226I, D226A, D226N, D226K, D226M, D226V, D226F, D226Y, D226E, D226G, G227S, G227T, G227C, G227L, G227P, G227H, G227Q, G227W, G227R, G227I, G227A, G227N, G227K, G227M, G227V, G227F, G227Y, G227E, G227D, Y275S, Y275T, Y275C, Y275L, Y275P, Y275H, Y275Q, Y275W, Y275R, Y275I, Y275A, Y275N, Y275K, Y275M, Y275V, Y275F, Y275G, Y275E and Y275D.One In a embodiment, the amino acid-sensitive polypeptide in optical probe of the present invention may include mutation selected from the following: F77L, A100G, D121E, D121S, D121T, D121V, D226E, D226N and Y275F.

In one embodiment, optical probe of the present invention includes that proline sensitivity polypeptide or its functional variety and optics are living Property polypeptide or its functional variety, wherein optical activity polypeptide or its functional variety are located at proline sensitivity polypeptide or its functional variety Sequence in, the proline sensitivity polypeptide includes the mutation at the following site: F77, A100, T102, D121, Y150, D226 and Y275, number correspond to the overall length of proline sensitivity polypeptide.In the above-described embodiment, proline sensitivity polypeptide can be Sequence shown in SEQ ID NO:1, or with it have 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, Include the mutation at the following site in the sequence of 99% sequence identity: F77, A100, T102, D121, Y150, D226 and Y275。

In the exemplary embodiment, B1-A-B2 formula optical probe of the present invention can melt for 121/122 site of Atu2422 When conjunction has cpYFP and has F77L, A100G, D121E, D121S, D121T, D121V, D226E, D226N or Y275F mutation The probe of formation, as shown in SEQ ID NO:16-24.In an exemplary embodiment, optical probe of the present invention has SEQ Sequence shown in ID NO:16-24 is made from it.

Optical probe provided by the invention includes any or its variant in amino acid sequence SEQ ID NO:10-24.One In a embodiment, optical probe provided by the invention include with it is any in amino acid sequence SEQ ID NO:10-24 have 35%, 40%, the sequence of 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity.In preferred embodiment In, optical probe provided by the invention includes to be substantially similar or identical with any in amino acid sequence SEQ ID NO:10-24 Sequence.In preferred embodiment, optical probe provided by the invention includes SEQ ID NO:23 or is made from it.

The present invention also provides fused polypeptides, include optical probe described herein and other polypeptides.In some embodiments, Optical probe described herein also includes the other polypeptides merged therewith.Other polypeptides described herein do not influence the property of optical probe Matter.In some embodiments, other polypeptides are located at the N-terminal and/or C-terminal of the optical probe.In some embodiments, His polypeptide includes the polypeptide that optical probe is navigated to different organelles or subcellular organelle, the label for being used to purify or is used to exempt from The label of epidemic disease trace.There can be connector between optical probe and other polypeptides in fused polypeptide described herein.

Subcellular organelle described herein includes cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, golgiosome, lyase Body and peroxisome etc..In some embodiments, the label for the label of purifying or for immunoblotting includes 6 Histidine (6*His), glutathione S-transferase (GST), Flag.

The present invention also provides the nucleic acid sequences or its complementary series that encode optical probe described herein or fused polypeptide.One In a embodiment, the present invention provides a kind of nucleic acid sequence, encodes any shown amino acid sequence in SEQ ID NO:10-24 Column.In one embodiment, nucleic acid sequence of the present invention includes any in nucleotide sequence SEQ ID NO:25-26 or its change Body.In the preferred embodiment, the present invention provides a kind of nucleic acid sequence, comprising in nucleotide sequence SEQ ID NO:25-26 Any sequence with 99%, 95%, 90%, 80%, the 70% or 50% phase same sex.In another preferred embodiment, this hair It is bright that a kind of nucleic acid sequence is provided, comprising with any core being substantially similar or identical in nucleotide sequence SEQ ID NO:25-26 Nucleotide sequence.

It may include encoding optical probe of the present invention the invention further relates to the complementary series of above-mentioned nucleic acid sequence or its variant Or segment, the nucleic acid sequence or its complementary series of analog, derivative, soluble fragments and variant of fusion protein.

Amino acid sequence and nucleic acid sequence in the present invention are preferably provided with unpack format, are more preferably purified to Matter.

The present invention also provides the nucleic acid sequence of the present invention comprising being operatively connected with expression control sequence or its mutually The expression vector of complementary series, nucleic acid sequence encoding optical probe of the present invention or fused polypeptide.In some embodiments, Expression vector is selected from prokaryotic expression carrier, carrier for expression of eukaryon and viral vectors.In some embodiments, prokaryotic expression carrier Such as it is operatively connected to obtain by plasmid pRSETb and nucleic acid sequence as described herein.In some embodiments, expression control Sequence includes replication orgin, promoter, enhancer, operon, terminator, ribosome bind site.

The present invention also provides the cell comprising expression vector of the present invention, which includes and expression control sequence The nucleic acid sequence of the present invention being operatively connected or its complementary series.The cell is expressed optical probe described herein or is melted Close polypeptide.

The present invention also provides include proline optical probe described herein or fused polypeptide or method as described herein preparation Proline optical probe or fused polypeptide detection kit.The kit detects proline.

The present invention provides the method for preparing optical probe described herein, comprising: provides and visits comprising expressing optics described herein The cell of needle or the carrier of fused polypeptide cultivates the cell under conditions of cell expression, and separation optical probe or Fused polypeptide.

In one embodiment, the method for preparing proline optical probe described herein or fused polypeptide includes: 1) will The expression vector for encoding proline optical probe described herein is transferred in host cell；2) it is being suitble to the expression vector to express Under conditions of cultivate the host cell, 3) separation proline optical probe.

The present invention also provides the methods of proline in test sample, comprising: makes optical probe described herein or fused polypeptide Or the optical probe or fused polypeptide of method preparation as described herein are contacted with sample, and the variation of detection optics active peptides. The detection can in vivo, in vitro, subcellular or in situ carry out.The sample such as blood.

The method that proline in quantitative sample is also provided herein, comprising: make optical probe described herein or fused polypeptide or The optical probe or fused polypeptide of method preparation as described herein are contacted with sample, detect variation and the root of optics active peptides According to the proline in the quantitative sample of the variation of optical activity polypeptide.

The present invention also provides the methods of screening compounds (such as drug), comprising: makes optical probe described herein or fusion The optical probe or fused polypeptide of polypeptide or method as described herein preparation are contacted with candidate compound, detect optics active peptides Variation, and variation screening compounds according to optical activity polypeptide.The method can screening compounds with high throughput.

The present invention also provides dried meat prepared by proline optical probe described herein or fused polypeptide or method as described herein The application of propylhomoserin optical probe or fused polypeptide in proline positions in real time.

Beneficial effects of the present invention: proline optical probe provided by the invention is easy to mature, and fluorescence dynamic change is big, special It is anisotropic good, and can be expressed in cell by the method for genetic manipulation, outer in the cell it can position in real time, is high-throughput, is fixed Amount detection proline, eliminates time-consuming processing sample step.Experiment effect shows that proline optics provided herein is visited Highest response for proline reaches 4 times or more of control, and can cytoplasm, mitochondria, nucleus, endoplasmic reticulum, Cell is positioned in the subcellular structures such as lysosome and golgiosome, qualitative and quantitative analysis, and high throughput can be carried out Screening compound and blood in proline quantitative detection.

Detailed description of the invention

Present invention will be further explained below with reference to the attached drawings and examples.

Fig. 1 is that the SDS-PAGE of exemplary proline optical probe described in embodiment 1 schemes；

It illustratively includes cpYFP and the protein-bonded proline optical probe pair of proline that Fig. 2, which is as described in example 2, Proline responds variation diagram；

It illustratively includes cpGFP and the protein-bonded proline optical probe pair of proline that Fig. 3, which is described in embodiment 3, Proline responds variation diagram；

It illustratively includes cpBFP and the protein-bonded proline optical probe pair of proline that Fig. 4, which is as described in example 4, Proline responds variation diagram；

Fig. 5 is illustratively to visit comprising cpmApple and the protein-bonded proline optics of proline described in embodiment 5 Variation diagram is responded for proline；

Fig. 6 is illustrative in the protein-bonded site 12 0/121,121/122,121/ of proline described in embodiment 6 123,324/330,325/330 or 326/330 fusions have titration of the proline optical probe of cpYFP to various concentration proline Curve；

Fig. 7 A is that illustrative merge in the protein-bonded site 12 1/122 of proline described in embodiment 6 has cpYFP's Histogram of the proline optical probe to the specific detection of 20 kinds of amino acid；

Fig. 7 B is to be fused to obtained by the protein-bonded N-terminal of proline or C-terminal described in embodiment 6 with fluorescin Fusion protein compare, fluorescin is located at specificity of the probe to proline of the protein-bonded site 12 1/122 of proline Histogram；

Fig. 8 is that illustrative merge in the protein-bonded site 12 1/122 of proline described in embodiment 7 has cpYFP simultaneously And have the proline optical probe of mutation to dried meat ammonia in the site F77, A100, T102, D121, Y150, D226, G227 or Y275 The histogram of acid response；

Fig. 9 is the fluorescent spectroscopic properties figure of exemplary proline optical probe described in embodiment 8；

Figure 10 is titration curve of the exemplary proline optical probe described in embodiment 8 to various concentration proline；

Figure 11 is column of the exemplary proline optical probe described in embodiment 8 to the specific detection of 20 kinds of amino acid Figure；

Figure 12 is the subcellular organelle positioning of exemplary proline optical probe described in embodiment 9 in mammalian cells Photo；

Figure 13 is described in embodiment 9 to exemplary proline optical probe different subcellular organelles in mammalian cells Interior proline transdermal delivery carries out the schematic diagram of dynamic monitoring；

Figure 14 is exemplary proline optical probe described in embodiment 10 in the high-throughput compound of living cells level progress The point diagram of screening；

Figure 15 is that exemplary proline optical probe described in embodiment 11 carries out the proline in mouse and human blood Quantitative histogram.

Specific embodiment

When providing several value or ranges, terms used herein " about " refers to the number value or range in given number value or range Within 20%, within 10% and within 5%.

Terms used herein "comprising", " comprising " and its equivalent form include " containing " and " by ... form " contain Justice, such as the composition of "comprising" X can only be made of X or can contain other materials, such as X+Y.

Terms used herein " proline sensitivity polypeptide " or " proline response polypeptide ", which refer to, generates the more of response to proline Peptide, the response include with the chemistry of the relevant polypeptide of the sensitivity interaction of polypeptide, biology, electricity or physiologic parameters Any response.Response includes small variation, for example, variation and, for example, the polypeptide in the direction of the amino acid or peptide fragment of polypeptide The variation of level-one, second level or tertiary structure, including for example protonate, the variation of electrochemical potential and/or conformation." conformation " is molecule In comprising side group molecule level-one, the three-dimensional arrangement of second level and tertiary structure；When the three-dimensional structure of molecule changes, structure As changing.The example of conformation change includes being changed into beta sheet from alpha-helix or being changed into alpha-helix from beta sheet.It can be with Understand, as long as the fluorescence of fluorescin part is changed, detectable change needs not be conformational change.It is described herein Proline sensitivity polypeptide may also include its functional variety.The functional variety of proline sensitivity polypeptide includes but is not limited to can be with dried meat Propylhomoserin interaction is to occur the variant with the same or similar variation of parent's proline sensitivity polypeptide.

Proline sensitivity polypeptide of the present invention includes but is not limited to proline binding protein or has with it 90% or more same The variant of source property.Amino acid binding protein of the present invention can derive from Agrobacterium, such as Agrobacterium fabrum.This Invention exemplary amino acid binding protein Atu2422 is abc transport protein family, is made of two structural domains, two structural domains Between be attached by three flexible amino acid peptide chains.Proline binding protein can incude the variation of concentration of proline, in dried meat The protein-bonded space conformation of proline can also change during propylhomoserin concentration dynamic change.

Terms used herein " optical probe " refers to the proline sensitivity polypeptide with optical activity peptide fusion.Invention human hair It is existing, generated structure after proline sensitivity polypeptide such as proline binding protein in specific manner combines the proline of physiological concentration It can cause the conformation change of optical activity polypeptide (such as fluorescin) as changing, and then lead to the optical of optical activity polypeptide Matter changes.Standard curve is drawn by the fluorescence of the fluorescin measured under different concentration of proline, can detecte and divide Analyse the presence and/or level of proline.Exemplary Atu2422 albumen is as shown in SEQ ID NO:1.It is visited when describing optics of the present invention When needle (such as when describing site locating for optical activity polypeptide or mutational site), the numbering amino acid residues referred to refer to SEQ ID NO:1.But the other similar protein-bonded corresponding residue numbering of proline as known to those skilled in the art.

In optical probe of the invention, it is quick that optical activity polypeptide (such as fluorescin) is operationally fused to proline Feel in polypeptide." optical activity polypeptide " based on protein is the polypeptide with transmitting fluorescence capability.Fluorescence is that optical activity is more A kind of optical property of peptide can be used as the means for detecting the responsiveness of optical probe of the invention.As used herein, term " photoluminescent property " refers to the molar extinction coefficient under appropriate excitation wavelength, fluorescence quantum efficiency, excitation spectrum or emission spectrum Shape, excitation wavelength maximum value and launch wavelength maximum value, the amplitude of two different wave lengths excitation, the transmitting of two different wave lengths Amplitude ratio, lifetime of excited state or fluorescence anisotropy.Any one of these properties between active and inactive state Measurable difference is sufficient to the effectiveness of fluorescent protein substrate of the invention in determination of activity.Measurable difference can pass through The amount of any quantitative fluorescence property is determined to determine, for example, the product of the fluorescence volume or fluorescence of certain wave strong point on emission spectrum Point.Preferably, selection protein substrate is to have the fluorescent characteristic for being easy to distinguish under the conformational state of un-activation and activation.This The text optical activity polypeptide may also include its functional variety.The functional variety of optical activity polypeptide includes but is not limited to that can send out The raw variant with the same or similar photoluminescent property variation of parent's optical activity polypeptide.

" connector " or " bonding pad " refers to the amino acid or core that two parts are connected in polypeptide of the present invention, protein or nucleic acid Nucleotide sequence.Illustratively, in the present invention bonding pad aminoterminal of proline sensitivity polypeptide and optical activity polypeptide amino acid Number selection is 0-3, and the amino acid number selection of c-terminus is 0-2；When recombination optical probe is as basic unit When connecting with functional protein, the amino acid or c-terminus in recombination optical probe can be merged.Joint sequence can be one or more The short peptide chain of a flexible amino acid composition.

Terms used herein " chromophore ", " fluorogen " are synonymous with " fluorescin ", refer to exciting light irradiation under issue it is glimmering The protein of light.Basic detection means of the fluorescin as bio-science field, such as field of biotechnology are commonly green Fluorescin GFP and the cyclic annular blue fluorescent protein (cpBFP) reset derived by the protein mutation, the cyclic annular green reset Fluorescin (cpGFP), cyclic annular yellow fluorescence protein (cpYFP) reset etc.；There are also the common red fluorescences of the art Albumen RFP, and by the protein derived cyclic annular albumen reset come out, such as cpmApple, cpmOrange, cpmKate etc..Show It is any shown in the sequence such as SEQ ID NO:2-9 of example property fluorescin.

Green fluorescent protein GFP is extracted from Victoria's luminescent jellyfish (Aequorea Victoria) , by 238 Amino acid profiles, molecular weight is about 26kDa.GFP is to form unique barrel-like structure by 12 beta sheet chains, The tripeptides that adds lustre to (Ser65-Tyr66-Gly67) is wrapped in it.When in the presence of oxygen, it is sub- that it can spontaneously form p- hydroxy benzenes Chromophore's structure of methylimidazole quinoline ketone and generate fluorescence.GFP generates fluorescence and does not need co-factor, and fluorescence is highly stable, It is a kind of good imaging tool.GFP is there are two excitation peak, and the main peak of 395nm can produce the transmitting light of 508nm, and acromion The transmitting light for the 503nm that the exciting light irradiation of 475nm can then generate.Exemplary cpGFP is as shown in SEQ ID NO:6

Yellow fluorescence protein YFP is derived from green fluorescent protein GFP, and amino acid sequence and GFP homology are up to 90% More than, YFP is modified in that the 203rd amino acids sport tyrosine (T203Y) by threonine compared to GFP key.Compared to The red shift of wavelength of the main excitation peak of original AvGFP, YFP is to 514nm and launch wavelength then changes into 527nm.On this basis Carrying out rite-directed mutagenesis (S65T) to the 65th amino acids of YFP can get fluorescence enhancement type yellow fluorescence protein EYFP.CpYFP be by The original N-terminal and C-terminal of GFP manufactures a new N by one section of small peptide chain link flexible, in the nearly chromophore position original GFP End and C-terminal, using former 145th~238 amino acids part as the N-terminal of new albumen, former 1st~144 amino acids are as new egg White C-terminal by 5~9 there is small peptide chain link flexible to obtain between two segments.In the present invention, nearly chromophore position is excellent It is selected as at Y144 and N145 amino acids；It is described have short peptide chain flexible be preferably VDGGSGGTG or GGSGG.It is exemplary The sequence of cpYFP is as shown in SEQ ID NO:2.

Red fluorescent protein RFP is extracted from the coral in ocean, and wild RFP is that oligomeric proteins are unfavorable In the amalgamation and expression of organism, the red fluorescent protein of different colours wave band has then further been derived on the basis of RFP, The most commonly used is mCherry and mKate etc..Exemplary cpmKate is as shown in SEQ ID NO:4 or 8.It is exemplary MCherry is as shown in SEQ ID NO:5.

In other embodiments, fluorescin can also be amino acid sequence blue-fluorescence as shown in SEQ ID NO:7 Albumen cpBFP, amino acid sequence orange fluorescence albumen cpmOrange, amino acid sequence such as SEQ as shown in SEQ ID NO:3 One of apple DsRed cpmApple shown in ID NO:9 is a variety of.

Proline optical probe of the present invention includes proline sensitivity polypeptide B, for example, proline binding protein or its Variant and optical activity polypeptide A, such as fluorescin.Optical activity polypeptide A is inserted into proline sensitivity polypeptide B, by B points For two parts B1 and B2, the probe structure of B1-A-B2 formula is formed；Proline sensitivity polypeptide B and proline interaction cause The optical signalling of optical activity polypeptide A becomes strong.

In optical probe of the invention, optical activity polypeptide can be located at or be fused to any of proline sensitivity polypeptide Position.In one embodiment, optical activity polypeptide with the direction N-C be located at the direction N-C proline sensitivity polypeptide it is any Position.Specifically, optical activity polypeptide is located at the flexible region of proline sensitivity polypeptide, and the flexible region refers to protein Some specific such as loop domain structures present in higher structure, these structural domains compared to protein other are advanced Structure has higher mobility and flexibility, and the region can be after the protein and ligand binding, space structure conformation Dynamic change occurs.Heretofore described flexible region refers mainly to the position of fusion region in proline binding protein, Such as the region amino acid residue 117-123,249-259 and 317-330.Illustratively, optical activity polypeptide is located at proline combination At the site selected from the following of the amino acid sequence of albumen: 117/118,117/119,117/120,117/121,118/119, 118/120,118/121,119/120,119/121,120/121,120/122,120/123,121/122,121/123,122/ 123,249/250,249/251,249/252,249/253,249/254,249/255,249/256,249/257,249/258, 249/259,250/251,250/252,250/253,250/254,250/255,250/256,250/257,250/258,250/ 259,251/252,251/253,251/254,251/255,251/256,251/257,251/258,251/259,252/253, 252/254,252/255,252/256,252/257,252/258,252/259,253/254,253/255,253/256,253/ 257,253/258,253/259,254/255,254/256,254/257,254/258,254/259,255/256,255/257, 255/258,255/259,256/257,256/258,256/259,257/258,257/259,258/259,323/330,324/ 330,325/330,326/327,326/328,326/329,326/330,327/328,327/329,327/330,328/329, 328/330 and 329/330.In the preferred embodiment, optical activity polypeptide is located at the protein-bonded amino acid sequence of proline 120/121,121/122,121/123,324/330,325/330 or 326/330 at.As shown in NO:10~15 SEQ ID.

When mentioning certain more peptide or protein, term used herein " variant " or " mutant " include having the polypeptide or egg White identical function but the different variant of sequence.These variants include but is not limited to: lacking in the sequence of more peptide or proteins Losing, be inserted into and/or replace one or more, (usually 1-30, preferably 1-20, more preferably 1-10 is a, most preferably 1-5 It is a) amino acid, and it is one or several (usually within 20, preferably in its carboxyl terminal and/or amino terminal addition More preferably be within 5 within 10) amino acid obtain sequence.It is not intended to be restricted by theory, amino acid residue changes Overall configuration and function without changing polypeptide or protein, i.e. function conservative variants.For example, in the art, with performance phase When close or similar amino acid is replaced, the function of more peptide or proteins is not usually changed.In the art, performance is similar Amino acid often refers to the amino acid residues with similar side chain, has in this field and explicitly defines.These families include having alkali The amino acid (such as lysine, arginine, histidine) of property side chain, amino acid (such as aspartic acid, paddy with acid side-chain Propylhomoserin), amino acid with uncharged polar side chain (such as glycine, asparagine, glutamine, serine, Soviet Union's ammonia Acid, tyrosine, cysteine), amino acid with non-polar sidechain (such as alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan), it is (such as threonine, valine, different with β-branched building block amino acid Leucine) and amino acid (such as tyrosine, phenylalanine, tryptophan, histidine) with aromatic side chain.For another example, in ammonia Base end and/or carboxyl terminal, which add one or several amino acid generally also, will not change the function of more peptide or proteins.For being permitted The conserved amino acid of mostly common known nongenetic coding amino acid replaces known in the art.Other undoded amino acids are guarded Substitution can be determined compared with the property of the amino acid genetically encoded based on its physical property.Those skilled in the art are public Know, in gene cloning operation, it is often necessary to design suitable restriction enzyme site, this certainly will be in expressed more peptide or protein ends One or more incoherent residues are introduced, and this has no effect on the activity of desired polypeptides or albumen.For another example melt to construct Hop protein, the expression for promoting recombinant protein obtain the recombinant protein being secreted into outside host cell automatically or conducive to recombinant protein Purifying, it is often necessary to which some amino acid are added to other suitable areas in the end N-, the end C- or the albumen of recombinant protein In domain, it may for example comprise but be not limited to, suitable joint peptide, signal peptide, leader peptide, end extension, glutathione S-transferase (GST), the albumen of maltose E binding protein, the label of albumin A, such as 6His or Flag or Xa factor or fibrin ferment or enterokinase Proteases site.The variant of more peptide or proteins can include: homologous sequence, allelic variant, natural mutation, lures at examples of conservative variations Lead mutant.These variants also may include with the sequence identity of more peptide or proteins be at least about 70%, at least about 75%, At least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% it is more Peptide or protein.

Optical probe of the present invention may include the proline sensitivity polypeptide with mutation.The mutation for example selected from F77, The mutation in the sites such as A100, T102, D121, Y150, D226, G227 and Y275.Illustratively, the mutation is selected from F77S, F77Y, F77C, F77L, F77P, F77H, F77Q, F77W, F77I, F77T, F77N, F77K, F77R, F77V, F77A, F77D, F77E, F77M, F77A, A100S, A100Y, A100C, A100L, A100P, A100H, A100Q, A100W, A100R, A100I, A100T, A100N, A100K, A100M, A100V, A100F, A100D, A100E, A100G, T102S, T102Y, T102C, T102L, T102P, T102H, T102Q, T102W, T102R, T102I, T102A, T102N, T102K, T102M, T102V, T102F, T102D, T102E, T102G, D121S, D121Y, D121C, D121L, D121P, D121H, D121Q, D121W, D121I, D121T, D121N, D121K, D121R, D121V, D121A, D121F, D121E, D121M, D121A, Y150S, Y150T, Y150C, Y150L, Y150P, Y150H, Y150Q, Y150W, Y150R, Y150I, Y150A, Y150N, Y150K, Y150M, Y150V, Y150F, Y150D, Y150E, Y150G, D226S, D226T, D226C, D226L, D226P, D226H, D226Q, D226W, D226R, D226I, D226A, D226N, D226K, D226M, D226V, D226F, D226Y, D226E, D226G, G227S, G227T, G227C, G227L, G227P, G227H, G227Q, G227W, G227R, G227I, G227A, G227N, G227K, G227M, G227V, G227F, G227Y, G227E, G227D, Y275S, Y275T, Y275C, Y275L, Y275P, Y275H, Y275Q, Y275W, Y275R, Y275I, Y275A, Y275N, Y275K, Y275M, Y275V, Y275F, Y275G, Y275E and Y275D.In one embodiment, the mutation is selected from F77L, A100G, D121E, D121S, D121T, D121V, D226E, D226N and Y275F.

In the exemplary embodiment, B1-A-B2 formula optical probe of the present invention can melt for 121/122 site of Atu2422 Conjunction has cpYFP and has selected from F77L, A100G, D121E, D121S, D121T, D121V, D226E, D226N and Y275F The probe formed when mutation, as shown in SEQ ID NO:16-24.

In two or more polypeptides or sequence of nucleic acid molecules, term " the phase same sex " or " phase same sex percentage " refer to and are comparing On window or specified region, using means known in the art such as sequence comparison algorithm, compared by manual alignment and visual inspection When compared with comparing maximum correspondence, two or more sequences or subsequence are identical or in which have certain percentage in specified region Amino acid residue or nucleotide it is identical (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% are identical).For example, being suitble to measurement percent sequence identity Optimization algorithm with sequence similarity percentage is 2.0 algorithm of BLAST and BLAST, respectively reference can be made to Altschul etc. (1977) (1990) J.Mol.Biol.215:403 such as Nucleic Acids Res.25:3389 and Altschul.

Terms used herein " functional variety ", " derivative " and " analog ", which refers to, to be kept substantially and original polypeptide or egg White (such as proline binding protein or fluorescin) identical biological function or active albumen.Polypeptide or egg of the invention The functional variety of white (such as proline binding protein or fluorescin), derivative or the like, which can be (i), one or more The substituted albumen of a conservative or non-conservative amino acid residue (preferably conservative amino acid), and such substituted ammonia Base acid residue, which can be, may not be by genetic code encoding, or (ii) in one or more amino acid residues have take For the albumen of group, or (iii) maturation protein and another compound (for example extend the compound of protein half-life, such as poly- Ethylene glycol) fusion is formed by albumen, or (iv) additional amino acid sequence be fused to this protein sequence and the albumen that is formed (such as Secretion sequence or for purifying the sequence of this albumen or proprotein sequence, or the fusion protein with the formation of antigen I gG segment). According to the teaching of this article, these functional varieties, derivative and analogue belong to scope known to those skilled in the art.

The difference of the analog and original polypeptide or albumen can be the difference on amino acid sequence, be also possible to not shadow The difference on the modified forms of sequence is rung, or is had both at the same time.These albumen include natural or induction genetic variation.Induction becomes Body can be obtained by various technologies, such as generate random mutagenesis by radiating or being exposed to mutagens, can also be lured by fixed point The technology of political reform or other known molecular biology obtains.

The analog further includes the analog with the residue (such as D- amino acid) different from natural L-amino acids, and Analog with non-naturally occurring or synthesis amino acid (such as β, gamma-amino acid).It should be understood that proline of the invention is quick Sense polypeptide is not limited to the above-mentioned representative albumen enumerated, variant, derivative and analogue.Modification (does not change level-one knot usually Structure) form include: internal or external albumen chemical derivative form such as acetylation or carboxylated.Modification further includes glycosylation, Albumen that is glycosylation modified and generating is carried out in the synthesis and processing of albumen or in further processing step such as those.It is this to repair Decorations are completed and can carrying out glycosylated enzyme (glycosylase or deglycosylation enzyme of such as mammal) by the way that albumen to be exposed to. Modified forms further include the sequence with phosphorylated amino acid residue (such as phosphotyrosine, phosphoserine, phosphothreonine). It further include being modified to improve its anti-proteolytic properties or optimize the albumen of solubility property.

The present invention also provides the preparation methods of above-mentioned proline optical probe, comprising the following steps: 1) will coding this paper The nucleic acid sequence of the proline optical probe is included in expression vector；2) expression vector is transferred in host cell；2) suitable It closes and cultivates the host cell under conditions of the expression vector is expressed, 3) separation proline optical probe.

Term used herein " nucleic acid " or " nucleotide " can be DNA form or rna form.DNA form include cDNA, Genomic DNA or artificial synthesized DNA.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand. When mentioning nucleic acid, terms used herein " variant " can be the variant that the allelic variant naturally occurred or non-natural occur.These Nucleotide variants include degeneracy variant, replace variant, deletion mutants and insertion variant.As known in the art, allelic variant is The alternative forms of one nucleic acid, it may be substitution, missing or the insertion of one or more nucleotide, but not from substantially changing Become the function of the albumen of its coding.It is at least about 70% that nucleic acid of the present invention, which may include with the sequence identity of the nucleic acid sequence, At least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% Or 100% nucleotide sequence.The invention further relates to the nucleic acid fragments hybridized with above-mentioned sequence.As used herein, " nucleic acid piece The length of section " at least contains 15 nucleotide, preferably at least 30 nucleotide, more preferably at least 50 nucleotide, preferably extremely It is more than few 100 nucleotide.Nucleic acid fragment can be used for the amplification technique (such as PCR) of nucleic acid.

The full length sequence of optical probe or fusion protein of the present invention or its segment can usually use PCR amplification method, artificial conjunction It is obtained at method or recombination method.For PCR amplification method, can disclosed nucleotide sequence design primer according to the present invention, and use city The library cDNA sold or by the library cDNA prepared by conventional method well known by persons skilled in the art as template, amplification and it is related Sequence.When nucleotide sequence is greater than 2500bp, 2~6 PCR amplifications are preferably carried out, then press the segment that each time expands Proper order is stitched together.The present invention is not particularly limited the program and system of the PCR amplification, normal using this field The PCR amplification program and system of rule.Related sequence can also be obtained in large quantity with recombination method.This is usually by its gram It is grand enter carrier, then be transferred to cell, then by conventional method from the host cell after proliferation separation obtained with purifying it is You Guan more Peptide or protein.In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In this hair In bright, when the nucleotide sequence of optical probe is less than 2500bp, artificial synthesis can be used to synthesize.It is described artificial synthesized Method is the artificial synthesis of the DNA of this field routine, without other particular/special requirements.In general, by first synthesizing multiple small fragments, Then it is attached the very long segment of available sequence again.At present, it is already possible to code book be obtained by chemical synthesis completely The DNA sequence dna of invention albumen (or its functional variety, derivative or the like).Then the DNA sequence dna can be introduced into this field In known various existing DNA moleculars (such as carrier) and cell.Mutation can be drawn by mutation the methods of PCR or chemical synthesis Enter in protein sequence of the present invention.

The present invention will encode the nucleotide sequence of the optical probe after the nucleotide sequence for obtaining coded optical probe It is included in expression vector, obtains recombinant expression carrier.The term as used herein " expression vector " and " recombinant vector " are used interchangeably, Refer to protokaryon or eukaryotic vector well known in the art, such as bacterial plasmid, bacteriophage, yeast plasmid, plant cell are viral, lactation is dynamic Object cell virus such as adenovirus, retrovirus or other carriers, these carriers can replicate in host and stablize expression, One important feature of these recombinant vectors is to usually contain expression control sequence.Terms used herein " expression control sequence " refers to Regulate and control target gene transcription, translation and expression the element that can be operatively connected with target gene, can be replication orgin, Promoter, marker gene or translation control element, including enhancer, operon, terminator, ribosome bind site etc., expression The selection of control sequence depends on host cell used.Applicable recombinant vector includes but is not limited to bacterium matter in the present invention Grain.In recombinant expression carrier, " being operatively connected " refers to the nucleotide sequence of purpose and adjusts sequence to allow nucleotides sequence The mode that list reaches connects.Those skilled in the art is known to be used to construct containing fusion protein coded sequence of the present invention and properly Transcription/translation control signal expression vector method.These methods include recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc..The DNA sequence dna can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to close At.The representative example of these promoters has: lac the or trp promoter of Escherichia coli；λ bacteriophage PL promoter；Eukaryon starting Attached bag includes CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retrovirus The promoter that LTR and some other known controllable gene are expressed in protokaryon or eukaryotic or its virus.Expression vector It further include the ribosome bind site and transcription terminator of translation initiation.In one embodiment, expression vector can be used Commercially available pRSETb carrier, without other particular/special requirements.Illustratively, the coding optics is visited respectively using BamHI and EcoRI The nucleotide sequence and expression vector of needle carry out double digestion, then connect to obtain recombinant expression carrier by the digestion products of the two. The present invention is not particularly limited the specific steps and parameter of digestion and connection, is using the step and parameter of this field routine It can.

After obtaining recombinant expression carrier, which is transformed into host cell, to generate the egg for including fusion protein White or peptide.Such transfer process can be carried out with the routine techniques well known to those skilled in the art such as conversion or transfection.Institute of the present invention The host cell stated be refer to receive and accommodate recombinant DNA molecules cell, be recombination amplification place, preferably by Body cell, which should meet, is easily obtained and is proliferated two conditions." host cell " of the invention may include that prokaryotic cell and eukaryon are thin Born of the same parents specifically include bacterial cell, yeast cells, insect cell and mammalian cell.It specifically can be Escherichia coli, streptomycete Belong to, the bacterial cell of salmonella typhimurium, fungal cell's such as yeast, plant cell, the insect cell of drosophila S2 or Sf9, CHO, COS, HEK293, HeLa cell or the zooblast of Bowes melanoma cells etc., including but not limited to above-mentioned Those host cells.The preferably various cells for being conducive to gene product expression or fermenting and producing of the host cell, such cell is It is well known in the art and commonly uses.Exemplary host cells used are e. coli jm109-DE3 bacterium in embodiments of the present invention Strain.Persons skilled in the art are aware that how to select carrier, promoter, enhancer and host cell appropriate.

The method of the present invention for being transferred to host cell is the method for this field routine, including calcium phosphate or calcium chloride Co-precipitation, DEAE- mannosan-mediation transfection, fat transfection, natural competence, the transfer of chemistry mediation or electroporation.Work as place When master is prokaryotes such as Escherichia coli, the method is preferably CaCl₂Method or MgCl₂Method processing, step used are ability Known in domain.When host cell is eukaryocyte, following DNA transfection method: calcium phosphate precipitation, conventional mechanical can be selected Method such as microinjection, electroporation, liposome packaging etc..

The present invention carries out amplification expression after expression vector is transferred to host cell, to the host cell for being transferred to expression vector Culture, isolated proline optical probe.The host cell amplification expression culture is using conventional method.According to institute Host cell species, culture medium used in culture can be various conventional mediums.It is being suitable for host cell growth Under the conditions of cultivated.

In the present invention, optical probe is expressed or is secreted into the cell, on cell membrane and is extracellular.If desired, can benefit The albumen of recombination is separated by various separation methods or purified with its physics, chemical and other characteristics.The present invention is to separation The method of the proline fluorescin is not particularly limited, using the separation method of the fusion protein of this field routine. These methods are well-known to those skilled in the art, including but not limited to: conventional renaturation process, salting-out method, centrifugation, Permeate broken bacterium, ultrasonic treatment, ultracentrifugation, sieve chromatography, adsorption chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) With the combination of various other liquid chromatography technologies and these methods.In one embodiment, the affinity chromatography of His label is utilized The separation of method progress optical probe.

The present invention also provides the proline optical probes to position in real time in proline, the quantization of quantitative detection and high pass Close the application in object screening.In one aspect, the proline optical probe preferably connects with the signal peptide of cell different parts It connects, is transferred in cell, by the power of fluorescence signal in detection cell, carry out the real-time positioning of proline；Pass through proline The quantitative detection that curve carries out corresponding proline is added dropwise in standard.It is according to dried meat ammonia that curve, which is added dropwise, in proline standard of the present invention Fluorescence signal of sour optical probe in the case where various concentration proline is drawn.Proline optical probe of the present invention It is directly transferred in cell, during proline real-time orientational and quantificational detection, does not need time-consuming sample handling processes, more Accurately.Proline optical probe of the present invention is added to cell training when carrying out high-throughput screening compound, by different compounds In nutrient solution, the variation of proline content is measured, to filter out on the influential compound of proline content variation.In the present invention Described in proline optical probe positioned in real time in proline, the application in quantitative detection and high-throughput screening compound, It is non-diagnosing and treating purpose, is not related to the diagnosing and treating of disease.

Herein, the form of concentration, content, percentage and the equal usable range of other numerical value indicates.It will also be appreciated that using This range format is intended merely to convenienct and succinct, it should is flexibly read as including the specifically mentioned number of range bound institute Value should also include all single numbers for including or subrange within the scope of this.

Embodiment

Proline optical probe provided by the invention is described in detail below with reference to embodiment, but cannot be it Be interpreted as limiting the scope of the present invention.

I. experimental material and reagent

Mainly using conventional genetic engineering molecular biology cloning process and cell culture and imaging side in embodiment Method etc., these methods are well known to those of ordinary skill in the art, such as: " the molecular biology of simple Ross Kai Musi etc. Laboratory reference handbook ", J. Pehanorm Brooker, D.W. Russell writes, and Huang Peitang etc. is translated: " Molecular Cloning:A Laboratory guide " (third edition, In August, 2002, Science Press publish, Beijing)；" animal cell culture: basic fundamental guide " the (the 5th of Fei Leixieni etc. Version), Zhang Jingbo, Xu Cunshuan etc. are translated；J.S. Boneface agriculture, " fine works Cell Biology Experiment guide " of the M. up to rope etc., Zhang Jing Wave etc. is translated.According to the following examples, slightly modified as the case may be of being not difficult succeeds those of ordinary skill in the art with transformation The implementation present invention, these modifications and transformation are all fallen in the range of the claim of this application.

PRSETb-cpYFP is based on used in embodiment, pRSETb- proline binding protein plasmid is big by East China science and engineering The building of experimental protein room is learned, pRSETb plasmid vector is purchased from Invitrogen company.All primers for PCR are by Shanghai The synthesis of JaRa biotechnology Co., Ltd is purified and is identified through mass spectrography correct.The expression plasmid constructed in embodiment is all By sequencing, sequencing is completed by Huada gene company and Jie Li sequencing company.Taq DNA used in each embodiment is poly- For synthase purchased from Dongsheng biology, pfu archaeal dna polymerase is purchased from TIANGEN Biotech (Beijing) Co., Ltd., and primeSTAR DNA is poly- Synthase is purchased from TaKaRa company, and three kinds of polymerases all incidentally give corresponding polymerase buffer and dNTP when buying.BamHI, The restriction enzymes such as BglII, HindIII, NdeI, XhoI, EcoRI, SpeI, T4 ligase, T4 phosphorylase (T4PNK) purchase From Fermentas company, when purchase, is accompanied with corresponding buffer etc..Transfection reagent Lip2000Kit is purchased from Invitrogen Company.The amino acid such as proline are purchased from Sigma company.Unless specifically stated otherwise, the chemical reagent such as inorganic salts are purchased from Sigma-Aldrich company.HEPES salt, ampicillin (Amp) and puromycin are purchased from Ameresco company.The detection of 96 holes Blackboard, 384 hole fluorescence detection blackboards are purchased from Grenier company.

DNA purification kit used in embodiment is purchased from BBI company (Canada), and the small pumping kit of ordinary plasmids is purchased from TIANGEN Biotech (Beijing) Co., Ltd..Clone strain Mach1 is purchased from Invitrogen company.Affinity chromatography column and de- Salt column packing is all from GE healthcare company.

The key instrument used in embodiment includes: that (U.S. Bio-Tek is public for 2 multi-function microplate reader of Biotek Synergy Department), X-15R high speed freezing centrifuge (Beckman company, the U.S.), the Microfuge22R table-type high-speed refrigerated centrifuge (U.S. Beckman company), PCR amplification instrument (German Biometra company), Ultrasonic Cell Disruptor (Ningbo Xin Zhi company), nucleic acid electrophoresis apparatus (Shen Neng betting office), sepectrophotofluorometer (Varian company, the U.S.), CO₂Constant temperature cell incubator (SANYO) is inverted glimmering Light microscope (Japanese Nikon company).

II. molecular biology method and cell experiment method

II.1 polymerase chain reaction (PCR):

1. target fragment expands PCR:

This method is mainly used for gene fragment amplification and bacterium colony PCR identification positive colony.The reaction system of the PCR amplification As shown in table 1, amplification program is as shown in table 2.

Table 1.PCR amplification reaction system

Table 2.PCR amplification program

2. long segment (> 2500bp) expands PCR:

The amplification of long segment used in the present invention, mainly Inverse PCR amplification carrier, in the following embodiments for obtaining A kind of technology of rite-directed mutagenesis.Inverse PCR primer is designed at variation position, wherein 5 ' nucleosides of the end comprising variation of a primer Acid sequence.Product after amplification just contains corresponding mutational site.Long segment amplification PCR reaction system is as shown in table 3, expands journey Sequence is as shown in table 4 or table 5.

3. long segment of table (> 2500bp) expands PCR reaction system

4. long segment of table (> 2500bp) expands PCR amplification program

5. long segment of table (> 2500bp) expands PCR amplification program

II.2 endonuclease endonuclease reaction:

The system for carrying out double digestion to plasmid vector is as shown in table 6, wherein n representative make system reach required for total volume plus The sterilizing ultrapure water μ L amount entered.

6. plasmid vector double digestion system of table

II.3DNA segment 5 ' holds phosphorylation reaction

The plasmid or genome ends extracted from microorganism all contains phosphate group, and PCR product does not have, therefore needs Phosphate group addition reaction is carried out to 5 ' end bases of PCR product, only end is contained phosphate group DNA molecular and could be connected It is reversed to answer.Phosphorylation reaction system is as shown in table 7, and wherein T4PNK is writing a Chinese character in simplified form for T4 polynueleotide kinase, for DNA points The addition reaction of 5 ' end phosphate groups of son.

7. phosphorylation reaction system of table

The connection of II.4 target fragment and carrier is reacted

Connection method between different segments and carrier difference, is employed herein three kinds of connection methods

1. the blunt end cloning of flat end short-movie section and linearized vector

The principle of this method is that the flat end products that PCR is obtained carry out phosphorus to 5 ' ends of DNA fragmentation under T4PNK effect After acidification reaction, is connected under the action of PEG4000 and T4DNA ligase with the carrier of linearisation and obtain recombinant plasmid.It is homologous It is as shown in table 8 to recombinate linked system.

8. flat terminal fragment coupled reaction system of table

2. the DNA fragmentation containing cohesive end and the connection containing blunt-ended vector segment

Cohesive end outstanding would generally be generated by the DNA fragmentation that restriction enzyme is cut, thus can with contain The blunt-ended vector segment of sequence complementation connects, and forms recombinant plasmid.Coupled reaction system is as shown in table 9, wherein PCR product The mass ratio of segment and carrier double enzyme digestion product is substantially between 2:1-6:1.

9. cohesive end coupled reaction system of table

3. the connection reaction of the DNA fragmentation product recirculation of 5 ' end phosphorylations after inverse PCR introducing rite-directed mutagenesis

The DNA fragmentation of 5 ' end phosphorylations is connected 3 ' ends of linearized vector and 5 ' ends by recirculation connection reaction Reaction obtains recombinant plasmid.Recirculation coupled reaction system is as shown in table 10.

10. recirculation coupled reaction system of table

The preparation and conversion of II.5 competent cell

The preparation of competent cell:

1. picking single colonie (such as Mach1) is inoculated in 5mL LB culture medium, 37 DEG C of shaking tables are stayed overnight.

2. the bacterium solution transferred species for taking 0.5-1mL to be incubated overnight is into 50mL LB culture medium, 37 DEG C, 220rpm culture 3 to 5 is small When, until OD600 reaches 0.5.

3. ice bath is pre-chilled cell 2 hours.

4. 4 DEG C, 4000rpm is centrifuged 10 minutes.

5. abandoning supernatant, cell is resuspended with the buffer of 5mL pre-cooling, resuspension buffer to final volume is added after uniformly is 50mL。

6. ice bath 45 minutes.

7. 4 DEG C of 4000rpm are centrifuged 10 minutes, bacterium is resuspended with the store buffer liquid that 5mL ice is pre-chilled.

8. putting 100 μ L bacterium solutions, -80 DEG C or liquid nitrogen cryopreservation in each EP pipe.

Buffer: CaCl is resuspended₂(100mM)、MgCl₂(70mM)、NaAc(40mM)

Store buffer liquid: 0.5mL DMSO, 1.9mL 80% glycerol, 10 × CaCl of 1mL₂(1M)、1mL 10×MgCl₂ (700mM)、1mL 10×NaAc(400mM)、4.6mL ddH₂O

The conversion of competent cell:

1. taking 100 μ L competent cells in melting on ice bath.

2. the connection product of proper volume is added, gently piping and druming is mixed, ice bath 30 minutes.The connection product being usually added into Volume is less than the 1/10 of competent cell volume.

3. bacterium solution is put into 42 DEG C of water-baths heat shock 90 seconds, it is transferred quickly to place 5 minutes in ice bath.

4. 500 μ L LB are added, cultivated 1 hour in 200rpm on 37 DEG C of constant-temperature tables.

5. bacterium solution 4000rpm is centrifuged 3 minutes, stays 200 μ L supernatants to blow thallus even, be spread evenly across containing appropriate antibiotic Agar plate surface, plate in 37 DEG C of constant incubators be inverted overnight.

The expression of II.6 protein, purifying and fluorescence detection

1. expression vector (such as proline optical probe expression vector based on pRSETb) is transformed into JM109 (DE3) in cell, it is inverted overnight incubation, picks from the plate and is cloned into 250ml conical flask, be placed in 37 DEG C of shaking tables, 220rpm The IPTG (1M) of 1/1000 (v/v) is added to OD=0.4-0.8 in culture, 18 DEG C inducing expression 24-36 hours.

2. after the completion of inducing expression, bacterium is received in 4000rpm, centrifugation in 30 minutes, bacterium is resuspended in the phosphate buffer that 50mM is added Body precipitating, ultrasonication to thallus are clarified.9600rpm, 4 DEG C are centrifuged 20 minutes.

Albumen is obtained by the affinity chromatography column purification of self-chambering 3. being centrifuged supernatant, the albumen after affinity chromatography is again The albumen being dissolved in 20mM MOPS buffer (pH 7.4) or phosphate buffer PBS is obtained by the desalting column of self-chambering.

4. purifying albumen after SDS-PAGE is identified, using measurement buffer (100mM HEPES, 100mM NaCl, PH 7.3) or phosphate buffer PBS dilution probe at final concentration of 5-10 μM of protein solution.With measurement buffer Proline is configured to the liquid storage of final concentration of 1M by (20mM MOPS, pH 7.4) or phosphate buffer PBS.

5. taking the protein solution of 5 μM of 100 μ l, 37 DEG C are incubated 5 minutes, are separately added into after proline mixes to final concentration of 100mM utilizes light absorption of the Multifunction fluorescent microplate reader measurement albumen at 340nm.

6. taking the protein solution of 1 μM of 100 μ l, 37 DEG C are incubated 5 minutes, and proline titration is added, measures the 485nm of albumen The fluorescence intensity that 528nm emits after fluorescence excitation.It is complete using Multifunction fluorescent microplate reader to fluorescence excitation, the transmitting measurement of sample At.

7. take the protein solution of 1 μM of 100 μ l, 37 DEG C are incubated 5 minutes, and proline is added, measure albumen absorption spectrum and Fluorescence spectrum.The measurement of the Absorption and fluorescence spectrum of sample is completed by spectrophotometer and sepectrophotofluorometer.

The transfection and fluorescence detection of II.7 mammalian cell

1. the proline optical probe plasmid based on pCDNA3.1+ is passed through transfection reagent Lipofectamine2000 (Invitrogen) it is transfected into HeLa, is placed in 37 DEG C, 5%CO₂Cell incubator in cultivate.It is given full expression to foreign gene Fluorescence detection is carried out after 24~36h.

2. after the completion of inducing expression, adherent HeLa cell is flushed three times with PBS, be placed in HBSS solution respectively into Row fluorescence microscope and microplate reader detection.

Embodiment 1: proline binding protein plasmid

Pass through proline binding protein (Atu2422 herein) gene in PCR amplification soil Agrobacterium gene, PCR product BamHI and EcoRI digestion is used after recycling after gel electrophoresis, while corresponding double digestion is carried out to pRSETb carrier.Connected with T4DNA After connecing enzyme connection, MachI is converted with product, the MachI of conversion is coated on LB plate (ampicillin 100ug/mL), is placed in 37 DEG C overnight incubation.After MachI transformant progress plasmid extraction will be grown, PCR identification is carried out.Positive plasmid is after being sequenced correctly Carry out subsequent plasmid construction.

Embodiment 2: the expression and detection of the cpYFP optical probe of different position of fusion

In the present embodiment, following site fusion cpYFP are selected based on pRSETb-Atu2422, are obtained corresponding PRSETb-Atu2422-cpYFP plasmid: 117/118,117/119,117/120,117/121,118/119,118/120,118/ 121,119/120,119/121,120/121,120/122,120/123,121/122,121/123,122/123,249/250, 249/251,249/252,249/253,249/254,249/255,249/256,249/257,249/258,249/259,250/ 251,250/252,250/253,250/254,250/255,250/256,250/257,250/258,250/259,251/252, 251/253,251/254,251/255,251/256,251/257,251/258,251/259,252/253,252/254,252/ 255,252/256,252/257,252/258,252/259,253/254,253/255,253/256,253/257,253/258, 253/259,254/255,254/256,254/257,254/258,254/259,255/256,255/257,255/258,255/ 259,256/257,256/258,256/259,257/258,257/259,258/259,323/330,324/330,325/330, 326/327,326/328,326/329,326/330,327/328,327/329,327/330,328/329,328/330 or 329/ 330。

Using PCR generate cpYFP DNA fragmentation, to the DNA fragmentation using 5 ' ends plus phosphorus operation after inactivate, simultaneously The pRSETb- proline binding protein linearized vector for containing different broken sites is generated by Inverse PCR amplification, will be linearized The cpYFP segment of the terminal phosphate of pRSETb-Atu2422 and 5 ' connect production under the action of PEG4000 with T4DNA ligase Raw recombinant plasmid, by these plates in Kodak multifunctional active imaging system, picking has yellow fluorescence in the case where the channel FITC excites Clone, by Beijing Liuhe Huada Genomics Technology Co., Ltd Shanghai branch company complete be sequenced.

Through sequencing it is correct after, by inducing expression in recombinant plasmid transformed to JM109 (DE3), and protein purification, pass through SDS-PAGE electrophoresis size is near 68Kda.It is pure containing His-tag to meet that pRSETb-Atu2422-cpYFP gives expression to for the size Change the size of the Atu2422-cpYFP fused protein of label.As a result as shown in Figure 1.

The Atu2422-cpYFP fused protein of purifying is subjected to proline response screening, 100mM proline will be contained The detection signal of fluorescence protein is merged divided by the detection signal for merging fluorescence protein of no proline.As a result as shown in Fig. 2, Testing result shows that responding optical probe more than 2 times to proline has 120/121,121/122,121/123,324/330, 325/330 and 326/330 site (as shown in SEQ ID NO 10-15) or the orresponding amino acid site of its family protein are implemented The optical probe of fusion.

Embodiment 3: the expression and detection of the cpGFP optical probe of different position of fusion

CpYFP is replaced with into cpGFP according to the method in embodiment 2, constructs proline green fluorescent protein fluorescence probe. As shown in figure 3, testing result shows that the optical probe for responding proline more than 2 times has 120/121,121/122,121/ The optics of fusion is implemented in 123,324/330,325/330 and 326/330 sites or the orresponding amino acid site of its family protein Probe.

Embodiment 4: the expression and detection of the cpBFP optical probe of different position of fusion

CpYFP is replaced with into cpBFP according to the method in embodiment 2, constructs proline blue fluorescent protein fluorescence probe. As shown in figure 4, testing result shows that the optical probe for responding proline more than 2 times has 120/121,121/122,121/ The optics of fusion is implemented in 123,324/330,325/330 and 326/330 sites or the orresponding amino acid site of its family protein Probe.

Embodiment 5: the expression and detection of the cpmApple optical probe of different position of fusion

CpYFP is replaced with into cpmApple according to the method in embodiment 2, building proline red fluorescent protein fluorescence is visited Needle.As shown in figure 5, testing result shows that responding optical probe more than 2 times to proline has 120/121,121/122, Fusion is implemented in 121/123,324/330,325/330 and 326/330 site or the orresponding amino acid site of its family protein Optical probe.

Embodiment 6: the performance of optical probe

For responding optical probe more than 2 times to proline obtained in embodiment 2, i.e., 120/121,121/122, 6 kinds of optical probes of fusion are implemented in 121/123,324/330,325/330 and 326/330 site, carry out the dried meat into concentration gradient Propylhomoserin detection, detection 420nm excite fluorescence intensity and 485nm at 528nm transmitting to excite fluorescence intensity ratio at 528nm transmitting Variation.6 kinds of proline light that position of fusion is 120/121,121/122,121/123,324/330,325/330 and 326/330 Learn the K of probe_d(binding constant) is respectively 18.6mM, 0.2mM, 15.5mM, 0.95mM, 0.38mM and 0.65mM, amplitude of variation Respectively 3.0 times, 4.5 times, 2.0 times, 2.9 times, 2.6 times and 2.0 times, as a result as shown in Figure 6.

The probe (Atu2422-121/122-cpYFP) for being 121/122 for position of fusion, carries out it to each amino acid Specific detection.Meanwhile with fluorescin cpYFP, cpGFP, cpBFP or cpmApple be fused to Atu2422 N-terminal or The obtained fusion protein of C-terminal compares probe that position of fusion is 121/122 to the specificity of proline as control.

The result shows that position of fusion be 121/122 probe to alanine, proline, valine, serine, threonine and Cysteine response all with higher, respectively 4.3 times, 4.5 times, 4.0 times, 3.2 times, 2.4 times and 3.0 times, such as Fig. 7 A institute Show.Meanwhile the fusion protein that fluorescin is fused to the N-terminal of Atu2422 or C-terminal obtains does not respond to proline, and melt The probe that coincidence point is 121/122 has about 4.5 times of response to proline, as shown in Figure 7 B.

Embodiment 7: the expression and detection of the cpYFP optical probe of mutation

Optical probe mutant is constructed on the basis of Atu2422-121/122-cpYFP.Matter is linearized by inverse PCR Grain pRSETb-Atu2422-121/122-cpYFP produces obtained PCR containing the base sequence for wanting mutational site in primer Object is under the action of PNK, T4DNA ligase are with PEG4000 plus phosphorus connects, and obtains F77, A100, T102, D121, Y150, The fixed point saturation mutation plasmid in this 8 sites D226, G227, Y275, and by the limited public affairs of Beijing six directions Hua Da Gene science share It takes charge of Shanghai branch company and completes sequencing.

As a result as shown in Figure 8.Fluorescence detection, which as the result is shown responds proline, has F77L, A100G more than 2 times, D121E, D121S, D121T, D121V, D226E, D226N and Y275F mutant.

The performance of 8. optical probe mutant of embodiment

Illustratively, the proline optical probe Atu2422-121/122-cpYFP-D226N of purifying is subjected to 0mM respectively After ten minutes with the processing of 500mM proline, the detection of fluorescence Spectra is carried out using sepectrophotofluorometer.

Measurement to excitation spectrum: exciting light is recorded with the excites scope of 350nm to 510nm and the launch wavelength of 530nm Spectrum, every 1nm read primary.The results show that there are two excitation peaks at about 420 and 490nm for probe, as shown in Figure 9 A.

Measurement to emission spectrum: fixed excitation wavelength is respectively 420nm and 490nm, records the transmitting light of 505-600nm Spectrum, every 1nm read primary.The results show that probe, after adding 500mM proline, fluorescence intensity is reduced under 420nm excitation 1.2 times for adding 0mM proline；Fluorescence intensity is reduced to 0.23 times of addition 0mM proline under 490nm excitation.Such as Fig. 9 B With shown in 9C.

The proline inspection of concentration gradient (0~100mM) is carried out to the Atu2422-121/122-cpYFP-D226N of purifying It surveys.The probe of purifying is handled after ten minutes, fluorescence intensity and 485nm excite 528nm at detection 420nm excitation 528nm transmitting The variation of fluorescence intensity ratio at transmitting.The results are shown in Figure 10, the K of proline optical probe_d(binding constant) is 2.8mM, Amplitude of variation is 5 times.

Atu2422-121/122-cpYFP-D226N and 20 kinds of amino acid are subjected to reactive detection, the results showed that it has There is good specificity, as shown in figure 11.

Embodiment 9: the subcellular organelle positioning of optical probe and performance of the optical probe in subcellular organelle

In the present embodiment, different localization signal peptide and optical probe Atu2422-121/122-cpYFP-D226N are used Fusion, optical probe is navigated in different organelles.

After optical probe plasmid transfection HeLa cell 36 hours that merge different localization signal peptides, is rinsed, set using PBS It carries out carrying out fluorescence detection under the channel FITC using inverted fluorescence microscope in HBSS solution.As a result as shown in figure 12.Dried meat ammonia Sour optical probe from different special localization signal peptide fusions by that can navigate to including cytoplasm, mitochondria, nucleus, height In the subcellular organelles such as dictyosome, peroxisome and lysosome.Fluorescence is all shown in different subcellular structures, and glimmering The distribution of light and intensity are different.

After optical probe plasmid transfection HeLa cell 36 hours of endochylema expression, is rinsed using PBS, be placed in HBSS solution 420nm excites fluorescence intensity and 485nm at 528nm transmitting to excite fluorescence intensity at 528nm transmitting in the middle detection 40min period The variation of ratio.As a result as shown in figure 13.10mM proline is added, is continued to test 20 minutes.Add the sample of proline 485/420 gradually increases, and highest can achieve 2.3 times, remains unchanged without adding the 485/420 of control group of proline for 1.

Embodiment 10: high-throughput screening compound is carried out based on optical probe in living cells

In the present embodiment, we are carried out using the HeLa cell of endochylema expression Atu2422-121/122-cpYFP-D226N High-throughput screening compound.

HeLa cell through transfecting is rinsed using PBS, is placed in HBSS solution (no proline) and is handled 1 hour, then makes It is handled 1 hour with 10 μM of compounds.Proline is added dropwise in each sample respectively.It is sent out using microplate reader record 420nm excitation 528nm Penetrate fluorescence intensity ratio variation at place's fluorescence intensity and 485nm excitation 528nm transmitting.The sample handled with unused any compound It is standardized as control.As a result as shown in figure 14.In the 2000 kinds of compounds used, most compounds is to dried meat It is minimum that propylhomoserin enters impact cell.In addition the intake ability for having 14 kinds of compounds to can be improved cell to proline has 10 kinds of changes Intake of the cell to proline can be substantially reduced by closing object.

Embodiment 11: the proline in optical probe quantitative detection blood

In this embodiment, using the Atu2422-121/122-cpYFP-D226N of purifying to the blood supernatant of mouse and people In proline analyzed.

After ten minutes by Atu2422-121/122-cpYFP-D226N and diluted blood supernatant mixed processing, enzyme is used It marks fluorescence intensity and 485nm at instrument detection 420nm excitation 528nm transmitting and excites fluorescence intensity ratio at 528nm transmitting.As a result such as Shown in Figure 15, the proline content in mouse blood is at 140 μM or so, and the proline content in human blood is at 160 μM or so.

As seen from the above embodiment, proline optical probe provided by the invention, molecular weight of albumen is relatively small and is easy to Maturation, fluorescence dynamic change is big, and specificity is good, and can be expressed in cell by the method for genetic manipulation, can be in cell Inside and outside real-time positioning, quantitative detection proline；And it is able to carry out high-throughput screening compound.

Other embodiment

Present specification describes many embodiments.It should be understood, however, that those skilled in the art are by reading this specification The various improvement without departing substantially from the spirit and scope of the present invention known, should also be included within the scope of the following claims.

Sequence table

<110>East China University of Science

<120>proline optical probe and its preparation method and application

<130> 191303 1CNCN

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<170> PatentIn version 3.5

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Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

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Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

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

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Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

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Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

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Asn Thr Phe Arg Thr Cys Gly Arg Asp Asp Gln Gln Gly Gly Ile Ala

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Gly Lys Tyr Leu Ala Asp His Phe Lys Asp Ala Lys Val Ala Ile Ile

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His Asp Lys Thr Pro Tyr Gly Gln Gly Leu Ala Asp Glu Thr Lys Lys

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Ala Ala Asn Ala Ala Gly Val Thr Glu Val Met Tyr Glu Gly Val Asn

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Val Gly Asp Lys Asp Phe Ser Ala Leu Ile Ser Lys Met Lys Glu Ala

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Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu His Thr Glu Ala Gly Leu

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

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Gly Asp Gly Ile Val Ser Asn Glu Leu Ala Ser Ile Ala Gly Asp Ala

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Val Glu Gly Thr Leu Asn Thr Phe Gly Pro Asp Pro Thr Leu Arg Pro

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Glu Asn Lys Glu Leu Val Glu Lys Phe Lys Ala Ala Gly Phe Asn Pro

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Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala Met Gln Ala Ile Ala Gly

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Ala Ala Lys Ala Ala Gly Ser Val Glu Pro Glu Lys Val Ala Glu Ala

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

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Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr Val Met Tyr Glu Trp Lys

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Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile Gln Gln Gly Ser

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

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Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser

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Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val

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Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp

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Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly

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Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys

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Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu

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Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro

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Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr

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Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu

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Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr

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

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Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly

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His Lys Leu Glu Tyr Asn

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Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr

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Ile Val Asp Ile Lys Leu Asp Ile Val Ser His Asn Glu Asp Tyr Thr

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Ile Val Glu Gln Cys Glu Arg Ala Glu Gly Arg His Pro Thr Gly Gly

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

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Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe Met Arg Phe Lys

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Val His Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly

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Glu Gly Glu Gly Arg Pro Tyr Glu Ala Phe Gln Thr Ala Lys Leu Lys

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Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro

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Gln Phe Thr Tyr Gly Ser Lys Ala Tyr Ile Lys His Pro Ala Asp Ile

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Pro Asp Tyr Phe Lys Leu Ser Phe Pro Glu Gly Phe Arg Trp Glu Arg

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Val Met Asn Phe Glu Asp Gly Gly Ile Ile His Val Asn Gln Asp Ser

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Ser Leu Gln Asp Gly Val Phe Ile Tyr Lys Val Lys Leu Arg Gly Thr

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Asn Phe Pro Pro Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp

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Glu Ala

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Met Gly Gly Arg Ser Lys Lys Pro Ala Lys Asn Leu Lys Met Pro Gly

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Val Tyr Tyr Val Asp Arg Arg Leu Glu Arg Ile Lys Glu Ala Asp Lys

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Glu Thr Tyr Val Glu Gln His Glu Val Ala Val Ala Arg Tyr Cys Asp

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Leu Pro Ser Lys Leu Gly His Lys Leu Asn Gly Gly Thr Gly Gly Ser

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Met Val Ser Lys Gly Glu Glu Leu Ile Lys Glu Asn Met His Met Lys

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Leu Tyr Met Glu Gly Thr Val Asn Asn His His Phe Lys Cys Thr Ser

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Glu Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg Ile Lys

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

115 120 125

Ser Phe Met Tyr Gly Ser Lys Thr Phe Ile Asn His Thr Gln Gly Ile

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Pro Asp Phe Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg

145 150 155 160

Val Thr Thr Tyr Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp Thr

165 170 175

Ser Leu Gln Asp Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg Gly Val

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Asn Phe Pro Ser Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly Trp

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Glu Ala Ser Thr Glu Met Leu Tyr Pro Ala Asp Gly Gly Leu Glu Gly

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Arg Ser Asp Met Ala Leu Lys Leu Val Gly Gly Gly His Leu Ile Cys

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Asn Leu Lys Thr Thr Tyr Arg Ser Lys Lys

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Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe

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Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe

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Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr

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Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp

50 55 60

Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His

65 70 75 80

Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe

85 90 95

Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val

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Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys

115 120 125

Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys

130 135 140

Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly

145 150 155 160

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

165 170 175

His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val

180 185 190

Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser

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His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly

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

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

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

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

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

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

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Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Gln

100 105 110

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

115 120 125

Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile

130 135 140

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr

145 150 155 160

Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys

165 170 175

Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Asn

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

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

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Glu Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro

100 105 110

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

115 120 125

Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys

130 135 140

Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val

145 150 155 160

Thr Thr Leu Ser His Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His

165 170 175

Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Gly Gly Tyr Ile

180 185 190

Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg

195 200 205

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

210 215 220

Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu

225 230 235 240

Glu Tyr Asn

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<212> PRT

<213>artificial sequence

<400> 8

Met Ser Glu Leu Ile Thr Glu Asn Met His Met Lys Leu Tyr Met Glu

1 5 10 15

Gly Thr Val Asn Asn His His Phe Lys Cys Thr Ser Glu Gly Glu Gly

20 25 30

Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg Ile Lys Val Val Glu Gly

35 40 45

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

50 55 60

Gly Ser Lys Thr Phe Ile Asn His Thr Gln Gly Ile Pro Asp Phe Phe

65 70 75 80

Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Val Thr Thr Tyr

85 90 95

Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp Thr Ser Leu Gln Asp

100 105 110

Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg Gly Val Asn Phe Pro Ser

115 120 125

Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly Trp Glu Ala Ser Thr

130 135 140

Glu Met Leu Tyr Pro Ala Asp Gly Gly Leu Glu Gly Arg Ala Asp Met

145 150 155 160

Ala Leu Lys Leu Val Gly Gly Gly His Leu Ile Cys Asn Leu Lys Thr

165 170 175

Thr Tyr Arg Ser Lys Lys Pro Ala Lys Asn Leu Lys Met Pro Gly Val

180 185 190

Tyr Tyr Val Asp Arg Arg Leu Glu Arg Ile Lys Glu Ala Asp Lys Glu

195 200 205

Thr Tyr Val Glu Gln His Glu Val Ala Val Ala Arg Tyr Cys Asp Leu

210 215 220

Pro Ser Lys Leu Gly His Lys Leu Asn

225 230

<210> 9

<211> 242

<212> PRT

<213>artificial sequence

<400> 9

Val Ser Glu Arg Met Tyr Pro Glu Asp Gly Ala Leu Lys Ser Glu Ile

1 5 10 15

Lys Lys Gly Leu Arg Leu Lys Asp Gly Gly His Tyr Ala Ala Glu Val

20 25 30

Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr

35 40 45

Ile Val Asp Ile Lys Leu Asp Ile Val Ser His Asn Glu Asp Tyr Thr

50 55 60

Ile Val Glu Gln Cys Glu Arg Ala Glu Gly Arg His Ser Thr Gly Gly

65 70 75 80

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

85 90 95

Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe Met Arg Phe Lys

100 105 110

Val His Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly

115 120 125

Glu Gly Glu Gly Arg Pro Tyr Glu Ala Phe Gln Thr Ala Lys Leu Lys

130 135 140

Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro

145 150 155 160

Gln Phe Met Tyr Gly Ser Lys Ala Tyr Ile Lys His Pro Ala Asp Ile

165 170 175

Pro Asp Tyr Phe Lys Leu Ser Phe Pro Glu Gly Phe Arg Trp Glu Arg

180 185 190

Val Met Asn Phe Glu Asp Gly Gly Ile Ile His Val Asn Gln Asp Ser

195 200 205

Ser Leu Gln Asp Gly Val Phe Ile Tyr Lys Val Lys Leu Arg Gly Thr

210 215 220

Asn Phe Pro Pro Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp

225 230 235 240

Glu Ala

<210> 10

<211> 596

<212> PRT

<213>artificial sequence

<400> 10

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

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

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Tyr Asn Ser Asp Asn Val Tyr Ile

115 120 125

Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg

130 135 140

His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln

145 150 155 160

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

165 170 175

Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp

180 185 190

His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly

195 200 205

Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 11

<211> 596

<212> PRT

<213>artificial sequence

<400> 11

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

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

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

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

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

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

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

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

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

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

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 12

<211> 595

<212> PRT

<213>artificial sequence

<400> 12

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

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

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

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

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

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

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

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

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

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

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gln

355 360 365

Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp Ala

370 375 380

Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu Ala

385 390 395 400

Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val Met

405 410 415

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

420 425 430

Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu His

435 440 445

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

450 455 460

Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala Ser

465 470 475 480

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

485 490 495

Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys Ala

500 505 510

Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala Met

515 520 525

Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro Glu

530 535 540

Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu Gly

545 550 555 560

Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr Val

565 570 575

Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile Gln

580 585 590

Gln Gly Ser

595

<210> 13

<211> 591

<212> PRT

<213>artificial sequence

<400> 13

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

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

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Asp Gln Gln Gly Gly Ile Ala

115 120 125

Gly Lys Tyr Leu Ala Asp His Phe Lys Asp Ala Lys Val Ala Ile Ile

130 135 140

His Asp Lys Thr Pro Tyr Gly Gln Gly Leu Ala Asp Glu Thr Lys Lys

145 150 155 160

Ala Ala Asn Ala Ala Gly Val Thr Glu Val Met Tyr Glu Gly Val Asn

165 170 175

Val Gly Asp Lys Asp Phe Ser Ala Leu Ile Ser Lys Met Lys Glu Ala

180 185 190

Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu His Thr Glu Ala Gly Leu

195 200 205

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

210 215 220

Gly Asp Gly Ile Val Ser Asn Glu Leu Ala Ser Ile Ala Gly Asp Ala

225 230 235 240

Val Glu Gly Thr Leu Asn Thr Phe Gly Pro Asp Pro Thr Leu Arg Pro

245 250 255

Glu Asn Lys Glu Leu Val Glu Lys Phe Lys Ala Ala Gly Phe Asn Pro

260 265 270

Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala Met Gln Ala Ile Ala Gly

275 280 285

Ala Ala Lys Ala Ala Gly Ser Val Glu Pro Glu Lys Val Ala Glu Ala

290 295 300

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

305 310 315 320

Glu Lys Gly Asp Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys

325 330 335

Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu

340 345 350

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

355 360 365

Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln

370 375 380

Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu

385 390 395 400

Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu

405 410 415

Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu

420 425 430

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

435 440 445

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

450 455 460

Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro

465 470 475 480

Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Val Met Tyr Glu Trp

565 570 575

Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile Gln Gln Gly Ser

580 585 590

<210> 14

<211> 592

<212> PRT

<213>artificial sequence

<400> 14

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

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

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Asp Gln Gln Gly Gly Ile Ala

115 120 125

Gly Lys Tyr Leu Ala Asp His Phe Lys Asp Ala Lys Val Ala Ile Ile

130 135 140

His Asp Lys Thr Pro Tyr Gly Gln Gly Leu Ala Asp Glu Thr Lys Lys

145 150 155 160

Ala Ala Asn Ala Ala Gly Val Thr Glu Val Met Tyr Glu Gly Val Asn

165 170 175

Val Gly Asp Lys Asp Phe Ser Ala Leu Ile Ser Lys Met Lys Glu Ala

180 185 190

Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu His Thr Glu Ala Gly Leu

195 200 205

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

210 215 220

Gly Asp Gly Ile Val Ser Asn Glu Leu Ala Ser Ile Ala Gly Asp Ala

225 230 235 240

Val Glu Gly Thr Leu Asn Thr Phe Gly Pro Asp Pro Thr Leu Arg Pro

245 250 255

Glu Asn Lys Glu Leu Val Glu Lys Phe Lys Ala Ala Gly Phe Asn Pro

260 265 270

Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala Met Gln Ala Ile Ala Gly

275 280 285

Ala Ala Lys Ala Ala Gly Ser Val Glu Pro Glu Lys Val Ala Glu Ala

290 295 300

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

305 310 315 320

Glu Lys Gly Asp Pro Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp

325 330 335

Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val

340 345 350

Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro

355 360 365

Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe

370 375 380

Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val

385 390 395 400

Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu

405 410 415

Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Val Met Tyr Glu

565 570 575

Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile Gln Gln Gly Ser

580 585 590

<210> 15

<211> 593

<212> PRT

<213>artificial sequence

<400> 15

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

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

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Asp Gln Gln Gly Gly Ile Ala

115 120 125

Gly Lys Tyr Leu Ala Asp His Phe Lys Asp Ala Lys Val Ala Ile Ile

130 135 140

His Asp Lys Thr Pro Tyr Gly Gln Gly Leu Ala Asp Glu Thr Lys Lys

145 150 155 160

Ala Ala Asn Ala Ala Gly Val Thr Glu Val Met Tyr Glu Gly Val Asn

165 170 175

Val Gly Asp Lys Asp Phe Ser Ala Leu Ile Ser Lys Met Lys Glu Ala

180 185 190

Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu His Thr Glu Ala Gly Leu

195 200 205

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

210 215 220

Gly Asp Gly Ile Val Ser Asn Glu Leu Ala Ser Ile Ala Gly Asp Ala

225 230 235 240

Val Glu Gly Thr Leu Asn Thr Phe Gly Pro Asp Pro Thr Leu Arg Pro

245 250 255

Glu Asn Lys Glu Leu Val Glu Lys Phe Lys Ala Ala Gly Phe Asn Pro

260 265 270

Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala Met Gln Ala Ile Ala Gly

275 280 285

Ala Ala Lys Ala Ala Gly Ser Val Glu Pro Glu Lys Val Ala Glu Ala

290 295 300

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

305 310 315 320

Glu Lys Gly Asp Pro Lys Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala

325 330 335

Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn

340 345 350

Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr

355 360 365

Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser

370 375 380

Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met

385 390 395 400

Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp

405 410 415

Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Val Met Tyr

565 570 575

Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile Gln Gln Gly

580 585 590

Ser

<210> 16

<211> 596

<212> PRT

<213>artificial sequence

<400> 16

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

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

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

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

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

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

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

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

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

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

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 17

<211> 596

<212> PRT

<213>artificial sequence

<400> 17

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

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

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Gly Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

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

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

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

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

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

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

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

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 18

<211> 596

<212> PRT

<213>artificial sequence

<400> 18

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

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

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Glu Tyr Asn Ser Asp Asn Val Tyr

115 120 125

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

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

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

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

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

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

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

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 19

<211> 596

<212> PRT

<213>artificial sequence

<400> 19

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

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

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Ser Tyr Asn Ser Asp Asn Val Tyr

115 120 125

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

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

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

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

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

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

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

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 20

<211> 596

<212> PRT

<213>artificial sequence

<400> 20

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

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

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Thr Tyr Asn Ser Asp Asn Val Tyr

115 120 125

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

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

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

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

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

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

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

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 21

<211> 596

<212> PRT

<213>artificial sequence

<400> 21

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

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

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Val Tyr Asn Ser Asp Asn Val Tyr

115 120 125

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

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

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

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

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

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

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

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 22

<211> 596

<212> PRT

<213>artificial sequence

<400> 22

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

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

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

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

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

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

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

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

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

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

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Glu Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 23

<211> 596

<212> PRT

<213>artificial sequence

<400> 23

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

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

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

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

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

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

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

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

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

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

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asn Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 24

<211> 596

<212> PRT

<213>artificial sequence

<400> 24

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

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

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

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

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

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

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

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

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

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

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Phe Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 25

<211> 1791

<212> DNA

<213>artificial sequence

<400> 25

atggatgtcg tgatcgctgt cggcgcaccg ctgaccggcc cgaacgctgc tttcggcgct 60

cagatccaga agggtgccga acaggctgcg aaagacatca atgctgccgg cggtatcaat 120

ggcgagcaga ttaagatcgt gctgggcgac gacgtatccg accccaagca gggtatttcg 180

gttgccaaca aattcgttgc tgacggcgtg aaattcgttg tcggtcactt caactcgggt 240

gtttccattc cggcatcgga agtttatgcc gaaaacggca ttctcgaaat cacgcccgct 300

gcgaccaacc cggtctttac cgagcgtggc ctgtggaaca ccttccgcac ctgcggccgt 360

gactacaaca gcgacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggcc 420

aacttcaaga tccgccacaa cgtcgaggac ggcagcgtgc agctcgccga ccactaccag 480

cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcttc 540

cagtccgtcc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 600

gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaacgtgga tggcggtagc 660

ggtggcaccg gcagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 720

ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 780

acctacggca agctgaccct gaagctgatc tgcaccaccg gcaagctgcc cgtgccctgg 840

cccaccctcg tgaccaccct cggctacggc ctgaagtgct tcgcccgcta ccccgaccac 900

atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 960

atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 1020

accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 1080

gggcacaagc tggagtacaa cgaccagcag ggcggcattg ccggcaagta cctggccgat 1140

catttcaagg acgccaaggt cgccatcatt cacgacaaga cgccttatgg tcagggtctt 1200

gccgatgaaa ccaaaaaggc tgccaatgct gccggcgtga ctgaggtcat gtatgaaggc 1260

gtcaacgtcg gcgacaagga cttctccgcg ctgatctcga agatgaagga agccggcgtt 1320

tccatcatct actggggcgg cctgcacacc gaagccggcc tgatcatccg ccaggcggct 1380

gaccagggtc tgaaggccaa gctcgtttcg ggcgacggta ttgtctcgaa cgaacttgct 1440

tccatcgccg gcgacgccgt cgagggcacg ctgaacacct tcggccctga tccgacgctg 1500

cgcccggaaa acaaggaact ggtagagaag ttcaaggccg ccggcttcaa cccggaagcc 1560

tacacgctct actcctatgc cgcgatgcag gcgattgcag gcgcagcgaa ggctgcgggt 1620

tccgtggagc cggaaaaggt tgccgaagcc ctgaagaagg gctccttccc gaccgcactc 1680

ggcgaaatct ccttcgatga gaagggcgac ccgaagcttc ccggctacgt catgtacgaa 1740

tggaagaagg gtccggacgg caagttcacc tacatccagc agggcagcta a 1791

<210> 26

<211> 1791

<212> DNA

<213>artificial sequence

<400> 26

atggatgtcg tgatcgctgt cggcgcaccg ctgaccggcc cgaacgctgc tttcggcgct 60

cagatccaga agggtgccga acaggctgcg aaagacatca atgctgccgg cggtatcaat 120

ggcgagcaga ttaagatcgt gctgggcgac gacgtatccg accccaagca gggtatttcg 180

gttgccaaca aattcgttgc tgacggcgtg aaattcgttg tcggtcactt caactcgggt 240

gtttccattc cggcatcgga agtttatgcc gaaaacggca ttctcgaaat cacgcccgct 300

gcgaccaacc cggtctttac cgagcgtggc ctgtggaaca ccttccgcac ctgcggccgt 360

gactacaaca gcgacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggcc 420

aacttcaaga tccgccacaa cgtcgaggac ggcagcgtgc agctcgccga ccactaccag 480

cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcttc 540

cagtccgtcc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 600

gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaacgtgga tggcggtagc 660

ggtggcaccg gcagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 720

ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 780

acctacggca agctgaccct gaagctgatc tgcaccaccg gcaagctgcc cgtgccctgg 840

cccaccctcg tgaccaccct cggctacggc ctgaagtgct tcgcccgcta ccccgaccac 900

atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 960

atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 1020

accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 1080

gggcacaagc tggagtacaa cgaccagcag ggcggcattg ccggcaagta cctggccgat 1140

catttcaagg acgccaaggt cgccatcatt cacgacaaga cgccttatgg tcagggtctt 1200

gccgatgaaa ccaaaaaggc tgccaatgct gccggcgtga ctgaggtcat gtatgaaggc 1260

gtcaacgtcg gcgacaagga cttctccgcg ctgatctcga agatgaagga agccggcgtt 1320

tccatcatct actggggcgg cctgcacacc gaagccggcc tgatcatccg ccaggcggct 1380

gaccagggtc tgaaggccaa gctcgtttcg ggcaacggta ttgtctcgaa cgaacttgct 1440

tccatcgccg gcgacgccgt cgagggcacg ctgaacacct tcggccctga tccgacgctg 1500

cgcccggaaa acaaggaact ggtagagaag ttcaaggccg ccggcttcaa cccggaagcc 1560

tacacgctct actcctatgc cgcgatgcag gcgattgcag gcgcagcgaa ggctgcgggt 1620

tccgtggagc cggaaaaggt tgccgaagcc ctgaagaagg gctccttccc gaccgcactc 1680

ggcgaaatct ccttcgatga gaagggcgac ccgaagcttc ccggctacgt catgtacgaa 1740

tggaagaagg gtccggacgg caagttcacc tacatccagc agggcagcta a 1791

Claims

1. a kind of proline sensitivity polypeptide or its functional variety, wherein proline sensitivity polypeptide sequence shown in SEQ ID NO:1 It arranges, or has the sequence of 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity with it In include the mutation at the following site: F77, A100, T102, D121, Y150, D226 and Y275.

2. a kind of optical probe, comprising proline sensitivity polypeptide or its functional variety and optical activity polypeptide or its functional variety, Wherein optical activity polypeptide or its functional variety are located in the sequence of proline sensitivity polypeptide or its functional variety, the proline Sensitive polypeptide includes the mutation at following site: F77, A100, T102, D121, Y150, D226 and Y275, and number corresponds to In the overall length of proline sensitivity polypeptide.

3. optical probe as claimed in claim 2, wherein proline sensitivity polypeptide sequence shown in SEQ ID NO:1 It arranges, or has the sequence of 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity with it In include the mutation at the following site: F77, A100, T102, D121, Y150, D226 and Y275.

4. optical probe as claimed in claim 1 or 2, wherein optical activity polypeptide is located at being selected from for proline sensitivity polypeptide Between residue below: residue 117-123, residue 249-259 and residue 323-330, number correspond to proline sensitivity polypeptide Overall length.

5. a kind of nucleic acid sequence encodes optical probe described in any one of claim 2-4.

6. a kind of expression vector, it includes the nucleic acid sequences as claimed in claim 5 being operatively connected with expression control sequence.

7. a kind of host cell, it includes described in claim 5 nucleic acid sequence or expression vector as claimed in claim 6.

8. a kind of method for preparing optical probe described in any one of claim 2-4, comprising the following steps:

(1) expression vector described in claim 5 is transferred in host cell,

(2) host cell is cultivated under conditions of being suitble to the expression vector to express, and

(3) optical probe is separated by the host cell.

9. optical probe described in any one of claim 2-4 or the optical probe of claim 8 the method preparation are being examined The application in proline in sample.

10. a kind of detection kit, it includes described in any one of claim 2-4 optical probe or claim 8 described in The optical probe of method preparation.