CN106905418A - A kind of histidine fluorescence probe and its preparation method and application - Google Patents

Abstract

The invention provides a kind of histidine fluorescence probe, including the fluorescin A showed to the sensitive polypeptide B of histidine and to histidine；Described fluorescin A is inserted into polypeptide B, and it is two parts of B1 and B2 that B is divided to, and forms the probe structure of B1 A B2 formulas；The polypeptide B and histidine interact causes fluorescin A fluorescence signals to change；The polypeptide B is HBP and its mutant.The histidine fluorescence probe molecular weight of albumen that the present invention is provided is relatively small and is easy to ripe, fluorescence dynamic change is big, specificity is good, and can be expressed in cell difference subcellular fraction by the method for genetic manipulation, can outer high flux, quantitative determination histidine in the cell.

Description

A kind of histidine fluorescent probe and its preparation method and application

technical field

The invention relates to the technical field of bioengineering, in particular to a histidine fluorescent probe and its preparation method and application.

Background technique

Histidine is one of the 20 basic amino acids and is an important amino acid. Since the pKa of its side chain is 6.0 (Nelson DL et al., Germany: Springer 2009), its side chain can be deprotonated in a neutral environment The state becomes the protonated state. Based on this property, histidine residues can act as both proton acceptors and proton donors in many intracellular reactions (Rebek J et al., Struct Chem 1:129–131 1990; Polgár L. et al., CellMol Life Sci 62:2161–2172 2005). Moreover, it contains a highly reactive imidazole ring, which plays an important role in the transport of metal ions in biological systems (Creighton TE. et al., The encyclopedia of molecular biology 1999; Kusakari Y et al., Curr Eye Res[J] 1997, :600-604.). Therefore, the selective determination of histidine in biological fluids is of great significance to the study of biochemistry.

Studies have shown that abnormal histidine content is usually associated with some diseases, including chronic kidney disease (Watanabe M et al., American Journal of Clinical Nutrition 2008,87(6):1860-1866), acute liver failure (Rama Rao KV et al., The American Journal of Pathology 2010,176(3):1400-1408), rheumatoid arthritis (Gerber DA et al., The Journal of Clinical Investigation 1975,55(6):1164-1173), advanced liver cirrhosis (The American Journal of Medicine 73(2):A29), AIDS (Jones AL et al., Immunol Cell Biol 2005,83(2):106-118), asthma (Morgan WT et al., Biochemical Medicine and Metabolic Biology 1986,36(2):210-213 ) and malaria (Sullivan DJ et al., Science 1996, 271(5246): 219-222), etc. Studies have shown (Watanabe M etc., American Journal of Clinical Nutrition 2008,87(6):1860-1866), the histidine content in the blood plasma of patients with chronic kidney disease is very low; corresponding research (Rama Rao KV etc., The American Journal of Pathology 2010,176(3):1400-1408) showed that histidine is a substance known to inhibit glutamine transport in mitochondria, which blocked oxidative stress and mitochondrial permeability changes in patients with acute liver failure and the formation of cerebral edema. In addition, high levels of histidine in serum or urine can cause metabolic disorders such as histidineemia.

It is precisely because histidine has the above-mentioned important functions that the detection of histidine content is also particularly important. The common detection method of histidine is capillary electrophoresis (Li X-t et al., Chem Res Chin Univ 2013,29(3):434-438; Meng J et al., The Analyst 2010,135(7):1592-1599), highly efficient Liquid chromatography (Tateda N etc., Analytical sciences: the international journal of the Japan Society for Analytical Chemistry 2001,17(6):775-778; Wadud S etc., Journal of chromatography B, Analytical technologies in the biomedical and life sciences 2002, 767(2):369-374), UV-visible spectrophotometry (Hortala MA et al., J Am Chem Soc 2003,125(1):20-21; PuF et al., Anal Chem 2010,82(19):8211-8216 ; Du J et al, Chemical communications (Cambridge, England) 2013,49(47):5399-5401; Engeser M et al, Chemical Communications 1999, (13):1191-1192) and fluorescence spectrometry (Engeser M et al, Chemical Communications 1999, (13): 1191-1192), but these detection methods have great defects in the study of live cells, which require time-consuming sample processing: cell disruption, separation, extraction and purification, etc., and cannot be used for real-time analysis of intact cells. accurate detection.

Contents of the invention

In view of this, the object of the present invention is to provide a histidine fluorescent probe for real-time localization inside and outside cells, high-throughput, and quantitative detection of histidine.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides a histidine fluorescent probe, including a histidine-sensitive polypeptide B and fluorescent protein A; the fluorescent protein A is inserted into the polypeptide B, and B is divided into polypeptide B1 and polypeptide B2 Part, forming the probe structure of B1-A-B2 formula;

The polypeptide B is HBP and its mutants.

Preferably, the amino acid sequence of the HBP is shown in SEQ ID NO.2.

Preferably, the fluorescent protein A is yellow fluorescent protein cpYFP, and the amino acid sequence of the yellow fluorescent protein cpYFP is shown in SEQ ID NO.3.

Preferably, the fluorescent protein A is replaced by green fluorescent protein cpGFP with amino acid sequence as shown in SEQ ID NO.4 or SEQ ID NO.10, blue fluorescent protein with amino acid sequence as shown in SEQ ID NO.5 or SEQ ID NO.11 Protein cpBFP, amino acid sequence as shown in SEQ ID NO.6 cyan fluorescent protein cpTFP, amino acid sequence as shown in SEQ ID NO.7 orange fluorescent protein cpmOrange, amino acid sequence as shown in SEQ ID NO.8 apple red fluorescent protein cpmApple, The amino acid sequence is one of red fluorescent protein cpmKate shown in SEQ ID NO.9 or SEQ ID NO.13 and the red fluorescent protein mcherry shown in SEQ ID NO.12.

Preferably, the fluorescent protein A is inserted into the polypeptide B to form a probe structure of the B1-A-B2 formula, and the insertion sites are 89/90, 89/91, 89/92, 89/93, 90/ 91, 90/92, 90/93, 91/92, 91/93, 92/93, 185/186, 185/187, 185/188, 185/189, 185/190, 185/191, 185/192, 185/193, 186/187, 186/188, 186/189, 186/190, 186/191, 186/192, 186/193, 187/188, 187/189, 187/190, 187/191, 187/ 192, 187/193, 188/189, 188/190, 188/191, 188/192, 188/193, 189/190, 189/191, 189/192, 189/193, 190/191, 190/192, 190/193, 191/192, 191/193 or 192/193 loci.

Preferably, the insertion sites of fluorescent protein A into polypeptide B are 90/91, 91/92, 186/191, 187/191, 189/190, 189/191, 189/193, 190/191, 190/ At 193, the corresponding amino acid sequence of the probe structure of the formula B1-A-B2 is shown in SEQ ID NO.14-22.

The present invention also provides the nucleotide sequence encoding the B1-A-B2 type probe.

The present invention also provides a preparation method for the above-mentioned histidine fluorescent probe, comprising the following steps: 1) connecting the nucleotide sequence encoding the B1-A-B2 type probe with the pRSETb vector to obtain a recombinant expression vector for Escherichia coli ; 2) transferring the recombinant expression vector of Escherichia coli to the host cell; 3) cultivating the host cell and isolating the histidine fluorescent probe.

The present invention also provides a histidine detection kit comprising the above-mentioned histidine fluorescent probe.

The present invention also provides the application of the above-mentioned histidine fluorescent probe in real-time histidine localization, quantitative detection and high-throughput compound screening.

Beneficial effects of the present invention: the histidine fluorescent probe provided by the present invention includes a histidine-sensitive polypeptide B and fluorescent protein A; the fluorescent protein A is inserted into polypeptide B, and B is divided into polypeptide B1 and The two parts of the polypeptide B2 form a probe structure of the B1-A-B2 formula; the B1-A-B2 formula histidine fluorescent probe provided by the present invention is easy to mature, has large dynamic changes in fluorescence, good specificity, and can pass The method of gene manipulation is expressed in cells, and can be used for real-time positioning, high-throughput, and quantitative detection of histidine inside and outside cells. Time-consuming sample handling steps are eliminated. The experimental results show that the histidine fluorescent probe provided by this application has a maximum response of more than 6 times to histidine, and can be detected in the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, outer membrane, inner membrane, and Golgi apparatus. Localization detection of cells in subcellular structures such as lysosomes and lysosomes; and high-throughput compound screening.

Description of drawings

Fig. 1 is the SDS-PAGE analysis figure of histidine fluorescent probe in embodiment 1;

Figure 2 is a graph showing the response of the histidine fluorescent probe formed by the yellow fluorescent protein cpYFP at different insertion sites of HBP to histidine;

Figure 3 is a diagram showing the response of histidine fluorescent probes formed by the blue fluorescent protein cpBFP at different insertion sites of HBP to histidine;

Figure 4 is a graph showing the response of the histidine fluorescent probe formed by the apple red fluorescent protein cpmApple at different insertion sites of HBP to histidine;

Fig. 5 is the fluorescence spectrum characteristic figure of histidine fluorescent probe;

Fig. 6 is the titration curve of different histidine fluorescent probes to different concentrations of histidine;

Fig. 7 is the subcellular organelle localization analysis figure of histidine fluorescent probe in mammalian cell;

Figure 8 is a quantitative analysis diagram of histidine in different subcellular organelles with histidine fluorescent probe Hisensor D;

Figure 9 is a dynamic analysis diagram of histidine transmembrane transport in different subcellular organelles by the histidine fluorescent probe Hisensor D;

Figure 10 is a high-throughput compound screening analysis diagram based on histidine fluorescent probe Hisensor D at the living cell level;

Fig. 11 is a quantitative analysis diagram of histidine fluorescent probe Hisensor D in culture medium and blood.

detailed description

The present invention provides a histidine fluorescent probe, comprising a histidine-sensitive polypeptide B and a histidine-expressing fluorescent protein A; the fluorescent protein A is inserted into the polypeptide B, and B is divided into Two parts, B1 and B2, form a probe structure of the formula B1-A-B2; the interaction between the polypeptide B and histidine causes the fluorescence signal of the fluorescent protein A to become stronger; the polypeptide B is HBP and its mutants.

In the present invention, the HBP protein is derived from Escherichia coli (Escherichia coli), or from the HBP protein that has more than 90% homology between Salmonella and HBP protein, and contains two typical α/β proteins that periplasmic binding proteins have. The structure of the globular domain connected by a hinge can bind histidine. HBP protein can sense the change of histidine concentration in the periplasm, and the spatial conformation of HBP protein will also change greatly during the dynamic change of histidine concentration. The conformational change of the fluorescent protein caused by the conformational change caused by the specificity of HBP protein binding to physiological concentrations of histidine, which in turn leads to a change in the fluorescence of the fluorescent protein, and the fluorescence of the fluorescent protein measured at different histidine concentrations A standard curve is prepared to detect and analyze the presence and/or level of histidine.

In the present invention, the nucleotide sequence encoding the HBP protein is preferably shown in SEQ ID NO.1, and the amino acid sequence of the HBP protein is preferably shown in SEQ ID NO.2.

The fluorescent protein A described in the present invention is a protein capable of displaying fluorescence, and the fluorescent protein A is preferably yellow fluorescent protein cpYFP, and the amino acid sequence of the yellow fluorescent protein cpYFP is shown in SEQ ID NO.3. In the present invention, the yellow fluorescent protein cpYFP connects the original N-terminal and C-terminal of GFP through a flexible short peptide chain, creates a new N-terminal and C-terminal at the position of the original GFP near the chromophore, and converts the original The 145th-238th amino acid part is used as the N-terminal of the new protein, and the original 1-144th amino acid is used as the C-terminal of the new protein, and the two fragments are connected by 5-9 flexible short peptide chains. In the present invention, the positions close to the chromophore are preferably Y144 and N145 amino acids; the flexible short peptide chain is preferably VDGGSGGTG or GGSGG.

In other specific embodiments of the present invention, the fluorescent protein A can also preferably be a green fluorescent protein cpGFP with an amino acid sequence such as SEQ ID NO.4 or SEQ ID NO.10, an amino acid sequence such as SEQ ID NO.5 or SEQ ID NO .11 blue fluorescent protein cpBFP, amino acid sequence as shown in SEQ ID NO.6 cyan fluorescent protein cpTFP, amino acid sequence as shown in SEQ ID NO.7 orange fluorescent protein cpmOrange, amino acid sequence as shown in SEQ ID NO.8 One of apple red fluorescent protein cpmApple, red fluorescent protein cpmKate shown in SEQ ID NO.9 or SEQ ID NO.13 with amino acid sequence and red fluorescent protein mcherry shown in SEQ ID NO.12.

Green fluorescent protein GFP was originally extracted from Aequorea Victoria. It consists of 238 amino acids and has a molecular weight of about 26kDa. GFP is a unique barrel-shaped structure formed by 12 β-fold chains, which is wrapped with a chromogenic tripeptide (Ser65-Tyr66-Gly67). In the presence of oxygen, it will spontaneously form the chromophore structure of p-hydroxybenzylidene imidazolidinone to produce fluorescence. GFP does not require cofactors to produce fluorescence, and its fluorescence is very stable, making it a good imaging tool. GFP has two excitation peaks, the main peak at 395nm can produce 508nm emission light, and the shoulder peak 475nm excitation light can produce 503nm emission light.

In the present invention, the red fluorescent protein cpmKate is initially extracted from corals in the ocean. The wild RFP is an oligomeric protein that is not conducive to the fusion expression of organisms, and different colors are further derived on the basis of RFP. band of red fluorescent proteins, the most commonly used are mcherry and mKate.

In the present invention, the fluorescent protein A is inserted into the polypeptide B to form a probe structure of the B1-A-B2 formula, and the insertion site is located in the flexible region of the polypeptide B. Some specific structures such as ring domains, these domains have higher mobility and flexibility than other high-level structures of proteins, and this region can dynamically change the spatial structure conformation after the protein binds to the ligand . The flexible region mentioned in the present invention mainly refers to the region where the insertion site is located in the HBP protein, such as the 89-93 and 185-193 regions. The insertion site of the present invention is located at 89/90, 89/91, 89/92, 89/93, 90/91, 90/92, 90/93, 91/92, 91/93, 92 of the amino acid sequence of the HBP protein /93, 185/186, 185/187, 185/188, 185/189, 185/190, 185/191, 185/192, 185/193, 186/187, 186/188, 186/189, 186/190 , 186/191, 186/192, 186/193, 187/188, 187/189, 187/190, 187/191, 187/192, 187/193, 188/189, 188/190, 188/191, 188 /192, 188/193, 189/190, 189/191, 189/192, 189/193, 190/191, 190/192, 190/193, 191/192, 191/193 or 192/193 sites; preferred The ones are 90/91, 91/92, 186/191, 187/191, 189/190, 189/191, 189/193, 190/191 or 190/193. In the present invention, when the insertion site is preferably 90/91, 91/92, 186/191, 187/191, 189/190, 189/191, 189/193, 190/191 or 190/193, the corresponding The amino acid sequence of the probe structure of formula B1-A-B2 is shown in SEQ ID NO.14-22.

The present invention also provides the nucleotide sequence encoding the probe of the above formula B1-A-B2.

The present invention also provides a preparation method for the above-mentioned histidine fluorescent probe, comprising the following steps: 1) linking the nucleotide sequence encoding the B1-A-B2 type probe with the pRSETb vector to obtain recombinant expression in Escherichia coli 2) transferring the recombinant expression vector of Escherichia coli into host cells; 3) cultivating the host cells and isolating the histidine fluorescent probe.

In the present invention, the synthesis of the nucleotide sequence encoding the B1-A-B2 type probe adopts the method of PCR amplification or artificial synthesis. When the PCR amplification method is used, primers are designed according to the nucleotide sequence described in the present invention, and a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art is used as a template to amplify. When the nucleotide sequence is larger than 2500bp, it is preferred to carry out 2-6 times of PCR amplification, and then splice together the fragments amplified in each time in the correct order. The present invention has no special limitation on the PCR amplification procedure and system, and the conventional PCR amplification procedure and system in the field can be used.

In the present invention, when the nucleotide sequence of the B1-A-B2 type probe is less than 2500bp, it is preferably synthesized by artificial synthesis. The artificial synthesis method is a conventional DNA artificial synthesis method in the art, without other special requirements. Specifically, the full-length sequence can be obtained by first synthesizing multiple small fragments and then connecting them.

After obtaining the nucleotide sequence encoding the B1-A-B2 formula probe, the present invention will encode the

The nucleotide sequence of the B1-A-B2 type probe is connected with the pRSETb carrier to obtain the Escherichia coli recombinant expression vector. In the present invention, the pRSETb vector can be a commercially available pRSETb vector without other special requirements. In the embodiment of the present invention, it is preferred to use BamHI and HindIII to perform double enzyme digestion on the nucleotide sequence encoding the B1-A-B2 type probe and the pRSETb vector respectively, and then the product after double enzyme digestion of the two The recombinant expression vector of Escherichia coli was obtained by ligation. The present invention has no special limitation on the specific steps and parameters of the double enzyme digestion and ligation, and conventional steps and parameters in the art can be used. In the present invention, after obtaining the recombinant expression vector of Escherichia coli, the recombinant expression vector of Escherichia coli is transferred to a host cell. The host cell in the present invention refers to a cell capable of receiving and accommodating recombinant DNA molecules, and is a place for recombinant gene amplification. Ideal recipient cells should meet the two conditions of easy acquisition and proliferation. The "host cell" of the present invention may include prokaryotic cells and eukaryotic cells, specifically bacterial cells, yeast cells, insect cells and mammalian cells. Specific examples may be bacterial cells of Escherichia coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells of Drosophila S2 or Sf9, CHO, COS, HEK293, HeLa cells, or Bowes melanoma cells animal cells, etc., including but not limited to those host cells mentioned above. The host cells are preferably various cells that are conducive to gene product expression or fermentation production. Such cells are well known and commonly used in the art. In the embodiments of the present invention, the host cells are preferably Escherichia coli JM109-DE3 strain.

The method for transferring host cells described in the present invention is a conventional method in the art, including calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemical mediated Conducted transfer or electroporation. When the host is prokaryote such as Escherichia coli, the method is preferably CaCl ₂ method or MgCl ₂ method, and the steps used are well known in the art. When the host cells are eukaryotic cells, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

In the present invention, after the Escherichia coli expression vector is transferred into the host cell, the host cell transformed with the Escherichia coli expression vector is amplified, expressed and cultured, and the histidine fluorescent probe is isolated and obtained. The expansion and expression culture of the host cells can be carried out by conventional methods. The medium used in the culture may be various conventional mediums depending on the kind of host cells used. The culture is carried out under conditions suitable for the growth of the host cells. In the present invention, the histidine fluorescent protein is expressed in the cell, on the cell membrane, or secreted outside the cell. The method for isolating the histidine fluorescent protein is not particularly limited in the present invention, and conventional separation methods for fusion proteins in the art can be used. Specifically, in the present invention, conventional renaturation treatment, salting-out method, centrifugation, osmotic bacteria destruction, supertreatment, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) can be used. And various other liquid chromatography methods and the combination of these methods. It is preferably carried out by affinity chromatography with a His-tag tag.

The present invention also provides the application of the histidine fluorescent probe in real-time histidine localization, quantitative detection and high-throughput compound screening. In the present invention, the histidine fluorescent probe is preferably linked with signal peptides in different parts of the cell, transferred into the cell, and the real-time positioning of histidine is performed by detecting the strength of the fluorescent signal in the cell; The histidine standard addition curve was used for the quantitative detection of the corresponding histidine. The histidine standard dropping curve described in the present invention is drawn according to the fluorescence signals of the histidine fluorescent probe at different concentrations of histidine. The histidine fluorescent probe of the present invention is directly transferred into cells, and in the process of real-time positioning and quantitative detection of histidine, no time-consuming sample processing process is required, which is more accurate. When the histidine fluorescent probe of the present invention is used for high-throughput compound screening, different compounds are added to the cell culture medium to measure the change of histidine content, so as to screen out compounds that affect the change of histidine content. The application of the histidine fluorescent probe described in the present invention in real-time histidine localization, quantitative detection and high-throughput compound screening is for non-diagnostic and therapeutic purposes, and does not involve the diagnosis and treatment of diseases.

The histidine fluorescent probes provided by the present invention will be described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.

Experimental Materials and Reagents

In the embodiments, conventional genetic engineering molecular biology cloning methods, cell culture and imaging methods, etc. are mainly used, and these methods are well known to those of ordinary skill in the art, for example: "Molecular Biology Experimental Reference" by Jane Roskems et al. Handbook", written by J. Sambrook, D.W. Russell, translated by Huang Peitang, etc.: "Molecular Cloning Experiment Guide" (third edition, August 2002, published by Science Press, Beijing); Animal Cell Culture: A Guide to Basic Techniques (Fifth Edition), translated by Zhang Jingbo, Xu Cunshuan, etc.; J.S. Bonifesnon, M. Dassault, etc.'s "Experimental Guide to Cell Biology", translated by Zhang Jingbo, etc. According to the following examples, those skilled in the art can easily implement the present invention by slightly modifying and transforming according to specific conditions, and these modifications and transforms all fall within the scope of the claims of the present application.

The pRSETb-cpYFP-based and pRSETb-HBP plasmids used in the examples were constructed by the Protein Laboratory of East China University of Science and Technology, and the pRSETb plasmid vector was purchased from Invitrogen. All primers used for PCR were synthesized, purified and identified by mass spectrometry by Shanghai Jierui Bioengineering Technology Co., Ltd. The expression plasmids constructed in the examples have all been sequenced, and the sequence determination was completed by Huada Gene Company and Jerry Sequencing Company. The Taq DNA polymerase used in each example was purchased from Dongsheng Bio, the pfu DNA polymerase was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and the primeSTAR DNA polymerase was purchased from TaKaRa Company. The three polymerases were purchased with a free corresponding Polymerase buffer and dNTPs. Restriction enzymes such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, and SpeI, T4 ligase, and T4 phosphorylase (T4 PNK) were purchased from Fermentas, and corresponding buffers were included with the purchase. Transfection reagent Lip2000Kit was purchased from Invitrogen. Histidine, etc. were purchased from Sigma. Unless otherwise stated, chemical reagents such as inorganic salts were purchased from sigma-aldrich company. HEPES salt, ampicillin (Amp) and puromycin were purchased from Ameresco; 96-well detection black plate and 384-well fluorescence detection black plate were purchased from Grenier Company.

The DNA purification kit used in the examples was purchased from BBI (Canada), and the common plasmid mini-prep kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Cloning strain Mach1 was purchased from Invitrogen. Nickel column affinity chromatography column and desalting column fillers are from GE healthcare company.

The main instrument used in the embodiment: Biotek Synergy 2 multifunctional microplate reader (U.S. Bio-Tek company), X-15R high-speed refrigerated centrifuge (U.S. Beckman company), Microfuge22R desktop high-speed refrigerated centrifuge (U.S. Beckman company), PCR amplification instrument (Biometra Company of Germany), ultrasonic breaker (Ningbo Xinzhi Company), nucleic acid electrophoresis instrument (Shenergy Gaming Company), fluorescence spectrophotometer (Varian Company of the United States), CO ₂ constant temperature cell incubator (SANYO), Inverted fluorescence microscope (Nikon, Japan).

II. Conventional molecular biology method and cell experiment method used in the embodiment

(1) Polymerase chain reaction (PCR):

1. Target fragment amplification PCR:

This method is mainly used for gene fragment amplification and colony PCR to identify positive clones. The reaction system of the PCR amplification is shown in Table 1, and the amplification program is shown in Table 2.

Table 1. PCR amplification reaction system

Table 2. PCR amplification program 2. Long fragment (>2500bp) amplification PCR:

The long-segment amplification used in the present invention is mainly an inverse PCR amplification vector, which is a technique for obtaining site-directed mutagenesis in the following examples. Design reverse PCR primers at the variation site, and the 5' end of one of the primers contains the variation nucleotide sequence. The amplified product contains the corresponding mutation site. The PCR reaction system for long fragment amplification is shown in Table 3, and the amplification program is shown in Table 4 or Table 5.

Table 3. Long fragment (>2500bp) amplification PCR reaction system

Table 4. Long fragment (>2500bp) amplification PCR amplification program

Table 5. Long fragment (>2500bp) amplification PCR amplification program

(2) Endonuclease digestion reaction:

The system for double enzyme digestion of the plasmid vector is shown in Table 6, where n represents the amount of sterilized ultrapure water μL that needs to be added to make the system reach the total volume.

Table 6 Plasmid vector double enzyme digestion system

(3) Phosphorylation reaction at the 5' end of the DNA fragment

Plasmids or genomes extracted from microorganisms contain phosphate groups, but PCR products do not. Therefore, it is necessary to perform phosphate group addition reaction on the 5' terminal base of PCR products. Only DNA molecules with phosphate groups at the ends can occur. Link reaction. The phosphorylation reaction system is shown in Table 7, wherein T4 PNK is the abbreviation of T4 polynucleotide kinase, which is used for the addition reaction of the phosphate group at the 5' end of the DNA molecule.

Table 7. Phosphorylation reaction system

(4) Ligation reaction of target fragment and carrier

There are differences in the connection methods between different fragments and carriers, and three connection methods are used in the present invention

1. Blunt end ligation of blunt-ended short fragments and linearized vectors

The principle of this method is that the blunt-end product obtained by PCR is phosphorylated on the 5' end of the DNA fragment under the action of T4 PNK, and then ligated with the linearized vector under the action of PEG4000 and T4 DNA ligase to obtain a recombinant plasmid. The homologous recombination ligation system is shown in Table 8.

Table 8. Homologous recombination ligation system

2. Ligation of DNA fragments containing cohesive ends and vector fragments containing cohesive ends

DNA fragments cleaved by restriction endonucleases usually produce overhanging cohesive ends, so they can be ligated with vector fragments containing complementary cohesive ends to form recombinant plasmids. The connection reaction system is shown in Table 9.

Table 9. Ligation reaction system

Note: The mass ratio of the PCR product fragments to the vector double-enzyme digestion products is roughly between 2:1 and 6:1.

3. Ligation reaction of self-circularization of DNA fragment products phosphorylated at the 5' end after inverse PCR introduction of site-directed mutagenesis

The phosphorylated DNA fragment at the 5' end was ligated with the 3' end and the 5' end of the linearized vector through a self-circularization ligation reaction to obtain a recombinant plasmid. The self-cyclization ligation reaction system is shown in Table 10.

Table 10. Self-cyclization ligation reaction system

(5) Preparation and transformation of competent cells

Preparation of Competent Cells:

1. Pick a single colony (such as Mach1) and inoculate it in 5 mL of LB medium, shake it overnight at 37°C.

2. Take 0.5-1ml of the overnight cultured bacterial solution and transfer it to 50mL LB medium, culture at 37°C, 220rpm for 3 to 5h, until the OD600 reaches 0.5.

3. Pre-cool the cells in an ice bath for 2 hours.

4. Centrifuge at 4000 rpm for 10 minutes at 4°C.

5. Discard the supernatant, suspend the cells with 5ml of pre-cooled resuspension buffer, and then add the resuspension buffer until the final volume is 50mL.

6. Ice bath for 45 minutes.

7. Centrifuge at 4000rpm at 4°C for 10min, and resuspend the bacteria with 5mL of ice-cold storage buffer.

8. Put 100 μL of bacterial solution in each EP tube, and freeze at -80°C or liquid nitrogen.

Resuspension buffer: CaCl2 (100mM), MgCl2 (70mM), NaAc (40mM)

Storage Buffer: 0.5 mL DMSO, 1.9 mL 80% Glycerol, 1 mL 10×CaCl ₂ (1M), 1 mL 10×MgCl ₂ (700 mM), 1 mL 10× NaAc (400 mM), 4.6 mL ddH ₂ O Transformation:

1. Thaw 100 μl of competent cells on an ice bath.

2. Add an appropriate volume of the ligation product, mix gently by pipetting, and ice-bath for 30 minutes. Usually the volume of ligation product added is less than 1/10 of the volume of competent cells.

3. Put the bacterial solution into a 42°C water bath for 90 seconds, then quickly transfer to an ice bath for 5 minutes.

4. Add 500 μl LB, and incubate for 1 hour on a constant temperature shaker at 37°C at 200 rpm.

5. Centrifuge the bacterial solution at 4000 rpm for 3 minutes, leave 200 μl of supernatant to blow the bacterial cells evenly, spread evenly on the surface of the agar plate containing appropriate antibiotics, and place the plate upside down in a constant temperature incubator at 37°C overnight.

(6) Protein expression, purification and fluorescence detection

1. Transform the pRSETb-based histidine probe plasmid into JM109 (DE3), culture it upside down overnight, pick the clones from the plate into a 250ml Erlenmeyer flask, place it on a shaker at 37°C, and cultivate it at 220rpm until OD= 0.4-0.8, add 1/1000 (v/v) IPTG (1M), induce expression at 18°C for 24-36 hours.

2. After the induced expression is completed, collect the bacteria by centrifugation at 4000 rpm for 30 minutes, add 50 mM phosphate buffer to resuspend the bacterial pellet, and ultrasonically break until the bacterial cells are clarified. Centrifuge at 9600rpm at 4°C for 20min.

3. The centrifuged supernatant was purified by a self-installed nickel column affinity chromatography column to obtain protein, and the protein after nickel column affinity chromatography was obtained by self-installed desalting column and dissolved in 20mM MOPS buffer (pH 7.4) or phosphate Protein in buffer PBS.

4. After the purified HBP mutant protein is identified by SDS-PAGE, use assay buffer (100mM HEPES, 100mM NaCl, pH 7.3) or phosphate buffered saline PBS to dilute the probe into a protein solution with a final concentration of 5-10 μM. Histidine was prepared as a stock solution at a final concentration of 1 M in assay buffer (20 mM MOPS, pH 7.4) or phosphate buffered saline PBS.

Take 100 μl of 5 μM protein solution, incubate at 37°C for 5 min, add histidine and mix until the final concentration is 100 mM, and measure the light absorption of the protein at 340 nm by using a multifunctional fluorescent microplate reader.

Take 100 μl of 1 μM fluorescent probe solution, incubate at 37° C. for 5 minutes, add histidine for titration, and measure the fluorescence intensity emitted by the protein at 528 nm after fluorescence excitation at 485 nm. The fluorescence excitation and emission measurement of the samples were completed with a multifunctional fluorescent microplate reader.

Take 100 μl of 1 μM fluorescent probe solution, incubate at 37° C. for 5 minutes, add histidine, and measure the absorption spectrum and fluorescence spectrum of the probe protein. The determination of the absorption spectrum and fluorescence spectrum of the sample is accomplished by a spectrophotometer and a fluorescence spectrophotometer.

(7) Mammalian cell fluorescence detection

1. The pCDNA3.1+-based histidine probe plasmid was transfected into HeLa by the transfection reagent Lipofectamine2000 (Invitrogen), and cultured in a cell incubator at 37° C. and 5% CO ₂ . Fluorescence detection was performed after 24-36 hours after the exogenous gene was fully expressed.

2. After the induced expression was completed, the adherent HeLa cells were washed three times with PBS, placed in HBSS solution, and detected by fluorescence microscope and microplate reader respectively.

Example 1

Construction of pRSETb-HBP plasmid

The HisJ gene in the Escherichia coli gene was amplified by PCR, and the PCR product was recovered after gel electrophoresis and digested with BamHI and HindIII, and the pRSETb vector was subjected to the same double digestion. After ligation with T4 DNAligase, the ligation product was transformed into MachI, and the transformed MachI was spread on LB plates (ampicillin 100ug/mL), and cultured at 37°C overnight. After plasmid extraction of the growing MachI transformants, PCR identification was performed. After the positive plasmid is correctly sequenced, the subsequent plasmid construction is carried out.

The primer sequences for constructing the pRSETb-HBP plasmid are shown in Seq ID NO.39-40.

Plasmid construction and detection of different insertion sites of pRSETb-HBP-cpFP fluorescent probe

In this example, we selected 89/90, 89/91, 89/92, 89/93, 90/91, 90/92, 90/93, 91/92 based on the HBP crystal structure based on pRSETb-HBP , 91/93, 92/93, 185/186, 185/187, 185/188, 185/189, 185/190, 185/191, 185/192, 185/193, 186/187, 186/188, 186 /189, 186/190, 186/191, 186/192, 186/193, 187/188, 187/189, 187/190, 187/191, 187/192, 187/193, 188/189, 188/190 , 188/191, 188/192, 188/193, 189/190, 189/191, 189/192, 189/193, 190/191, 190/192, 190/193, 191/192, 191/193, 192 /193. Among them, there are 90/91, 91/92, 186/191, 187/191, 189/190, 189/191, 189/193, 190/191, 190/193 positions (such as shown in SEQ ID NO 14-22) or the corresponding amino acid positions of its family proteins.

The DNA fragment of cpYFP was generated by PCR, and the DNA fragment was inactivated by adding phosphorous at the 5' end. At the same time, the pRSETb-HBP linearization vector containing different breakage sites was amplified by reverse PCR, and the linearized pRSETb -HBP and the cpYFP fragment phosphorylated at the 5' end were ligated under the action of PEG4000 and T4 DNA ligase to generate recombinant plasmids. These plates were placed on the Kodak multifunctional in vivo imaging system, and the clones with yellow fluorescence under the excitation of the FITC channel were picked. The Shanghai Branch of Beijing Liuhe Huada Gene Technology Co., Ltd. completed the sequencing.

The primers used to generate the DNA fragment of cpYFP by PCR are shown in Seq ID NO.41-42.

The primers used for the reverse amplification to generate the linearized vector are shown in Seq ID NO.23-38.

After the sequencing was correct, the recombinant plasmid was transformed into JM109 (DE3) to induce expression, and the protein was purified, and the size of the protein was around 60KDa by SDS-PAGE electrophoresis. This size is consistent with the size of the HBP-cpYFP fusion protein expressed by pRSETb-HBP-cpYFP and containing the His-tag purification tag. The result is shown in Figure 1.

The purified HBP-cpYFP fusion protein was screened for histidine response, and the detection signal of the fusion fluorescent protein containing 100 mM histidine was divided by the detection signal of the fusion fluorescent protein without histidine.

The results are shown in Figure 2, and the test results show that 90/91, 91/92, 186/191, 187/191, 189/190, 189/191, 189/193, 190/ 191, 190/193, 186/191, 197/191, 189/191, 190/191, the fluorescence intensity at 420nm excitation and 528nm emission increases with the concentration of histidine. The fluorescence intensity at 485nm excitation 528nm emission of 189/190, 189/191, 189/193, 190/191, 190/193 decreased with the concentration of histidine. Among them, the fluorescence intensity of 189/191 and 190/191 samples at 420nm excitation and 528nm emission and 485nm excitation and 528nm emission fluorescence intensity showed opposite changes with the concentration of histidine.

Example 2

According to the method in Example 1, cpYFP was replaced by blue fluorescent protein cpBFP, which was fused into HBP to construct a histidine blue fluorescent protein fluorescent probe. The results are shown in Figure 3. Fluorescence detection results showed that 90/91, 186/191, 187/191, 189/190, 189/191, 190/191, and 190/193 responded more than 3 times to histidine.

Example 3

According to the method in Example 1, cpYFP was replaced by apple red fluorescent protein cpmApple, which was fused into HBP to construct a histidine red fluorescent protein fluorescent probe. The times are 90/91, 91/92, 186/191, 187/191, 189/190, 189/191, 189/193, 190/191, 190/192, 190/193, 191/193.

The results of Examples 1-3 show that the linker regions 89-93 and 185-193 of HBP are suitable for fusion with cpFP fluorescent protein to obtain a fusion fluorescent protein histidine probe responsive to histidine.

Example 4. Detection of HBP-cpYFP fluorescent probe properties

After the purified HBP-cpYFP was treated with 0 mM and 100 mM histidine for 10 min, the fluorescence spectrum was detected using a fluorescence spectrophotometer. Determination of excitation spectrum: fixed emission at 530nm, detection of excitation spectrum in the range of 380-510nm; determination of emission spectrum, fixed excitation at 485nm, detection of emission spectrum in the range of 510-550nm. The spectral curve of the HBP-cpYFP fluorescent probe is shown in Figure 5. The above results show that the fluorescent spectral properties of the HBP-cpYFP fluorescent protein are similar to the fluorescent spectral properties of the cpYFP fluorescent protein (Nagai, T., etc., Proc Natl Acad Sci U S A.2001 , V.98(6), pp.3197-3202).

Among them, four probes Hisensor A, Hisensor B, Hisensor C and Hisensor D whose detection range for histidine is in the range of 0.1 μM to 1 mM are selected to detect histidine in a concentration gradient (0 to 1 mM). After the purified HBP-cpYFP was treated for 10 minutes, the changes in the ratio of the fluorescence intensity at 420nm excitation at 528nm emission to the fluorescence intensity at 485nm excitation at 528nm emission were detected. The K _d (binding constant) of the four histidine fluorescent probes were in turn: 4μM, 13μM, 3.4μM and 22μM, the range of change is 4.2 times, 4.5 times, 3.4 times and 6.2 times, the results are shown in Figure 6. In this way, a more suitable histidine probe can be selected for quantitative detection according to the histidine content level in the sample.

Example 5. Localization of Hisensor D Fluorescent Probe in Different Subcellular Organelles and Quantitative Analysis of Histidine in Subcellular Organelles

In this example, we used different localization signal peptides to fuse with Hisensor D to localize the Hisensor D histidine fluorescent protein probe to different organelles.

After the plasmids of Hisensor D gene fused with different localization signal peptides were transfected into HeLa cells for 36 hours, washed with PBS, placed in HBSS solution and detected under the FITC channel of an inverted fluorescence microscope. We found that Hisensor D can be localized to subcellular organelles including cytoplasm, nucleus, mitochondria, inner membrane, outer membrane, Golgi apparatus, and lysosome by fusion with different specific localization signal peptides. The results are shown in FIG. 7 . Fluorescence is shown in different subcellular structures, and the distribution and intensity of the fluorescence are different.

In this example, we used the histidine probe Hisensor D to quantitatively analyze the histidine in the cytoplasm and mitochondria respectively.

After the HeLa cells transfected with the Hisensor D gene were washed with PBS, they were placed in HBSS solution and the ratio of the fluorescence intensity at 420nm excitation at 528nm emission to the fluorescence intensity at 485nm excitation at 528nm emission was detected by a microplate reader. The corresponding histidine concentration was measured by the histidine standard drop curve (Figure 6) of Hisensor D, and the results are shown in Figure 8. It was found that the histidine content in the cell cytoplasm was about 150 μM, and the histidine in the mitochondria The content is around 50μM.

Example 6. Dynamic analysis of histidine transmembrane transport in different subcellular organelles by the histidine fluorescent probe Hisensor D

In this example, we use the histidine probe Hisensor D to dynamically detect and analyze the histidine in the cytoplasm and mitochondria respectively.

The HeLa cells transfected with the Hisensor D gene were washed with PBS, placed in HBSS solution (without histidine) for 2 hours, and histidine was added dropwise at 2 minutes. Using a microplate reader to record the ratio of the fluorescence intensity at 420nm excitation at 528nm emission and the fluorescence intensity at 485nm excitation at 528nm emission, the results are shown in Figure 9. It is found that extracellular histidine can quickly enter the cell, and the intracellular Histidine content reached the physiological maximum.

Example 7. High-throughput compound screening based on histidine fluorescent probe Hisensor D at the living cell level

In this example, we performed high-throughput compound screening using HeLa cells expressing the histidine probe Hisensor D in the cytoplasm.

The HeLa cells transfected with the Hisensor D gene were washed with PBS, treated in HBSS solution (without histidine) for 1 hour, and then treated with 10 μM compound for 1 hour. Histidine was added dropwise. Use a microplate reader to record the change in the ratio of the fluorescence intensity at 420nm excitation and 528nm emission to the fluorescence intensity at 485nm excitation and 528nm emission. Samples not treated with any compound were used as the standard. The results are shown in Figure 10, we found that among the cells treated with 500 compounds, most of the compounds had little effect on the entry of histidine into the cells. There are 9 compounds that can improve the uptake of histidine by cells, and another 6 compounds can significantly reduce the uptake of histidine by cells.

Example 8. Hisensor D fluorescent probe quantitatively detects histidine in DMEM medium and blood supernatant

In this implementation, the purified Hisensor D fluorescent protein was also used to analyze the histidine in the DMEM medium of cells and the blood supernatant of mice.

After the Hisensor D fluorescent protein was mixed with the diluted DMEM medium and blood supernatant for 10 min, the ratio of the fluorescence intensity at 420nm excitation at 528nm emission to the fluorescence intensity at 485nm excitation at 528nm emission was detected by a microplate reader. The results are shown in Figure 11. It was found that the histidine content in the DMEM medium was about 790 μM, and the histidine content in the mouse blood was about 80 μM.

It can be seen from the above examples that the histidine fluorescent probe provided by the present invention has a relatively small protein molecular weight and is easy to mature, with large dynamic changes in fluorescence and good specificity, and can be expressed in cells by genetic manipulation. Real-time localization and quantitative detection of histidine inside and outside; and high-throughput compound screening.

The above is only a preferred embodiment of the present invention, and it should be pointed out that for those of ordinary skill in the art, some improvements and modifications can be made without departing from the principle of the present invention. It should be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> East China University of Science and Technology

<120> A histidine fluorescent probe and its preparation method and application

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<160> 51

<170> PatentIn version 3.3

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Met Ala Ile Pro Gln Asn Ile Arg Ile Gly Thr Asp Pro Thr Tyr Ala

1 5 10 15

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

20 25 30

Asp Leu Ala Lys Glu Leu Cys Lys Arg Ile Asn Thr Gln Cys Thr Phe

35 40 45

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

50 55 60

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

65 70 75 80

Glu Ile Ala Phe Thr Asp Lys Leu Tyr Ala Ala Asp Ser Arg Leu Val

85 90 95

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

100 105 110

Lys Arg Val Gly Val Leu Gln Gly Thr Thr Gln Glu Thr Phe Gly Asn

115 120 125

Glu His Trp Ala Pro Lys Gly Ile Glu Ile Val Ser Tyr Gln Gly Gln

130 135 140

Asp Asn Ile Tyr Ser Asp Leu Thr Ala Gly Arg Ile Asp Ala Ala Phe

145 150 155 160

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

165 170 175

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

180 185 190

Gly Val Gly Thr Gly Met Gly Leu Arg Lys Glu Asp Asn Glu Leu Arg

195 200 205

Glu Ala Leu Asn Lys Ala Phe Ala Glu Met Arg Ala Asp Gly Thr Tyr

210 215 220

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

225 230 235

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

His Lys Leu Glu Tyr Asn

245

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

1 5 10 15

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

20 25 30

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

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

<210> 5

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

1 5 10 15

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

20 25 30

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

<210> 6

<211> 241

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

1 5 10 15

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

20 25 30

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

85 90 95

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

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

Phe Ser Tyr Gly Val Met Val Phe Ala Arg Tyr Pro Asp His Met Lys

165 170 175

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

<210> 7

<211> 242

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<400> 7

Val Ser Glu Arg Met Tyr Pro Glu Asp Gly Val 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 Pro Thr Gly Gly

65 70 75 80

Arg 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 Thr 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> 8

<211> 242

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<400> 8

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> 9

<211> 250

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<400> 9

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

130 135 140

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Asn Leu Lys Thr Thr Tyr Arg Ser Lys Lys

245 250

<210> 10

<211> 238

<212> PRT

<213> Artificial sequence

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Tyr Asn Ser His Asn Val Tyr Ile Met Gly Asp Lys Pro Lys Asn Gly

145 150 155 160

Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Thr Ala Ala Arg Ile Thr His Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 11

<211> 239

<212> PRT

<213> Artificial sequence

<400> 11

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Asn Phe Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn

145 150 155 160

Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser

165 170 175

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

180 185 190

Pro Val Leu Leu Pro Asp Ser His Tyr Leu Ser Thr Gln Ser Ala Leu

195 200 205

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

210 215 220

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

225 230 235

<210> 12

<211> 236

<212> PRT

<213> Artificial sequence

<400> 12

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

1 5 10 15

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

20 25 30

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

35 40 45

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

100 105 110

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

195 200 205

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

210 215 220

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

225 230 235

<210> 13

<211> 233

<212> PRT

<213> Artificial sequence

<400> 13

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> 14

<211> 485

<212> PRT

<213> Artificial sequence

<400> 14

Met Ala Ile Pro Gln Asn Ile Arg Ile Gly Thr Asp Pro Thr Tyr Ala

1 5 10 15

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

20 25 30

Asp Leu Ala Lys Glu Leu Cys Lys Arg Ile Asn Thr Gln Cys Thr Phe

35 40 45

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

50 55 60

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

65 70 75 80

Glu Ile Ala Phe Thr Asp Lys Leu Tyr Ala Ala Tyr Asn Ser Asp Asn

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Asn Asp Ser Arg Leu Val Val Ala Lys Asn Ser Asp Ile Gln Pro Thr

340 345 350

Val Glu Ser Leu Lys Gly Lys Arg Val Gly Val Leu Gln Gly Thr Thr

355 360 365

Gln Glu Thr Phe Gly Asn Glu His Trp Ala Pro Lys Gly Ile Glu Ile

370 375 380

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

385 390 395 400

Arg Ile Asp Ala Ala Phe Gln Asp Glu Val Ala Ala Ser Glu Gly Phe

405 410 415

Leu Lys Gln Pro Val Gly Lys Asp Tyr Lys Phe Gly Gly Pro Ser Val

420 425 430

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

435 440 445

Glu Asp Asn Glu Leu Arg Glu Ala Leu Asn Lys Ala Phe Ala Glu Met

450 455 460

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

465 470 475 480

Asp Val Tyr Gly Gly

485

<210> 15

<211> 485

<212> PRT

<213> Artificial sequence

<400> 15

Met Ala Ile Pro Gln Asn Ile Arg Ile Gly Thr Asp Pro Thr Tyr Ala

1 5 10 15

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

20 25 30

Asp Leu Ala Lys Glu Leu Cys Lys Arg Ile Asn Thr Gln Cys Thr Phe

35 40 45

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

50 55 60

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

65 70 75 80

Glu Ile Ala Phe Thr Asp Lys Leu Tyr Ala Ala Asp Tyr Asn Ser Asp

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Val Glu Ser Leu Lys Gly Lys Arg Val Gly Val Leu Gln Gly Thr Thr

355 360 365

Gln Glu Thr Phe Gly Asn Glu His Trp Ala Pro Lys Gly Ile Glu Ile

370 375 380

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

385 390 395 400

Arg Ile Asp Ala Ala Phe Gln Asp Glu Val Ala Ala Ser Glu Gly Phe

405 410 415

Leu Lys Gln Pro Val Gly Lys Asp Tyr Lys Phe Gly Gly Pro Ser Val

420 425 430

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

435 440 445

Glu Asp Asn Glu Leu Arg Glu Ala Leu Asn Lys Ala Phe Ala Glu Met

450 455 460

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

465 470 475 480

Asp Val Tyr Gly Gly

485

<210> 16

<211> 481

<212> PRT

<213> Artificial sequence

<400> 16

Met Ala Ile Pro Gln Asn Ile Arg Ile Gly Thr Asp Pro Thr Tyr Ala

1 5 10 15

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

20 25 30

Asp Leu Ala Lys Glu Leu Cys Lys Arg Ile Asn Thr Gln Cys Thr Phe

35 40 45

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

50 55 60

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

65 70 75 80

Glu Ile Ala Phe Thr Asp Lys Leu Tyr Ala Ala Asp Ser Arg Leu Val

85 90 95

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

100 105 110

Lys Arg Val Gly Val Leu Gln Gly Thr Thr Gln Glu Thr Phe Gly Asn

115 120 125

Glu His Trp Ala Pro Lys Gly Ile Glu Ile Val Ser Tyr Gln Gly Gln

130 135 140

Asp Asn Ile Tyr Ser Asp Leu Thr Ala Gly Arg Ile Asp Ala Ala Phe

145 150 155 160

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

165 170 175

Lys Asp Tyr Lys Phe Gly Gly Pro Ser Val Lys Tyr Asn Ser Asp Asn

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Gly

<210> 17

<211> 482

<212> PRT

<213> Artificial sequence

<400> 17

Met Ala Ile Pro Gln Asn Ile Arg Ile Gly Thr Asp Pro Thr Tyr Ala

1 5 10 15

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

20 25 30

Asp Leu Ala Lys Glu Leu Cys Lys Arg Ile Asn Thr Gln Cys Thr Phe

35 40 45

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

50 55 60

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

65 70 75 80

Glu Ile Ala Phe Thr Asp Lys Leu Tyr Ala Ala Asp Ser Arg Leu Val

85 90 95

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

100 105 110

Lys Arg Val Gly Val Leu Gln Gly Thr Thr Gln Glu Thr Phe Gly Asn

115 120 125

Glu His Trp Ala Pro Lys Gly Ile Glu Ile Val Ser Tyr Gln Gly Gln

130 135 140

Asp Asn Ile Tyr Ser Asp Leu Thr Ala Gly Arg Ile Asp Ala Ala Phe

145 150 155 160

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

165 170 175

Lys Asp Tyr Lys Phe Gly Gly Pro Ser Val Lys Asp Tyr Asn Ser Asp

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Tyr Asn Phe Gly Val Gly Thr Gly Met Gly Leu Arg Lys Glu Asp Asn

435 440 445

Glu Leu Arg Glu Ala Leu Asn Lys Ala Phe Ala Glu Met Arg Ala Asp

450 455 460

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

465 470 475 480

Gly Gly

<210> 18

<211> 485

<212> PRT

<213> Artificial sequence

<400> 18

Met Ala Ile Pro Gln Asn Ile Arg Ile Gly Thr Asp Pro Thr Tyr Ala

1 5 10 15

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

20 25 30

Asp Leu Ala Lys Glu Leu Cys Lys Arg Ile Asn Thr Gln Cys Thr Phe

35 40 45

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

50 55 60

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

65 70 75 80

Glu Ile Ala Phe Thr Asp Lys Leu Tyr Ala Ala Asp Ser Arg Leu Val

85 90 95

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

100 105 110

Lys Arg Val Gly Val Leu Gln Gly Thr Thr Gln Glu Thr Phe Gly Asn

115 120 125

Glu His Trp Ala Pro Lys Gly Ile Glu Ile Val Ser Tyr Gln Gly Gln

130 135 140

Asp Asn Ile Tyr Ser Asp Leu Thr Ala Gly Arg Ile Asp Ala Ala Phe

145 150 155 160

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

165 170 175

Lys Asp Tyr Lys Phe Gly Gly Pro Ser Val Lys Asp Glu Lys Tyr Asn

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Leu Glu Tyr Asn Leu Phe Gly Val Gly Thr Gly Met Gly Leu Arg Lys

435 440 445

Glu Asp Asn Glu Leu Arg Glu Ala Leu Asn Lys Ala Phe Ala Glu Met

450 455 460

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

465 470 475 480

Asp Val Tyr Gly Gly

485

<210> 19

<211> 484

<212> PRT

<213> Artificial sequence

<400> 19

Met Ala Ile Pro Gln Asn Ile Arg Ile Gly Thr Asp Pro Thr Tyr Ala

1 5 10 15

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

20 25 30

Asp Leu Ala Lys Glu Leu Cys Lys Arg Ile Asn Thr Gln Cys Thr Phe

35 40 45

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

50 55 60

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

65 70 75 80

Glu Ile Ala Phe Thr Asp Lys Leu Tyr Ala Ala Asp Ser Arg Leu Val

85 90 95

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

100 105 110

Lys Arg Val Gly Val Leu Gln Gly Thr Thr Gln Glu Thr Phe Gly Asn

115 120 125

Glu His Trp Ala Pro Lys Gly Ile Glu Ile Val Ser Tyr Gln Gly Gln

130 135 140

Asp Asn Ile Tyr Ser Asp Leu Thr Ala Gly Arg Ile Asp Ala Ala Phe

145 150 155 160

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

165 170 175

Lys Asp Tyr Lys Phe Gly Gly Pro Ser Val Lys Asp Glu Lys Tyr Asn

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Leu Glu Tyr Asn Phe Gly Val Gly Thr Gly Met Gly Leu Arg Lys Glu

435 440 445

Asp Asn Glu Leu Arg Glu Ala Leu Asn Lys Ala Phe Ala Glu Met Arg

450 455 460

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

465 470 475 480

Val Tyr Gly Gly

<210> 20

<211> 482

<212> PRT

<213> Artificial sequence

<400> 20

Met Ala Ile Pro Gln Asn Ile Arg Ile Gly Thr Asp Pro Thr Tyr Ala

1 5 10 15

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

20 25 30

Asp Leu Ala Lys Glu Leu Cys Lys Arg Ile Asn Thr Gln Cys Thr Phe

35 40 45

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

50 55 60

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

65 70 75 80

Glu Ile Ala Phe Thr Asp Lys Leu Tyr Ala Ala Asp Ser Arg Leu Val

85 90 95

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

100 105 110

Lys Arg Val Gly Val Leu Gln Gly Thr Thr Gln Glu Thr Phe Gly Asn

115 120 125

Glu His Trp Ala Pro Lys Gly Ile Glu Ile Val Ser Tyr Gln Gly Gln

130 135 140

Asp Asn Ile Tyr Ser Asp Leu Thr Ala Gly Arg Ile Asp Ala Ala Phe

145 150 155 160

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

165 170 175

Lys Asp Tyr Lys Phe Gly Gly Pro Ser Val Lys Asp Glu Lys Tyr Asn

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Leu Glu Tyr Asn Val Gly Thr Gly Met Gly Leu Arg Lys Glu Asp Asn

435 440 445

Glu Leu Arg Glu Ala Leu Asn Lys Ala Phe Ala Glu Met Arg Ala Asp

450 455 460

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

465 470 475 480

Gly Gly

<210> 21

<211> 485

<212> PRT

<213> Artificial sequence

<400> 21

Met Ala Ile Pro Gln Asn Ile Arg Ile Gly Thr Asp Pro Thr Tyr Ala

1 5 10 15

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

20 25 30

Asp Leu Ala Lys Glu Leu Cys Lys Arg Ile Asn Thr Gln Cys Thr Phe

35 40 45

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

50 55 60

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

65 70 75 80

Glu Ile Ala Phe Thr Asp Lys Leu Tyr Ala Ala Asp Ser Arg Leu Val

85 90 95

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

100 105 110

Lys Arg Val Gly Val Leu Gln Gly Thr Thr Gln Glu Thr Phe Gly Asn

115 120 125

Glu His Trp Ala Pro Lys Gly Ile Glu Ile Val Ser Tyr Gln Gly Gln

130 135 140

Asp Asn Ile Tyr Ser Asp Leu Thr Ala Gly Arg Ile Asp Ala Ala Phe

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Lys Leu Glu Tyr Asn Phe Gly Val Gly Thr Gly Met Gly Leu Arg Lys

435 440 445

Glu Asp Asn Glu Leu Arg Glu Ala Leu Asn Lys Ala Phe Ala Glu Met

450 455 460

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

465 470 475 480

Asp Val Tyr Gly Gly

485

<210> 22

<211> 483

<212> PRT

<213> Artificial sequence

<400> 22

Met Ala Ile Pro Gln Asn Ile Arg Ile Gly Thr Asp Pro Thr Tyr Ala

1 5 10 15

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

20 25 30

Asp Leu Ala Lys Glu Leu Cys Lys Arg Ile Asn Thr Gln Cys Thr Phe

35 40 45

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

50 55 60

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

65 70 75 80

Glu Ile Ala Phe Thr Asp Lys Leu Tyr Ala Ala Asp Ser Arg Leu Val

85 90 95

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

100 105 110

Lys Arg Val Gly Val Leu Gln Gly Thr Thr Gln Glu Thr Phe Gly Asn

115 120 125

Glu His Trp Ala Pro Lys Gly Ile Glu Ile Val Ser Tyr Gln Gly Gln

130 135 140

Asp Asn Ile Tyr Ser Asp Leu Thr Ala Gly Arg Ile Asp Ala Ala Phe

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Lys Leu Glu Tyr Asn Val Gly Thr Gly Met Gly Leu Arg Lys Glu Asp

435 440 445

Asn Glu Leu Arg Glu Ala Leu Asn Lys Ala Phe Ala Glu Met Arg Ala

450 455 460

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

465 470 475 480

Tyr Gly Gly

<210> 23

<211> 16

<212>DNA

<213> Artificial sequence

<400> 23

aacagacggg ccaccg 16

<210> 24

<211> 16

<212>DNA

<213> Artificial sequence

<400> 24

tttaacagac gggcca 16

<210> 25

<211> 16

<212>DNA

<213> Artificial sequence

<400> 25

atctttaaca gacggg 16

<210> 26

<211> 16

<212>DNA

<213> Artificial sequence

<400> 26

ttcatcttta acagac 16

<210> 27

<211> 16

<212>DNA

<213> Artificial sequence

<400> 27

tttttcatct ttaaca 16

<210> 28

<211> 16

<212>DNA

<213> Artificial sequence

<400> 28

cagtttttca tcttta 16

<210> 29

<211> 16

<212>DNA

<213> Artificial sequence

<400> 29

aaacagtttt tcatct 16

<210> 30

<211> 16

<212>DNA

<213> Artificial sequence

<400> 30

gccaaacagt ttttca 16

<210> 31

<211> 16

<212>DNA

<213> Artificial sequence

<400> 31

aaagatgaaa aactgt 16

<210> 32

<211> 16

<212>DNA

<213> Artificial sequence

<400> 32

gatgaaaaac tgtttg 16

<210> 33

<211> 16

<212>DNA

<213> Artificial sequence

<400> 33

gaaaaactgt ttggcg 16

<210> 34

<211> 16

<212>DNA

<213> Artificial sequence

<400> 34

aaactgtttg gcgtag 16

<210> 35

<211> 16

<212>DNA

<213> Artificial sequence

<400> 35

ctgtttggcg taggga 16

<210> 36

<211> 16

<212>DNA

<213> Artificial sequence

<400> 36

tttggcgtag ggaccg 16

<210> 37

<211> 16

<212>DNA

<213> Artificial sequence

<400> 37

ggcgtaggga ccggca 16

<210> 38

<211> 16

<212>DNA

<213> Artificial sequence

<400> 38

gtagggaccg gcatgg 16

<210> 39

<211> 28

<212>DNA

<213> Artificial sequence

<400> 39

cccggatccg atggcgattc cgcaaaac 28

<210> 40

<211> 26

<212>DNA

<213> Artificial sequence

<400> 40

cccaagcttt tagccaccat aaacat 26

<210> 41

<211> 18

<212>DNA

<213> Artificial sequence

<400> 41

tacaacagcg acaacgtc 18

<210> 42

<211> 18

<212>DNA

<213> Artificial sequence

<400> 42

gttgtactcc agcttgtg 18

Claims (10)

1. A histidine fluorescent probe, characterized in that it comprises a histidine-sensitive polypeptide B and a fluorescent protein A; the fluorescent protein A is inserted into the polypeptide B, and the polypeptide B is divided into polypeptide B1 and polypeptide B1. Two parts of B2, forming a probe with the structure of B1-A-B2; The polypeptide B is HBP and its mutants. 2. The histidine fluorescent probe according to claim 1, wherein the amino acid sequence of the HBP is as shown in SEQ ID NO.2. 3. The histidine fluorescent probe according to claim 1, wherein the fluorescent protein A is yellow fluorescent protein cpYFP, and the amino acid sequence of the yellow fluorescent protein cpYFP is shown in SEQ ID NO.3. 4. the histidine fluorescent probe according to claim 3, is characterized in that, described yellow fluorescent protein cpYFP is replaced with aminoacid sequence such as shown in SEQ ID NO.4 or SEQ ID NO.10 green fluorescent protein cpGFP, amino acid The sequence is blue fluorescent protein cpBFP as shown in SEQ ID NO.5 or SEQ ID NO.11, the amino acid sequence is cyan fluorescent protein cpTFP as shown in SEQ ID NO.6, and the amino acid sequence is orange fluorescent protein cpmOrange as shown in SEQ ID NO.7 , amino acid sequence as shown in SEQ ID NO.8 apple red fluorescent protein cpmApple, amino acid sequence as shown in SEQ ID NO.9 or SEQ ID NO.13 red fluorescent protein cpmKate and amino acid sequence as shown in SEQ ID NO.12 red fluorescence One of the proteins mcherry. 5. the histidine fluorescent probe according to claim 1, is characterized in that, described fluorescent protein A inserts polypeptide B to form the probe structure of B1-A-B2 formula, and the position of described insertion is polypeptide B 89/90, 89/91, 89/92, 89/93, 90/91, 90/92, 90/93, 91/92, 91/93, 92/93, 185/186, 185/187, 185/ 188, 185/189, 185/190, 185/191, 185/192, 185/193, 186/187, 186/188, 186/189, 186/190, 186/191, 186/192, 186/193, 187/188, 187/189, 187/190, 187/191, 187/192, 187/193, 188/189, 188/190, 188/191, 188/192, 188/193, 189/190, 189/ 191, 189/192, 189/193, 190/191, 190/192, 190/193, 191/192, 191/193 or 192/193 loci. 6. The histidine fluorescent probe according to claim 5, wherein the insertion sites of the fluorescent protein A inserted into the polypeptide B are 90/91, 91/92, 186/191, 187/191, 189 /190, 189/191, 189/193, 190/191, and 190/193, the amino acid sequences of the corresponding probe structures of the B1-A-B2 formula are shown in SEQ ID NO.14-22. 7. The nucleotide sequence encoding the B1-A-B2 formula probe of claim 1. 8. The preparation method of the histidine fluorescent probe described in any one of claims 1 to 6, comprising the following steps: 1) connecting the nucleotide sequence according to claim 7 to the pRSETb vector to obtain a recombinant expression vector for Escherichia coli; 2) transferring the Escherichia coli recombinant expression vector into a host cell; 3) Cultivate the host cells, and isolate the histidine fluorescent probe. 9. A histidine detection kit comprising the histidine fluorescent probe according to any one of claims 1-6. 10. The histidine fluorescent probe according to any one of claims 1 to 6 or the histidine fluorescent probe prepared by the preparation method according to claim 9 in real-time positioning, quantitative detection and high-throughput of histidine Application of compound screening for non-disease diagnostic or therapeutic purposes.