CN106905418B - Histidine fluorescent probe and preparation method and application thereof - Google Patents

Histidine fluorescent probe and preparation method and application thereof Download PDF

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CN106905418B
CN106905418B CN201710036611.5A CN201710036611A CN106905418B CN 106905418 B CN106905418 B CN 106905418B CN 201710036611 A CN201710036611 A CN 201710036611A CN 106905418 B CN106905418 B CN 106905418B
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杨弋
赵玉政
胡晗阳
顾燕芳
徐磊
陶荣坤
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Abstract

The invention provides a histidine fluorescent probe, which comprises a polypeptide B sensitive to histidine and a fluorescent protein A expressing the histidine; the fluorescent protein A is inserted into the polypeptide B, and the B is divided into two parts, namely B1 and B2, so that a probe structure of a B1-A-B2 formula is formed; the interaction of the polypeptide B and histidine leads to the change of fluorescent signal of the fluorescent protein A; the polypeptide B is HBP and a mutant thereof. The histidine fluorescent probe provided by the invention has relatively small molecular weight, is easy to mature, has large fluorescence dynamic change and good specificity, can be expressed in different subcells of cells by a gene operation method, and can detect histidine quantitatively at high flux inside and outside the cells.

Description

Histidine fluorescent probe and preparation method and application thereof
Technical Field
The invention relates to the technical field of biological engineering, in particular to a histidine fluorescent probe and a preparation method and application thereof.
Background
Histidine is one of the 20 basic amino acids, an important amino acid, and its side chain can be changed from an unprotonated state to a protonated state in a neutral environment due to its pKa of 6.0(Nelson DL et al, Germany: Springer 2009). According to this property, histidine residues act as both proton acceptors and proton donors in many intracellular reactions (Rebek J et al, Struct Chem 1: 129-1311990; Polg r L. et al, CellMol Life Sci 62: 2161-21722005). Furthermore, it contains highly reactive imidazole rings and 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 for biochemical studies.
Studies have shown that abnormalities in histidine content are often associated with several diseases, including chronic kidney disease (Watanabe M et AL, American Journal of Clinical Nutrition 2008,87(6): 1860-. Studies have shown (Watanabe M et al, American Journal of clinical Nutrition 2008,87(6):1860-1866) that plasma histidine levels in patients with chronic kidney disease are low; a corresponding study (Rama Rao KV et al, The American Journal of Pathology 2010,176(3):1400-1408) showed that histidine is a known substance that inhibits glutamine transport in mitochondria, blocking oxidative stress, mitochondrial permeability changes and The formation of cerebral edema in patients with acute liver failure. In addition, high levels of histidine in serum or urine can cause metabolic disorders such as histidine acidemia.
It is because histidine plays an important role as described above that the detection of the content of histidine is also particularly important. Common methods for detecting histidine are capillary electrophoresis (Li X-t et al, Chem Res Chin Univ 2013,29(3): 434-438; Meng J et al, The analysis 2010,135(7):1592-1599), high performance liquid chromatography (Tateda N et al, Analytical science: The internal J ournal of The Japan Society for Analytical Chemistry 2001,17(6): 775-778; Wadudu S et al, Journal of chromatographic B, Analytical technologies in The biological and life sciences 2002,7 (762) -369374), ultraviolet visible spectrophotometry (Hortala MA et al, J Am Chem Soc 2003, 125-1) 20-3521, PuF, 120-3519; Chemical engineering 2010, 8219; 9; fluorescent Spectrometry 120, 120-120; 9; 80; 9; 7-8247; 7; 9; 80; 7; 8247-120; 9; 7; 8247-120; 7; 9; 7; 8247-120; 7; 8247; 7, chemical Communications 1999, (13):1191-1192), but these detection methods have great drawbacks in live cell studies and require time-consuming sample handling procedures: cell disruption, separation, extraction, purification and the like, and can not detect the whole cells accurately in real time.
Disclosure of Invention
In view of the above, the present invention aims to provide a histidine fluorescent probe for real-time localization, high-throughput and quantitative detection of histidine inside and outside cells.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a histidine fluorescent probe, which comprises a polypeptide B sensitive to histidine and a fluorescent protein A; the fluorescent protein A is inserted into the polypeptide B, and the B is divided into two parts, namely a polypeptide B1 and a polypeptide B2, so as to form a probe structure of a B1-A-B2 formula;
the polypeptide B is HBP and a mutant thereof.
Preferably, the amino acid sequence of 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 one of a green fluorescent protein cpGFP shown by an amino acid sequence shown in SEQ ID No.4 or SEQ ID No.10, a blue fluorescent protein cpGFP shown by an amino acid sequence shown in SEQ ID No.5 or SEQ ID No.11, a cyan fluorescent protein cpTFP shown by an amino acid sequence shown in SEQ ID No.6, an orange fluorescent protein cpmOrange shown by an amino acid sequence shown in SEQ ID No.7, an apple red fluorescent protein cpmAple shown by an amino acid sequence shown in SEQ ID No.8, a red fluorescent protein cpmKate shown by an amino acid sequence shown in SEQ ID No.9 or SEQ ID No.13 and a red fluorescent protein mcherry shown by an amino acid sequence shown in SEQ ID No. 12.
Preferably, the fluorescent protein A is inserted into the polypeptide B to form a probe structure of the formula B1-A-B2, 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 of the polypeptide B, 189/190, 189/191, 189/192, 189/193, 190/191, 190/192, 190/193, 191/192, 191/193 or 192/193.
Preferably, when the insertion site of the fluorescent protein A into the polypeptide B is 90/91, 91/92, 186/191, 187/191, 189/190, 189/191, 189/193, 190/191 and 190/193, the amino acid sequence of the corresponding probe structure of B1-A-B2 formula is shown as SEQ ID No. 14-22.
The invention also provides a nucleotide sequence for coding the probe with the B1-A-B2 formula.
The invention also provides a preparation method of the histidine fluorescent probe, which comprises the following steps: 1) connecting the nucleotide sequence encoding the B1-A-B2 type probe with a pRSETb vector to obtain an escherichia coli recombinant expression vector; 2) transferring the recombinant expression vector of the escherichia coli into a host cell; 3) host cells were cultured and histidine fluorescent probe was isolated.
The invention also provides a histidine detection kit comprising the histidine fluorescent probe.
The invention also provides application of the histidine fluorescent probe in real-time positioning and quantitative detection of histidine and high-throughput compound screening.
The invention has the beneficial effects that: the histidine fluorescent probe provided by the invention comprises a polypeptide B sensitive to histidine and a fluorescent protein A; the fluorescent protein A is inserted into the polypeptide B, and the B is divided into two parts, namely a polypeptide B1 and a polypeptide B2, so as to form a probe structure of a B1-A-B2 formula; the B1-A-B2 type histidine fluorescent probe provided by the invention is easy to mature, has large fluorescence dynamic change and good specificity, can be expressed in cells by a gene operation method, and can be used for real-time positioning, high-flux and quantitative detection of histidine inside and outside the cells. Time consuming sample handling steps are eliminated. The experimental effect shows that the highest response of the histidine fluorescent probe provided by the application reaches more than 6 times, and the histidine fluorescent probe can be used for positioning and detecting cells in subcellular structures such as cytoplasm, mitochondria, nucleus, endoplasmic reticulum, outer cell membrane, inner cell membrane, Golgi apparatus, lysosome and the like; and allows high throughput screening of compounds.
Drawings
FIG. 1 is an SDS-PAGE analysis of the histidine fluorescent probe in example 1;
FIG. 2 is a graph showing the histidine fluorescent probe formed by the yellow fluorescent protein cpYFP at different insertion sites of HBP in response to histidine;
FIG. 3 is a graph showing the change of histidine fluorescent probe formed by the blue fluorescent protein cppBFP at different HBP insertion sites in response to histidine;
FIG. 4 is a graph showing the change of histidine fluorescent probe formed by apple red fluorescent protein cpmApple at different HBP insertion sites in response to histidine;
FIG. 5 is a graph showing fluorescence spectrum properties of a histidine fluorescent probe;
FIG. 6 is a titration curve of different histidine fluorescent probes for different concentrations of histidine;
FIG. 7 is a graph of subcellular organelle localization analysis of histidine fluorescent probes in mammalian cells;
FIG. 8 is a graph of the quantitative analysis of histidine in different subcellular organelles by the histidine fluorescent probe Hisensor D;
FIG. 9 is a graph showing the dynamic analysis of trans-membrane transport of histidine by Hisensor D, a histidine fluorescence probe, in different subcellular organelles;
FIG. 10 is a diagram of a high throughput compound screening assay based on Hisensor D, a histidine fluorescent probe, at the viable cell level;
FIG. 11 is a graph showing the quantitative analysis of histidine in the medium and blood by the histidine fluorescent probe Hisensor D.
Detailed Description
The invention provides a histidine fluorescent probe, which comprises a polypeptide B sensitive to histidine and a fluorescent protein A expressing the histidine; the fluorescent protein A is inserted into the polypeptide B, and the B is divided into two parts, namely B1 and B2, so that a probe structure of a B1-A-B2 formula is formed; the interaction of the polypeptide B and histidine causes the fluorescent signal of the fluorescent protein A to become stronger; the polypeptide B is HBP and a mutant thereof.
The HBP protein is derived from Escherichia coli (Escherichia coli), or derived from HBP protein with over 90 percent of homology between salmonella and the HBP protein, and contains a structure that two alpha/beta globular domains typical of periplasmic binding protein are connected through a hinge, and can be combined with histidine. The HBP protein can sense the change of histidine concentration in periplasm, and the spatial conformation of the HBP protein is also greatly changed in the dynamic change process of the histidine concentration. The conformation change of the fluorescent protein caused by the conformation change generated after the binding of histidine with physiological concentration is specifically carried out on the HBP protein, so that the fluorescence of the fluorescent protein is changed, a standard curve is drawn by virtue of the fluorescence of the fluorescent protein measured under different histidine concentrations, and the existence and/or the level of histidine are/is further detected and analyzed.
The nucleotide sequence of the HBP protein coded in the invention is preferably shown as SEQ ID NO.1, and the amino acid sequence of the HBP protein is preferably shown as SEQ ID NO. 2.
The fluorescent protein A is a protein capable of displaying fluorescence, the fluorescent protein A is preferably yellow fluorescent protein cpYFP, and the amino acid sequence of the yellow fluorescent protein cpYFP is shown as SEQ ID NO. 3. In the invention, the original N end and C end of GFP are connected through a section of flexible short peptide chain, a new N end and C end are manufactured at the position of a near chromophore of the original GFP, the amino acid part at the positions of 145-238 th positions is used as the N end of the new protein, the amino acid at the positions of 1-144 th positions is used as the C end of the new protein, and the two sections are connected through 5-9 flexible short peptide chains to obtain the yellow fluorescent protein cpYFP. In the present invention, the proximal chromophore position is preferably at amino acids Y144 and N145; the short peptide chain with flexibility is preferably VDGGSGGTG or GGSGG.
In another embodiment of the present invention, the fluorescent protein a may also be preferably one of a green fluorescent protein cpGFP having an amino acid sequence shown in SEQ ID No.4 or SEQ ID No.10, a blue fluorescent protein cpBFP having an amino acid sequence shown in SEQ ID No.5 or SEQ ID No.11, a cyan fluorescent protein cpTFP having an amino acid sequence shown in SEQ ID No.6, an orange fluorescent protein cpmaorange fluorescent protein cpmange having an amino acid sequence shown in SEQ ID No.7, an apple red fluorescent protein cpmApple having an amino acid sequence shown in SEQ ID No.8, a red fluorescent protein cckate having an amino acid sequence shown in SEQ ID No.9 or SEQ ID No.13, and a red fluorescent protein mcherry having an amino acid sequence shown in SEQ ID No. 12.
The green fluorescent protein GFP was originally extracted from Victoria luminifera (Aequorea Victoria), and was composed of 238 amino acids and had a molecular weight of approximately 26 kDa. GFP is a unique barrel-shaped structure formed by 12 beta-folded chains, and a chromogenic tripeptide (Ser65-Tyr66-Gly67) is wrapped in the GFP. When in the presence of oxygen, it spontaneously forms a chromophore structure of p-hydroxybenzylideneimidazolidinone to generate fluorescence. GFP produces fluorescence without the need for cofactors, and fluorescence is very stable and a good imaging tool. GFP has two excitation peaks, the main peak at 395nm can generate 508nm emission, and the excitation light irradiation at the shoulder 475nm can generate 503nm emission.
In the invention, the red fluorescent protein cpmKate is originally extracted from coral in the sea, wild RFP is oligomeric protein which is not beneficial to the fusion expression of organisms, and then red fluorescent protein with different color bands is further derived on the basis of RFP, wherein mcherry and mKate are the most commonly used.
In the invention, the fluorescent protein A is inserted into the polypeptide B to form a probe structure of the formula B1-A-B2, the insertion site is located in a flexible region of the polypeptide B, the flexible region refers to specific structures such as a ring domain and the like existing in a higher-order structure of the protein, the domains have higher mobility and flexibility compared with other higher-order structures of the protein, and the region can dynamically change the spatial structure conformation after the protein is combined with a ligand. The flexible region 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 sites of the present invention are located at 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; preferred 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 amino acid sequence of the corresponding probe structure of formula B1-A-B2 is shown in SEQ ID Nos. 14 to 22.
The present invention also provides a nucleotide sequence encoding the probe of the above-mentioned formula B1-A-B2.
The invention also provides a preparation method of the histidine fluorescent probe, which comprises the following steps: 1) connecting the nucleotide sequence encoding the B1-A-B2 type probe with a pRSETb vector to obtain an escherichia coli recombinant expression vector; 2) transferring the recombinant expression vector of the escherichia coli into a host cell; 3) host cells were cultured and histidine fluorescent probe was isolated.
In the invention, the nucleotide sequence of the probe encoding the B1-A-B2 is synthesized by a PCR amplification method or an artificial synthesis method. When the PCR amplification method is used, a primer is designed based on 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 the DNA. When the nucleotide sequence is more than 2500bp, 2-6 times of PCR amplification are preferably carried out, and then the amplified fragments are spliced together according to the correct sequence. The PCR amplification procedure and system of the present invention is not particularly limited, and conventional PCR amplification procedures and systems in the art may be used.
In the present invention, it is preferable that the nucleotide sequence of the probe of the B1-A-B2 type is synthesized by an artificial synthesis method when it is less than 2500 bp. The artificial synthesis method is a conventional artificial synthesis method of DNA in the field, and has no other special requirements. Specifically, the full-length sequence can be obtained by synthesizing a plurality of small fragments and then connecting the small fragments.
After obtaining the nucleotide sequence of the probe with the coding B1-A-B2, the invention codes the nucleotide sequence
The nucleotide sequence of the probe of B1-A-B2 type is connected with pRSETb vector to obtain the recombinant expression vector of Escherichia coli. In the invention, the pRSETb vector can be a commercially available pRSETb vector without other special requirements. In the embodiment of the invention, preferably, BamHI and HindIII are respectively adopted to carry out double digestion on the nucleotide sequence for coding the probe with the B1-A-B2 type and the pRSETb vector, and then products obtained after the double digestion are connected to obtain the recombinant expression vector of escherichia coli. The invention has no special limitation on the specific steps and parameters of double enzyme digestion and connection, and the conventional steps and parameters in the field can be adopted. After the escherichia coli recombinant expression vector is obtained, the escherichia coli recombinant expression vector is transferred into a host cell, the host cell refers to a cell capable of receiving and accommodating recombinant DNA molecules, is a place for recombinant gene amplification, and an ideal receptor cell should meet two conditions of easy acquisition and proliferation. The "host cells" of the present invention may include prokaryotic and eukaryotic cells, including in particular bacterial cells, yeast cells, insect cells and mammalian cells. Specific examples thereof include bacterial cells of Escherichia coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells of Drosophila S2 or Sf9, animal cells of CHO, COS, HEK293, HeLa cells, or Bowes melanoma cells, and the like, including but not limited to those host cells described above. The host cell is preferably any cell which facilitates the expression of the gene product or the fermentative production of the gene product, such cells being well known and commonly used in the art, and in the present example the host cell is preferably Escherichia coli JM109-DE3 strain.
The methods described herein for transferring into host cells are conventional in the art and include calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipofectionNatural competence, chemically mediated transfer or electroporation. When the host is a prokaryote such as E.coli, the method is preferably CaCl2Method or MgCl2Methods, the steps used are well known in the art. When the host cell is a eukaryotic cell, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.
After the escherichia coli expression vector is transferred into a host cell, the host cell transferred into the escherichia coli expression vector is subjected to amplification expression culture, and the histidine fluorescent probe is obtained through separation. The host cell is amplified and expressed by a conventional method. The medium used in the culture may be various conventional media depending on the kind of the host cell used. The culturing is performed under conditions suitable for growth of the host cell. In the present invention, the histidine fluorescent protein is expressed in a cell, on a cell membrane, or secreted out of the cell. The method for separating the histidine fluorescent protein is not particularly limited in the invention, and a conventional fusion protein separation method in the field can be adopted. Specifically, in the present invention, conventional renaturation treatment, salting out method, centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography methods and combinations thereof can be used. Preferably by affinity chromatography using a His-tag.
The invention also provides application of the histidine fluorescent probe in real-time positioning and quantitative detection of histidine and high-throughput compound screening. In the invention, the histidine fluorescent probe is preferably connected with signal peptides at different parts of a cell, transferred into the cell, and used for positioning histidine in real time by detecting the intensity of a fluorescent signal in the cell; and (4) carrying out quantitative detection on corresponding histidine by using a histidine standard dripping curve. The standard histidine dripping curve is drawn according to fluorescence signals of a histidine fluorescent probe under the condition of different concentrations of histidine. The histidine fluorescent probe is directly transferred into cells, and a time-consuming sample processing process is not needed in the real-time positioning and quantitative detection process of histidine, so that the histidine fluorescent probe is more accurate. When the histidine fluorescent probe is used for screening high-throughput compounds, different compounds are added into a cell culture solution, and the change of the histidine content is measured, so that the compounds which have influence on the change of the histidine content are screened. The application of the histidine fluorescent probe in real-time positioning and quantitative detection of histidine and high-throughput compound screening is non-diagnosis and treatment purposes, and does not relate to diagnosis and treatment of diseases.
The histidine fluorescent probe provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Test materials and reagents
In the examples, the conventional molecular biological cloning methods of genetic engineering and cell culture and imaging methods are mainly used, and these methods are well known to those skilled in the art, for example: briefly, Rous Kames et al, handbook of molecular biology laboratory references, J. SammBruk, D.W. Lassel, Huang Pentang et al: molecular cloning guidelines (third edition, 8 months 2002, published by scientific Press, Beijing); animal cell culture basic technical guidance (fifth edition), chapter calm, slow-release bolt, and so on, of Feremenoni et al; J.S. Bonifis Nong, M. Dasuo et al, eds of cell biology laboratory Manual, chapter Silent et al. Those of ordinary skill in the art will readily appreciate that modifications and variations may be made to the present invention as described in the following examples, and that such modifications and variations are within the scope of the claims of the present application.
The pRSETb-cpYFP-based pRSETb-HBP plasmid used in the examples was constructed by the protein laboratory of the university of east China's university and the pRSETb plasmid vector was purchased from Invitrogen. All primers used for PCR were synthesized, purified and identified correctly by Mass Spectrometry by Shanghai Czeri bioengineering technology, Inc. The expression plasmids constructed in the examples were subjected to sequencing, which was performed by Huada Gene Co and Jelie sequencing Co. Taq DNA polymerase used in each example was purchased from Dongpeng organisms, pfu DNA polymerase was purchased from Tiangen Biochemical technology (Beijing) Ltd, and primeSTAR DNA polymerase was purchased from TaKaRa, and the three polymerases were purchased with the corresponding polymerase buffer and dNTP. Restriction enzymes such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, SpeI, T4 ligase, and T4 phosphorylase (T4 PNK) were purchased from Fermentas, and supplied with buffers. The transfection reagent Lip2000Kit was purchased from Invitrogen. Histidine and the like were purchased from Sigma. Unless otherwise stated, chemical agents such as inorganic salts are available from sigma-aldrich. HEPES salts, ampicillin (Amp) and puromycin were purchased from Ameresco; a96-well detection blackboard and a 384-well fluorescence detection blackboard are purchased from Grenier company.
The DNA purification kit used in the examples was purchased from BBI (Canada) and the general plasmid minipump kit was purchased from Tiangen Biochemical technology (Beijing) Ltd. The cloning strain Mach1 was purchased from Invitrogen. The nickel column affinity chromatography column and the desalting column packing are both from GE healthcare.
The main instruments used in the examples: biotek Synergy 2 multifunctional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge22R desk-top high-speed refrigerated centrifuge (Beckman, USA), PCR amplification instrument (Biometra, Germany), ultrasonication instrument (Ningbo Xinzhi Co.), nucleic acid electrophoresis instrument (Shenneng Bo Co.), fluorescence spectrophotometer (Varian, USA), CO2Isothermal cell culture chamber (SANYO), inverted fluorescence microscope (japan nikon).
General molecular biology methods and cell Experimental methods used in the examples
Polymerase Chain Reaction (PCR):
1. and (3) target fragment amplification PCR:
the method is mainly used for gene fragment amplification and colony PCR identification of positive clones. The reaction system for the PCR amplification is shown in Table 1, and the amplification procedure is shown in Table 2.
TABLE 1 PCR amplification reaction System
Figure BDA0001212247630000081
Figure BDA0001212247630000082
Table 2 PCR amplification procedure 2. long fragment (>2500bp) amplification PCR:
the long-fragment amplification used in the present invention, mainly the inverse PCR amplification vector, is a technique for obtaining site-directed mutagenesis in the following examples. Reverse PCR primers are designed at the variant site, wherein the 5' end of one primer comprises a variant nucleotide sequence. The amplified product contains the corresponding mutation site. The long fragment amplification PCR reaction system is shown in Table 3, and the amplification procedure is shown in Table 4 or Table 5.
TABLE 3 Long fragment (>2500bp) amplification PCR reaction System
Figure BDA0001212247630000091
TABLE 4 Long fragment (>2500bp) amplification PCR amplification procedure
Figure BDA0001212247630000092
TABLE 5 Long fragment (>2500bp) amplification PCR amplification procedure
Figure BDA0001212247630000093
(II) endonuclease enzyme digestion reaction:
the system of double digestion of the plasmid vector is shown in Table 6, where n represents the amount of sterilized ultrapure water μ L to be added to bring the system to the total volume.
TABLE 6 plasmid vector double digestion System
Figure BDA0001212247630000094
(III) phosphorylation reaction of 5' end of DNA fragment
The ends of plasmids or genomes extracted from microorganisms contain phosphate groups, and PCR products do not contain phosphate groups, so that phosphate group addition reaction is needed to be carried out on 5' end bases of the PCR products, and only DNA molecules with phosphate groups at the ends can carry out ligation reaction. The phosphorylation reaction system is shown in table 7, wherein T4 PNK is abbreviated as T4 polynucleotide kinase, and is used for addition reaction to the 5' phosphate group of DNA molecule.
TABLE 7 phosphorylation reaction System
Figure BDA0001212247630000101
(IV) ligation of the fragment of interest and the vector
The ligation methods differ between different fragments and vectors, and three ligation methods are used in the present invention
1. Blunt-ended short fragment and blunt-ended ligation of linearized vector
The principle of the method is that after the blunt end product obtained by PCR phosphorylates the 5' end of a DNA fragment under the action of T4 PNK, the blunt end product is connected with a 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
Figure BDA0001212247630000102
2. Ligation of DNA fragment containing cohesive Ends and vector fragment containing cohesive Ends
DNA fragments cut by restriction endonucleases will generally produce overhanging sticky ends and can therefore be ligated with sticky end vector fragments containing sequence complementarity to form recombinant plasmids. The ligation reaction system is shown in Table 9.
TABLE 9 ligation reaction System
Figure BDA0001212247630000111
Note: the mass ratio of the PCR product fragment to the vector double-enzyme digestion product is approximately between 2:1 and 6: 1.
3. Ligation reaction of 5' end phosphorylated DNA fragment product self cyclization after introduction of site-directed mutagenesis by inverse PCR
And (3) carrying out self-cyclization ligation on the DNA fragment with 5 ' end phosphorylation to carry out ligation reaction on the 3 ' end and the 5 ' end of the linearized vector to obtain the recombinant plasmid. The self-cyclized ligation reaction system is shown in Table 10.
TABLE 10 self-cyclizing ligation reaction System
Figure BDA0001212247630000112
(V) preparation and transformation of competent cells
Preparation of competent cells:
1. a single colony (e.g., Mach1) was picked and inoculated into 5mL LB medium and shaken overnight at 37 ℃.
2. 0.5-1mL of overnight-cultured broth was transferred to 50mL LB medium and cultured at 37 ℃ and 220rpm for 3-5 hours until OD600 reached 0.5.
3. The cells were pre-cooled in an ice bath for 2 h.
Centrifugation was carried out at 4000rpm for 10min at 4.4 ℃.
5. The supernatant was discarded, the cells were suspended in 5mL of pre-cooled resuspension buffer and after homogenization the resuspension buffer was added to a final volume of 50 mL.
6. Ice-cooling for 45 min.
Centrifugation at 4000rpm for 10min at 7.4 ℃ resuspended bacteria with 5mL of ice-chilled storage buffer.
8. Each EP tube was filled with 100. mu.L of the bacterial solution and frozen at-80 ℃ or with liquid nitrogen.
Resuspension buffer CaCl2(100mM), MgCl2(70mM), NaAc (40mM)
Storage buffer 0.5mL DMSO, 1.9mL 80% glycerol, 1mL 10 × CaCl2(1M)、1mL 10×MgCl2(700mM)、1mL 10×NaAc(400mM)、4.6mL ddH2And (3) O conversion:
1. 100 μ l of competent cells were thawed on an ice bath.
2. The ligation product was added in the appropriate volume, gently whipped and mixed well, and ice-cooled for 30 min. The ligation product is typically added in a volume less than 1/10 the volume of competent cells.
3. The bacterial liquid is put into a water bath with the temperature of 42 ℃ for 90 seconds through heat shock, and is quickly transferred into an ice bath for 5 min.
4. 500. mu.l of LB was added and the mixture was incubated for 1hour at 37 ℃ on a constant temperature shaker for 200 rotations.
5. Centrifuging the bacterial liquid at 4000rpm for 3min, leaving 200 μ l of supernatant, blowing the thallus uniformly, uniformly coating on the surface of an agar plate containing proper antibiotics, and inverting the plate in a constant-temperature incubator at 37 ℃ overnight.
(VI) expression, purification and fluorescence detection of proteins
1. The pRSETb-based histidine probe plasmid was transformed into JM109(DE3), cultured overnight by inversion, picked from the plate and cloned into a 250ml Erlenmeyer flask, placed in a shaker at 37 ℃ and cultured at 220rpm until the OD is 0.4-0.8, added with 1/1000(v/v) of IPTG (1M), and induced to express at 18 ℃ for 24-36 hours.
2. After induction expression is finished, centrifuging at 4000rpm for 30min to collect bacteria, adding 50mM phosphate buffer solution to resuspend bacteria precipitation, and carrying out ultrasonic crushing until the bacteria are clear. 9600rpm, and centrifuging at 4 ℃ for 20 min.
3. The centrifuged supernatant is purified by a self-contained nickel column affinity chromatography column to obtain protein, and the protein after the nickel column affinity chromatography is subjected to a self-contained desalting column to obtain the protein dissolved in 20mM MOPS buffer (pH 7.4) or phosphate buffer PBS.
4. After the purified HBP mutant protein was identified by SDS-PAGE, the probe was diluted to a protein solution with a final concentration of 5-10. mu.M using assay buffer (100mM HEPES, 100mM NaCl, pH 7.3) or phosphate buffered saline PBS. Histidine was formulated as a stock solution at a final concentration of 1M in assay buffer (20mM MOPS, pH 7.4) or phosphate buffered saline PBS.
100 mul of 5 muM protein solution is taken, incubated for 5min at 37 ℃, added with histidine respectively and mixed evenly until the final concentration is 100mM, and the light absorption of the protein under 340nm is measured by a multifunctional fluorescence microplate reader.
100 μ l of 1 μ M fluorescent probe solution was incubated at 37 ℃ for 5min, histidine was added for titration, and the fluorescence intensity emitted at 528nm after 485nm fluorescence excitation of the protein was measured. The fluorescence excitation and emission measurement of the sample are completed by using a multifunctional fluorescence microplate reader.
100. mu.l of 1. mu.M fluorescent probe solution was incubated at 37 ℃ for 5min, histidine was added, and the absorption spectrum and fluorescence spectrum of the probe protein were measured. The measurement of the absorption spectrum and the fluorescence spectrum of the sample is performed by a spectrophotometer and a fluorescence spectrophotometer.
(VII) mammalian cell fluorescence detection
1. The pCDNA3.1+ -based histidine probe plasmid was transfected into HeLa by the transfection reagent Lipofectamine2000(Invitrogen) and placed at 37 ℃ with 5% CO2Cultured in a cell culture box. And carrying out fluorescence detection after the exogenous gene is fully expressed for 24-36 h.
2. After the induction expression is finished, the adherent HeLa cells are washed three times by PBS and placed in HBSS solution for detection by a fluorescence microscope and a microplate reader respectively.
Example 1
Construction of pRSETb-HBP plasmid
The HisJ gene in the E.coli gene was amplified by PCR, the PCR product was recovered after gel electrophoresis and digested with BamHI and HindIII, while the same double digestion was performed on the pRSETb vector. After ligation with T4 DNAlagase, the ligation products were transformed into MachI, which was plated on LB plates (ampicillin 100ug/mL) and incubated overnight at 37 ℃. After plasmid extraction of the MachI transformant, PCR identification is carried out. And (4) carrying out subsequent plasmid construction after the positive plasmid is sequenced correctly.
The construction primer sequence of the pRSETb-HBP plasmid is shown as Seq ID No. 39-40.
plasmid construction and detection of different insertion sites of pRSETb-HBP-cpFP fluorescent probe
In this example, pRSETb-HBP-based plasmid was selected from 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 on the basis of crystal structure of HBP, 189/192, 189/193, 190/191, 190/192, 190/193, 191/192, 191/193, 192/193. Wherein the corresponding amino acid positions of 90/91, 91/92, 186/191, 187/191, 189/190, 189/191, 189/193, 190/191 and 190/193 (shown as SEQ ID NO 14-22) or family proteins thereof are detected to respond more than 3 times to histidine.
Generating a DNA fragment of cpYFP by utilizing PCR, inactivating the DNA fragment after using a phosphate adding operation at the 5 'end, generating a pRSETb-HBP linearized vector containing different fracture sites by reverse PCR amplification, connecting the linearized pRSETb-HBP and the cpYFP fragment phosphorylated at the 5' end under the action of PEG4000 and T4 DNA ligase to generate recombinant plasmids, placing the plates in a Kodak multifunctional living body imaging system, selecting a clone with yellow fluorescence under the excitation of an FITC channel, and completing sequencing by Shanghai Bingkoku corporation of Beijing Liuhe Dagaku Gene science and technology Limited.
Primers used for generating a DNA fragment of cpYFP by PCR are shown in Seq ID No. 41-42.
Primers used for reverse amplification to generate linearized vectors are shown in Seq ID No. 23-38.
After sequencing was correct, the recombinant plasmid was transformed into JM109(DE3) for induction of expression and the protein was purified and electrophoresed by SDS-PAGE in the size range of 60 kDa. The size of the fusion protein is consistent with the size of HBP-cpYFP fusion protein containing His-tag purification label expressed by pRSETb-HBP-cpYFP. The results are shown in FIG. 1.
The purified HBP-cpYFP fusion protein was subjected to histidine-response screening, and the detection signal of the fusion fluorescent protein containing 100mM histidine was divided by the detection signal of the fusion fluorescent protein without histidine.
Results the results are shown in fig. 2, and the results of the assay showed 90/91, 91/92, 186/191, 187/191, 189/190, 189/191, 189/193, 190/191, 190/193 responses to histidine over 3-fold. The fluorescence intensity at 420nm excitation 528nm emission of 186/191, 197/191, 189/191, 190/191 increased with histidine concentration. The fluorescence intensity at 528nm excitation of 485nm excitation from 189/190, 189/191, 189/193, 190/191, 190/193 decreased with histidine concentration. Of these 189/191, 190/191 showed opposite changes in fluorescence intensity at 420nm excitation 528nm emission and at 485nm excitation 528nm emission with histidine concentration.
Example 2
The cpYFP was replaced with the blue fluorescent protein cpBFP and fused to HBP to construct a histidine blue fluorescent protein fluorescent probe according to the method in example 1. As shown in FIG. 3, the results of fluorescence detection showed 90/91, 186/191, 187/191, 189/190, 189/191, 190/191 and 190/193 responses to histidine by more than 3-fold.
Example 3
The cpYFP was replaced with apple red fluorescent protein cpmpile as in example 1, and fused to HBP to construct a histidine red fluorescent protein fluorescent probe, as shown in fig. 4, the results of fluorescence detection showed 90/91, 91/92, 186/191, 187/191, 189/190, 189/191, 189/193, 190/191, 190/192, 190/193, 191/193 in response to histidine over 3-fold.
The results of examples 1-3 demonstrate that the linker regions 89-93 and 185-193 of HBP are suitable for fusing cpFP fluorescent protein to obtain a fused fluorescent protein histidine probe responding to histidine.
Example 4 detection of HBP-cpYFP fluorescent Probe Properties
The purified HBP-cpYFP was treated with 0mM histidine and 100mM histidine, respectively, for 10min, and then fluorescence spectrum was detected using a fluorescence spectrophotometer. Measurement of excitation spectra: fixing the emission at 530nm, and carrying out excitation spectrum detection in a 380-510 nm range; and (3) measuring an emission spectrum, fixing excitation at a 485nm position, and detecting the emission spectrum in a range of 510-550 nm. The spectrum curve of HBP-cpYFP fluorescent probe is shown in FIG. 5, and the above results indicate that the fluorescence spectrum property of HBP-cpYFP fluorescent protein is similar to that of cpYFP fluorescent protein (Nagai, T. et al, Proc Natl Acad Sci U S A.2001, V.98(6), pp.3197-3202).
Four probes Hisensor A, Hisensor B, Hisensor C and Hisensor D with histidine detection range of 0.1 mu M-1 mM are selected for histidine detection with concentration gradient (0-1 mM). Respectively processing purified HBP-cpYFP for 10min, and detecting fluorescence at 528nm emission part excited by 420nmThe change of the ratio of the intensity to the fluorescence intensity at 528nm of 485nm excitation of the 4 histidine fluorescent probesd(binding constant) was 4. mu.M, 13. mu.M, 3.4. mu.M and 22. mu.M in this order, and the range of change was 4.2-fold, 4.5-fold, 3.4-fold and 6.2-fold in this order, the results are shown in FIG. 6. Therefore, the more suitable histidine probe can be selected for quantitative detection according to the content level of histidine in the sample.
Example 5 localization of Hisensor D fluorescent probes in different subcellular organelles and quantification of histidine within subcellular organelles
In this example, we used different localization signal peptides to fuse with Hisensor D, and localized the Hisensor D histidine fluorescent protein probe to different organelles.
After transfection of HeLa cells with 36hours by plasmids of Hisensor D gene fused with different localization signal peptides, the cells were washed with PBS and then placed in HBSS solution for fluorescence detection under FITC channel using 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, lysosome by fusion with different specific localization signal peptides. As a result, as shown in FIG. 7, fluorescence was observed in different subcellular structures, and the distribution and intensity of fluorescence were different from each other.
In this example, we used the histidine probe Hisensor D to quantify histidine in the cytosol and mitochondria, respectively.
HeLa cells transfected with Hisensor D gene were washed with PBS, and then placed in HBSS solution to measure the ratio of the fluorescence intensity at 528nm excitation at 420nm to the fluorescence intensity at 528nm excitation at 485nm using a microplate reader. As a result of the measurement of the histidine concentration according to the histidine standard addition curve (FIG. 6) of Hisensor D, it was found that the content of histidine in the cytoplasm was about 150. mu.M and the content of histidine in the mitochondria was about 50. mu.M, as shown in FIG. 8.
Example 6 dynamic analysis of transmembrane transport of histidine within different subcellular organelles by Hisensor D histidine fluorescent Probe
In this example, we performed a kinetic assay of histidine in the cytosol and mitochondria, respectively, using the histidine probe Hisensor D.
HeLa cells transfected with Hisensor D gene were washed with PBS, treated with HBSS solution (without histidine) for 2hours, and then histidine was added dropwise at 2 min. The change of the ratio of the fluorescence intensity at 528nm emission under 420nm excitation to the fluorescence intensity at 528nm emission under 485nm excitation was recorded by a microplate reader, and the results are shown in fig. 9, and it was found that extracellular histidine can rapidly enter cells, and the content of histidine in the cells reached the physiological maximum within about 4 min.
Example 7 high-throughput Compound screening at viable cell level based on Hisensor D, a histidine fluorescent Probe
In this example, we performed high-throughput compound screening using HeLa cells expressed cytoplasmic with histidine probe Hisensor D.
HeLa cells transfected with Hisensor D gene were washed with PBS, treated with HBSS solution (without histidine) for 1hour, and then treated with 10. mu.M compound for 1 hour. Histidine was added dropwise separately. The change of the ratio of the fluorescence intensity at 528nm excitation of 420nm to the fluorescence intensity at 528nm excitation of 485nm was recorded by a microplate reader. Samples not treated with any compound were used as standards. As shown in fig. 10, we found that most of the compounds had little effect on entry of histidine into the cells in the cells treated with 500 compounds. 9 compounds can improve the uptake capacity of cells to histidine, and 6 compounds can obviously reduce the uptake of histidine by cells.
Example 8 quantitative determination of histidine in DMEM Medium and blood supernatant Using Hisensor D fluorescent Probe
In this example, the cellular DMEM medium and histidine in mouse blood supernatant were also analyzed using purified Hisensor D fluorescent protein, respectively.
Mixing Hisensor D fluorescent protein with diluted DMEM culture medium and blood supernatant for 10min, and detecting the ratio of fluorescence intensity at 528nm excitation of 420nm to fluorescence intensity at 528nm excitation of 485nm by using a microplate reader. As shown in FIG. 11, it was found that the histidine content in the DMEM medium was about 790. mu.M, and the histidine content in the blood of the mice was about 80. mu.M.
The embodiments show that the histidine fluorescent probe provided by the invention has relatively small protein molecular weight, is easy to mature, has large fluorescence dynamic change and good specificity, can be expressed in cells by a gene operation method, and can be used for positioning and quantitatively detecting histidine inside and outside the cells in real time; and enables high throughput screening of compounds.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
SEQUENCE LISTING
<110> university of east China's college of science
<120> histidine fluorescent probe, and preparation method and application thereof
<130>1
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<170>PatentIn version 3.3
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accacccagg agacgttcgg taatgaacat tgggcaccaa aaggcattga aatcgtctcg 420
tatcaggggc aggacaacat ttattctgac ctgactgccg gacgtattga tgccgcgttc 480
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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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<213> Artificial sequence
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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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<213> Artificial sequence
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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
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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
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Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Gly Gly Tyr Ile
180 185 190
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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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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 155160
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
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<213> Artificial sequence
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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 LysLeu 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
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<213> Artificial sequence
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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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<213> Artificial sequence
<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
3540 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 200205
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 GlyGlu 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
IleAsp 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 AspGly 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 9095
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 250255
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 410415
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 gggcca16
<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 (7)

1. A histidine fluorescent probe is characterized by comprising a polypeptide B sensitive to histidine and a fluorescent protein A; the fluorescent protein A is inserted into the polypeptide B, and the polypeptide B is divided into two parts, namely a polypeptide B1 and a polypeptide B2, so as to form a probe with a B1-A-B2 type structure;
the polypeptide B is HBP;
the amino acid sequence of HBP is shown in SEQ ID NO. 2;
the insertion sites of the fluorescent protein A and the polypeptide B are 89/90, 89/91, 89/92, 89/93, 90/91, 90/92, 90/93, 91/92, 91/93 and 92/93.
2. The histidine fluorescent probe as claimed in 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 as SEQ ID No. 3.
3. The histidine fluorescence probe of claim 2, wherein the yellow fluorescent protein cpYFP is replaced by one of a green fluorescent protein cppGFP with an amino acid sequence shown in SEQ ID No.4 or SEQ ID No.10, a blue fluorescent protein cppBFP with an amino acid sequence shown in SEQ ID No.5 or SEQ ID No.11, a cyan fluorescent protein cppTFP with an amino acid sequence shown in SEQ ID No.6, a yellow fluorescent protein cpmOrange with an amino acid sequence shown in SEQ ID No.7, an apple red fluorescent protein cpmApplele with an amino acid sequence shown in SEQ ID No.8, a red fluorescent protein cpmKate with an amino acid sequence shown in SEQ ID No.9 or SEQ ID No.13, and a red fluorescent protein mCherry with an amino acid sequence shown in SEQ ID No. 12.
4. A nucleotide sequence encoding the probe of claim 1 of the formula B1-a-B2.
5. The method for preparing the histidine fluorescent probe as claimed in any one of claims 1 to 3, comprising the following steps:
1) connecting the nucleotide sequence of claim 4 with a pRSETb vector to obtain an Escherichia coli recombinant expression vector;
2) transferring the recombinant expression vector of the escherichia coli into a host cell;
3) and culturing the host cell, and separating to obtain the histidine fluorescent probe.
6. A histidine detection kit comprising the histidine fluorescent probe as claimed in any one of claims 1 to 3.
7. The use of the histidine fluorescent probe as claimed in any one of claims 1 to 3 or the histidine fluorescent probe prepared by the preparation method as claimed in claim 5 for real-time histidine localization, quantitative detection and high-throughput compound screening for non-disease diagnosis or treatment purposes.
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