CN108473973A - Including the composition of the excretion body of load albumen and preparation and the method for delivering the composition - Google Patents

Abstract

The present invention relates to for largely generating the excretion body comprising cargo protein method, the carrier for being used to prepare excretion body, the excretion body comprising cargo protein prepared by this method and cargo protein is loaded to by using the excretion body thus prepared method in cytosol.According to the method provided by the present invention for preparing the excretion body comprising cargo protein, the excretion body of load cargo protein can be generated with high yield so that it can be widely applied to disease treatment using the excretion body.

Description

Including the composition of the excretion body of load albumen and preparation and delivering the composition Method

Technical field

Cross reference to related applications

This application claims the South Korea patent application No.10-2016-0126335 submitted for 30th in September in 2016,2016 9 The South Korea patent application No.10-2016-0126921 that the moon is submitted on the 30th, the South Korea patent application submitted on the 30th of September in 2016 No.10-2016-0126961, the South Korea patent application No.10-2016-0127486 submitted on October 4th, 2016,2016 10 The South Korea patent application No.10-2016-0132616 that the moon is submitted on the 13rd, on 2 10th, 2017 South Korea patent applications submitted The content of the priority of No.10-2017-0018637, each of which application is incorporated herein by reference.

Load is prepared the present invention relates to the composition of the excretion body comprising load albumen, using light binding proteins specific The method of the excretion body of cargo protein and the method that cargo protein is delivered to cytosol using the excretion body thus prepared.

Background technology

Human body is made of about 200 kinds of 100,000,000,000,000 cells, and wherein physiological activity is adjusted by the effect of various albumen to remain raw Life.

The double-layer structure film that cell is made of phosphatide surrounds, and which prevent foreign substances to enter cell, opens so far Most of protein drugs of hair cannot enter cell by cell membrane, and can act on outside, or act on cell membrane On receptor show physiologic effect so that signal to be delivered into the cell.

Cytosol has a large amount of interact to adjust the albumen of physiological activity.It therefore, once can be by protein drug It is delivered into cell, that is, enters cytosol, it will be able to more effectively control cell activity.

Recently, cargo protein is directly delivered into cell through cell membrane to establish a kind of method actively being studied always In.It, can when preparing and the recombinant protein of cargo protein and nexin transduction domain (PTD) (passing through the peptide of cell membrane) being administered To pass through cell membrane to enter cytosol (Fig. 1).The example of PTD is HIV-1TAT, HSV VP22, Antp, dfTAT and Hph-1. It is generated by combining fusion protein prepared by PTDs and cargo protein to be used as recombinant protein, and needs separation process at this time.So And this process has the following problems：Refolding proteins are inappropriate, active reduction, the transfer of albumen non-specificity, cause to exempt from vivo Epidemic disease reaction risk is big, of high cost and low yield.

The cargo protein being conjugated with various nano particles can enter cytosol (figure by endocytosis across cell membrane 2).At this point, the example of nano particle be gold NP, liposome NP, magnetism NP and polymerize NP.The separation of nano particle and cargo protein It occurs mainly in the lysosome in cell, therefore cargo protein is decomposed in lysosome and lost activity.Or it is molten in cytoplasm Nano particle is difficult to detach with cargo protein in glue and the toxicity of nano particle may be another problem.

Excretion body (exosome) is the vesicles of the membrane structure with 50~200nm sizes, and excretion body is secreted cell Outside, it includes the albumen transduceed for intercellular signal, DNA and RNA.

Excretion body is found in for the first time only to be left by removing intracellular protein in the erythrocyte maturation later stage in red blood cell During hemoglobin.According to the observation under electron microscope, it was confirmed that excretion body is not detached by plasma membrane not instead of directly, It is extracellular from intracellular specific region (being known as multivesicular body (MVB)) discharge.That is, as MVB and plasma membrane fusion, such title It is extracellular (Fig. 3) for the vesica discharge of excretion body.

Not yet clearly announce the molecular mechanism that excretion body generates, but known various immunocytes, including bone-marrow-derived lymphocyte, T leaching Bar cell, Dendritic Cells, megacaryocyte, macrophage, stem cell and tumour cell, generate when it is survived and secrete excretion Body.

Excretion body includes albumen, DNA and RNA in various kinds of cell.It is included in these excretion bodies from what is secreted out of in cell Substance can import other cells again by fusion or endocytosis, and as intercellular courier.By analyzing from thin These substances being included in excretion body secreted out of in born of the same parents, so that it may to be diagnosed to be specified disease.

Excretion body further includes various types of microRNA.It has been reported that by detect its presence or absence of and its it is rich The method (KR 10-2010-0127768A) for spending to diagnose the illness.International patent publications NO WO2009-015357A describe logical That crosses excretion body of the detection in the sample (blood, saliva, tears etc.) of cancer patient predicts and diagnoses specified disease side Method.Particularly, the excretion body obtained from the patient with specified disease (pulmonary disease) is analyzed, and is had been described in detail specific Relationship between microRNA and pulmonary disease.In addition to diagnosing pulmonary disease, also continuing research to establish one kind by making With the specific protein being included in excretion body come the method for diagnosis of kidney disease.

Excretion body can also include antigen.In antigen presenting cell (APC), Antigenic Peptide is loaded in including multivesicular body The intracellular compartment of membrane structure in MHC (major histocompatibility complex) II class molecules in.Therefore, its excretion is derived from Body also has Antigenic Peptide-MHC II class compounds.Therefore, Antigenic Peptide is presented to CD4+T leaching by excretion body as immunogenic carrier Bar cell, so as to induce immune response, such as T lymphopoiesis.The molecule such as MHCI of immune response can be stimulated Class and heat shock protein (HSP) concentrate in excretion body so that excretion body can be used for increasing or decreasing immune response to treat Cancer or autoimmune disease.

Invention content

Solution to the problem

The present invention provides the composition for including the excretion body for being loaded with cargo protein.

In another embodiment, present invention offer is a kind of being loaded with cargo protein using light binding proteins specific to prepare Excretion body method.

In further embodiment, present invention offer is a kind of being delivered to cytosol using excretion body by cargo protein Method.

Description of the drawings

Fig. 1 illustrates the side of the recombinant protein delivering goods albumen by cargo protein and nexin transduction domain (PTD) Method (Steven R.et al.Protein transduction:unrestricted delivery into all cells Trends in Cell Biology,2000)。

Fig. 2 illustrates that cargo protein is delivered to born of the same parents by endocytosis using the compound of nano particle and cargo protein Method (the Munish Chanana et al.Physicochemical properties of protein- of matter colloidal sol coated gold nanoparticles in biological fluids and cells before and after proteolytic digestion.Angew.Chem.Int.Ed.2013)。

Fig. 3 illustrates process (the Graca Raposo and Willem that excretion body is detached and discharged from multivesicular body (MVB) Stoorvogel.Extracellular vesicles:Exosomes,microvesicles,and friends.Cell Biology 200(4),373-383,2013)。

Fig. 4 is illustrated by by delivering siRNA in targeting excretion body body come the process (Alvarez- for the treatment of cancer Erviti,L.et al.Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes.Nature Biotechnology 29,341-345,2011)。

Fig. 5 illustrates the preparation of the excretion body (EXPLOR) of the carrying albumen of smooth science of heredity design according to the present invention Journey.

Fig. 6 illustrates when to the illumination of EXPLOR stopping, the fusion protein of cargo protein and light binding proteins specific Separation process inside excretion body.

Fig. 7 is illustrated according to blue light illumination, in the conversion for importing CIBN-EGFP-CD9 genes and mCherry-CRY2 genes In HEK293T cells, the change in location of mCherry albumen in the cell.

Fig. 8 illustrates to obtain the experimental arrangement of EXPLOR according to the present invention.

Fig. 9 illustrates cargo protein (mCherry albumen) changes of contents according to blue light strength, captured in excretion body Measurement result.

Figure 10 illustrate using comprising cargo protein (mCherry albumen) excretion body processing target cell (HT1080) it Afterwards, the result of study of cargo protein is imported in target cell, the wherein left side indicates the target cell of unused excretion body processing, the right table Show the target cell handled with excretion body.

Figure 11 is one group of fluorescent image (a), illustrates to handle target with the excretion body comprising cargo protein (mCherry albumen) After cell (HT1080), the result of study of cargo protein is imported in target cell；And figure (b), it illustrates by excretion body Handle the comparison result of the apoptotic cell ratio of induction.

Figure 12 illustrates according to blue light illumination, is importing GIGANTEA-EGFP-CD9 genes and mCherry-FKF1LOV It converts in HEK293T cells, the change in location of mCherry albumen in the cell.

Figure 13 illustrates expression and the product of the luciferase-mCherry fusion proteins measured by fluorescence imaging (a) Uciferase activity and molecular amounts (b) in cell：

Control：The HEK293T cells being not processed；

Superposition：Only import the HEK293T cells of luciferase-mCherry-CRY2；

XP：It is led by using the commercially available carrier XPACK (Systems Biosciences) designed for excretion body load technology Enter the HEK293T cells of XPACK- luciferases-mCherry；

EXPLOR：Import the HEK293T of luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 according to the present invention Cell；

Figure 14 illustrates uciferase activity (a) and molecular amounts (b) in the excretion body of generation：

NEG：The excretion body that the HEK293T cells being not processed generate；

Superposition：Import the excretion body that the HEK293T cells of luciferase-mCherry-CRY2 generate；

XP：By using the commercially available carrier XPACK (Systems Biosciences) designed for excretion body load technology Import the excretion body generated in the HEK293T cells of XPACK- luciferases-mCherry；

EXPLOR：Import the HEK293T of luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 according to the present invention The excretion body that cell generates；

There is light：By cultivating 72 hours excretion bodies generated under 200 μ W blue light illuminations,

It is unglazed：By cultivating 72 hours excretion bodies generated under no light condition.

Figure 15 illustrates the load efficiency of cargo protein in the excretion body of above-mentioned generation.

Figure 16 illustrates the transfer efficiency that cargo protein is transported to target cell (HeLa) using excretion body：

Control：The excretion body that the HEK293T cells being not processed generate；

Superposition：Import the excretion body that the HEK293T cells of luciferase-mCherry-CRY2 generate；

XP：It is led by using the commercially available carrier XPACK (Systems Biosciences) designed for excretion body load technology Enter the excretion body that the HEK293T cells of XPACK- luciferases-mCherry generate；

EXPLOR：Import luciferase-mCherry-CRY2 and thin according to the HEK293T of CIBN-EGFP-CD9 of the present invention The excretion body that born of the same parents generate；

There is light：By cultivating 72 hours excretion bodies generated under 200 μ W blue light illuminations,

It is unglazed：By cultivating 72 hours excretion bodies generated under no light condition.

Figure 17 illustrates the expression position of luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells It sets, shows that they share identical position to express.

Figure 18 illustrates the expression position of Cre-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, table It is bright they share identical positions and express.

Figure 19 a illustrate Cre:The ZsGreen of EXPLOR processing inductions is in pCAG-loxP-STOP-loxP-ZsGreen winks When the HT1080 cells that transfect in expression (engineer's scale, 40 μm)：

It is negative:EXPLOR：The excretion body of unsupported cre is as negative control；

Cre:EXPLOR：Load the excretion body of cre；And

pCMV-Cre：The transfection of pCMV-Cre carriers is used as positive control.

Figure 19 b illustrate to use Cre:The ZsGreen of EXPLOR processing inductions is in pCAG-loxP-STOP-loxP- Expression (engineer's scale, 40 μm) in the HeLa cells that ZsGreen is transiently transfected：

It is negative:EXPLOR：The excretion body of unsupported cre is as negative control；

Cre:EXPLOR：Load the excretion body of cre；And

pCMV-Cre：The transfection of pCMV-Cre carriers is used as positive control.

Figure 20 illustrates to use Cre:The ZsGreen of EXPLOR processing inductions is in pCAG-loxP-STOP-loxP- Expression (engineer's scale, 100 μm) in the Primary rat Fetal neurons that ZsGreen is transiently transfected：

Control:EXPLOR：The excretion body of unsupported cre is as negative control；And

Cre:EXPLOR：Load the excretion body of cre.

Figure 21 illustrates to use Cre:ZsGreen is with pCAG-loxP-STOP-loxP- for EXPLOR processing inductions Expression (engineer's scale, 100 μm) in the transgenic mice of eNpHR3.0-EYFP genes：

Control:EXPLOR：The excretion body of unsupported cre is as negative control；

Cre:EXPLOR：Load the excretion body of cre；

Hip：Hippocampus；And

Th：Thalamus.

Figure 22 is illustrated in the transgenic mice with pCAG-loxP-STOP-loxP-eNpHR3.0-EYFP genes The ImmunohistochemistryResults Results of NEuN/GFAP：

Pink colour：Neuronal specificity nucleoprotein；NEuN, positive neuron；And

It is red：Glial fibrillary acid protein；GFAP, astrocyte.

Object lens, 40 ×, engineer's scale, 20 μm.

Figure 23 illustrates the expression position of Cas9-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, Show that they share identical position to express.

Figure 24 illustrates the generation of the DNA construct of the excretion body for generating load C as9.

Figure 25 illustrates the measurement result of cargo protein (CRISPR-Cas9 albumen) content captured in excretion body.

Figure 26 illustrates the expression position of GBA-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, table It is bright they share identical positions and express.

Figure 27 illustrates endogenous GBA and GBA-mCherry-CRY2 fusion protein in GBA-MCH-CRY2 and CIBN- EGFP-CD9 transiently transfect HEK293T cells, rat primary astroglia, people's primary astroglial cells and Ge Xie at Expression in fibrocyte.

Figure 28 illustrates the measurement result of cargo protein (GBA albumen) content captured in excretion body.

Figure 29 illustrates a kind of enzyme activity of the β-glucocerebrosidase (cargo protein (GBA albumen)) captured in excretion body Property measurement result：

Exo- is natural：The sources HEK293T excretion body；

Exo-GBA：Excretion body including β-glucocerebrosidase.

Figure 30 illustrates that GBA- excretions body to fibroblastic handling result from Gaucher patient, shows to use The processing of GBA- excretion bodies is significantly induction of the enzymatic activity in β-glucocerebrosidase deficient cells.

Figure 31 illustrates the generation of the DNA construct of the excretion body for generating load PTEN and stablizes expression load PTEN Excretion body cell generation.

Figure 32 illustrates the expression position of luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells It sets, shows that they share identical position to express.

Figure 33 illustrates the quantified results of the uciferase activity based on luciferase molecules quantity.

Figure 34 illustrates the expression position of PrxI/II-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells It sets, shows that they share identical position to express.

Prx I:Peroxiredoxin I (peroxiredoxin I)

Prx II:Peroxiredoxin II (peroxiredoxin II)

Figure 35 is illustrated in H₂O₂The protection that the excretion body of PrxI/II is loaded in the oxidative stress and cytotoxicity of induction is made With.

Nothing：H₂O₂The group of processing；

Cre:EXPLOR：The excretion body of load C re；

PrxI:EXPLOR：Load the excretion body of PrxI；And

PrxII:EXPLOR：Load the excretion body of PrxII.

Figure 36 illustrates the expression position of MyoD-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, Show that they share identical position to express.

Figure 37 illustrates the excretion body of load MyoD to the handling result of fat stem cell, show MyoD- excretions body (gram Grand #A6) processing 6 days after induction of cell Proliferation.

Figure 38 illustrates to stablize the generation of the cell of the excretion body of expression load p53.

Figure 39 illustrates the measurement result of cargo protein (p53 albumen) content captured in excretion body.

Stablize cell：Stablize the cell of the excretion body of expression load p53；

mcherry：Load the excretion body of mcherry；

p53：Load the excretion body of p53.

Figure 40 illustrates the measurement result of the p53 transcriptional activities using luciferase reporter gene, shows at adriamycin-place Using the processing of the excretion body of load p53 induction of the transcriptional activity of p53 in the HeLa cells of reason.

Figure 41 illustrates the generation of the DNA construct of the excretion body for generating load HMGB1 and stablizes expression load The generation of the cell of the excretion body of HMGB1.

Figure 42 illustrates expression positions of the srI κ B-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, Show that they share identical position to express.

Figure 43 illustrates srIkB-mCherry:The processing of EXPLOR substantially reduces tumor necrosis factor α in Hela cells and lures The DNA of the transposition and NF- κ B p65 subunits led is combined.

Figure 44 illustrates for the excretion body for loading srIkB to be administered into the progression of disease after rheumatoid arthritis animal model Analysis.

Figure 45 illustrates that the survival of the group of the excretion body processing with load srIkB in the sepsis model that LPS- is induced is bent Line.

Without excretion body：The group of only LPS processing；

Natural excretion body：The group handled using the excretion body from HEK293T；

SrIkB excretion bodies：Use the group of the excretion body processing of load srIkB.

Figure 46 illustrates pYSTAT3 intracellular antibody-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells Expression position, show that they share identical positions and express.

It is thin that Figure 47 illustrates that pYSTAT3 intracellular antibodies are delivered to target by the excretion body using load pYSTAT3 intracellular antibodies Born of the same parents' is intracellular.

Figure 48 illustrates the expression position of Bax-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, table It is bright they share identical positions and express.

Figure 49 illustrates the processing induced cytochrome c of the excretion body of load Bax from the quick of Hela cell Mitochondrias Release.

Figure 50 illustrates the expression position of AIMP-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, Show that they share identical position to express.

Figure 51 illustrates the measurement result of cargo protein (AIMP albumen) content captured in excretion body.

Figure 52 illustrates the expression position of mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, shows it Share identical position and express.

Best mode for carrying out the invention

The present invention provides the compound for including the excretion body for being loaded with cargo protein.

In another embodiment, present invention offer is a kind of being loaded with cargo protein using light binding proteins specific to prepare Excretion body method.

In the mode further implemented, it is molten that cargo protein will be delivered to cytoplasm by a kind of use excretion body of present invention offer The method of glue.

In another embodiment, the present invention provides the method for largely generating the excretion body comprising fusion protein, The fusion protein is made of excretion body specific marker object and cargo protein.

The present invention provides include the goods for being isolated from excretion body film to largely generating by using light binding proteins specific The method of the excretion body of object albumen.

The present invention also provides the carrier for being used to prepare excretion body, which can be used for the preparation of excretion body.

Invention further provides the methods imported cargo protein by using above-mentioned excretion body in cytosol.

In one embodiment, the present invention provides the pharmaceutical composition comprising the excretion body for being loaded with cargo protein and its Preparation method.

In a preferred embodiment, cargo protein is the super repressor I kB proteins for inhibiting NF- κ B, (Bcl-2- is related by Bax X protein), Peroxiredoxin I, Peroxiredoxin II, cre recombinase, Cas9 (CRISPR correlation eggs It is white 9), Cpf1 (from Prey irrigate Pseudomonas (Prevotella) and Francisella (Francisella) CRISPR 1) Or GBA (β-glucocerebrosidase).

The present invention provides the excretion bodies including cargo protein, and internal various diseases can be used for by delivering goods albumen The treatment of disease.For example, excretion body can be prepared to include the albumen or siRNA with active anticancer, cancer cell is then handled To carry out treatment of cancer (Fig. 4).

For including the excretion body of the cargo protein for treating disease, need effectively to prepare excretion body to have appropriate bear The cargo protein of load.Korean Patent Publication No.2004-0015508 describes the excretion body being used to prepare comprising specific antigen Method.Precisely, the method for cargo protein is discharged by using excretion body which depict a kind of, wherein will encode specific anti- In former gene Insertion Into Host Cell system and the albumen of quiding gene stablizes expression in cell line, which passes through excretion body It extracellularly discharges, and excretion body is used as to the method for vaccine.

However excretion body is self-assembling formation in the cell.Therefore, though coding cargo protein gene be inserted into it is endogenous Property generate excretion body cell in, it is difficult to thus prepare the excretion body for the albumen for being included in expression.

The present invention provides the methods for producing more efficiently at the excretion body for including cargo protein.As a result, the present inventor By being expressed in large quantities by excretion body specific marker object and cargo protein in the cell that high concentration endogenous generates excretion body The fusion protein of composition is successfully prepared the excretion body (Fig. 5) for effectively including cargo protein.

According to the above method, cargo protein is attached on excretion body film.Therefore, by excretion body specific marker object and cargo Albumen expresses the fusion protein of composition in the cell that high concentration generates excretion body, then passes through radiation-induced fusion protein Connection.Then, fusion protein is imported in excretion body by the effect of excretion body specific marker object.Irradiation is terminated after importing When, fusion protein is separated into cargo protein and light binding proteins specific in excretion body.As a result, can effectively prepare and include It is isolated from the excretion body (Fig. 6) of the free cargo protein of fusion protein.

The cargo protein loaded in the present invention in excretion body includes but not limited to natural or non-native protein, clipped form Or mutant form.The example of cargo protein is listed in down, but not limited to this.

[table 1]

＜ enzymes ＞

Enzyme is to speed up the living things catalysis molecule chemically reacted in biologic artifact.Enzyme and its Binding Capacity, and pass through reduction Its activation energy promotes reaction rate.Enzyme can be divided into following a few classes：Protease, nuclease, hydrolase, kinases, phosphatase and other The enzyme of type.

The target protein loaded in excretion body in the present invention includes enzyme and its instrumentality.It lists in the following description The example of target protein, but not limited to this.

Protease and its inhibitor

MMP and TIMP

Matrix metalloproteinase (MMP), also known as stromatin (matrixin), are Ca-dependent endopeptidases containing zinc.MMP energy It enough degrades various extracellular matrix proteins, and the known cutting for participating in cell surface receptor, apoptosis ligand (such as FAS ligands) Release and chemotactic factor (CF)/cell factor inactivation.MMP is recognized as playing a major role in cell behavior, such as cell Proliferation, Migrate (adherency/dispersion), differentiation, angiogenesis, apoptosis and host defense.

Matrix metalloproteinase is inhibited by specific endogenous metalloproteinase tissue depressant (TIMPs), the inhibitor Include the family of four kinds of protease inhibitors：TIMP1, TIMP2, TIMP3 and TIMP4.

The balance of MMP and TIMP with various physiology or pathologic process (such as form generation, angiogenesis, tissue repair, Hepatic sclerosis, arthritis and transfer) it plays an important role in relevant tissue remodeling.MMP-2 and MMP-9 is considered very heavy in transfer It wants.MMP-1 is considered critically important in rheumatoid arthritis and osteoarthritis.The imbalance balanced between MMP and TIMP is also The feature of acute and chronic angiocardiopathy.

Including the excretion body of MMPs and TIMPs by high concentration generate excretion body cell in great expression by excretion body It is prepared by the fusion protein of specific marker object and target protein composition.The excretion body for being loaded with MMPs or TIMPs can be used for treating MMP Relevant disease, including rheumatoid arthritis.

Caspase and its inhibitor

Caspase (cysteine-aspartic protease, cysteine aspartase or cysteine dependence Aspartic acid orients protease) it is a protease family, it plays an important role in apoptosis, programmed cell is dead It dies and dies (pyroptosis) and necrosis including Apoptosis, cell coke.These form of cell death are for protecting organisms from Stress signal and pathogenic challenge it is extremely important.Caspase also works in inflammation, it directly acts on proinflammatory disease Cell factor, such as pro-IL1 β.Exactly these signaling molecules allow immunocyte to raise to infected cell or tissue.Guang Its protease also has the function of that other are determined, such as cell Proliferation, tumor suppression, cell differentiation, neurodevelopment, aixs cylinder induction And aging.

The reason of Caspase defect has been confirmed as tumor development.Tumour growth can be due to the combination of many factors Occur, include the mutation of cell cycle gene, this eliminates the limitation to cell growth；And the mutation of apoptotic proteins, it is such as logical The Caspase for crossing in excrescent cell inducing cell death to respond.

On the contrary, the excessive activation of certain Caspases such as caspase-3 mRNA can cause excessive programmed cell dead It dies.This is found in several neurodegenerative diseases of nerve cell loss, such as Alzheimer disease.It is related to handling inflammatory signals Caspase it is also related with disease.These Caspases activation deficiency can increase neurological susceptibility of the organism to infection, because It may not be activated for suitable immune response.The important function that Caspase plays in cell death and disease causes Using Caspase as the research of drug target.For example, inflammatory Caspase -1 and leading to autoimmune disease It is related.

Including the excretion body of Caspase and its inhibitor passes through the big scale in the cell that high concentration generates excretion body It is prepared up to the fusion protein being made of excretion body specific marker object and target protein.It is loaded with Caspase or its inhibitor Excretion body can be used for treating and the relevant disease of Caspase, including neurodegenerative disease or autoimmune disease.

Cathepsin and its inhibitor

Cathepsin is the protease being found in all animals and other organisms.This family about more than ten A member, they are distinguishing on the albumen of structure, catalyst mechanism and their cuttings.Most of member is in lysosome It was found that low pH under be activated.Therefore, the activity of this family is almost in these organelles.

Cathepsin and cancer, apoplexy, Alzheimer's disease, arthritis, Ebola virus, COPD, Chronic periodontal Inflammation, pancreatitis are related with some eye diseases including keratoconus.Especially for cancer, cathepsin D is A kind of mitogen, it reduces the anti-tumor immune response of decaying chemotactic factor (CF), to inhibit the function of dendritic cells.Tissue Cathepsin B and L participate in substrate degradation and cell invasion.

Excretion body including cathepsin and its inhibitor passes through the big scale in the cell that high concentration generates excretion body It is prepared up to the fusion protein being made of excretion body specific marker object and target protein.It is loaded with cathepsin or its inhibitor Excretion body can be used for treating various cathepsin relevant diseases, including cancer and Alzheimer's disease.

Nuclease

Cre recombinases

Cre recombinases are the albumen detached by P1 bacteriophages, and induce weight by detecting two different regions loxP Group.LoxP is the DNA fragmentation with 34bp, by two 13bp palindromic sequences on both ends and the 8bp unsymmetric kernels in centre Introns form.Cre recombinases are attached to palindromic sequence, change the introns of DNA after cutting, then recombinant DNA.Based on interval The directionality of son is cut off between two different regions loxP or inversion DNA sequence dna.If the direction in the regions loxP it is identical or On the contrary, excision or inversion then occur respectively.

[being lacked by the DNA of Cre recombinases]

One of the representative example that Cre recombinases utilize is the mutation period that can inhibit specific gene and the tissue of expression Conditional gene knockout mouse.The mouse that the technology is inserted by generating loxP between the front end and end of specific target gene, It mates with the transgenic mice of expression Cre, or Cre recombinases is directly acted on into specific cells, it is thin to remove certain separation Specific target gene in born of the same parents.Conditional gene knockout mouse can effectively determine the work(of specific gene by expressing this gene Can, this all has mortality during embryonic development early stage, Late Embryogenesis or adult.

The present invention provides the excretion bodies for being loaded with Cre recombination zymoproteins, and confirm that Cre recombination zymoproteins are delivered to target The cytosol of cell.The result shows that the excretion body for being loaded with the present invention of Cre recombination zymoproteins can be used for conditional gene behaviour Make.

Crispr/Cas9

CRISPR-Cas9 is a kind of artificial restriction enzyme based on RNA so that is carried out by the specific region of repressor gene DNA corrections are possibly realized.Recently, it is received significant attention as the key element of genetic engineering.

CRISPR is a kind of palindromic sequence, is the short palindrome repetitive sequence (Clustered in Regularity interval Regularly-interspaced short palindromic repeats) abbreviated form and the bacterium that arrives of first observed Acquired immune system.First, Cas9 albumen identifies and limits intrusive viruses.Then the virus sequence of limitation is inserted into CRISPR In sequence, and combining virus and the sequence of CRISPR are transcribed into RNA.The RNA is used to form Cas9 compounds.By this After process, " the CRISPR+ virus sequences " of transcription is combined with Cas9, and quickly removes identical invasion than individual Cas9 Virus.Target sequence is limited by the combination of target sequence and Cas9 compounds, which can be applied in genetic engineering.

Cpf1 is egg of the Cas9 albumen in the restriction endonuclease CRISPR-Cas9 systems with above-mentioned engineering with identity function In vain.As shown below, different from Cas9, Cpf1 identification prototype intervening sequences adjoin motif (PAM, protospace adjacent Motif) sequence.It can be used for the region of Cas9 None- identifieds, and particularly it is more practical, because of individually crispr RNA (CrRNA) it can work.TracrRNA is also needed in the case of Cas9.

[comparison between Cas9 and Cpf1 albumen]

The present invention provides the excretion bodies for being loaded with Cas9 or Cpf1 albumen, and confirm Cas9 or Cpf1 protein deliveries extremely The cytosol of target cell.The result shows that be loaded with Cas9 or Cpf1 albumen the present invention excretion body can be used for removing, add or Change the segment of DNA sequence dna.

Caspase activates DNA enzymatic

It is by DFFB bases in the mankind that Caspase, which activates DNA enzymatic (CAD) or DNA fragmentation factor subunit β (DFFB), Because of the albumen of coding.It decomposing D NA and promotes cell differentiation in apoptosis process.It is typically to inhibit non-live by ICAD Property monomer.It is cut before dimerization.

Apoptosis is the process of cell death that toxic and/or useless cell is removed during mammalian development, carefully Born of the same parents' apoptotic process is along with the contraction of cell and nucleus and fragmentation and chromosomal DNA degradation at nucleosomal units.DNA pieces The sectionization factor (DFF) is the heterodimeric protein of 40-kD (DFFB) subunits and 45-kD (DFFA) subunit.DFFA is Guang day albumen The substrate of enzyme -3, and in apoptosis process trigger DNA fragmentation.When DFFA is cracked by Caspase -3, DFF quilts Activation.Active component DFFB dissociation of the cutting segment of DFFA from DFF.It has been found that DFFB triggers DNA pieces during Apoptosis Sectionization and Chromatin condensation.

By high concentration generate excretion body cell in great expression by excretion body specific marker object and target protein group At fusion protein come prepare include Caspase activate DNA enzymatic excretion body.It is loaded with the outer of Caspase activation DNA enzymatic Body is secreted to can be used for adjusting the Apoptosis in different system.

Hydrolase

Include the lysosomal enzyme of β-glucocerebrosidase

Lysosomal storage disease is since lysosome congenital deficiency leads to the disease of the storage of lysosomal degradation substance.It is common One of lysosomal storage disease be Gaucher disease (Gaucher disease), by lysosomal enzyme β-glucocerebrosidase (GBA) base Because of defect induction.

By storing glucocerebrosidase/glucose vaginula ammonia on macrophage lysosome, GBA lacks induction liver, spleen, bone The dysfunctions such as marrow.It also can induce hematological abnormality, such as anaemia, decrease of platelet, Neuroleptic Leukocytopenia, hepatosplenomegaly (gepatolientalny), osteoclastic, central nervous system injury etc..

At present to the treatment of Gaucher disease be by be injected intravenously think by praise the enzyme of (cerezyme, a kind of GBA analogs) and replace For therapy.However, this kind of protein drug has the shortcomings that a variety of, such as efficiency caused by half-life short, antibody tormation in blood It is low, be difficult to be delivered to lysosome and neurogenicity Gaucher disease etc. cannot be applied.

The present invention provides the excretion bodies for being loaded with GBA (β-glucocerebrosidase) albumen, and confirm GBA (β-glucose brain glycosides Lipase) albumen is delivered to the cytosol of target cell.The result shows that being loaded with this hair of GBA (β-glucocerebrosidase) albumen Bright excretion body can be used for treating Gaucher disease.

Kinases and phosphatase

Mitogen-activated kinase modulator：P38MAP kinases

P38 blocking effect of mitogen activated protein kinases is a kind of Mitogen-actived protein kinase (MAPK), responds stress stimulation, Such as irradiation of cell factor, ultraviolet light, heat shock and osmotic shock.P36MAP kinases participates in cell differentiation, apoptosis and autophagy.

P38MAP kinases (MAPK) participate in control to cell factor and stress cell response signal cascade react. Seek P38 inhibitor to autoimmune disease and the possible therapeutic effect of inflammatory process.

Including the excretion body of p38MAPK and its inhibitor by high concentration generate excretion body cell in great expression by It is prepared by the fusion protein of excretion body specific marker object and target protein composition.Load p38MAPK or the excretion body of its inhibitor It can be used for treating p38MAPK relevant diseases, including autoimmune disease.

Repressor kappa B kinases (IKK)

I kappa b kinases (IKK) are to participate in promoting the multienzyme complex to the cell response of inflammation.I kappa b kinase compounds are upstreams A part for NF- κ B signal transductory cascades.The nuclear localization signal that I κ B α (kB inhibitor) albumen passes through masking NF- kB proteins (NLS) and it is made to keep inactive state in cytoplasm so that NF- κ B transcription factors inactives.IKK inhibition of phosphorylation I κ B α Albumen.This phosphorylation causes I κ B α to be dissociated from NF- κ B.Free NF- κ B move to nucleus and activate at least 150 now The expression of gene；Some of them are anti-apoptotics.

Activation for Nuclear factor κB-kB (NF- κ B) family member, I kappa b kinase activity is crucial, in lymph It plays an important role in Culture in vitro.The activation of typical NF- κ B accesses starts to the various responses for promoting inflammatory stimulus Release (such as tumor necrosis factor (TNF) or white of (lipopolysaccharides for being included in pathogen surface expression) or proinflammatory cytokine Cytokine -1 (IL-1)).After immunocyte stimulation, signal transduction cascades the activation for causing IKK compounds, the spy of this event Sign is that NEMO is combined with homologous kinase subunit IKK- α and IKK- β.

Although having function adaptability in inflammatory stimulus reaction, the imbalance of NF- κ B signals transduction is in various diseases It is utilized in diseased state.In atherosclerosis, asthma, rheumatoid arthritis, inflammatory bowel disease and multiple sclerosis Observe that NF- kB activities caused by the I κ B α phosphorylations mediated by composing type IKK increase in development.Particularly, composing type NF- κ B Activity promotes lasting Inflammatory Signal Transduction on a molecular scale, and chronic inflammation is converted into phenotype.In addition, NF- κ B are simultaneously Apoptosis and the ability of promotion lymphocyte continued propagation and proliferation is inhibited to explain it close with multiple types cancer System.

Including the excretion body of IKK by high concentration generate excretion body cell in great expression by excretion body specificity mark The fusion protein that object and target protein form is remembered to prepare.The excretion body for being loaded with IKK can be used for treating NF- κ B relevant diseases, including Cancer.

PTEN phosphatases

Phosphatase and tensin homologue (PTEN) are identified as tumor suppressor protein.The mutation of this gene is many One step of cancer development.The albumen is comprising tensin spline structure domain and is similar to dual specificity protein tyrosine phosphatase The catalyst structure domain of enzyme.Different from most of Protein-tyrosine-phosphatases, the albumen is preferentially by phosphoinositide substrate dephosphorylation. Its negative regulator endocellular phosphorus acyl inositol -3,4, the Intracellular levels of 5- triphosphoric acids, and pass through negative regulator Akt/PKB signal paths Play the function of tumor inhibitor.

PTEN lacks or is mutated closely related with cancer, the formation of non-cancerous tumor and autism.Especially in the tumor development phase Between, the mutation of PTEN and missing can make it lose enzymatic activity, increase so as to cause cell Proliferation and cell death is reduced.PTEN frequencies Numerous genetic inactivation betides collagen blastoma, carcinoma of endometrium and prostate cancer；In many other tumor types such as lung Find that PTEN expression is reduced in cancer and breast cancer.In addition, PTEN is mutated the various cancer genetic predispositions also resulted in.

The mutation of PTEN genes results in other several diseases, such as cowden's syndrome (Cowden syndrome), feature It is the non-carcinomatous tumor for occurring to be known as hamartoma.These diseases include that spot is received and raises-Lai Li-Lu Waer kappa syndromes (Bannayan-Riley-Ruvalcaba syndrome) and proteus sample syndrome (Proteus-like syndrome). Disease is referred to as PTEN hamartomas or PHTS caused by PTEN mutation.The mutation of these syndromes is caused to lead to generation Albumen nonfunctional is not present.This deficient protein allows cell to divide in an uncontrolled fashion and hinder the dead of damaged cell It dies, causes tumour growth.

Including the excretion body of PTEN by the cell that high concentration generates excretion body great expression by excretion body specificity It is prepared by the fusion protein of marker and target protein composition.The excretion body for being loaded with PTEN can be used for treating different types of cancer.

Janus kinases

Janus kinases (JAK) is intracellular nonreceptor tyrosine kinase family, passes through JAK-STAT access transducer cells The factor mediates signal.Due to the member of I types and II cytokines receptor families do not have catalytic kinase activity, they according to Rely JAK Family Tyrosine Kinases phosphorylation and activates the downstream albumen for participating in its signal transduction pathway.Receptor and its respectively Cell factor/ligand binding after, undergo conformation change, keep two JAK close enough with phosphorylation each other.

The conformation change that JAK autophosphorylations induce its own internal, can pass through further phosphorylation and activation The transcription factor of referred to as STATs (signal transduction and transcription swash the factor) carrys out signal in transducer cell.The STATs of activation divides from receptor From and form dimer before being transferred to nucleus, the transcription of selected genes is adjusted there.

Some examples using the molecule of JAK/STAT signal paths be colony stimulating factor, prolactin, growth hormone and Many cell factors.Developing JAK inhibitor for treat psoriasis, rheumatoid arthritis, polycythemia vera, Baldness, primary thrombocytosis, ulcerative colitis, marrow metaplasia and leucoderma with myelofibrosis.

Excretion body including JAK and its inhibitor by high concentration generate excretion body cell in great expression by excretion It is prepared by the fusion protein of body specific marker object and target protein composition.The excretion body for being loaded with JAK or its inhibitor can be used for controlling Treat JAK relevant diseases, including cancer.

It is other

Ubiquitin ligase

Ubiquitin ligase (also referred to as E3 ubiquitin ligases) is a kind of albumen, raises the E2 ubiquitin combination for being loaded with ubiquitin Enzyme, identify protein substrate and assist or directly catalysis ubiquitin from E2 to protein substrate transfer.Ubiquitin passes through isopeptide bond and target protein On lysine connection.E3 ligases and target protein and E2 enzyme interactings, therefore assign specificity of the substrate to E2.

The ubiquitination of E3 ligases adjusts multiple fields, and such as cell transport, DNA is repaired and signal transduction, is given birth in cell Have far-reaching significance in object.E3 ligases be also the cell cycle control crucial participant, mediated cell cycle element with And the degradation of cell cycle cyclin-dependent kinase inhibitors.

E3 ubiquitinbond enzyme adjustments homeostasis, cell cycle and DNA repair approach, and therefore, these many albumen participate in Various cancers, including famous MDM2, BRCA1 and Von Hippel-Lindau tumor inhibitors.For example, in gastric cancer, nephrocyte It is found that the mutation of MDM2 in cancer and liver cancer (s), by increasing MDM2 promoters to the affinity of Sp1 transcription factors, causes MDM2mRNA transcriptions increase, to make MDM2 concentration lack of proper care.

Including the excretion body of ubiquitin ligase by high concentration generate excretion body cell in great expression by excretion body Specific marker goes the fusion protein formed with target protein to prepare.The excretion body for being loaded with ubiquitin ligase can be used for treating ubiquitin Change relevant disease, including cancer.

Luciferase

Luciferase is the common name of a kind of oxidizing ferment for generating bioluminescence, usually different from luminescent protein.Luciferase It is widely used in biotechnology, microexamination and is used as reporter gene, and many application identical with fluorescin. However, different from fluorescin, luciferase does not need external light source, but needs to add fluorescein (consumable bottom really Object).

All luciferases are classified as oxidoreducing enzyme (EC 1.13.12.-), it means that they pass through binding molecule Oxygen acts on single donor.Since luciferase is from many incoherent different protein families, ununified machine System, because any mechanism all relies on the combination of luciferase and luciferin.However, the luciferin of characterizations all so far Enzyme-luciferin reaction is all shown needs molecular oxygen in some stage.

In biological study, luciferase is typically used as intelligencer and is transfected under the control of interested promoter to assess Transcriptional activity in the cell of genetic constructs containing luciferase genes.In addition, being converted into fluorescein under the activity of certain enzyme Preceding fluorescent molecular (pro-luminescent molecules) can be used for detect coupling or two step luciferase assays in enzyme Activity.Such substrate has been used for detection caspase activity and Cytochrome P450 activity etc..

Luciferase can also be used for detecting cellular ATP levels in cell viability measurement or kinase activity measure.Fluorescein Enzyme can be used as ATP sensor proteins by biotinylation.Biotinylation is by combining strepto- Avidin-Biotin compound will Luciferase is fixed on cell surface.This allows luciferase to detect the outflow of ATP in cell, and will have by bioluminescence Effect shows the real-time release of ATP.Increase luminous intensity, luciferase by changing certain amino acid residues in protein sequence It can be extraly sensitiveer to the detection of ATP.

Can be used the cell line injection of expressing luciferase carry out entire animal imaging (be known as in vivo or once in a while in vitro at Picture).Different types of cell (such as stem cell, T cell) can be designed with expressing luciferase, allow it to use sensitive Ccd camera (CCD camera) Noninvasive visualization is carried out in mobiles.The technology has been used to track Tumour occurs and response of the tumour to treatment in animal model.

The present invention is prepared for being loaded with the excretion body of luciferase protein, and confirms that luciferase protein is delivered to target cell Cytosol.The result shows that the excretion body for being loaded with the present invention of luciferase protein can be used for cell viability analysis, kinases is lived Property analysis and entire animal imaging.

Peroxiredoxin

Peroxiredoxin (Prx) is antioxidase representative in cytoplasm, in mammalian cell The water-solubility protein of middle acquisition 0.1~0.8%.Prx has in cell is reduced into H by receiving 2e- by hydroperoxides₂O With the effect of ROH-.By participating in H₂O₂The formation and removing of (nmol concentration), Prx also participate in cell Proliferation, differentiation, death and Cell signalling.Quantity based on cysteine amino acids, Prx are more specifically classified as 1-Cys Prx or 2-Cys Prx. In addition, the difference based on structure and mechanism, 2-Cys prx are subdivided into " typical case " or " atypia ".All three Prx are in Cys- It is variant in terms of redox during the second of SOH formation.Prx I-Prx IV are typical 2-Cys Prx, Prx V right and wrong Typical 2-Cys Prc, and Prx VI are 1-Cys Prx.Certain 2-Cys Prx form oligomer.

By adjusting the H that growth factor and TNF-α generate in cell₂O₂Concentration, Prx I and II participate in receptor signal and turn The activation of pathway.Particularly, Prx II, which have, protects cells from cell death induction factor such as serum starvation, neural acyl The effect of the stimulation of amine and Etoposide (epotoside).

In normal cell, Prx I maintain the activity of PTEN phosphatases by inhibiting the oxidation of PTEN phosphatases.But In the case of oxidative stress is increased, H₂O₂Prx is detached from PTEN by irreversible oxidation, to inhibit the activity of PTEN.Cause This, tumour is induced by sustained activation cell proliferation signals such as Akt.

The quantity variation of Prx has significant relationship with disease in cell.In cancer, artery sclerosis, respiratory inflammation, bone During loose, fat, degenerative dementia development of matter etc., the variation of active oxygen quantity has close contact.

The present invention provides the excretions for being loaded with Peroxiredoxin I or Peroxiredoxin II albumen Body, and confirm Peroxiredoxin I or Peroxiredoxin II albumen be delivered to target cell cytoplasm it is molten Glue.The result shows that being mounted with the excretion of the present invention of Peroxiredoxin I or Peroxiredoxin II albumen Body can be used for therapeutic activity oxygen relevant disease.

＜ transcription factors ＞

Transcription factor is that albumen of the transcription from the mRNA of DNA is adjusted in eucaryote.Transcription factor and basal transcription tune Section, organismal development, the response of intercellular signal or environment, cell cycle are controlled it is related with pathogenesis.

The target protein being supported in excretion body of the present invention includes transcription factor and its regulator (enhancer or repressor). The example of target protein is listed in following description, but not limited to this.

Transcription factor and its regulator

NF-kB regulators, super repressor IkB

NF- κ B are to induce the central transcription factor of inflammatory response, and adjust inflammation-related gene and be especially in various cells Expression in immunocyte.Therefore, the NF- κ B signals Signal Transduction Pathways of overacfivity can become in selective depression immunocyte The effective therapeutic strategy for the chronic inflammatory diseases that can not be cured, such as rheumatoid arthritis, septicemia and psoriasis.Separately Outside, the activation of NF- κ B has the function of inhibiting Apoptosis by increasing the expression of cloning.From this effect From the point of view of, the reason of sustained activation of NF- κ B signal Signal Transduction Pathways is anticancer drug drug resistance in cancer, and then reduce anti- The therapeutic effect of cancer drug.

Most of NF- κ B by with the inhibition protein I κ B of NF- κ B in normal cell in conjunction with by be in unactivated state.By Various stimulants such as TNF-α and I kappa b kinases (IKK) compound of LPS activation are by I κ B phosphorylations.Then the I κ B of phosphorylation are general Elementization is simultaneously finally degraded by proteasome.By the degradation of I κ B, the NF- κ B (p50/p65) being incorporated on I κ B pass through nuclear membrane.It is logical Later, NF- κ B (p50/p65) activate mRNA to transcribe by the promoter region of syncaryon target gene.This is inducing cell The critical elements of the factor and the immune response of inflammatory mediator (such as iNOS, COX-2, NO, PGE2, TNF-α and IL-1) transcription (Lappas etc., Biol.Reprod.67：668673,2002).

Super repressor I κ B are S32A the and S36A mutant forms of I κ B, can continue inhibit NF- κ B because it not by I kappa b kinase phosphorylations, and do not degraded by proteasome.Therefore, very big as the potentiality for treating various diseases associated with inflammation.

The present invention provides the excretion bodies for being loaded with super repressor I kB proteins, and confirm that super repressor I kB proteins are delivered to The cytosol of target cell.The result shows that be loaded with super repressor I kB proteins the present invention excretion body can be used for treating it is inflammatory Disease.

MyoD

MyoD is a kind of albumen to play a crucial role in adjusting muscle differentiation.MyoD belongs to referred to as muscle-derived regulatory factor (MRF) protein family.Known MyoD has binding interactions with hundreds of muscle cdna promoters and allows sarcoblast Proliferation.One of major function of MyoD is by enhancing the transcription of p21 and myogenin by cell from the cell cycle It removes.

Including the excretion body of MyoD albumen by the cell that high concentration generates excretion body great expression it is special by excretion body It is prepared by the fusion protein of specific labels and target protein composition.The excretion body for being loaded with MyoD can be used for treating sarcoblast correlation Disease.

Tbx18 (T-box transcription factors 18)

The member for the transcription factor family guarded on Tbx18 coding evolutions, plays vital work in embryonic development With.Tbx18 is characterized in that there are DNA combination T-box structural domains, and its specificity T bx1 subfamily that belong to vertebrate. Tbx18 serves as transcription repressor by the activating transcription factor in antagonism T-box families.It was developed in tissue and the various of organ Tbx18 is needed in journey, tissue and organ include heart and coronary vasodilator, ureter and backbone.In sinoatrial node (SAN) head zone It is also required to Tbx18.

Tbx18 transductions are a kind of methods for opening gene in cardiac muscle cell, as the method for treating certain arrhythmia cordis. In the heart of health, pacemaker of the sinus node cells as heart, and make the regular bounce of heart.Sick sinus syndrome The problem of be that SA knot functions are abnormal, lead to cardiac arrhythmia.Tbx18 expression is entered into atrial muscle cell using adenovirus, by the heart Room myocyte is converted into the SA nodal cells for starting heartbeat.Tbx18 can be treat arrhythmia cordis several genes form of therapy it One.

Including the excretion body of Tbx18 albumen by the cell that high concentration generates excretion body great expression it is special by excretion body It is prepared by the fusion protein of specific labels and target protein composition.The excretion body for being loaded with Tbx18 albumen can be used for treating ill sinus Room knot syndrome.

P53

Known cancer Proteins p53 is the defender of genome, because it preserves genome by preventing genome mutation Stability.When DNA is damaged, p53 can activate DNA repair protein.In addition, p53 can be by will protect the cell cycle The G1/S point of adjustment that identifies in DNA damage is held to inhibit to grow.In the case of DNA damage and unrepairable, p53 can induce thin Born of the same parents' apoptosis.Finally, p53 is most important to the aging response of short end grain.P53 responses it is countless stress and be activated, stress include DNA Damage, oxidative stress, osmotic shock, oncogene expression ribonucleotide consumption and lacked of proper care.

If p53 is impaired, tumor suppression is severely damaged.The people that only P53 gene functions copy there are one heredity is very Tumour may be suffered from adult early stage.The content for increasing p53 may be the solution treated tumour or prevent its diffusion.

Including the excretion body of p53 albumen by high concentration generate excretion body cell in great expression it is special by excretion body Property marker and the fusion protein of target protein composition prepare.Be loaded with p53 albumen excretion body can be used for treating it is various types of Cancer.

HMGB1 (high mobility group protein B 1)

Such as histone, HMGB1 is one of most important Chromatin Protein.In nucleus, HMGB1 and nucleosome turn Record the factor and histone interaction.This nucleoprotein tissue DNA simultaneously adjusts transcription.In conjunction with rear, HMGB1 curved DNAs, this is advantageous In the combination of other albumen.It also interacts with nucleosome so that the DNA accumulated is loose and remold chromatin.

HMGB1 is secreted by immunocyte by amitosis approach.The macrophage and monocyte of activation secrete HMGB1 Cell factor medium as inflammation.The antibody for neutralizing HMGB1 is assigned for arthritis, colitis, ischaemic, septicemia etc. The damage of period and the protective effect of tissue damage.

Including the excretion body of HMGB1 albumen by the cell that high concentration generates excretion body great expression it is special by excretion body It is prepared by the fusion protein of specific labels and target protein composition.Be loaded with HMGB1 albumen excretion body can be used for treating it is inflammatory Disease.

NeuroD1

Neurogenicity differentiation 1 (also referred to as β 2) is the transcription factor of NeuroD types.Its promoter by combining the box containing E Shared core sequence 5'-CANNTG-3' carrys out mediate transcription activation.It helps to adjust several cell differentiation approach.It promotes early Phase retinal ganglial cells, inner ear sensory neuron, granular cell, the pancreas for forming cerebellum or the dentate fascia cellular layer of hippocampus Endocrine islet cell and small intestine enteroendocrine cell.

Including the excretion body of NeuroD1 albumen by high concentration generate excretion body cell in great expression by excretion body It is prepared by the fusion protein of specific marker object and target protein composition.The excretion body for being loaded with NeuroD1 albumen can be used for adjusting god It is developed through member.

The relevant macrophage of tumour (TAM) is a kind of cell belonging to macrophage system.They are located near tumor mass Or in tumor mass.For TAM from the monocyte or fixing organization macrophage of cycle, they are formed in many tumor types The major leukocyte that Medium Culture is found infiltrates object.The poor prognosis of TAM and breast cancer, oophoroma, glioma and lymphoma type It is related, it is related with colon cancer and the preferable prognosis of gastric cancer, and all with the good and undesirable prognosis of lung cancer and prostate cancer It is related.

It is two kinds of main phenotypes, M1 and M2 that TAM, which is divided to,.M1TAM inhibits cancer progression, and M2TAMs promotes cancer progression. Several transcription factors are related with the transformation of M2 macrophages to M1 macrophages.The target protein packet being supported in excretion body of the present invention It includes and converts related transcription factor with the M2 to M1 of macrophage.The example of target protein is listed in the following description, but not It is limited to this.

IRF5

IRF 5 is the member of interferon regulatory factor (a kind of transcription factor).Its virus-mediated interferon activation and Cell growth, differentiation, apoptosis and immune system activity play an important roll in adjusting.IRF5 by directly with DNA or other eggs White interaction carrys out work.

Whether IRF5 promotes or inhibits inflammation as molecular switch, control macrophage.Block the production of IRF in macrophage Life can help to treat extensive autoimmune disease, and raise IRF5 levels can help to treat immune system it is weak or impaired People.

Including the excretion body of IRF5 albumen by the cell that high concentration generates excretion body great expression it is special by excretion body It is prepared by the fusion protein of specific labels and target protein composition.Be loaded with IRF5 albumen excretion body can be used for it is huge from M2 to M1 Phagocyte converts, for treating various types of cancers.

IRF3

IRF 3 is the member of interferon regulatory factor, is one group of transcription factor.IRF 3 includes functional domain, core output letter Number, DNA binding domain, C-terminal IRF associated domains and several regulatory sites.It is in the cytoplasm of non-infected cells with the shape of inactivation Formula exists.When virus infection, double-stranded RNA or when Toll-like receptor signal transduction, IKBKE and 1 kinases of TBK are by its phosphorylation.This Lead to dimerization and nuclear location.IRF 3 can be with gene expression program different in activating macrophage.

Including the excretion body of IRF3 albumen by the cell that high concentration generates excretion body great expression it is special by excretion body It is prepared by the fusion protein of specific labels and target protein composition.Be loaded with IRF3 albumen excretion body can be used for it is huge from M2 to M1 Phagocyte converts, for treating various types of cancers.

STAT1

Signal transduction and activating transcription factor 1 are transcription factors, are the members of stat protein family.STAT1 can be several Kind ligand such as interferon-' alpha ', interferon gamma, epidermal growth factor, platelet derived growth factor or interleukin-6 activation.

After I types IFN is combined with cell surface receptor, Jak kinases is activated and phosphorylation STAT1 and STAT2.STAT dimerization Change and is combined to form the compound of referred to as ISGF3 transcription factors with ISGF3G/IRF-9.The response member of ISGF3 combinations IFN stimulations Element is to activate the transcription of the gene of IFN stimulations.

Response II types IFN, STAT1 are tyrosine and serine phosphorylation compound.It forms homodimer and is activated with IFN γ Sequence combine to drive the expression of target gene, inducing cell antiviral state.

Including the excretion body of STAT1 albumen by the cell that high concentration generates excretion body great expression it is special by excretion body It is prepared by the fusion protein of specific labels and target protein composition.Be loaded with STAT1 albumen excretion body can be used for treating it is various types of The cancer of type.

SOCS3

Suppressor of cytokine signaling is the member of the STAT inhibitor of STAT inductions.The STAT of STAT inductions inhibits Agent is the cytokine induction type negative regulator agent of cytokine signaling.SOCS3 by cytokine profiles such as IL6, IL10 and IFN-γ induces.

The overexpression of SOCS3 inhibits the insulin signal transduction in adipose tissue and liver, but does not inhibit in muscle Insulin signal transduction.But SOCS3 is lacked in the skeletal muscle of mouse can prevent obesity.Increase since ceramide synthesizes Add, SOCS3 additionally aids leptin resistance and insulin resistance.Studies have shown that removal SOCS genes can prevent the pancreas in obesity Insulin resistance.SOCS3 albumen can be combined with JAK2 kinases and be inhibited the activity of JAK2 kinases.

Including the excretion body of SOCS3 albumen by the cell that high concentration generates excretion body great expression it is special by excretion body It is prepared by the fusion protein of specific labels and target protein composition.Be loaded with SOCS3 albumen excretion body can be used for treating it is various types of The cancer of type.

＜ antibody ＞

Antibody is the albumen for identifying and being combined with its specific antigen by the Y-shaped antibody tip variable regions Fab.Antibody can To inhibit the activity of target antigen albumen by connection.

The target protein being supported in excretion body of the present invention includes antibody and antibody related peptide.It lists in the following description The example of target protein, but not limited to this.

Antibody and related peptide

PySTAT3 intracellular antibodies

STAT (signal transduction and transcription) is the transcription factor having determined：STAT1、STAT2、STAT3、STAT4、 STAT5 (STAT5A and STAT5B) and STAT6.STAT3 albumen has the C-terminal transcriptional activation domain (major phosphate of STAT3 Change site is 727 residue of 705 residue of tyrosine and serine).Tyrosine phosphorylation and subsequent STAT3 dimerizations promote to thin The activation of transport and the transcription of karyon.

JAK/STAT3 signal transduction pathways in the activation of the interferon signal transduction of growth factor-induced be identified and It participates in proliferation, differentiation, apoptosis, angiogenesis, tumour generation and is immunized.Therefore, for developing anticancer drug, STAT3 albumen can Using the good target spot as single medicine or combined therapy drug.

The present invention is prepared for being loaded with the excretion body of pYSTAT3 intracellular antibodies, and confirms that pYSTAT3 intracellular antibodies are delivered to The cytosol of target cell.The result shows that the excretion body for being loaded with the present invention of pYSTAT3 intracellular antibodies can be used for treating cancer.

The other ＞ of ＜

The relevant albumen of Apoptosis

Apoptosis (apoptosis) is the process of damaged cell to be removed by various factors, and abnormal cell withers Induction tumour is died to generate.Based on this reason, the research about inducing apoptosis of tumour cell is as the strategy for removing tumour And actively it is in progress.Chromatin condensation caused by cellular atrophy, apoptotic body are formed and DNA fragmentation is the feature of Apoptosis. Apoptosis is induced by two kinds of different approaches；One is inherent approach by mitochondria, the other is passing through death receptor External approach.Apoptosis is by various adjustings, such as promotees the activating of Apoptosis Bcl-2 families, point of preceding Caspase Cut the fragmentation etc. with more Poly ADP-ribose polymerases (PARP).Especially belong to the Caspase of cysteine proteinase Preferment in the cell of normal proliferative, and by apoptosis-inducing signal activation, then by comprising cargo protein such as PARP has remarkable effect in Apoptosis.

The approach induction mammalian cell that most cells apoptotic stimulus object is controlled by Bcl-2 gene family members withers It dies, Bcl-2 gene families coding includes the homologous protein group of Apoptosis agonist and antagonist, such as Bcl-2 and Bcl-XL. Even if these members are discriminatively adjusted shared sequence homology structural domain if these members.During Apoptosis, bcl-2 Anti-apoptotic or rush cells apoptosis with Bax (with Bcl-2 with 21% homogeneity on protein level) is by homologous Regulate and control with heterodimer, the difference is that the ratio of Bcl-2 and Bax.

Promote apoptosis protein：Bax

Bax (the relevant X proteins of Bcl-2) is one of Bcl-2 protein families, i.e. so-called Bcl-2 samples albumen 4.Above-mentioned Bax It is combined with mitochondrial outer membrane, in the intermembrane space upper process of mitochondria, Bax has active cell apoptosis for 4 residues of C-terminal Effect.In NCBI (GenBank：NM_001291428, NP_001278357 etc.) on disclose about above-mentioned albumen and its gene The specific information of base sequence.

Bax is one of the Bcl-2 gene families that synthesis promotees apoptosis protein.Bax inhibits its turn by mutant p53 Record.It is well known that it is that Bax is expressed in the cell strain for cause blood cancer, colon cancer and the carcinoma of the rectum to be inserted into or lack Bax base sequences The reason of significant reduction.

Known Bax participates in thin caused by the apoptosis of neuronal development, the homeostasis of lymph and reproductive system, DNA damage Born of the same parents' death, ischemical reperfusion injury etc..

The present invention provides the excretion bodies for being loaded with Bax (the relevant X proteins of Bcl-2) albumen, and confirm Bax (Bcl-2 correlations X protein) in protein delivery to the cytosol of target cell.The result shows that being loaded with Bax (the relevant X proteins of Bcl-2) albumen The excretion body of the present invention can be used for treating cancer.

Anti-apoptotic proteins：BcL-xL

It is the transmembrane molecule in mitochondria by B cell lymphoma-super large (Bcl-xL) of 1 gene code of BCL2 samples.Bcl- XL is the member of Bcl-2 protein families, and playing anti-cell by preventing the release of mitochondria content such as cytochrome c withers The effect of albumen is died, the release of mitochondria content causes the activation of Caspase and eventually leads to apoptosis.

One generally acknowledged concept in Apoptosis field is that growth-promoting deposits the relative populations that Bcl-2 family proteins are deposited with antibiosis Determine whether cell can occur cell death；If there is more Bcl-xL, then hole is not for promoting Apoptosis molecule It is permeable, then cell survival.Similar with Bcl-2, it is thin that Bcl-xL participates in cancer by inhibiting the function of tumor suppressor p53 The survival of born of the same parents.

Including the excretion body of Bcl-xL albumen by high concentration generate excretion body cell in great expression by excretion body It is prepared by the fusion protein of specific marker object and target protein composition.The excretion body for being loaded with Bcl-xL albumen can be used for adjusting cell Apoptosis.

Other

Multifunctional signal molecule：AIMP (multifunctional protein of aminoacyl tRNA synthetase interaction)

The multifunctional protein 1 (AIMP1) of amide-tRNA synthesis enzyme interactings is non-catalytic group of more synthase complex Point.Stimulate the catalytic activity of cytoplasm arginyl-tRNA synthase.With inflammatory cytokine activity.By being combined with SMURF2 And inhibit the degradation that its SMAD7 is mediated to stablize SMURF2, negative regulator TGF-β signal transduction.By being lured in low glucose level It leads the secretion of glucagon and participates in the homeostasis of glucose.Promote dermal fibroblast proliferation and wound reparation.

By the migration of inducing endothelial cell at low concentrations and inducing endothelial cell apoptosis in higher concentrations, in angiogenesis In work.Inducing dendritic shape cell maturation and Adherence of Monocytes.It is adjusted by interacting degradation HIF-1A with PSMA7 Endothelial cell response.

The multifunctional protein 2 (AIMP2) of aminoacyl tRNA synthetase interaction is multi-ARS complex assembling Necessary to stability.Ubiquitination and the degradation for mediating FUBP 1 (activating transcription factor of MYC), lead to alveolar type II cells MYC necessary to differentiation is lowered.With recessive hereditary teenager's Parkinson's disease (autosomal- Recessive juvenile Parkinsonism), idiopathic parkinsonism and diffusivity lewy body disease (diffuse Lewy Body disease) intracerebral accumulation.

Including the excretion body of AIMP1 and AIMP2 albumen by high concentration generate excretion body cell in great expression by It is prepared by the fusion protein of excretion body specific marker object and target protein composition.The excretion body for being loaded with AIMP1 or AIMP2 albumen can For multi-functional adjusting.

Fluorescin (mCherry, GFP)

Fluorescin is the member of an albuminoid homologous in structure, they have unique characteristic, can oneself from its The chromophore of visible wavelength is formed in 3 amino acid sequences in the polypeptide sequence of oneself.Biologist will encode engineering fluorescence The gene (or geneic chimera) of albumen imports in living cells, then uses the position of fluorescence microscope gene outcome and moves State, this is common research practice.

The most common application of fluorescin is determined specific cells device in living cells or recombinant protein using fluorescin Position and dynamic are imaged.In order to be imaged specific organelle, the Protocols in Molecular Biology of standard is used to coding fluorescence egg White Gene Fusion is to coding known locations on the albumen of the specific cells device or the cDNA of peptide.This fusion is so that chimeric base Because a single polypeptide will be expressed as, a covalent linkage is formd between targeting motif and fluorescin.It is included in The plasmid of mosaic gene under suitable promoter control is used for transfection mammalian cell, and it is corresponding to generate then to express the gene Chimeric protein.Chimera is positioned at target cell device and fluorescence is therefore presented.By using fluorescence microscope, the shape of organelle State, dynamics and distribution can be imaged at any time.

MCherry is a kind of monomer fluorescence structure, has peak value excitation/emission at 587nm/610nm respectively.It is fast light It bleaches and stablizes.It is ripe quickly, t_0.5It it is 15 minutes, this allows it to visualize soon upon translation.

The albumen that green fluorescent protein (GFP) is made of 238 amino acid residues (26.9kDa), when being exposed to blue When light in ultraviolet ray range, emerald green fluorescence is shown.Although many other marine organisms have similar green glimmering Photoprotein, but GFP refers to traditionally being isolated from Victoria's multitube luminescent jellyfish (Aequorea victoria) first Albumen.GFP from Victoria's multitube luminescent jellyfish (A.victoria) has main excitation peak at 395nm wavelength, There is secondary excitation peak at 475nm.Its emission peak is located at 509nm, in the relatively low green portion of visible spectrum.

The present invention is prepared for being loaded with the excretion body of mCherry or GFP albumen, and confirms that mCherry or GFP albumen is passed It send to the cytosol of target cell.The result shows that the excretion body for being loaded with the present invention of mCherry or GFP albumen can be used for living carefully The positioning of excretion body and connection albumen and dynamic imaging in born of the same parents or animal.

Nucleic acid binding protein (such as RNP)

Nucleoprotein is any and the nucleic acid either relevant albumen of DNA or RNA structures.Deoxyribonucleoprotein (DNP) It is the compound of DNA and albumen.Typical example is nucleosome complexes, and wherein genomic DNA is surround in eukaryocyte core To form chromatin on the cluster of eight histones.During spermatogenesis, protamine replaces histone.In this compound Deoxyribonucleoprotein interaction adjusts compound to generate polyprotein, and the DNA intervened in the composite is surround or wound. Deoxyribonucleoprotein participates in adjusting DNA replication dna and transcription.

Ribonucleoprotein (RNP) is the compound of RNA and albumen.Telomerase, fornix ribonucleoprotein, ribonuclease P, HnRNP and small nut RNP (snRNP) and ribosomes are ribonucleoproteins.Ribonucleoprotein plays a protective role.MRNA is in cell In will not exist with free RNA molecule.They are always incorporated as ribonucleoprotein complexes with ribonucleoprotein knot and play work With.

Including the excretion body of DNP or RNP by the cell that high concentration generates excretion body great expression it is special by excretion body It is prepared by the fusion protein of specific labels and target protein composition.Be loaded with DNP or RNP excretion body can be used for science of heredity regulation and control or The excretion body of nucleic acid Transshipment Permitted.

Present invention demonstrates cargo protein is successfully delivered to target cell by using the excretion body comprising cargo protein Cytosol, and thus the present invention provides it is a kind of using excretion body effectively adjusted in cytosol Intracellular signals turn Lead the method to treat disease.

It is a further object to provide it is a kind of for prevent or treat diseases associated with inflammation comprising as activity at The pharmaceutical composition of the excretion body divided.

It is a further object to provide for prevent or treating cancer comprising excretion body as active constituent Pharmaceutical composition.

It is a further object to provide include as active constituent for prevent or treat oxygen relevant disease The pharmaceutical composition of excretion body.

It is a further object to provide include as work for generate target gene conditionity knockout allele The composition of the excretion body of property ingredient.

It is a further object to provide the combinations for including the excretion body as active constituent for manipulating DNA sequence dna Object.

It is a further object to provide for prevent or treat Gaucher disease comprising excretion as active constituent The pharmaceutical composition of body.

In order to develop the effective ways for being used to prepare the body of the excretion comprising cargo protein, tasted present inventor has performed various Examination.In our research process, inventor notices excretion body specific marker object (CD9, CD63, CD81 and CD82).This A little markers belong to four transmembrane proteins family, and typically 4 times of penetrating type memebrane proteins.The present inventor predicts, works as cargo protein When being conjugated on the memebrane protein of excretion body, cargo protein will include relatively easily inside excretion body.

It is abundant on excretion body film by cargo protein and particularly by expression in generating excretion body cell in high concentration And the fusion protein that can be formed with the excretion body marker of penetration cell film, can largely generate the excretion comprising cargo protein Body.

Particularly, the method for being used to prepare the excretion body comprising cargo protein of the present invention is characterized in that, is generating excretion The polynucleotides for the fusion protein that coding is made of excretion body specific marker object and cargo protein are imported in body cell.

At this point, in the excretion body of preparation, cargo protein melts with the excretion body specific marker object in embedded excretion body film It closes.

The cargo protein is combined with the memebrane protein of excretion body, and is not detached reaching after target cell, to understand Certainly this problem has carried out various trials, therefore develops a kind of technology, by the way that cargo protein to be temporarily conjugated with labelled protein To prepare the excretion body comprising cargo protein.For example, light binding proteins specific such as CIBN and CRY 2 can be used herein. Particularly, CIBN is expressed in the form of being merged with the CD9 as one of marker protein.Meanwhile CRY2 and cargo egg will be encoded The channel genes excretion body of white fusion protein generates in cell.Due to CD9, the CIBN-CD9 fusion proteins of expression may include In excretion body generates cell.At this point, when with blue LED light irradiating cell, the cargo expressed in cell is generated in excretion body The CRY2 structural domains of albumen-CRY2 fusion proteins are combined with the CIBN structural domains for merging CD9.As a result reversible " cargo egg is generated - CRY2-CIBN-CD9 fusion proteins in vain ".Due to CD9, this fusion protein may include inside excretion body.Once producing Include cargo protein excretion body and blue LED light irradiation terminate, then CIBN-CRY2 connections be destroyed and cargo protein with The form separated with excretion body film is retained in excretion body, and it includes the excretion body (Fig. 5~Figure 10) of cargo protein to lead to preparation.

The effect of this excretion body prepared by the method for the present invention and the conventional excretion body comprising target substance is completely not Together.Conventional excretion body is expressed as being fused on excretion body specific marker object, so that cargo protein is presented inside excretion body, Even if so that cargo protein is included in inside excretion body nor free, but being rendered as being attached on excretion body film, this table Bright cargo protein cannot from excretion body UF membrane, therefore only when excretion body fusion on the cell wall of target cell when could be by goods In object protein delivery to target cell.Moreover, even if after being fused on target cell, cargo protein is still conjugated in excretion body film On, therefore, cargo protein shows that the possibility of its effect is very low in target cell, however, the excretion body of the present invention presents one Free existing and not conjugated with the excretion body film cargo protein of kind.Therefore, when the endocytosis that this excretion body passes through target cell When effect enters cytosol, cargo protein will not be adhered on the film of excretion body, when excretion body decomposes in target cell, packet The cargo protein contained can be delivered and can be moved freely in cytosol colloidal sol in cytosol colloidal sol, this shows goods Object albumen in target cell cytoplasm colloidal sol fully active (Figure 11) with its physiological activity.

The combination level of cargo protein and labelled protein can change according to the intensity of light to be illuminated.Therefore, pass through The intensity for adjusting light, can control the concentration for the cargo protein collected in excretion body.

Preparing the method for excretion body comprising cargo protein using light binding proteins specific, there is not been reported, and for the first time by The present inventor proposes.

Particularly, the method for preparing the excretion body comprising cargo protein of the present invention comprises the steps of：(a) in excretion Body generates the polynucleotides and encoding fusion protein (fusion protein II) that encoding fusion protein (fusion protein I) is imported in cell Polynucleotides, wherein fusion protein I is made of excretion body specific marker object and the first smooth binding proteins specific, merges egg White II is made of cargo protein and the second smooth binding proteins specific that can be connect with the first smooth Specific binding proteins；(b) energy is used The light being conjugated between the first smooth binding proteins specific and the second smooth binding proteins specific irradiation excretion body is enough caused to generate thin Born of the same parents；(c) it completes to terminate irradiation after excretion body generates in excretion body produces cell.

Term " excretion body " in the present invention indicates the vesicles with plasma structure, originating from referred to as multivesicular body (MVB) it cell internal specific compartment and discharges or secretes from cell.

In the present invention, cargo protein is delivered to by target cell by self-contained cargo protein as the excretion body of carrier Or in tissue.At this point, the cargo protein that excretion body carries acts on target cell or tissue, to help to treat or diagnose specific disease Disease.

Term " excretion body generates cell " in the present invention indicates that the cell of excretion body can be generated.

In the present invention, it is unrestricted but preferably thin with bone-marrow-derived lymphocyte, T lymphocytes, dendron that excretion body generates cell For born of the same parents, megacaryocyte, macrophage, stem cell and tumour cell etc..For example, in the present invention, being used as excretion body and generating carefully The HEK293T cells of born of the same parents are a kind of immortalized cell lines.

Term " excretion body specific marker object " in the present invention indicates the albumen that excretion body film is rich in.

In the present invention, excretion body specific marker object is unrestricted, but preferably with CD9, CD63, CD81 and CD82 etc. For.For example, in a preferred embodiment of the invention, using CD9 as excretion body specific marker object.CD9、CD63、 CD81 and CD82 is 4 times of penetrating type memebrane proteins, when cargo protein is combined with the memebrane protein of excretion body, cargo protein is allowed to be easy Ground is present in excretion body.

Term " light binding proteins specific " in the present invention is also referred to as photoinduction heterodimer and forms albumen or photoinduction Homodimer formed albumen, refer to when irradiate specific wavelength light when, can by from different protein bindings formed heterodimer Or the albumen by forming homodimer with the protein binding of another same type.

In the present invention, light binding proteins specific is unrestricted, but preferably with photoinduction heterodimer formed albumen or CIB (cryptochrome-interaction base-helix-loop-helix protein), CIBN (the N-terminal structural domain of CIB), PhyB are (photosensitive Pigment B), PIF (phytochrome interaction factor), FKF1 (flavine combine, Kelch repetitive sequences, F-box 1), For GIGANTEA, CRY (cryptochrome) and PHR (plant hydrolyzed-enzyme homologous region) etc..

Particularly, when light binding proteins specific is that photoinduction heterodimer forms albumen, two types can be used Light binding proteins specific (the first and second smooth binding proteins specifics).When the first smooth binding proteins specific be CIB or When CIBN, the second smooth binding proteins specific can be CRY or PHR.When the first smooth binding proteins specific is PhyB, second Light binding proteins specific can be PIF.When the first smooth binding proteins specific is GIGANTEA, the specific binding of the second light Albumen can be FKF1.

For example, in a preferred embodiment of the invention, using CIBN as the first smooth binding proteins specific, using CRY2 is as the second smooth binding proteins specific.The wavelength of light used herein is the blue light of 460~490nm.The intensity of light is 20~50 μ W.

Meanwhile in order to confirm by the excretion body specific marker object expressed wherein and the first smooth binding proteins specific group At the first fusion protein expression and find out its position, labelled protein can be fused in fusion protein.For example, in this hair In bright preferred embodiment, fluorescin EGFP is inserted into the first fusion protein, wherein CIBN and CD9 or GIGANTEA and CD links together.Therefore, the expression pattern (expression and expression) of the first fusion protein and intracellular locations can pass through The expression of the first fusion protein of fluorescin EGFP is carried to study.

Term " cargo protein " in the present invention indicates a kind of albumen, is expressed as sewing with the second smooth binding proteins specific The fusion protein of conjunction, cargo protein is located in excretion body.

In the present invention, cargo protein is carried after being expressed in cell by excretion body.Cargo protein is unrestricted, but Preferably disease treatment albumen or medical diagnosis on disease albumen.For example, in a preferred embodiment of the invention, using with fluorescence MCherry as cargo protein.

The example of cargo protein of the present invention is selected from but not limited to matrix metalloproteinase (MMP) albumen, metalloproteinases group It knits inhibitor (TIMP) albumen, caspase protein, Caspase and inhibits albumen, histone zymoprotein or histone Enzyme inhibits albumen.

Wherein,

MMPs albumen is all if so, but being not limited to MMP1 albumen (SEQ ID：13)；

TIMPs albumen is all if so, but being not limited to TIMP1 albumen (SEQ ID NO：14), TIMP2 albumen (SEQ ID NO： 15), TIMP3 albumen (SEQ ID NO：Or TIMP4 albumen (SEQ ID NO 16)：17)；

Caspase protein is all if so, but being not limited to 1 albumen of Caspase (SEQ ID NO:18), Caspase 2 albumen (SEQ ID NO:19), caspase 3 albumen (SEQ ID NO:20), 4 albumen of Caspase (SEQ ID NO: 21), 5 albumen of Caspase (SEQ ID NO:22), 6 albumen of Caspase (SEQ ID NO:23), 7 egg of Caspase (SEQ ID NO in vain:24), 8 albumen of Caspase (SEQ ID NO:25), 9 albumen of Caspase (SEQ ID NO:26)、 10 albumen of Caspase (SEQ ID NO:27), 11 albumen of Caspase (SEQ ID NO:28), 12 egg of Caspase (SEQ ID NO in vain:29), 13 albumen of Caspase (SEQ ID NO:Or 14 albumen of Caspase (SEQ ID NO 30): 31)；

Caspase inhibits albumen all if so, but being not limited to inhibit with SEQ ID NO：The Guang day albumen that 18-31 is represented Zymoprotein inhibits albumen or any albumen for inhibiting Caspase.

Histone zymoprotein is such as, but is not limited to cathepsin A's albumen (SEQ ID NO:32), cathepsin B albumen (SEQ ID NO:33), cathepsin C's albumen (SEQ ID NO:34), cathepsin D's albumen (SEQ ID NO: 35), cathepsin E's albumen (SEQ ID NO:36), cathepsin F protein (SEQ ID NO:37), cathepsin G's egg (SEQ ID NO in vain:38), Cathepsin H's albumen (SEQ ID NO:39), cathepsin K albumen (SEQ ID NO:40)、 Cathepsin L 1 albumen (SEQ ID NO:41), proteinase cathepsin L2 albumen (SEQ ID NO:42), cathepsin O albumen (SEQ ID NO:43), cathepsin S albumen (SEQ ID NO:44), Cathepsin W albumen (SEQ ID NO:45) or Ctsz albumen (SEQ ID NO:46)；And

Cathepsin inhibits albumen all if so, but being not limited to inhibit with SEQ ID NO:The histone that 32-46 is represented Zymoprotein inhibits the albumen of albumen or any inhibiting cathepsin.

Another example of cargo protein is selected from but not limited to Cre recombinases, Cas albumen, Caspase in the present invention Activate DNA enzymatic (CAD) albumen, β-glucocerebrosidase (GBA), p38 mitogen-activated protein kinase, phosphatase and tensin Homologue (PTEN), Janus kinases (JAK), ubiquitin ligase, luciferase, Peroxiredoxin (Prx) I or II, Inhibit albumen, MyoD albumen, Tbx18 albumen, p53 albumen, High mobility group box-1 (HMGB1) albumen, the neuron of NF- κ B Property differentiation 1 (Neuro-D1) albumen, interferon regulatory factor 5 (IRF5) albumen, Interferon regulatory factor-3 (IRF3) albumen, letter Number transduction and transcriptional activation 1 (STAT1) albumen, Suppressor of cytokine signaling-3 (SOCS3) albumen, signal transduction and Transcriptional activation 2 (STAT2) albumen inhibits albumen (pySTAT3), Bax (the relevant X proteins of Bcl2), the B of pSTAT3 thin Born of the same parents' lymthoma-super large (Bcl-xL) albumen, aminoacyl tRNA synthetase interaction multifunctional protein (AIMP), mCherry albumen, Nucleoprotein green fluorescent protein (GFP) or combined with nucleic acid,

Wherein,

Cre recombinases in DNA by identifying the sites LoxP come the recombinant DNA between the sites LoxP comprising but not office It is limited to SEQ ID：The 9 Cre recombinases represented；

When Cas albumen is by guiding RNA to be combined with compound, there is restriction endonuclease or notch enzymatic activity.In some implementations In mode, Cas albumen is that Cas9 albumen is (all in this way with SEQ ID NO：The 10 Cas albumen represented) or its mutant or Cpf1 Albumen is (all in this way with SEQ ID NO：11 amino acid represented)；

CAD albumen is all in this way with SEQ ID NO：47 amino acid represented；

β-glucocerebrosidase (GBA) is all in this way with SEQ ID NO：12 amino acid represented；

P38 Mitogen-actived protein kinases (p38MAPKs) albumen is all p38- α in this way or its variant, and includes With SEQ ID NO：The amino acid that 48-51 is represented；

Repressor kappa B kinases (IKK) albumen is all in this way with SEQ ID NO：83 amino acid represented；

Nuclear factor-kappaB (NF- κ B) albumen is all in this way with SEQ ID NO：84 amino acid represented；

Phosphatase and tension element homologous protein (PTEN) are all in this way with SEQ ID NO：52 amino acid represented；

Janus kinases (JAK) albumen includes JAK1, JAK2, JAK3 and TYK2, and wherein JAK1 albumen is all in this way with SEQ ID NO：53 amino acid represented, JAK2 albumen are all in this way with SEQ ID NO：54 represent amino acid, JAK3 albumen it is all in this way with SEQ ID NO：55 amino acid represented, TYK2 albumen are all in this way with SEQ ID NO：56 amino acid represented；Ubiquitin ligase Albumen includes c-CBL, PRKN, RBX1, TRAF2 and Mdm2, and wherein ubiquitinbond zymoprotein is such as with SEQ ID NO：57 to 61 amino acid represented；

Luciferase protein is all in this way with SEQ ID NO：62 amino acid represented；

Peroxiredoxin (Prx) I or II has the function of cytotoxicity of the inhibition from oxidative stress, wherein Peroxiredoxin I is all in this way with SEQ ID NO：7 amino acid represented, oxide enzyme II are all in this way with SEQ ID NO：8 amino acid represented；

It is the super repressor I κ B for making NF- κ B inactivations by being combined with the NF- κ B in cytoplasm that NF- κ B, which inhibit albumen, In super repressor I kB proteins (S32A the and S36A mutant forms of I κ B) not by I kappa b kinases (IKK) phosphorylation, therefore it can be with It is continuous to inhibit NF- κ B, and NF- κ B inhibit albumen all in this way with SEQ ID NO：One of 1 to 5 amino acid, such as I κ represented B- α, I κ B- β, I κ B- ε, BCL-3 or its mutant；

MyoD albumen is all in this way with SEQ ID NO：63 amino acid represented；

Tbx18 albumen is all in this way with SEQ ID NO：64 amino acid represented；

P53 albumen is all in this way with SEQ ID NO：65 amino acid represented；

High mobility group box-1 (HMGB1) is all in this way with SEQ ID NO：66 amino acid represented；

It is all in this way with SEQ ID NO that neurogenicity breaks up 1 (Neuro-D1) albumen：67 amino acid represented；

Interferon regulatory factor 5 (IRF5) albumen is all in this way with SEQ ID NO：68 amino acid represented；

Interferon regulatory factor-3 (IRF3) albumen is all in this way with SEQ ID NO：69 amino acid represented；

Signal transducers and activators of transcription 1 (STAT1) albumen is all in this way with SEQ ID NO：70 amino acid represented；

Suppressor of cytokine signaling 3 (SOCS3) albumen is all in this way with SEQ ID NO：71 amino acid represented；

Direct signal-modulated 2 (STAT2) albumen is all in this way with SEQ ID NO：72 amino acid represented；

The albumen (pySTAT3) for inhibiting pSTAT3, includes being combined with pySTAT3 so that pySTAT3 was inactivated PySTAT3 intracellular antibody albumen, and it is all in this way with SEQ ID NO：73 amino acid represented or any egg for inhibiting pySTAT3 In vain；

Bax (the relevant X proteins of Bcl2) is all in this way with EQ ID NO：6 amino acid represented；

B cell lymphoma-super large (Bcl-xL) albumen is all in this way with SEQ ID NO：74 amino acid represented；

Aminoacyl tRNA synthetase interaction multifunctional protein (AIMPs) includes AIMP1 and AIMP2, wherein AIMP1 eggs It is white all in this way with EQ ID NO：The amino acid and AIMP2 albumen of 75 representatives are all in this way with SEQ ID NO：76 amino represented Acid；

MCherry albumen is all in this way with EQ ID NO：77 amino acid represented；

Green fluorescent protein (GFP) is all in this way with EQ ID NO：78 amino acid represented；

The nucleoprotein combined with nucleic acid includes that combined with DNA deoxyribonucleoprotein (DNP) or the ribose that is combined with RNA Nucleoprotein (RNP), wherein DNP includes RBBP4 or NAP1L4, and RNP includes Telomerase, heterologous nuclear ribonucleoprotein K (HNRNPK), and wherein all nucleosomes in this way of nucleoprotein, protamine, small nut RNP (snRNP), with SEQ ID NO：79-82 generations The amino acid of table or their mutant or any albumen combined with nucleic acid.

Term " culture " in the present invention indicates to grow the method for cell or microorganism in the environment of suitable control.

In the present invention, it by transformant culture 1~3 day, then changes culture medium into serum free medium, continues culture 2 ~5 days.

In the present invention, the method for cultivating transformant is known to the skilled in the art any method.

The culture medium herein refers to the notice culture medium for being widely used in animal cell culture, can select free city Sell the group for the culture medium composition that serum free medium, protein-free medium and chemical composition determine.

Above-mentioned serum free medium is used for animal cell culture, without cow's serum and with SFM4CHO (HyClone) and For EX-Cell (JHR Bioscience).Insulin-like growth factor I (IGF-I), ethanol amine, iron chloride and phosphatidyl courage Alkali can be added in culture medium, but always not be limited to this.

Above-mentioned protein-free medium is Zooblast culture medium, and the albumen of animal origin is removed from culture medium (especially High-molecular-weight protein, particularly molecular weight be at least the albumen of 10kDa), protein-free medium can be ProCHO (Lonza) and PF-Cho (HyClone), but it is not always limited to this.

The culture medium that above-mentioned chemical composition determines is Zooblast culture medium, does not include any animal sources component, but The component all determined with chemical constitution.Chemical composition determine culture medium can be CDM4CHO (HyClone), PowerCHO2CD (Lonza) and CD-optiCHO (Life Technologies), but not always it is limited to this.

Term " the first fusion protein " in the present invention is indicated through excretion body specific marker object and the first light specificity Binding protein in conjunction with and formed fusion protein.

In the present invention, excretion body specific marker object and the first optical specificity knot included in the first fusion protein Putting in order for hop protein is unrestricted, as long as when the first fusion protein is first photosensitive when being expressed during excretion body generates cell Binding proteins specific is located towards the direction inside excretion body.For example, the N-terminal of the first smooth binding proteins specific can be with The C-terminal of excretion body specific marker object is conjugated.

Form the excretion body specific marker object and the first smooth binding proteins specific phase directly with one another of the first fusion protein It can be connected even or by connector, above-mentioned connector is unrestricted, as long as the first fusion protein is expressed in excretion body generates cell, and is in Now towards the first smooth binding proteins specific inside excretion body, it is preferred that the peptide linker being made of amino acid, and more Preferably flexible peptide linker.The peptide linker can be expressed by using expression vector, wherein encode the nucleic acid of the connector with Other nucleic acid connection of each structural domain is encoded in frame.

Term " the second fusion protein " indicates the fusion that the wherein second smooth binding proteins specific and cargo protein are combined Albumen.

In the present invention, the arrangement of the second smooth binding proteins specific and cargo protein included in the second fusion protein It is sequentially unrestricted, if the second fusion protein be located in excretion body and in excretion body generates cell with the first fusion protein First smooth binding proteins specific region combines.For example, the N-terminal of cargo protein can be with the second smooth binding proteins specific C-terminal is conjugated.

It forms the second smooth binding proteins specific of the second fusion protein and cargo protein is directly connected to each other or can be by connecing Head connection, above-mentioned connector is unrestricted, as long as the second fusion protein is located in excretion body and in excretion body generation cell with the First smooth binding proteins specific region of one fusion protein combines, it is preferred that the peptide linker being made of amino acid, and More preferably flexible peptide linker.The peptide linker can be expressed by using expression vector, wherein encoding the nucleic acid of the connector It is connect with other nucleic acid for encoding each structural domain in frame.

In addition, above-mentioned each fusion protein may include polypeptide, at least one amino acid residue in the sequence which has Amino acid residue in the wild-type amino acid sequence for each structural domain that each fusion protein being different from includes.In albumen and polypeptide Amino acid exchange without change molecule overall activity be well known to those skilled in the art.Most common exchange is present in Ala/Ser、Val/Ile、Asp/Glu、Thr/Ser、Ala/Gly、Ala/Thr、Ser/Asn、Ala/Val、Ser/Gly、Thy/ Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly.Furthermore it is possible to include by In amino acid sequence mutation or modification and with improving to heat or the structural stability of pH or the egg of increased protein active In vain.

Finally, above-mentioned fusion protein or polypeptide including each domain fusion protein can be ripe by those skilled in the art Prepared by the chemical peptide symthesis method known, or be prepared by the following method.The gene for encoding each structural domain passes through PCR (polymerases Chain reaction) it expands or is synthesized by conventional method well known to those skilled in the art.It will be in the gene cloning to expression vector And it expresses.

Meanwhile the polynucleotides of each fusion protein are encoded by being imported in generating cell in excretion body, it can be in excretion body It generates in cell and expresses each fusion protein.At this point, polynucleotides are imported by conventional method well known to those skilled in the art Excretion body produces in cell.For example, expression vector can be used for importing.

Term " expression vector " in the present invention is the recombinant vector that target peptide can be expressed in host cell.The carrier It refer to the gene construct for including the key modulator for being operably connected to expressing gene insert.Expression vector includes expression Control element, such as initiation codon, terminator codon, promoter and operon.Generally by initiation codon and termination codon Son is interpreted as a part for the nucleotide sequence of coding polypeptide.When gene construct is imported into and is present in the coding in frame When in sequence, they are considered as working.The promoter of carrier can be composing type or induction type.

Term " being operably connected " in the present invention indicates the expression of nucleic acid regulatory sequence that ought usually work and coding The state when nucleic acid sequence of cargo protein or RNA are by functional link connection.For example, promoter and coding albumen or RNA Nucleic acid sequence be operably connected to influence coded sequence expression.This field can be passed through by being linked with expression vector functionality Recombinant DNA technology known to technical staff realizes, especially by using the conventional enzyme well known to those skilled in the art can be with Realize locus specificity DNA cuttings and link.

The expression vector may include for the signal sequence of fused polypeptide to be discharged to promote albumen from cell culture medium Middle separation.In order to effectively translate the nucleic acid sequence of insertion, specific initial signal may be necessary.These signals include ATG Initiation codon and its flanking sequence.In some cases, Exogenous translational control signal should be provided, wherein may be risen including ATG Beginning codon.It can be various natural origins and synthesis source that these Exogenous translationals, which control signal and initiation codon,.By leading Expression efficiency can be improved by entering suitable transcriptional enhancer or translational enhancer.

In a preferred embodiment of the invention, expression vector can express the cargo protein being conjugated with label, to confirm Insertion of the cargo protein in excretion body.The label of this paper is used to confirm the presence of cargo protein, can be conjugated to and second The opposite region in the conjugated region of light binding proteins specific.For example, using such as red fluorescent protein and green fluorescent protein Fluorescin it is conjugated as the C-terminal of label and cargo protein.

The cargo protein prepared as described above is expressed in excretion body generates cell.Once generating excretion body, it is with regard to research It is no to detect fluorescent protein tag, it can be confirmed that in excretion body cargo protein presence.

Term " light " in the present invention indicates the light to be irradiated, to make to generate the first light expressed in cell in excretion body Binding proteins specific and the second smooth binding proteins specific temporarily combine.

As described above, the first smooth binding proteins specific is expressed as be conjugated with excretion body specific marker object first Fusion protein, and the second smooth binding proteins specific is expressed as the second fusion protein being conjugated with cargo protein.When light irradiates When generating cell to excretion body, the first smooth binding proteins specific is combined with the second smooth binding proteins specific, as a result, temporary shape At including the-the second photosynthetic binding proteins specific of the-the first smooth binding proteins specific of excretion body specific marker object-cargo egg White fusion protein compound.When generating excretion body in generating cell in excretion body, due to excretion body specific marker object, goods Object albumen can be connect with excretion body.At this point, cargo protein is present in excretion body, and stop illumination after the generation of excretion body When penetrating, the first smooth binding proteins specific is detached with the second smooth binding proteins specific, and is therefore included in excretion body Cargo protein is discharged as a part for excretion body together with excretion body.In order to which cargo protein is more effectively delivered to excretion In vivo, preferably by light break it is irradiated to cell rather than continuously irradiating cell.That is, when intermittently irradiation light, first The conjugated and separation of light binding proteins specific and the second smooth binding proteins specific repeats, and can so increase cargo protein and lead Enter the possibility of excretion body.

Simultaneously, it is sufficient to the optical wavelength root for inducing the first smooth binding proteins specific to be combined with the second smooth binding proteins specific Change according to the type of the first and second smooth binding proteins specifics.Induce the first smooth binding proteins specific and the second light specificity The optical wavelength that binding protein combines depends on the type of albumen.Therefore, as it is known by the man skilled in the art, can select appropriate Optical wavelength.For example, in order to which CRY2 is connected to CIBN, wavelength is that the light of 460~490nm is preferred.If illumination is less than 10 Minute, then CRY2 and CIBN are separated from each other.When PhyB is combined with PIF, the light for being 650nm with wavelength irradiates 10 minutes.Work as wave When the light of a length of 750nm irradiates 5 minutes, PhyB and PIF are separated from each other.When FKF1 is combined with GIGANTEA, it is with wavelength The light of 460nm irradiates 30 minutes.In a preferred embodiment of the invention, it is irradiated with the light that wavelength is 460~490nm to induce The combination of CIBN and CRY2.

In a preferred embodiment of the invention, CRY2/mCherry fusion proteins and CIB/CD9 fusion proteins exist It is expressed in HEK293T, immortalized cell line generates a large amount of excretion bodies.As a result, it has been found that equally distributed in cytosol MCherry albumen is distributed in blue light illumination in cell membrane and endosome spline structure film (Fig. 7).When FKF1/mCherry is merged Albumen observes similar result (Figure 12) when being expressed in HEK293T cells with GIGANTEA/CD9 fusion proteins. CRY2/mCherry fusion proteins and CIBN/CD9 fusion proteins are expressed in HEK293T cells, then with blue light illumination and are adjusted Luminous intensity.As a result, when the light irradiation with 20~50 μ W of intensity position, the horizontal highest for the mCherry albumen collected in excretion body (Fig. 9).The excretion body detached from cell is acted on into HT1080 cells with the concentration of about 250 μ g/ml.As a result, excretion body pair HT1080 cells do not show any specific cytotoxicity, and confirm that mCherry albumen delivers in its cytosol (Figure 10).

In order to compare the efficiency that the efficiency of cargo protein importing excretion body and excretion body in conventional method are transferred to target cell, XPACK carriers are used for conventional method, and by the expression vector of CRY2/mCherry fusion proteins and CIBN/CD9 fusion proteins It imports in HEK293T cells.Then, compare the generation of cargo protein in excretion body.As a result, it was confirmed that when the method for using the present invention When, it is significantly high (Figure 15) to import efficiency.By from excretion body generate cell detach excretion body act on target cell (HeLa) with Compare the expression of cargo protein.When the excretion body detached using method through the invention, the expression of cargo protein is thin in target Highest (Figure 16) in born of the same parents.

In another preferred embodiment of the present invention, the present invention provides the carrier for generating excretion body, packets It includes：(a) include fusion protein (the first fusion egg for encoding excretion body specific marker object and the first smooth binding proteins specific First expression vector of polynucleotides in vain)；(b) the second expression vector, it includes multiple cloning sites and coding and above-mentioned the The polynucleotides of second smooth binding proteins specific of one smooth binding proteins specific connection, can import to multiple cloning sites and compile The polynucleotides of code cargo protein.

In the carrier provided by the present invention for producing excretion body, excretion body specific marker object, the first light specificity Binding protein, excretion body generate cell and the second smooth binding proteins specific is same as described above.

In the present invention, term " transformed cells generated for excretion body " expression can by expressing the first fusion protein Generate the cell of excretion body, wherein imported coding excretion body specific marker object and the first smooth binding proteins specific melts The polynucleotides of hop protein (the first fusion protein).

In the present invention, the second expression vector includes encoding the polynucleotides of the second smooth binding proteins specific and adjacent Multiple cloning sites.When the polynucleotides for encoding cargo protein are inserted into multiple cloning sites, it is expressed as including the second light specificity The fusion protein (the second fusion protein) of binding protein and cargo protein.

It is provided by the invention prepare excretion body carrier can include component, solution or device in it is one or more, It cannot be only used for generating the transformed cells and expression vector of excretion body, it may also be used for import expression vector；It is outer for cultivating generation Secrete the transformed cells of body；And for detaching and purifying the excretion body generated from the transformed cells for generating excretion body.For example, also May include be suitable for import expression vector buffer solution and culture generate excretion body transformed cells necessary to culture medium and Container.

Term " Cas albumen " in the present invention indicates key protein in CRISPR/Cas systems, when Cas albumen and is known as When two kinds of RNA of CRSPR RNA (crRNA) and trans-activation crRNA (tracrRNA) form compound, Cas albumen forms work Property endonuclease or nickase.

Term " guiding RNA " instruction can form compound with Cas albumen and guide Cas albumen to target in the present invention The target DNA specific RNA of DNA.

In the present invention, above-mentioned guiding RNA can be by the essential part of fusion crRNA and tracrRNA, by CRSPR Two kinds of RNA of RNA (CrRNA) and trans-activation crRNA (TracrRNA) or single stranded RNA (SgRNA) are made.

Above-mentioned guiding RNA can be the dual RNA (daul RNA) for including crRNA and tracrRNA.If above-mentioned RNA packets The essential part and target complementary portion of crRNA and tracrRNA are included, then any guiding RNA can be used for the present invention.It is above-mentioned CrRNA can hybridize with target DNA.

Above-mentioned guiding RNA can the ends 5' of crRNA include one in the ends 5' of single-stranded guiding RNA or dual RNA Or multiple other nucleotide.

It is desirable that the ends 5' that above-mentioned guiding RNA is capable of crRNA in the single-stranded ends 5' for guiding RNA or dual RNA include Two other guanylic acids.Guiding RNA can with RNA or coding guiding RNA DNA form be delivered to cell or Organism.It is the RNA that can be detached to guide RNA, the RNA being included in viral vectors, or is encoded in the carrier.It is ideal Ground, above-mentioned carrier is unrestricted, but can be viral vectors, plasmid vector or agrobacterium vector.

The DNA of coding guiding RNA can be the carrier for the DNA sequence dna for including coding guiding RNA.For example, being wrapped by transfecting The Plasmid DNA for including the sequence and promoter of the guiding RNA or coding guiding RNA of separation, can will be oriented to RNA delivery to cell or life Object.According to other methods, RNA delivery can will be guided to cell or organism by using virus-mediated gene delivery.

It, can be by using known in industry when guiding RNA to be transfected into cell or organism with the rna form detached Any in-vitro transcription system carry out in-vitro transcription come prepare guiding RNA.It is desirable that RNA will be oriented in the form of the RNA detached Rather than it is delivered to cell in the form of the plasmid of the sequence including coding guiding RNA.In the present invention, the term " RNA of separation (isolated RNA) " can be replaced by " naked rna (naked RNA) ".Because the RNA of separation does not need cloning procedure, can To save cost and time.It is, however not excluded that using Plasmid DNA or virus-mediated gene delivery for guiding RNA transfection.

The present invention provides through the excretion bodies including cargo protein prepared by the method for the present invention.

On the other hand, the present invention provides through the excretion bodies for including cre recombinases that the above method generates.

On the other hand, the present invention provides through the excretion bodies for including Cas9 albumen prepared by the above method.

On the other hand, the present invention provides through the outer comprising GBA (β-glucocerebrosidase) albumen of above method generation Secrete body.

On the other hand, include Peroxiredoxin (Prx) I or II the present invention provides through above method generation The excretion body of albumen.

On the other hand, the excretion body of the albumen including inhibiting NF-kB generated the present invention provides through the above method.

On the other hand, the present invention provides through the above method prepare comprising Bax (the relevant X proteins of Bcl-2) albumen Excretion body.

Include to be specifically bound by excretion body specific marker object and the first light by excretion body prepared by the above method The fusion protein (the first fusion protein) on excretion constitution film of albumen composition and by cargo protein and can be with the first optics Another fusion protein (the second fusion protein) of conjugated the second optical specificity binding protein composition of binding proteins specific.Cause This, when this excretion body is acted on target tissue cell, the second fusion protein included in excretion body can pass through plasma membrane Merge and be delivered to the cytosol of target tissue cell.

Including the excretion body of cargo protein can be used for the treatment of internal various diseases.For example, it includes conduct to prepare Cargo protein shows the excretion body of the protein polymer (such as antibody etc.) of active anticancer, is then acted on cancer cell. That is, excretion body may be used as the biocompatibility anticancer agent to work more better than conventional liposome.

The present invention also provides the drug components for preventing and treating diseases associated with inflammation, including inhibit egg with NF- κ B White excretion body.

Above-mentioned diseases associated with inflammation is unrestricted, but preferably with allergy, dermatitis, spy answer disease, conjunctivitis, periodontitis, rhinitis, Tympanitis, sphagitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease (Crohn ' s disease), colitis, pain Wind, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder Zhou Yan, tendonitis, tenosynovitis, peritendinitis, myositis, hepatitis, cystitis, ephritis, Sjogren syndrome (sjogren ' s syndrome), Multiple sclerosis, active chronic inflammation disease, septicemia and ulcerative colitis etc..

In the EXPERIMENTAL EXAMPLESThe of the present invention, present inventors have shown that using super repressor-I κ B:EXPLOR is pre-processed HeLa cells inhibit transfers of the NF- κ B activated from TNF-α to core, (are schemed by the inflammation inhibitory effect that TNF-α mediates with verifying 43. left).In addition, it was confirmed that the DNA of the NF- κ B of TNF-α activation, which is combined, to be suppressed (Figure 43 is right).In addition, present inventors have shown that Arthritic symptom mitigates in the mouse model of 3 retrobulbar injection collagen induced arthritis, to verify inflammation suppression Effect processed, therefore the super repressor I κ B in the present invention:EXPLOR may be used as drug component for preventing and treating inflammatory disease Disease.

The present invention also provides the drug components for preventing and treating cancer, including include Bax (Bcl-2 correlation X eggs Excretion body in vain).

Above-mentioned cancer is unrestricted, but preferred embodiment be breast cancer, colon cancer, lung cancer, Small Cell Lung Cancer, gastric cancer, liver cancer, Leukemia, osteocarcinoma, cancer of pancreas, cutaneum carcinoma, head or neck cancer, skin or mela-noma of choroid, cancer eye, peritoneal cancer, uterine cancer, ovary Cancer, the carcinoma of the rectum, cancer of anus and cervical carcinoma etc..

In the EXPERIMENTAL EXAMPLESThe of the present invention, present inventors have shown that using Bax::EXPLOR pre-processes HeLa cells and increases The release of cytochrome c is added, it is possible thereby to by Bax:EXPLOR is used as the drug component for preventing and treating cancer.

The present invention also provides for oxidation resistant drug component, including contain Peroxiredoxin (Prx) I or The excretion body of II.

In addition, the present invention provides the medicine groups of the excretion body including containing Peroxiredoxin (Prx) I or II Point, for preventing and treating active oxygen disease, with cancer, arteriosclerosis, respiratory disease, osteoporosis, obesity For disease and degenerative dementia disease etc..

In addition, the present invention provides for oxidation resistant components of cosmetics, including with Peroxiredoxin (Prx) the excretion body of I or II.

In addition, the present invention provides the components of cosmetics for skin anti-aging, including with peroxide redox The excretion body of enzyme (Prx) I or II.

In the EXPERIMENTAL EXAMPLESThe of the present invention, present inventors have shown that with Prx I/II::It is thin that EXPLOR pre-processes HeLa Born of the same parents statistically significantly inhibit the cytotoxicity caused by oxidative stress, to demonstrate to H₂O₂Induced oxidation stress be caused The inhibition of cytotoxicity, therefore PrxI/II::EXPLOR can be used as drug component and live for anti-oxidant or prevention and treatment Property oxygen, or as cosmetic composition be used for skin anti-oxidant or anti-aging.

The present invention also provides include have Cre recombinases outer for create that the condition of target gene knocks out allele Secrete the component of body.

In the EXPERIMENTAL EXAMPLESThe of the present invention, the present inventor passes through by pCAG-loxP-STOP-loxP-ZsGreen Detection ZsGreen reporter proteins expression after coding DNA is transfected into HT1080 and HeLa cells, it was confirmed that Cre::EXPLOR The expression of ZsGreen reporter proteins is transfected with pCMV-Cre carriers as positive right in the HT1080 cells and HeLa cells of processing According to result it is identical, to demonstrate the effect (Figure 19 a and 19b) of Cre recombinases.In addition, the present inventor can by implement with Above-mentioned identical experiment confirms ZsGreen in Cre:Expression (Figure 20) on the Primary mouse embryonic neuron of EXPOR processing.And And present inventors have shown that outside pCAG-lowP-STOP-loxP-eNpHR3.0-EYFP transgenic mice abdomens inject Cre:: After EXPLOR, EYFP is in Cre::It is expressed in EXPLOR processing groups, to verify Cre-EXPLOR functions (Figure 21) in vivo.In addition, Confirm the Cre in ImmunohistochemistryResults Results::EXPLOR mainly targets neuron in mouse brain by merging neuron areas, with Verify Cre::EXPLOR targets cell (Figure 22), and therefore Cre::EXPLOR may be used as striking for creating target gene condition Except the component of allele.

The present invention also provides the components for being engineered DNA sequence dna, including draw with Cas9 albumen and target DNA specificity Lead the excretion body of RNA (gRNA).

Aforementioned component is unrestricted, but mutation or correcting mutant of the preferred induction in normal sequence.Mutation can be natural The mutation of the mutation of generation or pathogenic microorganisms induction.In other words, when detecting pathogenic microorganisms and understand biological sample When infected, caused by mutation is the infection by pathogenic microorganisms.Pathogenic microorganisms is unrestricted, but can be viral or thin Bacterium.

In the EXPERIMENTAL EXAMPLESThe of the present invention, present inventors have shown that it includes CRISPR/Cas9 that can be prepared with high yield The excretion body of albumen.

Include comprising β-glucocerebrosidase (GBA) the present invention also provides the drug component for treating Gaucher disease Excretion body.

In the EXPERIMENTAL EXAMPLESThe of the present invention, present inventors have shown that passing through GBA::EXPLOR processing comes from Gaucher disease The cell of patient can restore the activity (Figure 30) of β-glucocerebrosidase (GBA), therefore GBA::EXPLOR may be used as treating The drug ingedient of Gaucher disease.

The method of excretion body produced according to the present invention comprising cargo protein can be prepared with high yield comprising cargo protein Excretion body.In addition, cargo protein be rendered as with excretion body UF membrane, therefore can be widely applied to treatment disease.

Invention mode

As illustrated in the examples below, reality of the invention and presently preferred embodiment are illustrative.However, should It recognizes, those skilled in the art can be repaiied within the spirit and scope of the present invention in view of the disclosure Change and improves.

Embodiment

Embodiment 1：Prepare excretion body

＜ 1-1 ＞ confirm the combination of CIBN and CRY2 to generate excretion body

By PcDNA3.1 (+) carrier comprising CIBN-EGFP-CD9 genes and include mCherry-CRY2 genes PcDNA3.1 (+) carrier imports HEK293T cells (excretion body generates cell) under no light condition, then will cultivate 24 hours. It changes culture medium into serum free medium, is then cultivated for 48 hours.It is 460~490nm's with wavelength after the completion of culture Blue light irradiating cell.It is red by using being shown in mCherry before and after Laser Scanning Confocal Microscope confirmation blue light irradiation The position (Fig. 7) of color fluorescence.

Fig. 7 is fluorescent image, is illustrated in the conversion for importing CIBN-EGFP-CD9 genes and mCherry-CRY2 genes In HEK293T cells, according to blue light illumination, the variation of mCherry albumen position in the cell.As shown in fig. 7, can draw in irradiation Before playing the blue light that light binding proteins specific CIBN and CRY2 are combined, mCherry albumen is evenly distributed in cytosol.So And after irradiating blue light, mCherry protein compressions are in film.It is by light specificity through analyzing this mCherry albumen and clustering Caused by the combination of binding protein CIBN and CRY2.

＜ 1-2 ＞ confirm the combination of the GIGANTEA and FKF1 for generating excretion body

Use in the present embodiment PcDNA3.1 (+) carrier comprising GIGANTEA-EGFP-CD9 genes and comprising PcDNA3.1 (+) carrier of mCherry-FKF1LOV genes.By with embodiment<1-1>Described in identical mode confirm Intracellular excretion body.(LOV in FKF1LOV above is the abbreviation in light-oxygen-voltage structure domain, is indicated in FKF1 protein In the structural domain that is combined with other protein by light, so FKF1 and FKF1LOV be actually herein defined as it is identical).

Similar to embodiment<1-1>, as shown in figure 12, irradiation can cause light binding proteins specific GIGANTEA and Before the blue light that FKF1 is combined, mCherry albumen is evenly distributed in cytosol.However, after irradiating blue light, MCherry protein concentrates in film.It is by light binding proteins specific GIGANTEA through analyzing this mCherry albumen and clustering Caused by combination with FKF1.

Embodiment 2：The influence that the generation of excretion body and luminous intensity generate excretion body

It is in wavelength by each expression vector for separately including CIBN-EGFP-CD9 genes and mCherry-CRY2 genes It is imported in HEK293T cells under the LED light of 460nm, intensity 0,5,20,50 and 200 μ W, then culture 24 hours.Then, will Culture medium is changed to serum free medium, is then further cultured for 48 hours.After the completion of culture, isolation medium, centrifugation (3000 × g, 15 minutes) to obtain the supernatant without cell fragment.The ExoQuick-TC excretion bodies precipitation of the volume of 5 times of supernatants is molten Liquid (System Biosciences, Mountain View, California, USA) is added in gained supernatant.After mixing, (1500 × g, 30 minutes) is centrifuged to obtain the excretion body of precipitation.The excretion body of acquisition is suspended in PBS, excretion is formed Liquid suspension.Using the syringe equipped with 27-G needles, excretion liquid suspension is filtered with 0.2 μm of filter.As a result, obtaining single big Small excretion body (Fig. 8).Then, excretion body lysate is prepared using lysis buffer, then carries out immunoblotting with relatively outer Secrete the amount (Fig. 9) of mCherry albumen in body.

Fig. 9 is immunoblotting assay image, shows and measures the cargo protein captured in excretion body according to blue light strength The result of (mCherry albumen) changes of contents.As shown in figure 9, when with the blue light illumination cells of 20~50 μ W intensity, excretion body The amount highest of middle cargo protein mCherry.From the above it can be confirmed that leading in the cohesive process of light binding proteins specific The intensity that control is irradiated to the light of cell is crossed, the content of the cargo protein captured in excretion body can be adjusted.

Embodiment 3：The effect of excretion body processing

It is in wavelength by each expression vector for separately including CIBN-EGFP-CD9 genes and mCherry-CRY2 genes 460nm, intensity are then extracted by mode same as Example 2 outer to be imported in HEK293T cells under the LED light of 50 μ W Secrete body.HT1080 cells are handled 24 hours with the concentration of 250 μ l/ml with the excretion body of extraction.By addition comprising 4%PFA and HT1080 cells are fixed on 10% gelatin gel by the 0.1M phosphate buffers (pH7.4) of 0.01%GA.By using liquid Nitrogen cools down the cell being attached on gelatin gel one day.At -120 DEG C, 45nm slices are cut with cryoultramicrotome. With anti-mCherry antibody and albumin A-gold immunostaining slice.With Tecnai G2 Spirit Twin tem observations MCherry albumen (Figure 10).

Figure 10 is electron microscope picture, and which show thin with the excretion body processing target comprising cargo protein (mCherry) The result of study of cargo protein is imported after born of the same parents (HT1080) in target cell, the wherein left side indicates that the target of unused excretion body processing is thin Born of the same parents, the right indicate the target cell handled with excretion body.As shown in Figure 10, it was confirmed that when the excretion body processing target cell with the present invention When, cargo protein is transferred in target cell.

Embodiment 4：The analysis of excretion body with cargo protein

It is in wavelength by each expression vector for separately including CIBN-EGFP-CD9 genes and mCherry-CRY2 genes 460nm, intensity then extract excretion to import HEK293T cells under the LED light of 50 μ W by mode same as Example 2 Body.HT1080 cells are handled 24 hours with the concentration of 250 μ l/ml with the excretion body of extraction.Then, it is confirmed under fluorescence microscope Red fluorescence in mCherry protein, and by LDH cell death measuring methods compare the cell handled with excretion body with The ratio (Figure 11) of dead cell between the cell of unused excretion body processing.

Figure 11 is one group of fluorescent image (a), illustrates to handle target with the excretion body comprising cargo protein (mCherry albumen) After cell (HT1080), the result of study of cargo protein is imported in target cell；And chart (b), it illustrates by excretion The comparison result of the apoptotic cell ratio of body processing induction.As shown in figure 11, it was confirmed that Apoptosis is handled by excretion body Induction

Embodiment 5：The generation of excretion body and cargo protein enter the comparison of the importing efficiency of the excretion body of generation

The confirmation of ＜ 5-1 ＞ excretion body generation efficiencies

In order to which the excretion body more of the invention generated with conventional method and cargo protein enter resulting excretion body Efficiency is imported, the expression that excretion body generates cargo protein in cell is studied by measuring uciferase activity therein.

According to conventional methods, using the commercially available carrier XPACK (Systems designed for excretion body load technology (XP) Biosciences XPACK- luciferases-mCherry) is imported into HEK293T cells.On the other hand, according to the method for the present invention, Luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 is imported in HEK293T cells (EXPLOR).Then, two kinds are measured Luciferase activity in cell is to compare the efficiency of two methods.According to manufacturer specification (Luciferase Assay Reagent, Promega luciferase activity) is measured.Then drawing result standard curve quantitatively calculates the quantity of excretion body in cell.

As shown in figure 14, it was confirmed that the method using light the binding proteins specific CIBN and CRY2 of the present invention enters excretion The importing efficiency of body is significantly higher than conventional method (XP) (Figure 14).

Expression of the ＜ 5-2 ＞ cargo proteins in the excretion body of generation

By embodiment<5-1>Cell culture 72 hours, then extract excretion body (Exoquick-TC, Systems biosciences).By measuring luciferase activity therein, (XP) or the method for the present invention are divided relatively by conventional method indirectly From excretion body in include cargo protein concentration.As shown in figure 15, it was confirmed that the method for the present invention compared with conventional method The excretion body (Figure 15) comprising notable lot cargo albumen can be generated.

＜ 5-3 ＞ cargo proteins import the comparison of efficiency

Based on embodiment<5-1>With<5-2>The uciferase activity of middle measurement calculates cargo egg by following mathematical formulae White importing efficiency (E).

[mathematical formulae 1]

Uciferase activity measured value/excretion body generates uciferase activity in cell and measures in the excretion body that E=is generated Value

As shown in figure 15, it was confirmed that the efficiency of the excretion body generated using the combination of the CRY2 and CIBN of the present invention is other 4 to 120 times of height (Figure 15) of comparative group.

Embodiment 6：Comparison of the excretion body to target cell transfer efficiency

In order to compare excretion body transfer efficiency, target cell is handled with the excretion body comprising cargo protein.Particularly, with 5 × 10⁹A excretion body processing HeLa cells 24 hours, then measure the fluorescence intensity expressed in cell.As shown in figure 16, it was demonstrated that this Fluorescence intensity in invention excretion body (EXPLOR) is significantly high (Figure 16).

Therefore, it was confirmed that it is thin can cargo protein to be more effectively delivered to target using the method for excretion body of the present invention Born of the same parents.

EXPERIMENTAL EXAMPLESThe

EXPERIMENTAL EXAMPLESThe 1：MMP (matrix metalloproteinase)

It has evaluated under 488nm wavelength blue lights in the cell of expression CIBN-EGFP-CD9 and MMPs-mCherry-Cry2 The load of MMP in the combination of CIBN and CRY2 and excretion body.

In order to largely generate the excretion body of load MMPs, establishes and stablize expression CIBN-EGFP-CD9 genes and MMP- The cell of mCherry-CRY2 genes, and excretion body is isolated and purified from culture supernatant by tangential flow filtration (TFF) method.

Functional selection is carried out to the excretion body for loading MMP in target cell：

Target cell is handled with evaluation function enzymatic activity with the excretion body of load MMP.

The excretion body of load MMP is administered to animal model by intraperitoneal or intravenous fashion, to show therapeutic effect.

EXPERIMENTAL EXAMPLESThe 2：TIMP (tissue inhibitors of metalloproteinases)

It has evaluated under 488nm wavelength blue lights in the cell of expression CIBN-EGFP-CD9 and TIMPs-mCherry-Cry2 The load of TIMPs in the combination of middle CIBN and CRY2 and excretion body.

In order to largely generate the excretion body of load TIMPs, establishes and stablize expression CIBN-EGFP-CD9 genes and TIMPs- The cell of mCherry-CRY2 genes, and excretion body is isolated and purified from culture supernatant by tangential flow filtration (TFF) method.

Functional selection is carried out to the excretion body for loading TIMP in target cell：

Target cell is handled with evaluation function enzymatic activity with the excretion body of load TIMPs.

The excretion body of load TIMP is administered to animal model by intraperitoneal or intravenous fashion, to show therapeutic effect.

EXPERIMENTAL EXAMPLESThe 3：Caspase

It has evaluated under 488nm wavelength blue lights expression CIBN-EGFP-CD9's and Caspase-mCherry-Cry2 In cell in the combination of CIBN and CRY2 and excretion body Caspase load.

In order to largely generate load Caspase excretion body, establish stablize expression CIBN-EGFP-CD9 genes and The cell of Caspase-mCherry-CRY2 genes, and detached from culture supernatant by tangential flow filtration (TFF) method pure It is outside the pale of civilization to secrete body.

Functional selection is carried out to the excretion body for loading Caspase in target cell：

Target cell is handled with evaluation function enzymatic activity with the excretion body of load Caspase.

The excretion body of load Caspase is administered to animal model by intraperitoneal or intravenous fashion, to show treatment Effect.

EXPERIMENTAL EXAMPLESThe 4：Cathepsin

It has evaluated under 488nm wavelength blue lights expression CIBN-EGFP-CD9's and cathepsin-mCherry-Cry2 The combination of CIBN and CRY2 and the load of excretion in-vivo tissue protease in cell.

In order to largely generate load cathepsin excretion body, establish stablize expression CIBN-EGFP-CD9 genes and The cell of cathepsin-mCherry-CRY2 genes, and detached from culture supernatant by tangential flow filtration (TFF) method pure It is outside the pale of civilization to secrete body.

Functional selection is carried out to the excretion body for loading cathepsin in target cell：

Target cell is handled with evaluation function enzymatic activity with the excretion body of load cathepsin.

By the way that the excretion body of load cathepsin is administered in intraperitoneal or vein to animal model, with display treatment effect Fruit.

EXPERIMENTAL EXAMPLESThe 5：Cre recombinant proteases

＜ 5-1 ＞ generate the excretion body (Cre of load C re recombinases::EXPLOR)

A. the Cre recombinases in excretion body are confirmed

Present inventors have shown that CIBN and CRY2 is in the cell of expression CIBN-EGFP-CD9 and Cre-mCherry-Cry2 Combination, with verify excretion body load have be recorded as SEQ ID NO：The Cre recombinases of 9 amino acid.In particular, unglazed Under the conditions of use transfection pcDNA3.1 (+) carrier training for including CIBN-EGFP-CD9 genes and Cre-mCherry-CRY2 genes After supporting 24 hours, excretion body generates cell HEK293T in Eagle culture medium of the Dulbecco improvement without fetal calf serum (FBS) (DMEM) it is further cultured in 48 hours.After culture, the red fluorescence from mCherry is studied by Laser Scanning Confocal Microscope and is existed Position before and after the blue light illumination of 488nm wavelength.This experiment be more than five times.

According to result, it was confirmed that combinations (Figure 18) of the Cre-mCherry-CRY2 (red) between CIBN-EGFP-CD9, To demonstrate load of the excretion body to Cre recombinases.

B. the excretion body (Cre of load C re recombinases is generated::EXPLOR)

Present inventor has performed following experiments to obtain the excretion body of load C re recombinases.

Particularly, the carrier including CIBN-EGRP-CD9 genes and Cre-mCherry-CRY2 genes is transfected into excretion Body generated on cell HEK293T, and by these cell culture 24 hours.After culture 24 hours, cell culture medium is changed into and is free of Fetal calf serum (FBS), and be further cultured for 48 hours under the blue light of 50 μ W power of 488nm wavelength.After culture, pass through centrifugation (2000 × g, 15 minutes) generates the supernatant for removing cell fragment from the culture medium of separation.5 times of volumes are added to supernatant ExoQuick-TC excretion bodies precipitation solution (System Biosciences, Mountain View, California, USA), And it is mixed 18 hours at 4 DEG C.Come by the mixture of the above-mentioned supernatant of centrifugation (1500 × g, 30 minutes) and ExoQuick-TC Suspension excretion body bead is to obtain the excretion body (Fig. 8) of suspension.

In addition, the excretion body for stablizing expression CIBN-EGFP-CD9 genes and 2 genes of CRE-mCherry-CRY is generated thin Born of the same parents HEK293T cultivates 48~72h under the blue light of 50 μ W power of 488nm wavelength in the culture medium of no fetal calf serum.Culture knot Shu Hou generates the supernatant for removing cell fragment by centrifugation (2000 × g, 15 minutes) from the culture medium of separation.In order to from Particle of the removal more than 200nm, is filtered with 0.2 μ l PES films (Corning) in supernatant.It is answered in identical supernatant With particle of tangential flow filtration (TFF) method removal less than 20nm, is concentrated from filtrate and refine excretion body.It is used in TFF Vivaflow 50-100kDa PES films (Sartorius).It is dense by rotating filtrate under 1.5~2 air pressures of TFF Contracting and refined excretion body.Then, by being centrifuged on AmiconUltra-0.5 (100kDa) (Millipore) filter (10000~14000g, 5 minutes) removes excretion body concentrate.Finally, by with it is reversed according to the preferred buffer solution of experiment purpose It centrifuges (10000~14000g, 5 minutes) and obtains excretion body.

Excretion body (the Cre that ＜ 5-2 ＞ pass through load C re recombinases::EXPLOR Cre recombinase functions) are confirmed

A. Cre is confirmed on HT1080 and HeLa cells::EXPLOR functions

Cre recombinases have the function of the recombinant DNA on the regions loxP.Present inventor has performed following experiments to study Cre::The function of EXPLOR.

Particularly, the pCAG-loxP-STOP-loxP-ZsGreen DNA encoded are transfected to HT1080 and HeLa cells simultaneously It washs after 6 hours.Then 0.25mg/ml Cre are used::EXPLOR or feminine gender::EXPLOR processing or transfection pCMV-Cre are carried Body.After culture 48 hours, the expression of the ZsGreen with green fluorescence is studied.In Cre::EXPLOR processing HT1080 and The expression of ZsGreen is confirmed in HeLa cells, this is different from feminine gender::HT1080 the and HeLa cells of EXPLOR processing, and The expression of the ZsGreen and the result of the pCMV-Cre carriers transfection in positive control are similar (Figure 19 a and 19b).

B. Cre is confirmed::Functions of the EXPLOR on Primary rat Fetal neurons

Following experiment is carried out to study Cre::Functions of the EXPLOR on Primary rat Fetal neurons.

Particularly, pCAG-loxP-STOP-loxP-ZsGreen coding DNAs are transfected to Primary rat Fetal neurons simultaneously It washs after 6 hours.Then by them in 0.15mg/ml Cre:It is cultivated on EXPLOR.After culture 48 hours, research has green The expression of the ZsGreen of color fluorescence.The experiment is at least repeated three times, it is confirmed that in Cre::EXPLOR is handled primary The expression (Figure 20) of ZsGreen on rat embryo neuron.

C. Cre is confirmed::In vivo functionalities of the EXPLOR on transgenic mice

The present inventor carries out following experiment to verify Cre:The functions of EXPLOR in vivo.

Particularly, 50 μ l CreEXPLOR (10mg/mL) are injected into pCAG-loxP-STOP- by being injected intraperitoneally In loxP-eNpHR3.0-EYFP transgenic mices.After injection, the fixed brain section of 4% formaldehyde is imaged by fluorescence microscope. Green fluorescence indicates the expression of eNpHR3.0-EYFP, blue-fluorescence indicator cells core.Cre is studied by Laser Scanning Confocal Microscope:: EXPLOR handles the expression of eNpHR3.0-EYFP on neuron in the incertitude zone (ZI) of mouse, and therefore in Cre::At EXPLOR The expression (Figure 21) of EYFP is confirmed in the pCAG-loxP-STOP-loxP-eNpHR3.0-EYFP transgenic mice groups of reason.

D. Cre is confirmed on transgenic mice::EXPLOR targets cell

In order to confirm Cre::Specific cell targetings of the EXPLOR in above-mentioned experiment in vivo, has carried out immuning tissue Learn experiment.Neuron is dyed with NeuN antibody specificities, astroglia is dyed with GFAP antibody specificities, from there through grinding It is mainly neuron to study carefully corresponding circle of sensation, it was confirmed that Cre:EXPLOR specifically targets the neuron (Figure 22) in mouse brain.

EXPERIMENTAL EXAMPLESThe 6：CRISPR-Cas9

＜ 6-1 ＞ generate the excretion body (Cas9 of load C as9::EXPLOR)

A. the Cas9 in excretion body is confirmed

Present inventors studied expression CIBN and CRY2 CIBN-EGFP-CD9 and Cas9-mCherry-CRY2 combination, With validation record in amino acid SEQ ID NO：The load of Cas9 in 10.

As shown in figure 23, pcDNA3.1 (+) carrier that Cas9-mCherry-CRY2 is inserted into has 11,890 base-pairs Length, and three protein portions by Cas9 of 5 ends with NLS sequences, with 45 base-pair joint sequences MCherry and cryptochrome 2 form, and are respectively provided with the joint sequence of 45 and 27 base-pairs.Each protein portion length point It Wei not 4194,699 and 1497 base-pairs.

Particularly, it includes CIBN-EGFP-CD9 genes and Cre-mCherry-CRY2 genes to be used under no light condition After transfecting pcDNA3.1 (+) carrier culture 24 hours, excretion body generates cell HEK293T and is free of tire ox what Dulbecco was improved It is further cultured for 48 hours in the Eagle culture mediums (DMEM) of serum (FBS).After culture, studied by Laser Scanning Confocal Microscope The position of the red fluorescence from mCherry before or after the blue light illumination of 488nm wavelength.This experiment is performed for more than five It is secondary.From there through confirming that CIBN-EGFP-CD9 is combined (Figure 23) with Cas9-mCherry-CRY2 under blue light stimulation, by Cas9 In protein load to excretion body.

B. the excretion body (Cas9 of load C as9 is generated::EXPLOR)

Present inventor has performed following experiments to obtain the excretion body of load C as9.

Particularly, the carrier comprising CIBN-EGRP-CD9 genes and Cre-mCherry-CRY2 genes is transfected into excretion Body generated on cell HEK293T, and by these cell culture 24 hours.After culture 24 hours, cell culture medium is changed into and is free of Fetal calf serum (FBS), and be further cultured for 48 hours under the blue light of 50 μ W power of 488nm wavelength.After culture, pass through centrifugation (2000 × g, 15 minutes) generates the supernatant for removing cell fragment from the culture medium of separation.5 times of volumes are added to supernatant ExoQuick-TC excretion bodies precipitation solution (System Biosciences, Mountain View, California, USA), And it is mixed 18 hours at 4 DEG C.Come by the mixture of the above-mentioned supernatant of centrifugation (1500 × g, 30 minutes) and ExoQuick-TC Suspension excretion body bead is to obtain the excretion body (Fig. 8) of suspension.

In addition, the excretion body for stablizing expression CIBN-EGFP-CD9 genes and 2 genes of CRE-mCherry-CRY is generated thin Born of the same parents HEK293T cultivates 48~72h under the blue light of 50 μ W power of 488nm wavelength in the culture medium of no fetal calf serum.Culture knot Shu Hou generates the supernatant for removing cell fragment by centrifugation (2000 × g, 15 minutes) from the culture medium of separation.In order to from Particle of the removal more than 200nm, is filtered with 0.2 μ l PES films (Corning) in supernatant.It is answered in identical supernatant With particle of tangential flow filtration (TFF) method removal less than 20nm, is concentrated from filtrate and refine excretion body.It is used in TFF Vivaflow 50-100kDa PES films (Sartorius).It is dense by rotating filtrate under 1.5~2 air pressures of TFF Contracting and refined excretion body.Then, by being centrifuged on Amicon Ultra-0.5 (100kDa) (Millipore) filter (10000~14000g, 5 minutes) removes liquid in excretion body concentrate.Finally, by preferably delaying with according to experiment purpose Fliud flushing reversely centrifuges (10000~14000g, 5 minutes) and obtains excretion body.Assess the amount (Figure 25) of Cas9 in excretion body.

Functional selection is carried out to the excretion body of load C as9 in target cell：

Target cell is handled with display functionality enzymatic activity with the excretion body of load C as9.

By the way that the excretion body of load C as9 is administered in intraperitoneal or vein to animal model, to show therapeutic effect.

EXPERIMENTAL EXAMPLESThe 7：The DNA enzymatic (CAD) of Caspase activation

It has evaluated under 488nm wavelength blue lights in the cell of expression CIBN-EGFP-CD9 and CAD-mCherry-Cry2 The load of CAD in the combination of CIBN and CRY2 and excretion body.

In order to largely generate the excretion body of load C AD, establishes and stablize expression CIBN-EGFP-CD9 genes and CAD- The cell of mCherry-CRY2 genes, and excretion body is isolated and purified from culture supernatant by tangential flow filtration (TFF) method.

Functional selection is carried out to the excretion body of load C AD in target cell：

With the excretion body processing target cell of load C AD with display function activity.

By the way that the excretion body of load C AD is administered in intraperitoneal or vein to animal model, to show therapeutic effect.

EXPERIMENTAL EXAMPLESThe 8：β-glucocerebrosidase

＜ 8-1 ＞ loads generate the excretion body (GBA of GBA::EXPLOR)

A. the GBA in excretion body is confirmed

Present inventors studied expression CIBN and CRY2 CIBN-EGFP-CD9 and Cas9-mCherry-CRY2 combination, With validation record in amino acid SEQ ID NO：The load of GBA in 12.

[table 2]

Particularly, it cultivates to have under no light condition and includes CIBN-EGFP-CD9 genes and Cre-mCherry-CRY2 bases The excretion body of pcDNA3.1 (+) carrier of the transfection of cause generated cell HEK293T after 24 hours, and excretion body generates cell HEK293T is further cultured for 48 hours in Eagle culture mediums (DMEM) of the Dulbecco improvement without fetal calf serum (FBS).It is training After supporting, the red fluorescence from mCherry is studied before and after the blue light illumination of 488nm wavelength by Laser Scanning Confocal Microscope Position.This experiment is performed for more than five times.From there through confirm blue light stimulation under CIBN-EGFP-CD9 and Cas9- MCherry-CRY2 combines (Figure 26), will be in GBA protein loads to excretion body.

B. the excretion body (GBA of load GBA is generated::EXPLOR)

Present inventor has performed following experiments to obtain the excretion body of load GBA.

Particularly, the carrier comprising CIBN-EGRP-CD9 genes and Cre-mCherry-CRY2 genes is transfected into excretion Body generated on cell HEK293T, and by these cell culture 24 hours.After culture 24 hours, cell culture medium is changed into and is free of Fetal calf serum (FBS), and in addition cultivated 48 hours under the blue light of 50 μ W power of 488nm wavelength.After culture, by from The heart (2000 × g, 15 minutes) generates the supernatant for removing cell fragment from the culture medium of separation.5 times of bodies are added to supernatant Long-pending ExoQuick-TC excretion bodies precipitation solution (System Biosciences, Mountain View, California, USA it), and at 4 DEG C mixes 18 hours.Pass through the mixed of the above-mentioned supernatant of centrifugation (1500 × g, 30 minutes) and ExoQuick-TC Object is closed to suspend excretion body bead to obtain the excretion body (Fig. 8) of suspension.

In addition, the excretion body for stablizing expression CIBN-EGFP-CD9 genes and 2 genes of CRE-mCherry-CRY is generated thin Born of the same parents HEK293T cultivates 48~72h under the blue light of 50 μ W power of 488nm wavelength in the culture medium of no fetal calf serum.Culture knot Shu Hou is generated from the culture medium of separation by centrifugation (2000 × g, 15 minutes) and is removed cell fragment supernatant.In order to from upper Particle of the removal more than 200nm, is filtered with 0.2 μ l PES films (Corning) in clear liquid.It is applied in identical supernatant Particle of tangential flow filtration (TFF) method removal less than 20nm, concentrates from filtrate and refines excretion body.It is used in TFF Vivaflow 50-100kDa PES films (Sartorius).It is dense by rotating filtrate under 1.5~2 air pressures of TFF Contracting and refined excretion body.Then, by being centrifuged on AmiconUltra-0.5 (100kDa) (Millipore) filter (10000~14000g, 5 minutes) removes liquid in excretion body concentrate.Finally, by preferably delaying with according to experiment purpose Fliud flushing reversely centrifuges (10000~14000g, 5 minutes) and obtains excretion body.

The excretion body of ＜ 8-2 ＞ loads GBA generates the measurement that GBA is expressed in cell

The present inventor carries out the expression of GBA in excretion body of the western blot to measure load GBA.

Particularly, the carrier comprising CIBN-EGRP-CD9 genes and GBA-mCherry-CRY2 genes is transfected into excretion Body generated on cell HEK293T, and by these cell culture 24 hours.It is cracked using MPER (mammalian proteins extracts reagent) HEK293T cells, and pass through western blot analysis albumen.By rat primary astroglia, people's primary astroglial cells With from Gaucher patient fibroblast (wherein since GBA gene unconventionalities cause β-glucocerebroside enzyme defect) crack with It carries out western blot and passes through western blot analysis albumen.

As a result, in addition to the fibroblast from Gaucher patient, including CIBN-EGRP-CD9 genes and GBA- It is observed in the HEK293T cells of mCherry-CRY2 genes, rat primary astroglia and people's primary astroglial cells To endogenous GBA (Figure 27).

In addition, being observed in the HEK293T cells including CIBN-EGRP-CD9 genes and GBA-mCherry-CRY2 genes To GBA-mCherry-CRY2 fusion proteins (151kDa), this shows GBA-mCherry-CRY2 fusion proteins load GBA's It is expressed in excretion body good (Figure 28).

Excretion body (the GBA that ＜ 8-3 ＞ pass through load GBA::EXPLOR) confirm GBA to from Gaucher patient's cell Activity

A. in excretion body GBA enzymatic activity

Present inventor has performed the experiments of β-glucocerebrosidase activity, to study the glucocerebroside of GBA in excretion body Degrading activity.

Particularly, the carrier comprising CIBN-EGRP-CD9 genes and Cre-mCherry-CRY2 genes is transfected into excretion Body generated on cell HEK293T, and by these cell culture 24 hours.After culture 24 hours, cell culture medium is changed into and is free of Fetal calf serum (FBS), and in addition cultivated 48 hours under the blue light of 50 μ W power of 488nm wavelength.After culture, by from The heart (2000 × g, 15 minutes) generates the supernatant for removing cell fragment from the culture medium of separation.5 times of bodies are added to supernatant Long-pending ExoQuick-TC excretion bodies precipitation solution (System Biosciences, Mountain View, California, USA it), and at 4 DEG C mixes 18 hours.Pass through the mixed of the above-mentioned supernatant of centrifugation (1500 × g, 30 minutes) and ExoQuick-TC Object is closed to suspend excretion body bead to obtain the excretion body of suspension.

In addition, the excretion body for stablizing expression CIBN-EGFP-CD9 genes and 2 genes of CRE-mCherry-CRY is generated thin Born of the same parents HEK293T cultivates 48~72h under the blue light of 50 μ W power of 488nm wavelength in the culture medium of no fetal calf serum.Culture knot Shu Hou generates the supernatant for removing cell fragment by centrifugation (2000 × g, 15 minutes) from the culture medium of separation.In order to from Particle of the removal more than 200nm, is filtered with 0.2 μ l PES films (Corning) in supernatant.It is answered in identical supernatant With particle of tangential flow filtration (TFF) method removal less than 20nm, is concentrated from filtrate and refine excretion body.It is used in TFF Vivaflow 50-100kDa PES films (Sartorius).It is dense by rotating filtrate under 1.5~2 air pressures of TFF Contracting and refined excretion body.Then, by being centrifuged on Amicon Ultra-0.5 (100kDa) (Millipore) filter (10000~14000g, 5 minutes) removes liquid in excretion body concentrate.Finally, by reversely centrifuge (10000~ 14000g, 5 minutes) obtain excretion body.Excretion body is cracked using MPER (mammalian proteins extracts reagent) and analyzes egg In vain.

Compared with the excretion body of load mCherry, the GBA of load GBA is observed::β-glucocerebrosidase of EXPLOR Enzymatic activity increases, to confirm that active GBA is loaded on excretion body (Figure 29).

B. β-glucocerebrosidase (GBA) is to the enzymatic activity from Gaucher patient's cell

Present inventor has performed following experiments, and GBA is used to confirm to work as::β-glucocerebrosidase is to source when EXPLOR processing From the recovery of the enzymatic activity of Gaucher patient's cell.

With in 60mm culture dishes 2 × 10⁵The density culture of a cell is originated from Gaucher patient fibroblast.Then, it uses mCherry::EXPLOR(2×10⁹A excretion body) or GBA::EXPLOR(1.2×10¹⁰A excretion body) it handles in serum-free That is cultivated in DMEM culture mediums is originated from Gaucher patient fibroblast.By using substrate 4-methyl umbelliferone base-β-D- pyrroles Glucopyranoside glycosides (4-MUG；Sigma fluorescence) is detected to measure the hydrolysing activity of GBA-mCh-CRY2.It is split comprising 50 μ l cells Solve 0.15% (v/v) Triton X-100 (Sigma), 0.8% (w/v) natrium taurocholicum (Sigma) and the 10mM 4-MUG of liquid 0.2ml citrate phosphate buffers (pH 0.5) in carry out enzyme reaction.After being incubated 1 hour at 37 DEG C, the 0.1M of 100ul is used Glycine and 0.1M NaOH (pH 10.3) stop enzymatic activity.Enzyme reaction production is measured under 365nm excitations, 460nm launching conditions Object 4-methyl umbelliferone (4-MU).

As a result, the GBA of load GBA::Activity of beta-glucosidase of the EXPLOR processing from Gaucher patient's cell is able to Restore (Figure 30).

EXPERIMENTAL EXAMPLESThe 9：Mitogen-activated kinase modulator：P38MAP kinases

Present inventors have shown that in expression CIBN-EGFP-CD9 and p38MAP kinases-under the 488nm wavelength blue lights The combination of CIBN and CRY2 in the cell of mCherry-Cry2, and demonstrate load of the p38MAP kinases in excretion body.

In order to largely generate load p38MAP kinases excretion body, establish stablize expression CIBN-EGFP-CD9 genes and The cell of p38MAP kinases-mCherry-CRY2 genes, and detached from culture supernatant by tangential flow filtration (TFF) method pure It is outside the pale of civilization to secrete body.

Functional selection is carried out to the excretion body for loading p38MAP kinases in target cell：

With the excretion body processing target cell of load p38MAP kinases with display function activity.

By the way that the excretion body of load p38MAP kinases is administered in intraperitoneal or vein to animal model, with display treatment effect Fruit.

EXPERIMENTAL EXAMPLESThe 10：Repressor kappa B kinases (IKK)

It has evaluated under 488nm wavelength blue lights in the cell of expression CIBN-EGFP-CD9 and IKK-mCherry-Cry2 The load of IKK in the combination of CIBN and CRY2 and excretion body.

In order to largely generate the excretion body of load IKK, establishes and stablize expression CIBN-EGFP-CD9 genes and IKK- The cell of mCherry-CRY2 genes, and excretion body is isolated and purified from culture supernatant by tangential flow filtration (TFF) method.

Functional selection is carried out to the excretion body for loading IKK in target cell：

With the excretion body processing target cell of load IKK with display function activity.

By the way that the excretion body of load IKK is administered in intraperitoneal or vein to animal model, to show therapeutic effect.

EXPERIMENTAL EXAMPLESThe 11：PTEN phosphatases

Present inventors have shown that under the 488nm wavelength blue lights in the cell of expression CIBN-EGFP-CD9 and PTEN-Cry2 The load of PTEN in the combination of CIBN and CRY2 and excretion body.

In order to largely generate the excretion body of load PTEN, establishes and stablize expression CIBN-EGFP-CD9 genes and PTEN- The cell (Figure 31) of CRY2 genes, and excretion body is isolated and purified from culture supernatant by tangential flow filtration (TFF) method.

Functional selection is carried out to the excretion body for loading PTEN in target cell：

With the excretion body processing target cell of load PTEN with display function activity.

By the way that the excretion body of load PTEN is administered in intraperitoneal or vein to animal model, to show therapeutic effect.

EXPERIMENTAL EXAMPLESThe 12：Janus kinases (JNK)

It has evaluated under 488nm wavelength blue lights in the cell of expression CIBN-EGFP-CD9 and JNK-mCherry-Cry2 The load of JNK in the combination of CIBN and CRY2 and excretion body.

In order to largely generate the excretion body of load JNK, establishes and stablize expression CIBN-EGFP-CD9 genes and JNK- The cell of mCherry-CRY2 genes, and excretion body is isolated and purified from culture supernatant by tangential flow filtration (TFF) method.

Functional selection is carried out to the excretion body for loading JNK in target cell：

With the excretion body processing target cell of load JNK with display function activity.

By the way that the excretion body of load JNK is administered in intraperitoneal or vein to animal model, to show therapeutic effect.

EXPERIMENTAL EXAMPLESThe 13：Ubiquitin ligase

It has evaluated under 488nm wavelength blue lights expression CIBN-EGFP-CD9's and ubiquitin ligase-mCherry-Cry2 In cell in the combination of CIBN and CRY2 and excretion body ubiquitin ligase load.

In order to largely generate load ubiquitin ligase excretion body, establish stablize expression CIBN-EGFP-CD9 genes and The cell of ubiquitin ligase-mCherry-CRY2 genes, and detached from culture supernatant by tangential flow filtration (TFF) method pure It is outside the pale of civilization to secrete body.

Functional selection is carried out to the excretion body for loading ubiquitin ligase in target cell：

With the excretion body processing target cell of load ubiquitin ligase with display function activity.

By the way that the excretion body of load ubiquitin ligase is administered in intraperitoneal or vein to animal model, with display treatment effect Fruit.

EXPERIMENTAL EXAMPLESThe 14：Luciferase

Present inventors have shown that in expression CIBN-EGFP-CD9 and luciferase-mCherry- under 488nm wavelength blue lights In the cell of Cry2 in the combination of CIBN and CRY2 and excretion body luciferase load (Figure 32).

In order to largely generate the excretion body of load luciferase, establishes and stablize expression CIBN-EGFP-CD9 genes and glimmering The cell of light element enzyme-mCherry-CRY2 genes, and isolated and purified from culture supernatant outside by tangential flow filtration (TFF) method Secrete body.

Based on luciferase molecules quantity, quantitative luciferase activity (Figure 33) is analyzed.

EXPERIMENTAL EXAMPLESThe 15：Peroxiredoxin

＜ 15-1 ＞ generate excretion body (the Prx I/II of load Prx I or Prx II:EXPLOR)

A. the Prx I/Prx II in excretion body are confirmed

Present inventors studied the knots of the CIBN-EGFP-CD9 and PrxI/II-mCherry-CRY2 of expression CIBN and CRY2 It closes, with validation record in amino acid SEQ ID NO：The load of Prx I or Prx II in 7 or 8.

Particularly, use includes CIBN-EGFP-CD9 genes and PrxI/II-mCherry-CRY2 bases under no light condition After transfection pcDNA3.1 (+) carrier culture 24 hours of cause, excretion body generates cell HEK293T and is free of tire in Dulbecco improvement It is additionally cultivated 48 hours in the Eagle culture mediums (DMEM) of cow's serum (FBS).After culture, pass through Laser Scanning Confocal Microscope Study position of the red fluorescence from mCherry before and after the blue light illumination of 488nm wavelength.This experiment is performed for more than five It is secondary.Thereby confirm that the aggregation (Figure 34) of the Prx I/II protein under blue light stimulus.Therefore, by confirming Prx I/II- The common location (yellow) of mCherry-CRY2 (red) and CIBN-EGFP-CD9 (green), by Prx I/II protein loads outside It secretes in body.

B. Prx I/II are generated::EXPLOR

Present inventor has performed following experiments to obtain the excretion body of load Prx I/II.

Particularly, the carrier comprising CIBN-EGRP-CD9 genes and Prx I/II-mCherry-CRY2 genes is transfected into Excretion body generated on cell HEK293T, and by these cell culture 24 hours.After culture 24 hours, cell culture medium is changed into Without fetal calf serum (FBS), and in addition cultivated 48 hours under the blue light of 50 μ W power of 488nm wavelength.After culture, lead to It crosses centrifugation (2000 × g, 15 minutes) and generates the supernatant for removing cell fragment from the culture medium of separation.It is added 5 to supernatant Times volume ExoQuick-TC excretion bodies precipitation solution (System Biosciences, Mountain View, California, USA), and mixed 18 hours at 4 DEG C.By centrifugation (1500 × g, 30 minutes) above-mentioned supernatant and The mixture of ExoQuick-TC suspends excretion body bead to obtain the excretion body (Fig. 8) of suspension.

＜ 15-2 ＞ confirm Prx I/II::The inhibition for the cytotoxicity that EXPLOR induces oxidative stress

Present inventor has performed following experiments to confirm Prx I/II::The cytotoxicity that EXPLOR induces oxidative stress Inhibition.

Particularly, after the serum free medium for changing HeLa cells, with the PrxI/II of 100 μ g/mL::EXPLOR processing And it cultivates 18 hours.Use H₂O₂(0,0.5,1mM) handles and continues culture 8 hours.WST is analyzed for analyzing cell activity.

Due to Prx I/II::The cytotoxicity of the pretreatment of EXPLOR, oxidative stress induction is suppressed significantly (Figure 35).

EXPERIMENTAL EXAMPLESThe 16：NF-κB

It has evaluated under 488nm wavelength blue lights in the cell of expression CIBN-EGFP-CD9 and NF- κ B-mCherry-Cry2 The load of NF- κ B in the combination of middle CIBN and CRY2 and excretion body.

In order to largely generate the excretion body of load NF- κ B, establishes and stablize expression CIBN-EGFP-CD9 genes and NF- κ B- The cell of mCherry-CRY2 genes, and excretion body is isolated and purified from culture supernatant by tangential flow filtration (TFF) method.

Functional selection is carried out to the excretion body for loading NF- κ B in target cell：

With the excretion body processing target cell of load NF- κ B with display function activity.

By the way that the excretion body of load NF- κ B is administered in intraperitoneal or vein to animal model, to show therapeutic effect.

EXPERIMENTAL EXAMPLESThe 17：MyoD

Present inventors have shown that in expression CIBN-EGFP-CD9 and MyoD-mCherry-Cry2 under the 488nm wavelength blue lights Cell in CIBN and CRY2 combination (Figure 36), and demonstrate the load of MyoD in excretion body.

In order to largely generate the excretion body of load MyoD, establishes and stablize expression CIBN-EGFP-CD9 genes and MyoD- The cell of mCherry-CRY2 genes, and excretion body is isolated and purified from culture supernatant by tangential flow filtration (TFF) method.

It is active (Figure 37) with display function with the excretion body processing target cell of load MyoD.

EXPERIMENTAL EXAMPLESThe 18：Tbx18 (T-box transcription factors 18)

It has evaluated under 488nm wavelength blue lights in the cell of expression CIBN-EGFP-CD9 and Tbx18-mCherry-Cry2 The load of Tbx18 in the combination of middle CIBN and CRY2 and excretion body.

In order to largely generate the excretion body of load Tbx18, establishes and stablize expression CIBN-EGFP-CD9 genes and Tbx18- The cell of mCherry-CRY2 genes, and excretion body is isolated and purified from culture supernatant by tangential flow filtration (TFF) method.

Functional selection is carried out to the excretion body for loading Tbx18 in target cell：

With the excretion body processing target cell of load Tbx18 with display function activity.

By the way that the excretion body of load Tbx18 is administered in intraperitoneal or vein to animal model, to show therapeutic effect.

EXPERIMENTAL EXAMPLESThe 19：p53

Present inventors have shown that in expression CIBN-EGFP-CD9 and p53-mCherry-Cry2 under the 488nm wavelength blue lights Cell in the combination (Figure 38) of CIBN and CRY2 and the load (Figure 39) of PTEN in excretion body.

In order to largely generate the excretion body of load p53, establishes and stablize expression CIBN-EGFP-CD9 genes and p53- The cell of mCherry-CRY2 genes, and excretion body is isolated and purified from culture supernatant by tangential flow filtration (TFF) method.

Target cell is handled to show transcriptional activity (Figure 40) with the excretion body of load p53.

By the way that the excretion body of load p53 is administered in intraperitoneal or vein to animal model, to show therapeutic effect.

EXPERIMENTAL EXAMPLESThe 20：HMGB1

Present inventors have shown that in expression CIBN-EGFP-CD9 and HMGB1-mCherry- under the 488nm wavelength blue lights In the cell of Cry2 in the combination of CIBN and CRY2 and excretion body HMGB1 load (Figure 41).

In order to largely generate the excretion body of load HMGB1, establishes and stablize expression CIBN-EGFP-CD9 genes and HMGB1- The cell of mCherry-CRY2 genes, and excretion body is isolated and purified from culture supernatant by tangential flow filtration (TFF) method.

Functional selection is carried out to the excretion body for loading HMGB1 in target cell：

With the excretion body processing target cell of load HMGB1 with display function activity.

By the way that the excretion body of load HMGB1 is administered in intraperitoneal or vein to animal model, to show therapeutic effect.

EXPERIMENTAL EXAMPLESThe 21：Super repressor I κ B

＜ 21-1 ＞ generate excretion body (the super repressor I κ B for loading super repressor I κ B:EXPLOR)

A. the super repressor I κ B in excretion body are confirmed

Present inventors studied the CIBN-EGFP-CD9 and super repressor I κ B-mCherry-CRY2 of expression CIBN and CRY2 Combination, with validation record in amino acid SEQ ID NO：The load of super repressor I κ B in 5.

Particularly, it cultivates to have under no light condition and includes CIBN-EGFP-CD9 genes and super repressor I κ B- The excretion body of transfection pcDNA3.1 (+) carrier of mCherry-CRY2 genes generated cell after HEK293T24 hours, the production of excretion body It is small that raw cell HEK293T additionally cultivates 48 in Eagle culture mediums (DMEM) of the Dulbecco improvement without fetal calf serum (FBS) When.After culture, the red fluorescence from mCherry is studied by Laser Scanning Confocal Microscope and is shone in the blue light of 488nm wavelength Penetrate front and back position.This experiment is performed for more than five times.Thereby confirm that the aggregation of the super repressor I kB proteins under blue light stimulus (Figure 42).Therefore, by confirming that super repressor I κ B-mCherry-CRY2 (red) and the total of CIBN-EGFP-CD9 (green) are determined Position (yellow), super repressor I kB proteins are loaded in excretion body.

B. super repressor I κ B are generated::EXPLOR

Present inventor has performed following experiments to obtain the excretion body for loading super repressor I κ B.

Particularly, the carrier comprising CIBN-EGRP-CD9 genes and super repressor I κ B-mCherry-CRY2 genes is turned It contaminates excretion body to generate on cell HEK293T, and by these cell culture 24 hours.After culture 24 hours, by cell culture medium It changes into and is free of fetal calf serum (FBS), and in addition cultivated 48 hours under the blue light of 50 μ W power of 488nm wavelength.Culture terminates Afterwards, the supernatant for removing cell fragment is generated from the culture medium of separation by centrifugation (2000 × g, 15 minutes).To supernatant Be added 5 times of volumes ExoQuick-TC excretion bodies precipitation solution (System Biosciences, Mountain View, California, USA), and mixed 18 hours at 4 DEG C.By centrifugation (1500 × g, 30 minutes) above-mentioned supernatant and The mixture of ExoQuick-TC suspends excretion body bead to obtain the excretion body (Fig. 8) of suspension.

In addition, the excretion body for stablizing expression CIBN-EGFP-CD9 genes and 2 genes of CRE-mCherry-CRY is generated thin Born of the same parents HEK293T cultivates 48~72h under the blue light of 50 μ W power of 488nm wavelength in the culture medium of no fetal calf serum.Culture knot Shu Hou generates the supernatant for removing cell fragment by centrifugation (2000 × g, 15 minutes) from the culture medium of separation.In order to from Particle of the removal more than 200nm, is filtered with 0.2 μ l PES films (Corning) in supernatant.It is answered in identical supernatant With particle of tangential flow filtration (TFF) method removal less than 20nm, is concentrated from filtrate and refine excretion body.It is used in TFF Vivaflow 50-100kDa PES films (Sartorius).It is dense by rotating filtrate under 1.5~2 air pressures of TFF Contracting and refined excretion body.Then, by being centrifuged on Amicon Ultra-0.5 (100kDa) (Millipore) filter (10000~14000g, 5 minutes) removes liquid in excretion body concentrate.Finally, by preferably delaying with according to experiment purpose Fliud flushing reversely centrifuges (10000~14000g, 5 minutes) and obtains excretion body.

＜ 21-2 ＞ confirm super repressor I κ B:The NF- kB activity inhibitions that EXPLOR mediates TNF-α

Present inventor has performed following experiments to confirm to use super repressor I κ B:The anti-inflammatory effect that the TNF-α of EXPLOR mediates Fruit.

Particularly, HeLa is in 100mg/mL mCherry:In EXPLOR or super repressor-I κ B-mCherry:At EXPLOR It is cultivated 3 hours in the culture medium of reason.Then, it is handled with TNF-α (10ng/mL) and continues to be incubated 30 minutes.With 4% paraformaldehyde After fixation, NF- κ Bp65 are dyed with 488 conjugation of antibodies of Alexa Fluor and are checked using Laser Scanning Confocal Microscope.In order to measure The combination activity of p65/c-Rel (NF-kB), according to manufacturer specification in TransAM NF-kB and AP-1 assay kits Karyorhexis object is used in (ActiveMotif, Carlsbad, CA, USA).Average value ± SEM (n=3) is presented in data, and uses Tukey's after tests (Tukey ' s post hoc test) are applied, and are tested by ANOVA and determined notable group of (* *, p <0.01)。

By with super repressor I κ B:EXPLOR pre-processes HeLa, and the NF- κ B of TNF-α activation are transported to nucleus and press down NF- κ BDNA processed combine (Figure 43).

＜ 21-3 ＞ confirm super repressor I κ B:Antiphlogistic effects of the EXPLOR to collagen-induced arthritis animal model

Present inventor has performed following experiments to suppress sub- I κ B to confirm to surpass:EXPLOR is small to collagen induced arthritis The antiphlogistic effects of mouse model.

Particularly, main model of rheumatoid arthritis to be used, collagen induced arthritis mouse model is to pass through The tail subcutaneous tissue of injecting ox typeⅡ Collagen and adjuvant to DBA/1 is immune and develops.It is lured to two collagens within every 2 days It leads arthritis mouse model and carries out the super repressor I κ B of 4 retrobulbar injections:EXPLOR.It is true according to the clinical score listed in table 3 Determine the progress of symptoms of rheumatoid arthritis.Mean clinical scores are the clinical scores from mouse foot according to above table Average value.

As super repressor I κ B:When EXPLOR retrobulbar injections are to collagen induced arthritis mouse model, rheumatoid Arthritic mice shows the reduction (Figure 44) of symptom.

[table 3]

Severity score	Phenotype marker
		0	There is no the sign of erythema and swelling
1	Erythema and mild swelling are confined to shank or ankle-joint
		2	Erythema and mild swelling extend to shank from ankle-joint
3	Erythema and moderate swelling extend to metatarsal joints from ankle-joint
		4	Erythema and serious swelling cover ankle, foot and toes or limbs are tetanic

The excretion body of ＜ 21-4 ＞ loads srIkB induces LPS the influence of sepsis model

In addition, as super repressor I κ B:When EXPLOR intraperitoneal injections induce septicopyemia disease mouse model to LPS, pyemia is small Mouse shows notable raised survival rate (Figure 45).

EXPERIMENTAL EXAMPLESThe 22：PySTAT3 intracellular antibodies

Present inventors have shown that in expression CIBN-EGFP-CD9 and pySTAT3-mCherry- under the 488nm wavelength blue lights In the cell of Cry2 in the combination of CIBN and CRY2 and excretion body pySTAT3 load (Figure 46).

In order to largely generate load pySTAT3 excretion body, establish stablize expression CIBN-EGFP-CD9 genes and The cell of pySTAT3-mCherry-CRY2 genes, and isolated and purified from culture supernatant by tangential flow filtration (TFF) method Excretion body.

With the excretion body processing target cell of load pySTAT3 with evaluation function activity.

By the way that the excretion body of load pySTAT3 is administered in intraperitoneal or vein to animal model, to show therapeutic effect.

EXPERIMENTAL EXAMPLESThe 23：The relevant X proteins of Bcl-2

＜ 23-1 ＞ generate the excretion body (Bax of load Bax::EXPLOR)

A. the Bax in excretion body is confirmed

Present inventors studied expression CIBN and CRY2d CIBN-EGFP-CD9 and Bax-mCherry-CRY2 combination, With validation record in amino acid SEQ ID NO：The load of Bax in 6.

Particularly, it cultivates to have under no light condition and includes CIBN-EGFP-CD9 genes and Bax-mCherry-CRY2 bases The excretion body of transfection pcDNA3.1 (+) carrier of cause generated cell HEK293T after 24 hours, and excretion body generates cell HEK293T It is additionally cultivated 48 hours in Eagle culture medium (DMEM) of the Dulbecco improvement without fetal calf serum (FBS).Terminate in culture Afterwards, position of the red fluorescence from mCherry before and after the blue light illumination of 488nm wavelength is studied by Laser Scanning Confocal Microscope. This experiment has been carried out more than five times.Thereby confirm that the aggregation (Figure 48) of the Bax albumen under blue light stimulus.Therefore, pass through card The combination of real Bax-mCherry-CRY2 (red) and CIBN-EGFP-CD9 (green), will be in Bax protein loads to excretion body.

B. Bax is generated::EXPLOR

Present inventor has performed following experiments to obtain the excretion body of load Bax.

Particularly, the carrier comprising CIBN-EGRP-CD9 genes and Bax-mCherry-CRY2 genes is transfected into excretion Body generated on cell HEK293T, and by these cell culture 24 hours.After culture 24 hours, cell culture medium is changed into and is free of Fetal calf serum (FBS), and in addition cultivated 48 hours under the blue light of 50 μ W power of 488nm wavelength.After culture, by from The heart (2000 × g, 15 minutes) generates the supernatant for removing cell fragment from the culture medium of separation.5 times of bodies are added to supernatant Long-pending ExoQuick-TC excretion bodies precipitation solution (System Biosciences, Mountain View, California, USA it), and at 4 DEG C mixes 18 hours.Pass through the mixed of the above-mentioned supernatant of centrifugation (1500 × g, 30 minutes) and ExoQuick-TC Object is closed to suspend excretion body bead to obtain the excretion body (Fig. 8) of suspension.

In addition, the excretion body for stablizing expression CIBN-EGFP-CD9 genes and 2 genes of Bax-mCherry-CRY is generated thin Born of the same parents HEK293T cultivates 48~72h under the blue light of 50 μ W power of 488nm wavelength in the culture medium of no fetal calf serum.Culture knot Shu Hou generates the supernatant for removing cell fragment by centrifugation (2000 × g, 15 minutes) from the culture medium of separation.In order to from Particle of the removal more than 200nm, is filtered with 0.2 μ l PES films (Corning) in supernatant.It is answered in identical supernatant With particle of tangential flow filtration (TFF) method removal less than 20nm, is concentrated from filtrate and refine excretion body.It is used in TFF Vivaflow 50-100kDa PES films (Sartorius).It is dense by rotating filtrate under 1.5~2 air pressures of TFF Contracting and refined excretion body.Then, by being centrifuged on Amicon Ultra-0.5 (100kDa) (Millipore) filter (10000~14000g, 5 minutes) removes liquid in excretion body concentrate.Finally, by preferably delaying with according to experiment purpose Fliud flushing reversely centrifuges (10000~14000g, 5 minutes) and obtains excretion body.

＜ 23-2 ＞ are confirmed by Bax::Apoptosis caused by EXPLOR

Bax is apoptosis regulators, therefore Bax overexpressions are by being combined release cytochrome c with mitochondrial membrane simultaneously Inducing cell apoptosis.Present inventors have shown that using Bax:EXPLOR secretory cell pigments c.

Particularly, HeLa is in 0.1mg/mL mCherry:In EXPLOR or include Bax-mCherry:The culture of EXPLOR It is cultivated 12 hours in base.After being fixed using 4% paraformaldehyde, in order to measure the secretion of cytochrome c, with Alexa Fluor HeLa is dyed and is imaged using Laser Scanning Confocal Microscope by antibody conjugated 647-, and by count cell number (20 μm of engineer's scale) come Analyze the ratio of cytochrome c.Average value ± SEM (n=3) is presented in data, and is applied using Tukey's after tests, And it is tested by ANOVA and determines notable group of (* *, p<0.01).

As a result, in Bax:Ratio is in mCherry in the HeLa of EXPLOR processing:Bigger is observed in the HeLa of EXPLOR processing The cytochrome c release (Figure 49) of amount.

EXPERIMENTAL EXAMPLESThe 24：BcL-xL

It has evaluated under 488nm wavelength blue lights in the cell of expression CIBN-EGFP-CD9 and BcL-xL-mCherry-Cry2 The load of BcL-xL in the combination of middle CIBN and CRY2 and excretion body.

In order to largely generate the excretion body of load BcL-xL, establishes and stablize expression CIBN-EGFP-CD9 genes and BcL- The cell of xL-mCherry-CRY2 genes, and excretion is isolated and purified from culture supernatant by tangential flow filtration (TFF) method Body.

Functional selection is carried out to the excretion body for loading BcL-xL in target cell：

With the excretion body processing target cell of load BcL-xL with display function activity.

By the way that the excretion body of load BcL-xL is administered in intraperitoneal or vein to animal model, to show therapeutic effect.

EXPERIMENTAL EXAMPLESThe 25：AIMP

Present inventors have shown that in expression CIBN-EGFP-CD9 and AIMP-mCherry-Cry2 under the 488nm wavelength blue lights Cell in the combination (Figure 50) of CIBN and CRY2 and the load (Figure 51) of pySTAT3 in excretion body.

In order to largely generate the excretion body of load AIMP, establishes and stablize expression CIBN-EGFP-CD9 genes and AIMP- The cell of mCherry-CRY2 genes, and excretion body is isolated and purified from culture supernatant by tangential flow filtration (TFF) method.

With the excretion body processing target cell of load AIMP with evaluation function activity.

By the way that the excretion body of load AIMP is administered in intraperitoneal or vein to animal model, to show therapeutic effect.

EXPERIMENTAL EXAMPLESThe 26：Mcherry (fluorescin)

Present inventors have shown that in the thin of expression CIBN-EGFP-CD9 and mCherry-Cry2 under the 488nm wavelength blue lights In born of the same parents in the combination (Figure 52) of CIBN and CRY2 and excretion body AIMP load.

In order to largely generate load mCherry excretion body, establish stablize expression CIBN-EGFP-CD9 genes and The cell of mCherry-CRY2 genes, and excretion body is isolated and purified from culture supernatant by tangential flow filtration (TFF) method.

EXPERIMENTAL EXAMPLESThe 27：Nucleic acid binding protein

It has evaluated under 488nm wavelength blue lights in expression CIBN-EGFP-CD9 and nucleic acid binding protein-mCherry-Cry2 Cell in CIBN and CRY2 combination and the protein-bonded load of excretion nucleic acid in vivo.

In order to largely generate the excretion body of load nucleic acid binding protein, establishes and stablize expression CIBN-EGFP-CD9 genes With the cell of nucleic acid binding protein-mCherry-CRY2 genes, and by tangential flow filtration (TFF) method from culture supernatant point From purifying excretion body.

Functional selection is carried out to the excretion body for loading nucleic acid binding protein in target cell：

With the excretion body processing target cell of load nucleic acid binding protein with display function activity.

By the way that the excretion body of load nucleic acid binding protein is administered in intraperitoneal or vein to animal model, with display treatment effect Fruit.

Those skilled in the art will recognize that concept and specific implementation mode disclosed in foregoing description can be easy to Ground is with the basis for making an amendment or designing other embodiment to realize that the identical purpose of the present invention, those skilled in the art will also recognize Know, these same embodiments are without departing from the spirit and scope of the present invention described in appended claims.

<110>Sai Er Simon Rexs Life Sciences（CELLEX LIFE SCIENCES, INCORPORATED）

<120>Including the composition of the excretion body of load albumen and preparation and the method for delivering the composition

<130> 2017FPO-09-017_PCT

<150> KR 10-2016-0126335

<151> 2016-09-30

<150> KR 10-2016-0126921

<151> 2016-09-30

<150> KR 10-2016-0126961

<151> 2016-09-30

<150> KR 10-2016-0127486

<151> 2016-10-04

<150> KR 10-2016-0132616

<151> 2016-10-13

<150> KR 10-2017-0018637

<151> 2017-02-10

<160> 84

<170> PatentIn version 3.2

<210> 1

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<213>People（Homo sapiens）

<400> 1

Met Phe Gln Ala Ala Glu Arg Pro Gln Glu Trp Ala Met Glu Gly Pro

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Arg Asp Gly Leu Lys Lys Glu Arg Leu Leu Asp Asp Arg His Asp Ser

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

35 40 45

Leu Gln Glu Ile Arg Leu Glu Pro Gln Glu Val Pro Arg Gly Ser Glu

50 55 60

Pro Trp Lys Gln Gln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu

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Ala Ile Ile His Glu Glu Lys Ala Leu Thr Met Glu Val Ile Arg Gln

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Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln

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Thr Pro Leu His Leu Ala Val Ile Thr Asn Gln Pro Glu Ile Ala Glu

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Ala Leu Leu Gly Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly

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Asn Thr Pro Leu His Leu Ala Cys Glu Gln Gly Cys Leu Ala Ser Val

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Gly Val Leu Thr Gln Ser Cys Thr Thr Pro His Leu His Ser Ile Leu

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Lys Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Ile

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His Gly Tyr Leu Gly Ile Val Glu Leu Leu Val Ser Leu Gly Ala Asp

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Val Asn Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala

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Val Asp Leu Gln Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly

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

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Thr Trp Gly Arg Pro Ser Thr Arg Ile Gln Gln Gln Leu Gly Gln Leu

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Thr Leu Glu Asn Leu Gln Met Leu Pro Glu Ser Glu Asp Glu Glu Ser

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Tyr Asp Thr Glu Ser Glu Phe Thr Glu Phe Thr Glu Asp Glu Leu Pro

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Tyr Asp Asp Cys Val Phe Gly Gly Gln Arg Leu Thr Leu

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Met Ala Gly Val Ala Cys Leu Gly Lys Ala Ala Asp Ala Asp Glu Trp

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Cys Asp Ser Gly Leu Gly Ser Leu Gly Pro Asp Ala Ala Ala Pro Gly

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Gly Pro Gly Leu Gly Ala Glu Leu Gly Pro Gly Leu Ser Trp Ala Pro

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

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

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Ser Ala Gly Thr Glu Tyr Met Asp Leu Gln Asn Asp Leu Gly Gln Thr

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Ala Leu His Leu Ala Ala Ile Leu Gly Glu Thr Ser Thr Val Glu Lys

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Leu Tyr Ala Ala Gly Ala Gly Leu Cys Val Ala Glu Arg Arg Gly His

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Thr Ala Leu His Leu Ala Cys Arg Val Gly Ala His Ala Cys Ala Arg

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Ala Leu Leu Gln Pro Arg Pro Arg Arg Pro Arg Glu Ala Pro Asp Thr

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

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Val Ala Leu Tyr Pro Asp Ser Asp Leu Glu Lys Glu Glu Glu Glu Ser

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Glu Glu Asp Trp Lys Leu Gln Leu Glu Ala Glu Asn Tyr Glu Gly His

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Thr Pro Leu His Val Ala Val Ile His Lys Asp Val Glu Met Val Arg

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Leu Leu Arg Asp Ala Gly Ala Asp Leu Asp Lys Pro Glu Pro Thr Cys

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Gly Arg Ser Pro Leu His Leu Ala Val Glu Ala Gln Ala Ala Asp Val

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

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Gly Gly Arg Thr Pro Leu Gly Ser Ala Met Leu Arg Pro Asn Pro Ile

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Leu Ala Arg Leu Leu Arg Ala His Gly Ala Pro Glu Pro Glu Gly Glu

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Asp Glu Lys Ser Gly Pro Cys Ser Ser Ser Ser Asp Ser Asp Ser Gly

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Asp Glu Gly Asp Glu Tyr Asp Asp Ile Val Val His Ser Ser Arg Ser

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Gln Thr Arg Leu Pro Pro Thr Pro Ala Ser Lys Pro Leu Pro Asp Asp

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Pro Arg Pro Val

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Met Asn Gln Arg Arg Ser Glu Ser Arg Pro Gly Asn His Arg Leu Gln

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

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Ser Gly Ser Ala Arg Arg Gly Arg Gly Gly Gly Gly Ala Ile Arg Val

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Arg Arg Pro Cys Trp Ser Gly Gly Ala Gly Arg Gly Gly Gly Pro Ala

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Trp Ala Val Arg Leu Pro Thr Val Thr Ala Gly Trp Thr Trp Pro Ala

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Leu Arg Thr Leu Ser Ser Leu Arg Ala Gly Pro Ser Glu Pro His Ser

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Pro Gly Arg Arg Pro Pro Arg Ala Gly Arg Pro Leu Cys Gln Ala Asp

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Pro Gln Pro Gly Lys Ala Ala Arg Arg Ser Leu Glu Pro Asp Pro Ala

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Gln Thr Gly Pro Arg Pro Ala Arg Ala Ala Gly Met Ser Glu Ala Arg

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Lys Gly Pro Asp Glu Ala Glu Glu Ser Gln Tyr Asp Ser Gly Ile Glu

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Ser Leu Arg Ser Leu Arg Ser Leu Pro Glu Ser Thr Ser Ala Pro Ala

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Ser Gly Pro Ser Asp Gly Ser Pro Gln Pro Cys Thr His Pro Pro Gly

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Pro Val Lys Glu Pro Gln Glu Lys Glu Asp Ala Asp Gly Glu Arg Ala

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Asp Ser Thr Tyr Gly Ser Ser Ser Leu Thr Tyr Thr Leu Ser Leu Leu

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Gly Gly Pro Glu Ala Glu Asp Pro Ala Pro Arg Leu Pro Leu Pro His

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Val Gly Ala Leu Ser Pro Gln Gln Leu Glu Ala Leu Thr Tyr Ile Ser

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

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Ala Val Leu Leu Cys Cys Leu Ala Leu Leu Pro Gln Glu Val Leu Asp

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Ile Gln Asn Asn Leu Tyr Gln Thr Ala Leu His Leu Ala Val His Leu

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

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Ala Leu Gln Asp Arg His Gly Asp Thr Ala Leu His Val Ala Cys Gln

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Arg Gln His Leu Ala Cys Ala Arg Cys Leu Leu Glu Gly Arg Pro Glu

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Pro Gly Arg Gly Thr Ser His Ser Leu Asp Leu Gln Leu Gln Asn Trp

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Gln Gly Leu Ala Cys Leu His Ile Ala Thr Leu Gln Lys Asn Gln Pro

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Leu Met Glu Leu Leu Leu Arg Asn Gly Ala Asp Ile Asp Val Gln Glu

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Gly Thr Ser Gly Lys Thr Ala Leu His Leu Ala Val Glu Thr Gln Glu

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Arg Gly Leu Val Gln Phe Leu Leu Gln Ala Gly Ala Gln Val Asp Ala

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Arg Met Leu Asn Gly Cys Thr Pro Leu His Leu Ala Ala Gly Arg Gly

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Leu Met Gly Ile Ser Ser Thr Leu Cys Lys Ala Gly Ala Asp Ser Leu

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Leu Arg Asn Val Glu Asp Glu Thr Pro Gln Asp Leu Thr Glu Glu Ser

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

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Leu Cys Thr Asp

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Met Pro Arg Cys Pro Ala Gly Ala Met Asp Glu Gly Pro Val Asp Leu

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Arg Thr Arg Pro Lys Ala Ala Gly Leu Pro Gly Ala Ala Leu Pro Leu

20 25 30

Arg Lys Arg Pro Leu Arg Ala Pro Ser Pro Glu Pro Ala Ala Pro Arg

35 40 45

Gly Ala Ala Gly Leu Val Val Pro Leu Asp Pro Leu Arg Gly Gly Cys

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Asp Leu Pro Ala Val Pro Gly Pro Pro His Gly Leu Ala Arg Pro Glu

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Ala Leu Tyr Tyr Pro Gly Ala Leu Leu Pro Leu Tyr Pro Thr Arg Ala

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Met Gly Ser Pro Phe Pro Leu Val Asn Leu Pro Thr Pro Leu Tyr Pro

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Met Met Cys Pro Met Glu His Pro Leu Ser Ala Asp Ile Ala Met Ala

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Thr Arg Ala Asp Glu Asp Gly Asp Thr Pro Leu His Ile Ala Val Val

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Gln Gly Asn Leu Pro Ala Val His Arg Leu Val Asn Leu Phe Gln Gln

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Gly Gly Arg Glu Leu Asp Ile Tyr Asn Asn Leu Arg Gln Thr Pro Leu

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His Leu Ala Val Ile Thr Thr Leu Pro Ser Val Val Arg Leu Leu Val

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Thr Ala Gly Ala Ser Pro Met Ala Leu Asp Arg His Gly Gln Thr Ala

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Ala His Leu Ala Cys Glu His Arg Ser Pro Thr Cys Leu Arg Ala Leu

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Leu Asp Ser Ala Ala Pro Gly Thr Leu Asp Leu Glu Ala Arg Asn Tyr

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Asp Gly Leu Thr Ala Leu His Val Ala Val Asn Thr Glu Cys Gln Glu

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

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Ile Lys Ser Gly Arg Ser Pro Leu Ile His Ala Val Glu Asn Asn Ser

275 280 285

Leu Ser Met Val Gln Leu Leu Leu Gln His Gly Ala Asn Val Asn Ala

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Gln Met Tyr Ser Gly Ser Ser Ala Leu His Ser Ala Ser Gly Arg Gly

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

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Leu Lys Asn Cys His Asn Asp Thr Pro Leu Met Val Ala Arg Ser Arg

340 345 350

Arg Val Ile Asp Ile Leu Arg Gly Lys Ala Thr Arg Pro Ala Ser Thr

355 360 365

Ser Gln Pro Asp Pro Ser Pro Asp Arg Ser Ala Asn Thr Ser Pro Glu

370 375 380

Ser Ser Ser Arg Leu Ser Ser Asn Gly Leu Leu Ser Ala Ser Pro Ser

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

405 410 415

Ala Pro Pro Asn Phe Phe Leu Pro Ser Pro Ser Pro Pro Ala Phe Leu

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Pro Phe Ala Gly Val Leu Arg Gly Pro Gly Arg Pro Val Pro Pro Ser

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Pro Ala Pro Gly Gly Ser

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<213>People（Homo sapiens）

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Met Phe Gln Ala Ala Glu Arg Pro Gln Glu Trp Ala Met Glu Gly Pro

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

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Gly Leu Asp Ala Met Lys Asp Glu Glu Tyr Glu Gln Met Val Lys Glu

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Leu Gln Glu Ile Arg Leu Glu Pro Gln Glu Val Pro Arg Gly Ser Glu

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Pro Trp Lys Gln Gln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu

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Ala Ile Ile His Glu Glu Lys Ala Leu Thr Met Glu Val Ile Arg Gln

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Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln

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Thr Pro Leu His Leu Ala Val Ile Thr Asn Gln Pro Glu Ile Ala Glu

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Ala Leu Leu Gly Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly

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Asn Thr Pro Leu His Leu Ala Cys Glu Gln Gly Cys Leu Ala Ser Val

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Gly Val Leu Thr Gln Ser Cys Thr Thr Pro His Leu His Ser Ile Leu

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Lys Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Ile

180 185 190

His Gly Tyr Leu Gly Ile Val Glu Leu Leu Val Ser Leu Gly Ala Asp

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Val Asn Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala

210 215 220

Val Asp Leu Gln Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly

225 230 235 240

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

245 250 255

Thr Trp Gly Arg Pro Ser Thr Arg Ile Gln Gln Gln Leu Gly Gln Leu

260 265 270

Thr Leu Glu Asn Leu Gln Met Leu Pro Glu Ser Glu Asp Glu Glu Ser

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Tyr Asp Thr Glu Ser Glu Phe Thr Glu Phe Thr Glu Asp Glu Leu Pro

290 295 300

Tyr Asp Asp Cys Val Phe Gly Gly Gln Arg Leu Thr Leu

305 310 315

<210> 6

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<212> PRT

<213>People（Homo sapiens）

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Met Asp Gly Ser Gly Glu Gln Pro Arg Gly Gly Gly Pro Thr Ser Ser

1 5 10 15

Glu Gln Ile Met Lys Thr Gly Ala Leu Leu Leu Gln Gly Phe Ile Gln

20 25 30

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

35 40 45

Pro Val Pro Gln Asp Ala Ser Thr Lys Lys Leu Ser Glu Cys Leu Lys

50 55 60

Arg Ile Gly Asp Glu Leu Asp Ser Asn Met Glu Leu Gln Arg Met Ile

65 70 75 80

Ala Ala Val Asp Thr Asp Ser Pro Arg Glu Val Phe Phe Arg Val Ala

85 90 95

Ala Asp Met Phe Ser Asp Gly Asn Phe Asn Trp Gly Arg Val Val Ala

100 105 110

Leu Phe Tyr Phe Ala Ser Lys Leu Val Leu Lys Ala Leu Cys Thr Lys

115 120 125

Val Pro Glu Leu Ile Arg Thr Ile Met Gly Trp Thr Leu Asp Phe Leu

130 135 140

Arg Glu Arg Leu Leu Gly Trp Ile Gln Asp Gln Gly Gly Trp Asp Gly

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Leu Leu Ser Tyr Phe Gly Thr Pro Thr Trp Gln Thr Val Thr Ile Phe

165 170 175

Val Ala Gly Val Leu Thr Ala Ser Leu Thr Ile Trp Lys Lys Met Gly

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<210> 7

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Met Ser Ser Gly Asn Ala Lys Ile Gly His Pro Ala Pro Asn Phe Lys

1 5 10 15

Ala Thr Ala Val Met Pro Asp Gly Gln Phe Lys Asp Ile Ser Leu Ser

20 25 30

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

35 40 45

Thr Phe Val Cys Pro Thr Glu Ile Ile Ala Phe Ser Asp Arg Ala Glu

50 55 60

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

65 70 75 80

His Phe Cys His Leu Ala Trp Val Asn Thr Pro Lys Lys Gln Gly Gly

85 90 95

Leu Gly Pro Met Asn Ile Pro Leu Val Ser Asp Pro Lys Arg Thr Ile

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Gln Ala Phe Gln Phe Thr Asp Lys His Gly Glu Val Cys Pro Ala Gly

165 170 175

Trp Lys Pro Gly Ser Asp Thr Ile Lys Pro Asp Val Gln Lys Ser Lys

180 185 190

Glu Tyr Phe Ser Lys Gln Lys

195

<210> 8

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<213>People（Homo sapiens）

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Met Ala Ser Gly Asn Ala Arg Ile Gly Lys Pro Ala Pro Asp Phe Lys

1 5 10 15

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

20 25 30

Tyr Lys Gly Lys Tyr Val Val Leu Phe Phe Tyr Pro Leu Asp Phe Thr

35 40 45

Phe Val Cys Pro Thr Glu Ile Ile Ala Phe Ser Asn Arg Ala Glu Asp

50 55 60

Phe Arg Lys Leu Gly Cys Glu Val Leu Gly Val Ser Val Asp Ser Gln

65 70 75 80

Phe Thr His Leu Ala Trp Ile Asn Thr Pro Arg Lys Glu Gly Gly Leu

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Asp Leu Pro Val Gly Arg Ser Val Asp Glu Ala Leu Arg Leu Val Gln

145 150 155 160

Ala Phe Gln Tyr Thr Asp Glu His Gly Glu Val Cys Pro Ala Gly Trp

165 170 175

Lys Pro Gly Ser Asp Thr Ile Lys Pro Asn Val Asp Asp Ser Lys Glu

180 185 190

Tyr Phe Ser Lys His Asn

195

<210> 9

<211> 343

<212> PRT

<213>Bacteriophage P1

<400> 9

Met Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro Ala Leu Pro Val

1 5 10 15

Asp Ala Thr Ser Asp Glu Val Arg Lys Asn Leu Met Asp Met Phe Arg

20 25 30

Asp Arg Gln Ala Phe Ser Glu His Thr Trp Lys Met Leu Leu Ser Val

35 40 45

Cys Arg Ser Trp Ala Ala Trp Cys Lys Leu Asn Asn Arg Lys Trp Phe

50 55 60

Pro Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu Leu Tyr Leu Gln Ala

65 70 75 80

Arg Gly Leu Ala Val Lys Thr Ile Gln Gln His Leu Gly Gln Leu Asn

85 90 95

Met Leu His Arg Arg Ser Gly Leu Pro Arg Pro Ser Asp Ser Asn Ala

100 105 110

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

115 120 125

Glu Arg Ala Lys Gln Ala Leu Ala Phe Glu Arg Thr Asp Phe Asp Gln

130 135 140

Val Arg Ser Leu Met Glu Asn Ser Asp Arg Cys Gln Asp Ile Arg Asn

145 150 155 160

Leu Ala Phe Leu Gly Ile Ala Tyr Asn Thr Leu Leu Arg Ile Ala Glu

165 170 175

Ile Ala Arg Ile Arg Val Lys Asp Ile Ser Arg Thr Asp Gly Gly Arg

180 185 190

Met Leu Ile His Ile Gly Arg Thr Lys Thr Leu Val Ser Thr Ala Gly

195 200 205

Val Glu Lys Ala Leu Ser Leu Gly Val Thr Lys Leu Val Glu Arg Trp

210 215 220

Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn Tyr Leu Phe Cys

225 230 235 240

Arg Val Arg Lys Asn Gly Val Ala Ala Pro Ser Ala Thr Ser Gln Leu

245 250 255

Ser Thr Arg Ala Leu Glu Gly Ile Phe Glu Ala Thr His Arg Leu Ile

260 265 270

Tyr Gly Ala Lys Asp Asp Ser Gly Gln Arg Tyr Leu Ala Trp Ser Gly

275 280 285

His Ser Ala Arg Val Gly Ala Ala Arg Asp Met Ala Arg Ala Gly Val

290 295 300

Ser Ile Pro Glu Ile Met Gln Ala Gly Gly Trp Thr Asn Val Asn Ile

305 310 315 320

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

325 330 335

Arg Leu Leu Glu Asp Gly Asp

340

<210> 10

<211> 469

<212> PRT

<213>Micrococcus scarlatinae（Streptococcus pyogenes）

<400> 10

Met His Ser Phe Pro Pro Leu Leu Leu Leu Leu Phe Trp Gly Val Val

1 5 10 15

Ser His Ser Phe Pro Ala Thr Leu Glu Thr Gln Glu Gln Asp Val Asp

20 25 30

Leu Val Gln Lys Tyr Leu Glu Lys Tyr Tyr Asn Leu Lys Asn Asp Gly

35 40 45

Arg Gln Val Glu Lys Arg Arg Asn Ser Gly Pro Val Val Glu Lys Leu

50 55 60

Lys Gln Met Gln Glu Phe Phe Gly Leu Lys Val Thr Gly Lys Pro Asp

65 70 75 80

Ala Glu Thr Leu Lys Val Met Lys Gln Pro Arg Cys Gly Val Pro Asp

85 90 95

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

100 105 110

His Leu Thr Tyr Arg Ile Glu Asn Tyr Thr Pro Asp Leu Pro Arg Ala

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Pro Gly Gly Asn Leu Ala His Ala Phe Gln Pro Gly Pro Gly Ile Gly

180 185 190

Gly Asp Ala His Phe Asp Glu Asp Glu Arg Trp Thr Asn Asn Phe Arg

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Gln Ala Ile Tyr Gly Arg Ser Gln Asn Pro Val Gln Pro Ile Gly Pro

260 265 270

Gln Thr Pro Lys Ala Cys Asp Ser Lys Leu Thr Phe Asp Ala Ile Thr

275 280 285

Thr Ile Arg Gly Glu Val Met Phe Phe Lys Asp Arg Phe Tyr Met Arg

290 295 300

Thr Asn Pro Phe Tyr Pro Glu Val Glu Leu Asn Phe Ile Ser Val Phe

305 310 315 320

Trp Pro Gln Leu Pro Asn Gly Leu Glu Ala Ala Tyr Glu Phe Ala Asp

325 330 335

Arg Asp Glu Val Arg Phe Phe Lys Gly Asn Lys Tyr Trp Ala Val Gln

340 345 350

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

355 360 365

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

370 375 380

Asn Thr Gly Lys Thr Tyr Phe Phe Val Ala Asn Lys Tyr Trp Arg Tyr

385 390 395 400

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

405 410 415

His Asp Phe Pro Gly Ile Gly His Lys Val Asp Ala Val Phe Met Lys

420 425 430

Asp Gly Phe Phe Tyr Phe Phe His Gly Thr Arg Gln Tyr Lys Phe Asp

435 440 445

Pro Lys Thr Lys Arg Ile Leu Thr Leu Gln Lys Ala Asn Ser Trp Phe

450 455 460

Asn Cys Arg Lys Asn

465

<210> 11

<211> 1300

<212> PRT

<213>Francisella tularensis（Francisella tularensis）

<400> 11

Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys

20 25 30

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

35 40 45

Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu

50 55 60

Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser

65 70 75 80

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

85 90 95

Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr

100 105 110

Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile

115 120 125

Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln

130 135 140

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

145 150 155 160

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

165 170 175

Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser

180 185 190

Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu

195 200 205

Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys

210 215 220

Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu

225 230 235 240

Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys

305 310 315 320

Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser

325 330 335

Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met

340 345 350

Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys

355 360 365

Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala

435 440 445

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

450 455 460

Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala

465 470 475 480

Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys

485 490 495

Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys

500 505 510

Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp

515 520 525

Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His

530 535 540

Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His

545 550 555 560

Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val

565 570 575

Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser

580 585 590

Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly

595 600 605

Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys

610 615 620

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

625 630 635 640

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

645 650 655

Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val

660 665 670

Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile

675 680 685

Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln

690 695 700

Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe

705 710 715 720

Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp

725 730 735

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

740 745 750

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

755 760 765

Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr

770 775 780

Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg

785 790 795 800

Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn

805 810 815

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

820 825 830

Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala

835 840 845

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

850 855 860

Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe

865 870 875 880

His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe

885 890 895

Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His

900 905 910

Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu

915 920 925

Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile

930 935 940

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

945 950 955 960

Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn

965 970 975

Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile

980 985 990

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

995 1000 1005

Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr

1010 1015 1020

Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe

1025 1030 1035 1040

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

1045 1050 1055

Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr Gly

1060 1065 1070

Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys Pro Val

1075 1080 1085

Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys

1090 1095 1100

Ser Gln Glu Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp

1105 1110 1115 1120

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

1125 1130 1135

Ala Ala Lys Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile

1140 1145 1150

Asn Phe Arg Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val

1155 1160 1165

Tyr Pro Thr Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu

1170 1175 1180

Tyr Gly His Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp

1185 1190 1195 1200

Lys Lys Phe Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln

1205 1210 1215

Met Arg Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro

1220 1225 1230

Val Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys

1235 1240 1245

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

1250 1255 1260

Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys

1265 1270 1275 1280

Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln

1285 1290 1295

Asn Arg Asn Asn

1300

<210> 12

<211> 536

<212> PRT

<213>People（Homo sapiens）

<400> 12

Met Glu Phe Ser Ser Pro Ser Arg Glu Glu Cys Pro Lys Pro Leu Ser

1 5 10 15

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

20 25 30

Ala Val Ser Trp Ala Ser Gly Ala Arg Pro Cys Ile Pro Lys Ser Phe

35 40 45

Gly Tyr Ser Ser Val Val Cys Val Cys Asn Ala Thr Tyr Cys Asp Ser

50 55 60

Phe Asp Pro Pro Thr Phe Pro Ala Leu Gly Thr Phe Ser Arg Tyr Glu

65 70 75 80

Ser Thr Arg Ser Gly Arg Arg Met Glu Leu Ser Met Gly Pro Ile Gln

85 90 95

Ala Asn His Thr Gly Thr Gly Leu Leu Leu Thr Leu Gln Pro Glu Gln

100 105 110

Lys Phe Gln Lys Val Lys Gly Phe Gly Gly Ala Met Thr Asp Ala Ala

115 120 125

Ala Leu Asn Ile Leu Ala Leu Ser Pro Pro Ala Gln Asn Leu Leu Leu

130 135 140

Lys Ser Tyr Phe Ser Glu Glu Gly Ile Gly Tyr Asn Ile Ile Arg Val

145 150 155 160

Pro Met Ala Ser Cys Asp Phe Ser Ile Arg Thr Tyr Thr Tyr Ala Asp

165 170 175

Thr Pro Asp Asp Phe Gln Leu His Asn Phe Ser Leu Pro Glu Glu Asp

180 185 190

Thr Lys Leu Lys Ile Pro Leu Ile His Arg Ala Leu Gln Leu Ala Gln

195 200 205

Arg Pro Val Ser Leu Leu Ala Ser Pro Trp Thr Ser Pro Thr Trp Leu

210 215 220

Lys Thr Asn Gly Ala Val Asn Gly Lys Gly Ser Leu Lys Gly Gln Pro

225 230 235 240

Gly Asp Ile Tyr His Gln Thr Trp Ala Arg Tyr Phe Val Lys Phe Leu

245 250 255

Asp Ala Tyr Ala Glu His Lys Leu Gln Phe Trp Ala Val Thr Ala Glu

260 265 270

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

275 280 285

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

290 295 300

Pro Thr Leu Ala Asn Ser Thr His His Asn Val Arg Leu Leu Met Leu

305 310 315 320

Asp Asp Gln Arg Leu Leu Leu Pro His Trp Ala Lys Val Val Leu Thr

325 330 335

Asp Pro Glu Ala Ala Lys Tyr Val His Gly Ile Ala Val His Trp Tyr

340 345 350

Leu Asp Phe Leu Ala Pro Ala Lys Ala Thr Leu Gly Glu Thr His Arg

355 360 365

Leu Phe Pro Asn Thr Met Leu Phe Ala Ser Glu Ala Cys Val Gly Ser

370 375 380

Lys Phe Trp Glu Gln Ser Val Arg Leu Gly Ser Trp Asp Arg Gly Met

385 390 395 400

Gln Tyr Ser His Ser Ile Ile Thr Asn Leu Leu Tyr His Val Val Gly

405 410 415

Trp Thr Asp Trp Asn Leu Ala Leu Asn Pro Glu Gly Gly Pro Asn Trp

420 425 430

Val Arg Asn Phe Val Asp Ser Pro Ile Ile Val Asp Ile Thr Lys Asp

435 440 445

Thr Phe Tyr Lys Gln Pro Met Phe Tyr His Leu Gly His Phe Ser Lys

450 455 460

Phe Ile Pro Glu Gly Ser Gln Arg Val Gly Leu Val Ala Ser Gln Lys

465 470 475 480

Asn Asp Leu Asp Ala Val Ala Leu Met His Pro Asp Gly Ser Ala Val

485 490 495

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

500 505 510

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

515 520 525

His Thr Tyr Leu Trp Arg Arg Gln

530 535

<210> 13

<211> 469

<212> PRT

<213>People（Homo sapiens）

<400> 13

Met His Ser Phe Pro Pro Leu Leu Leu Leu Leu Phe Trp Gly Val Val

1 5 10 15

Ser His Ser Phe Pro Ala Thr Leu Glu Thr Gln Glu Gln Asp Val Asp

20 25 30

Leu Val Gln Lys Tyr Leu Glu Lys Tyr Tyr Asn Leu Lys Asn Asp Gly

35 40 45

Arg Gln Val Glu Lys Arg Arg Asn Ser Gly Pro Val Val Glu Lys Leu

50 55 60

Lys Gln Met Gln Glu Phe Phe Gly Leu Lys Val Thr Gly Lys Pro Asp

65 70 75 80

Ala Glu Thr Leu Lys Val Met Lys Gln Pro Arg Cys Gly Val Pro Asp

85 90 95

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

100 105 110

His Leu Thr Tyr Arg Ile Glu Asn Tyr Thr Pro Asp Leu Pro Arg Ala

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Pro Gly Gly Asn Leu Ala His Ala Phe Gln Pro Gly Pro Gly Ile Gly

180 185 190

Gly Asp Ala His Phe Asp Glu Asp Glu Arg Trp Thr Asn Asn Phe Arg

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Gln Ala Ile Tyr Gly Arg Ser Gln Asn Pro Val Gln Pro Ile Gly Pro

260 265 270

Gln Thr Pro Lys Ala Cys Asp Ser Lys Leu Thr Phe Asp Ala Ile Thr

275 280 285

Thr Ile Arg Gly Glu Val Met Phe Phe Lys Asp Arg Phe Tyr Met Arg

290 295 300

Thr Asn Pro Phe Tyr Pro Glu Val Glu Leu Asn Phe Ile Ser Val Phe

305 310 315 320

Trp Pro Gln Leu Pro Asn Gly Leu Glu Ala Ala Tyr Glu Phe Ala Asp

325 330 335

Arg Asp Glu Val Arg Phe Phe Lys Gly Asn Lys Tyr Trp Ala Val Gln

340 345 350

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

355 360 365

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

370 375 380

Asn Thr Gly Lys Thr Tyr Phe Phe Val Ala Asn Lys Tyr Trp Arg Tyr

385 390 395 400

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

405 410 415

His Asp Phe Pro Gly Ile Gly His Lys Val Asp Ala Val Phe Met Lys

420 425 430

Asp Gly Phe Phe Tyr Phe Phe His Gly Thr Arg Gln Tyr Lys Phe Asp

435 440 445

Pro Lys Thr Lys Arg Ile Leu Thr Leu Gln Lys Ala Asn Ser Trp Phe

450 455 460

Asn Cys Arg Lys Asn

465

<210> 14

<211> 207

<212> PRT

<213>People（Homo sapiens）

<400> 14

Met Ala Pro Phe Glu Pro Leu Ala Ser Gly Ile Leu Leu Leu Leu Trp

1 5 10 15

Leu Ile Ala Pro Ser Arg Ala Cys Thr Cys Val Pro Pro His Pro Gln

20 25 30

Thr Ala Phe Cys Asn Ser Asp Leu Val Ile Arg Ala Lys Phe Val Gly

35 40 45

Thr Pro Glu Val Asn Gln Thr Thr Leu Tyr Gln Arg Tyr Glu Ile Lys

50 55 60

Met Thr Lys Met Tyr Lys Gly Phe Gln Ala Leu Gly Asp Ala Ala Asp

65 70 75 80

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

85 90 95

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

100 105 110

Gln Asp Gly Leu Leu His Ile Thr Thr Cys Ser Phe Val Ala Pro Trp

115 120 125

Asn Ser Leu Ser Leu Ala Gln Arg Arg Gly Phe Thr Lys Thr Tyr Thr

130 135 140

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

145 150 155 160

Lys Leu Gln Ser Gly Thr His Cys Leu Trp Thr Asp Gln Leu Leu Gln

165 170 175

Gly Ser Glu Lys Gly Phe Gln Ser Arg His Leu Ala Cys Leu Pro Arg

180 185 190

Glu Pro Gly Leu Cys Thr Trp Gln Ser Leu Arg Ser Gln Ile Ala

195 200 205

<210> 15

<211> 220

<212> PRT

<213>People（Homo sapiens）

<400> 15

Met Gly Ala Ala Ala Arg Thr Leu Arg Leu Ala Leu Gly Leu Leu Leu

1 5 10 15

Leu Ala Thr Leu Leu Arg Pro Ala Asp Ala Cys Ser Cys Ser Pro Val

20 25 30

His Pro Gln Gln Ala Phe Cys Asn Ala Asp Val Val Ile Arg Ala Lys

35 40 45

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

50 55 60

Pro Ile Lys Arg Ile Gln Tyr Glu Ile Lys Gln Ile Lys Met Phe Lys

65 70 75 80

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

85 90 95

Val Cys Gly Val Ser Leu Asp Val Gly Gly Lys Lys Glu Tyr Leu Ile

100 105 110

Ala Gly Lys Ala Glu Gly Asp Gly Lys Met His Ile Thr Leu Cys Asp

115 120 125

Phe Ile Val Pro Trp Asp Thr Leu Ser Thr Thr Gln Lys Lys Ser Leu

130 135 140

Asn His Arg Tyr Gln Met Gly Cys Glu Cys Lys Ile Thr Arg Cys Pro

145 150 155 160

Met Ile Pro Cys Tyr Ile Ser Ser Pro Asp Glu Cys Leu Trp Met Asp

165 170 175

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

180 185 190

Cys Ile Lys Arg Ser Asp Gly Ser Cys Ala Trp Tyr Arg Gly Ala Ala

195 200 205

Pro Pro Lys Gln Glu Phe Leu Asp Ile Glu Asp Pro

210 215 220

<210> 16

<211> 211

<212> PRT

<213>People（Homo sapiens）

<400> 16

Met Thr Pro Trp Leu Gly Leu Ile Val Leu Leu Gly Ser Trp Ser Leu

1 5 10 15

Gly Asp Trp Gly Ala Glu Ala Cys Thr Cys Ser Pro Ser His Pro Gln

20 25 30

Asp Ala Phe Cys Asn Ser Asp Ile Val Ile Arg Ala Lys Val Val Gly

35 40 45

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

50 55 60

Lys Gln Met Lys Met Tyr Arg Gly Phe Thr Lys Met Pro His Val Gln

65 70 75 80

Tyr Ile His Thr Glu Ala Ser Glu Ser Leu Cys Gly Leu Lys Leu Glu

85 90 95

Val Asn Lys Tyr Gln Tyr Leu Leu Thr Gly Arg Val Tyr Asp Gly Lys

100 105 110

Met Tyr Thr Gly Leu Cys Asn Phe Val Glu Arg Trp Asp Gln Leu Thr

115 120 125

Leu Ser Gln Arg Lys Gly Leu Asn Tyr Arg Tyr His Leu Gly Cys Asn

130 135 140

Cys Lys Ile Lys Ser Cys Tyr Tyr Leu Pro Cys Phe Val Thr Ser Lys

145 150 155 160

Asn Glu Cys Leu Trp Thr Asp Met Leu Ser Asn Phe Gly Tyr Pro Gly

165 170 175

Tyr Gln Ser Lys His Tyr Ala Cys Ile Arg Gln Lys Gly Gly Tyr Cys

180 185 190

Ser Trp Tyr Arg Gly Trp Ala Pro Pro Asp Lys Ser Ile Ile Asn Ala

195 200 205

Thr Asp Pro

210

<210> 17

<211> 224

<212> PRT

<213>People（Homo sapiens）

<400> 17

Met Pro Gly Ser Pro Arg Pro Ala Pro Ser Trp Val Leu Leu Leu Arg

1 5 10 15

Leu Leu Ala Leu Leu Arg Pro Pro Gly Leu Gly Glu Ala Cys Ser Cys

20 25 30

Ala Pro Ala His Pro Gln Gln His Ile Cys His Ser Ala Leu Val Ile

35 40 45

Arg Ala Lys Ile Ser Ser Glu Lys Val Val Pro Ala Ser Ala Asp Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

His Leu Cys Asn Tyr Ile Glu Pro Trp Glu Asp Leu Ser Leu Val Gln

130 135 140

Arg Glu Ser Leu Asn His His Tyr His Leu Asn Cys Gly Cys Gln Ile

145 150 155 160

Thr Thr Cys Tyr Thr Val Pro Cys Thr Ile Ser Ala Pro Asn Glu Cys

165 170 175

Leu Trp Thr Asp Trp Leu Leu Glu Arg Lys Leu Tyr Gly Tyr Gln Ala

180 185 190

Gln His Tyr Val Cys Met Lys His Val Asp Gly Thr Cys Ser Trp Tyr

195 200 205

Arg Gly His Leu Pro Leu Arg Lys Glu Phe Val Asp Ile Val Gln Pro

210 215 220

<210> 18

<211> 404

<212> PRT

<213>People（Homo sapiens）

<400> 18

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

1 5 10 15

Met Gly Glu Gly Thr Ile Asn Gly Leu Leu Asp Glu Leu Leu Gln Thr

20 25 30

Arg Val Leu Asn Lys Glu Glu Met Glu Lys Val Lys Arg Glu Asn Ala

35 40 45

Thr Val Met Asp Lys Thr Arg Ala Leu Ile Asp Ser Val Ile Pro Lys

50 55 60

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

65 70 75 80

Ser Tyr Leu Ala Gly Thr Leu Gly Leu Ser Ala Asp Gln Thr Ser Gly

85 90 95

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

100 105 110

Ala Pro Gln Ala Val Gln Asp Asn Pro Ala Met Pro Thr Ser Ser Gly

115 120 125

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

130 135 140

Trp Lys Gln Lys Ser Ala Glu Ile Tyr Pro Ile Met Asp Lys Ser Ser

145 150 155 160

Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Glu Glu Phe Asp Ser Ile

165 170 175

Pro Arg Arg Thr Gly Ala Glu Val Asp Ile Thr Gly Met Thr Met Leu

180 185 190

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

195 200 205

Ser Asp Met Thr Thr Glu Leu Glu Ala Phe Ala His Arg Pro Glu His

210 215 220

Lys Thr Ser Asp Ser Thr Phe Leu Val Phe Met Ser His Gly Ile Arg

225 230 235 240

Glu Gly Ile Cys Gly Lys Lys His Ser Glu Gln Val Pro Asp Ile Leu

245 250 255

Gln Leu Asn Ala Ile Phe Asn Met Leu Asn Thr Lys Asn Cys Pro Ser

260 265 270

Leu Lys Asp Lys Pro Lys Val Ile Ile Ile Gln Ala Cys Arg Gly Asp

275 280 285

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

290 295 300

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

305 310 315 320

Ala His Ile Glu Lys Asp Phe Ile Ala Phe Cys Ser Ser Thr Pro Asp

325 330 335

Asn Val Ser Trp Arg His Pro Thr Met Gly Ser Val Phe Ile Gly Arg

340 345 350

Leu Ile Glu His Met Gln Glu Tyr Ala Cys Ser Cys Asp Val Glu Glu

355 360 365

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

370 375 380

Gln Met Pro Thr Thr Glu Arg Val Thr Leu Thr Arg Cys Phe Tyr Leu

385 390 395 400

Phe Pro Gly His

<210> 19

<211> 452

<212> PRT

<213>People（Homo sapiens）

<400> 19

Met Ala Ala Pro Ser Ala Gly Ser Trp Ser Thr Phe Gln His Lys Glu

1 5 10 15

Leu Met Ala Ala Asp Arg Gly Arg Arg Ile Leu Gly Val Cys Gly Met

20 25 30

His Pro His His Gln Glu Thr Leu Lys Lys Asn Arg Val Val Leu Ala

35 40 45

Lys Gln Leu Leu Leu Ser Glu Leu Leu Glu His Leu Leu Glu Lys Asp

50 55 60

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

65 70 75 80

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

85 90 95

Gln Ala Phe Asp Ala Phe Cys Glu Ala Leu Arg Glu Thr Lys Gln Gly

100 105 110

His Leu Glu Asp Met Leu Leu Thr Thr Leu Ser Gly Leu Gln His Val

115 120 125

Leu Pro Pro Leu Ser Cys Asp Tyr Asp Leu Ser Leu Pro Phe Pro Val

130 135 140

Cys Glu Ser Cys Pro Leu Tyr Lys Lys Leu Arg Leu Ser Thr Asp Thr

145 150 155 160

Val Glu His Ser Leu Asp Asn Lys Asp Gly Pro Val Cys Leu Gln Val

165 170 175

Lys Pro Cys Thr Pro Glu Phe Tyr Gln Thr His Phe Gln Leu Ala Tyr

180 185 190

Arg Leu Gln Ser Arg Pro Arg Gly Leu Ala Leu Val Leu Ser Asn Val

195 200 205

His Phe Thr Gly Glu Lys Glu Leu Glu Phe Arg Ser Gly Gly Asp Val

210 215 220

Asp His Ser Thr Leu Val Thr Leu Phe Lys Leu Leu Gly Tyr Asp Val

225 230 235 240

His Val Leu Cys Asp Gln Thr Ala Gln Glu Met Gln Glu Lys Leu Gln

245 250 255

Asn Phe Ala Gln Leu Pro Ala His Arg Val Thr Asp Ser Cys Ile Val

260 265 270

Ala Leu Leu Ser His Gly Val Glu Gly Ala Ile Tyr Gly Val Asp Gly

275 280 285

Lys Leu Leu Gln Leu Gln Glu Val Phe Gln Leu Phe Asp Asn Ala Asn

290 295 300

Cys Pro Ser Leu Gln Asn Lys Pro Lys Met Phe Phe Ile Gln Ala Cys

305 310 315 320

Arg Gly Asp Glu Thr Asp Arg Gly Val Asp Gln Gln Asp Gly Lys Asn

325 330 335

His Ala Gly Ser Pro Gly Cys Glu Glu Ser Asp Ala Gly Lys Glu Lys

340 345 350

Leu Pro Lys Met Arg Leu Pro Thr Arg Ser Asp Met Ile Cys Gly Tyr

355 360 365

Ala Cys Leu Lys Gly Thr Ala Ala Met Arg Asn Thr Lys Arg Gly Ser

370 375 380

Trp Tyr Ile Glu Ala Leu Ala Gln Val Phe Ser Glu Arg Ala Cys Asp

385 390 395 400

Met His Val Ala Asp Met Leu Val Lys Val Asn Ala Leu Ile Lys Asp

405 410 415

Arg Glu Gly Tyr Ala Pro Gly Thr Glu Phe His Arg Cys Lys Glu Met

420 425 430

Ser Glu Tyr Cys Ser Thr Leu Cys Arg His Leu Tyr Leu Phe Pro Gly

435 440 445

His Pro Pro Thr

450

<210> 20

<211> 277

<212> PRT

<213>People（Homo sapiens）

<400> 20

Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu

1 5 10 15

Glu Pro Lys Ile Ile His Gly Ser Glu Ser Met Asp Ser Gly Ile Ser

20 25 30

Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile

35 40 45

Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe

115 120 125

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

130 135 140

Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile

145 150 155 160

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

165 170 175

Gly Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp

180 185 190

Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn

195 200 205

Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys

210 215 220

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

225 230 235 240

Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe

245 250 255

His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu

260 265 270

Leu Tyr Phe Tyr His

275

<210> 21

<211> 377

<212> PRT

<213>People（Homo sapiens）

<400> 21

Met Ala Glu Gly Asn His Arg Lys Lys Pro Leu Lys Val Leu Glu Ser

1 5 10 15

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

20 25 30

Asn Val Leu Asn Trp Lys Glu Glu Glu Lys Lys Lys Tyr Tyr Asp Ala

35 40 45

Lys Thr Glu Asp Lys Val Arg Val Met Ala Asp Ser Met Gln Glu Lys

50 55 60

Gln Arg Met Ala Gly Gln Met Leu Leu Gln Thr Phe Phe Asn Ile Asp

65 70 75 80

Gln Ile Ser Pro Asn Lys Lys Ala His Pro Asn Met Glu Ala Gly Pro

85 90 95

Pro Glu Ser Gly Glu Ser Thr Asp Ala Leu Lys Leu Cys Pro His Glu

100 105 110

Glu Phe Leu Arg Leu Cys Lys Glu Arg Ala Glu Glu Ile Tyr Pro Ile

115 120 125

Lys Glu Arg Asn Asn Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Thr

130 135 140

Glu Phe Asp His Leu Pro Pro Arg Asn Gly Ala Asp Phe Asp Ile Thr

145 150 155 160

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

165 170 175

Glu Asn Leu Thr Ala Arg Asp Met Glu Ser Ala Leu Arg Ala Phe Ala

180 185 190

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

195 200 205

Ser His Gly Ile Leu Glu Gly Ile Cys Gly Thr Val His Asp Glu Lys

210 215 220

Lys Pro Asp Val Leu Leu Tyr Asp Thr Ile Phe Gln Ile Phe Asn Asn

225 230 235 240

Arg Asn Cys Leu Ser Leu Lys Asp Lys Pro Lys Val Ile Ile Val Gln

245 250 255

Ala Cys Arg Gly Ala Asn Arg Gly Glu Leu Trp Val Arg Asp Ser Pro

260 265 270

Ala Ser Leu Glu Val Ala Ser Ser Gln Ser Ser Glu Asn Leu Glu Glu

275 280 285

Asp Ala Val Tyr Lys Thr His Val Glu Lys Asp Phe Ile Ala Phe Cys

290 295 300

Ser Ser Thr Pro His Asn Val Ser Trp Arg Asp Ser Thr Met Gly Ser

305 310 315 320

Ile Phe Ile Thr Gln Leu Ile Thr Cys Phe Gln Lys Tyr Ser Trp Cys

325 330 335

Cys His Leu Glu Glu Val Phe Arg Lys Val Gln Gln Ser Phe Glu Thr

340 345 350

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

355 360 365

Arg Tyr Phe Tyr Leu Phe Pro Gly Asn

370 375

<210> 22

<211> 434

<212> PRT

<213>People（Homo sapiens）

<400> 22

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

1 5 10 15

Met Phe Lys Gly Ile Leu Gln Ser Gly Leu Asp Asn Phe Val Ile Asn

20 25 30

His Met Leu Lys Asn Asn Val Ala Gly Gln Thr Ser Ile Gln Thr Leu

35 40 45

Val Pro Asn Thr Asp Gln Lys Ser Thr Ser Val Lys Lys Asp Asn His

50 55 60

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

65 70 75 80

His Gly Val Phe Asn Tyr Leu Ala Lys His Asp Val Leu Thr Leu Lys

85 90 95

Glu Glu Glu Lys Lys Lys Tyr Tyr Asp Thr Lys Ile Glu Asp Lys Ala

100 105 110

Leu Ile Leu Val Asp Ser Leu Arg Lys Asn Arg Val Ala His Gln Met

115 120 125

Phe Thr Gln Thr Leu Leu Asn Met Asp Gln Lys Ile Thr Ser Val Lys

130 135 140

Pro Leu Leu Gln Ile Glu Ala Gly Pro Pro Glu Ser Ala Glu Ser Thr

145 150 155 160

Asn Ile Leu Lys Leu Cys Pro Arg Glu Glu Phe Leu Arg Leu Cys Lys

165 170 175

Lys Asn His Asp Glu Ile Tyr Pro Ile Lys Lys Arg Glu Asp Arg Arg

180 185 190

Arg Leu Ala Leu Ile Ile Cys Asn Thr Lys Phe Asp His Leu Pro Ala

195 200 205

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

210 215 220

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

225 230 235 240

Met Glu Ser Val Leu Arg Ala Phe Ala Ala Arg Pro Glu His Lys Ser

245 250 255

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

260 265 270

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

275 280 285

Asp Thr Ile Phe Gln Ile Phe Asn Asn Arg Asn Cys Leu Ser Leu Lys

290 295 300

Asp Lys Pro Lys Val Ile Ile Val Gln Ala Cys Arg Gly Glu Lys His

305 310 315 320

Gly Glu Leu Trp Val Arg Asp Ser Pro Ala Ser Leu Ala Leu Ile Ser

325 330 335

Ser Gln Ser Ser Glu Asn Leu Glu Ala Asp Ser Val Cys Lys Ile His

340 345 350

Glu Glu Lys Asp Phe Ile Ala Phe Cys Ser Ser Thr Pro His Asn Val

355 360 365

Ser Trp Arg Asp Arg Thr Arg Gly Ser Ile Phe Ile Thr Glu Leu Ile

370 375 380

Thr Cys Phe Gln Lys Tyr Ser Cys Cys Cys His Leu Met Glu Ile Phe

385 390 395 400

Arg Lys Val Gln Lys Ser Phe Glu Val Pro Gln Ala Lys Ala Gln Met

405 410 415

Pro Thr Ile Glu Arg Ala Thr Leu Thr Arg Asp Phe Tyr Leu Phe Pro

420 425 430

Gly Asn

<210> 23

<211> 293

<212> PRT

<213>People（Homo sapiens）

<400> 23

Met Ser Ser Ala Ser Gly Leu Arg Arg Gly His Pro Ala Gly Gly Glu

1 5 10 15

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

20 25 30

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

35 40 45

Ile Phe Asn His Glu Arg Phe Phe Trp His Leu Thr Leu Pro Glu Arg

50 55 60

Arg Gly Thr Cys Ala Asp Arg Asp Asn Leu Thr Arg Arg Phe Ser Asp

65 70 75 80

Leu Gly Phe Glu Val Lys Cys Phe Asn Asp Leu Lys Ala Glu Glu Leu

85 90 95

Leu Leu Lys Ile His Glu Val Ser Thr Val Ser His Ala Asp Ala Asp

100 105 110

Cys Phe Val Cys Val Phe Leu Ser His Gly Glu Gly Asn His Ile Tyr

115 120 125

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

130 135 140

Gly Asp Lys Cys His Ser Leu Val Gly Lys Pro Lys Ile Phe Ile Ile

145 150 155 160

Gln Ala Cys Arg Gly Asn Gln His Asp Val Pro Val Ile Pro Leu Asp

165 170 175

Val Val Asp Asn Gln Thr Glu Lys Leu Asp Thr Asn Ile Thr Glu Val

180 185 190

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

195 200 205

Cys Tyr Ser Val Ala Glu Gly Tyr Tyr Ser His Arg Glu Thr Val Asn

210 215 220

Gly Ser Trp Tyr Ile Gln Asp Leu Cys Glu Met Leu Gly Lys Tyr Gly

225 230 235 240

Ser Ser Leu Glu Phe Thr Glu Leu Leu Thr Leu Val Asn Arg Lys Val

245 250 255

Ser Gln Arg Arg Val Asp Phe Cys Lys Asp Pro Ser Ala Ile Gly Lys

260 265 270

Lys Gln Val Pro Cys Phe Ala Ser Met Leu Thr Lys Lys Leu His Phe

275 280 285

Phe Pro Lys Ser Asn

290

<210> 24

<211> 303

<212> PRT

<213>People（Homo sapiens）

<400> 24

Met Ala Asp Asp Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser

1 5 10 15

Ala Asn Glu Asp Ser Val Asp Ala Lys Pro Asp Arg Ser Ser Phe Val

20 25 30

Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile

35 40 45

Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu

115 120 125

Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His

130 135 140

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

145 150 155 160

Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu

165 170 175

Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp

180 185 190

Asp Gly Ile Gln Ala Asp Ser Gly Pro Ile Asn Asp Thr Asp Ala Asn

195 200 205

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

210 215 220

Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp

225 230 235 240

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

245 250 255

Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg Val Ala Arg His

260 265 270

Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu Lys Lys Gln Ile

275 280 285

Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr Phe Ser Gln

290 295 300

<210> 25

<211> 479

<212> PRT

<213>People（Homo sapiens）

<400> 25

Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gly Glu Gln Leu Asp Ser

1 5 10 15

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

20 25 30

Arg Lys Gln Glu Pro Ile Lys Asp Ala Leu Met Leu Phe Gln Arg Leu

35 40 45

Gln Glu Lys Arg Met Leu Glu Glu Ser Asn Leu Ser Phe Leu Lys Glu

50 55 60

Leu Leu Phe Arg Ile Asn Arg Leu Asp Leu Leu Ile Thr Tyr Leu Asn

65 70 75 80

Thr Arg Lys Glu Glu Met Glu Arg Glu Leu Gln Thr Pro Gly Arg Ala

85 90 95

Gln Ile Ser Ala Tyr Arg Val Met Leu Tyr Gln Ile Ser Glu Glu Val

100 105 110

Ser Arg Ser Glu Leu Arg Ser Phe Lys Phe Leu Leu Gln Glu Glu Ile

115 120 125

Ser Lys Cys Lys Leu Asp Asp Asp Met Asn Leu Leu Asp Ile Phe Ile

130 135 140

Glu Met Glu Lys Arg Val Ile Leu Gly Glu Gly Lys Leu Asp Ile Leu

145 150 155 160

Lys Arg Val Cys Ala Gln Ile Asn Lys Ser Leu Leu Lys Ile Ile Asn

165 170 175

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

180 185 190

Pro Asp Glu Phe Ser Asn Gly Glu Glu Leu Cys Gly Val Met Thr Ile

195 200 205

Ser Asp Ser Pro Arg Glu Gln Asp Ser Glu Ser Gln Thr Leu Asp Lys

210 215 220

Val Tyr Gln Met Lys Ser Lys Pro Arg Gly Tyr Cys Leu Ile Ile Asn

225 230 235 240

Asn His Asn Phe Ala Lys Ala Arg Glu Lys Val Pro Lys Leu His Ser

245 250 255

Ile Arg Asp Arg Asn Gly Thr His Leu Asp Ala Gly Ala Leu Thr Thr

260 265 270

Thr Phe Glu Glu Leu His Phe Glu Ile Lys Pro His Asp Asp Cys Thr

275 280 285

Val Glu Gln Ile Tyr Glu Ile Leu Lys Ile Tyr Gln Leu Met Asp His

290 295 300

Ser Asn Met Asp Cys Phe Ile Cys Cys Ile Leu Ser His Gly Asp Lys

305 310 315 320

Gly Ile Ile Tyr Gly Thr Asp Gly Gln Glu Ala Pro Ile Tyr Glu Leu

325 330 335

Thr Ser Gln Phe Thr Gly Leu Lys Cys Pro Ser Leu Ala Gly Lys Pro

340 345 350

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

355 360 365

Ile Pro Val Glu Thr Asp Ser Glu Glu Gln Pro Tyr Leu Glu Met Asp

370 375 380

Leu Ser Ser Pro Gln Thr Arg Tyr Ile Pro Asp Glu Ala Asp Phe Leu

385 390 395 400

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

405 410 415

Glu Gly Thr Trp Tyr Ile Gln Ser Leu Cys Gln Ser Leu Arg Glu Arg

420 425 430

Cys Pro Arg Gly Asp Asp Ile Leu Thr Ile Leu Thr Glu Val Asn Tyr

435 440 445

Glu Val Ser Asn Lys Asp Asp Lys Lys Asn Met Gly Lys Gln Met Pro

450 455 460

Gln Pro Thr Phe Thr Leu Arg Lys Lys Leu Val Phe Pro Ser Asp

465 470 475

<210> 26

<211> 416

<212> PRT

<213>People（Homo sapiens）

<400> 26

Met Asp Glu Ala Asp Arg Arg Leu Leu Arg Arg Cys Arg Leu Arg Leu

1 5 10 15

Val Glu Glu Leu Gln Val Asp Gln Leu Trp Asp Ala Leu Leu Ser Arg

20 25 30

Glu Leu Phe Arg Pro His Met Ile Glu Asp Ile Gln Arg Ala Gly Ser

35 40 45

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

50 55 60

Arg Gly Ser Gln Ala Leu Pro Leu Phe Ile Ser Cys Leu Glu Asp Thr

65 70 75 80

Gly Gln Asp Met Leu Ala Ser Phe Leu Arg Thr Asn Arg Gln Ala Ala

85 90 95

Lys Leu Ser Lys Pro Thr Leu Glu Asn Leu Thr Pro Val Val Leu Arg

100 105 110

Pro Glu Ile Arg Lys Pro Glu Val Leu Arg Pro Glu Thr Pro Arg Pro

115 120 125

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

130 135 140

Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys

145 150 155 160

Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly

165 170 175

Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg

180 185 190

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

195 200 205

Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu Ala Gln Gln Asp His

210 215 220

Gly Ala Leu Asp Cys Cys Val Val Val Ile Leu Ser His Gly Cys Gln

225 230 235 240

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

245 250 255

Pro Val Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys

260 265 270

Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly

275 280 285

Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu

290 295 300

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

305 310 315 320

Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro

325 330 335

Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val

340 345 350

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

355 360 365

Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu

370 375 380

Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met

385 390 395 400

Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser

405 410 415

<210> 27

<211> 521

<212> PRT

<213>People（Homo sapiens）

<400> 27

Met Lys Ser Gln Gly Gln His Trp Tyr Ser Ser Ser Asp Lys Asn Cys

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Leu Leu Ala Glu Asp Leu Leu Ser Glu Glu Asp Pro Phe Phe Leu Ala

65 70 75 80

Glu Leu Leu Tyr Ile Ile Arg Gln Lys Lys Leu Leu Gln His Leu Asn

85 90 95

Cys Thr Lys Glu Glu Val Glu Arg Leu Leu Pro Thr Arg Gln Arg Val

100 105 110

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

115 120 125

Glu Asn Leu Lys Asp Met Ile Phe Leu Leu Lys Asp Ser Leu Pro Lys

130 135 140

Thr Glu Met Thr Ser Leu Ser Phe Leu Ala Phe Leu Glu Lys Gln Gly

145 150 155 160

Lys Ile Asp Glu Asp Asn Leu Thr Cys Leu Glu Asp Leu Cys Lys Thr

165 170 175

Val Val Pro Lys Leu Leu Arg Asn Ile Glu Lys Tyr Lys Arg Glu Lys

180 185 190

Ala Ile Gln Ile Val Thr Pro Pro Val Asp Lys Glu Ala Glu Ser Tyr

195 200 205

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

210 215 220

Glu Ala Leu Pro Gln Glu Ser Trp Gln Asn Lys His Ala Gly Ser Asn

225 230 235 240

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

245 250 255

Gln Gly Ala Ser Ala Asn Thr Leu Asn Ser Glu Thr Ser Thr Lys Arg

260 265 270

Ala Ala Val Tyr Arg Met Asn Arg Asn His Arg Gly Leu Cys Val Ile

275 280 285

Val Asn Asn His Ser Phe Thr Ser Leu Lys Asp Arg Gln Gly Thr His

290 295 300

Lys Asp Ala Glu Ile Leu Ser His Val Phe Gln Trp Leu Gly Phe Thr

305 310 315 320

Val His Ile His Asn Asn Val Thr Lys Val Glu Met Glu Met Val Leu

325 330 335

Gln Lys Gln Lys Cys Asn Pro Ala His Ala Asp Gly Asp Cys Phe Val

340 345 350

Phe Cys Ile Leu Thr His Gly Arg Phe Gly Ala Val Tyr Ser Ser Asp

355 360 365

Glu Ala Leu Ile Pro Ile Arg Glu Ile Met Ser His Phe Thr Ala Leu

370 375 380

Gln Cys Pro Arg Leu Ala Glu Lys Pro Lys Leu Phe Phe Ile Gln Ala

385 390 395 400

Cys Gln Gly Glu Glu Ile Gln Pro Ser Val Ser Ile Glu Ala Asp Ala

405 410 415

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

420 425 430

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

435 440 445

Phe Arg His Val Glu Glu Gly Ser Trp Tyr Ile Gln Ser Leu Cys Asn

450 455 460

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

465 470 475 480

Met Glu Ile Arg Gly Arg Lys Arg Thr Val Trp Gly Ala Lys Gln Ile

485 490 495

Ser Ala Thr Ser Leu Pro Thr Ala Ile Ser Ala Gln Thr Pro Arg Pro

500 505 510

Pro Met Arg Arg Trp Ser Ser Val Ser

515 520

<210> 28

<211> 373

<212> PRT

<213>People（Homo sapiens）

<400> 28

Met Ala Glu Asn Lys His Pro Asp Lys Pro Leu Lys Val Leu Glu Gln

1 5 10 15

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

20 25 30

Asn Val Leu Lys Leu Lys Glu Glu Asp Lys Gln Lys Phe Asn Asn Ala

35 40 45

Glu Arg Ser Asp Lys Arg Trp Val Phe Val Asp Ala Met Lys Lys Lys

50 55 60

His Ser Lys Val Gly Glu Met Leu Leu Gln Thr Phe Phe Ser Val Asp

65 70 75 80

Pro Gly Ser His His Gly Glu Ala Asn Leu Glu Met Glu Glu Pro Glu

85 90 95

Glu Ser Leu Asn Thr Leu Lys Leu Cys Ser Pro Glu Glu Phe Thr Arg

100 105 110

Leu Cys Arg Glu Lys Thr Gln Glu Ile Tyr Pro Ile Lys Glu Ala Asn

115 120 125

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

130 135 140

Leu Ser Leu Arg Tyr Gly Ala Asn Phe Asp Ile Ile Gly Met Lys Gly

145 150 155 160

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

165 170 175

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

180 185 190

His Gln Thr Ser Asp Ser Thr Phe Leu Val Leu Met Ser His Gly Thr

195 200 205

Leu His Gly Ile Cys Gly Thr Met His Ser Glu Lys Thr Pro Asp Val

210 215 220

Leu Gln Tyr Asp Thr Ile Tyr Gln Ile Phe Asn Asn Cys His Cys Pro

225 230 235 240

Gly Leu Arg Asp Lys Pro Lys Val Ile Ile Val Gln Ala Cys Arg Gly

245 250 255

Gly Asn Ser Gly Glu Met Trp Ile Arg Glu Ser Ser Lys Pro Gln Leu

260 265 270

Cys Arg Gly Val Asp Leu Pro Arg Asn Met Glu Ala Asp Ala Val Lys

275 280 285

Leu Ser His Val Glu Lys Asp Phe Ile Ala Phe Tyr Ser Thr Thr Pro

290 295 300

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

305 310 315 320

Arg Leu Ile Ser Cys Phe Arg Lys His Ala Cys Ser Cys His Leu Phe

325 330 335

Asp Ile Phe Leu Lys Val Gln Gln Ser Phe Glu Lys Ala Ser Ile His

340 345 350

Ser Gln Met Pro Thr Ile Asp Arg Ala Thr Leu Thr Arg Tyr Phe Tyr

355 360 365

Leu Phe Pro Gly Asn

370

<210> 29

<211> 341

<212> PRT

<213>People（Homo sapiens）

<400> 29

Met Ala Asp Glu Lys Pro Ser Asn Gly Val Leu Val His Met Val Lys

1 5 10 15

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

20 25 30

Asn Asn Val Leu Asn Thr Asp Glu Ile His Leu Ile Gly Lys Cys Leu

35 40 45

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

50 55 60

Thr Ala Gln Thr Ala Gly Lys Ile Phe Arg Glu His Leu Trp Asn Ser

65 70 75 80

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

85 90 95

Asn Met Pro Gly Leu Asn Ile Arg Asn Lys Glu Phe Asn Tyr Leu His

100 105 110

Asn Arg Asn Gly Ser Glu Leu Asp Leu Leu Gly Met Arg Asp Leu Leu

115 120 125

Glu Asn Leu Gly Tyr Ser Val Val Ile Lys Glu Asn Leu Thr Ala Gln

130 135 140

Glu Met Glu Thr Ala Leu Arg Gln Phe Ala Ala His Pro Glu His Gln

145 150 155 160

Ser Ser Asp Ser Thr Phe Leu Val Phe Met Ser His Ser Ile Leu Asn

165 170 175

Gly Ile Cys Gly Thr Lys His Trp Asp Gln Glu Pro Asp Val Leu His

180 185 190

Asp Asp Thr Ile Phe Glu Ile Phe Asn Asn Arg Asn Cys Gln Ser Leu

195 200 205

Lys Asp Lys Pro Lys Val Ile Ile Met Gln Ala Cys Arg Gly Asn Gly

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

His Asn Val Ser Trp Arg His Glu Thr Asn Gly Ser Val Phe Ile Ser

275 280 285

Gln Ile Ile Tyr Tyr Phe Arg Glu Tyr Ser Trp Ser His His Leu Glu

290 295 300

Glu Ile Phe Gln Lys Val Gln His Ser Phe Glu Thr Pro Asn Ile Leu

305 310 315 320

Thr Gln Leu Pro Thr Ile Glu Arg Leu Ser Met Thr Arg Tyr Phe Tyr

325 330 335

Leu Phe Pro Gly Asn

340

<210> 30

<211> 377

<212> PRT

<213>People（Homo sapiens）

<400> 30

Met Ala Glu Asp Lys His Asn Lys Asn Pro Leu Lys Met Leu Glu Ser

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Lys Asn Ser Thr Ser Ile Lys Ala Pro Glu Glu Thr Val Ala Gly Pro

85 90 95

Asp Glu Ser Val Gly Ser Ala Ala Thr Leu Lys Leu Cys Pro His Glu

100 105 110

Glu Phe Leu Lys Leu Cys Lys Glu Arg Ala Gly Glu Ile Tyr Pro Ile

115 120 125

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

130 135 140

Glu Phe Asp His Met Pro Pro Arg Asn Gly Ala Ala Leu Asp Ile Leu

145 150 155 160

Gly Met Lys Gln Leu Leu Glu Gly Leu Gly Tyr Thr Val Glu Val Glu

165 170 175

Glu Lys Leu Thr Ala Arg Asp Met Glu Ser Val Leu Trp Lys Phe Ala

180 185 190

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

195 200 205

Ser His Gly Ile Leu Asp Gly Ile Cys Gly Thr Met His Ser Glu Glu

210 215 220

Glu Pro Asp Val Leu Pro Tyr Asp Thr Ile Phe Arg Thr Phe Asn Asn

225 230 235 240

Arg Asn Cys Leu Ser Leu Lys Asp Lys Pro Lys Val Ile Ile Val Gln

245 250 255

Ala Cys Arg Gly Ala Asn Arg Gly Glu Leu Trp Val Ser Asp Ser Pro

260 265 270

Pro Ala Leu Ala Asp Ser Phe Ser Gln Ser Ser Glu Asn Leu Glu Glu

275 280 285

Asp Ala Val Tyr Lys Thr His Val Glu Lys Asp Phe Ile Ala Phe Cys

290 295 300

Ser Ser Thr Pro His Asn Val Ser Trp Arg Asp Ile Lys Lys Gly Ser

305 310 315 320

Leu Phe Ile Thr Arg Leu Ile Thr Cys Phe Gln Lys Tyr Ala Trp Cys

325 330 335

Cys His Leu Glu Glu Val Phe Arg Lys Val Gln Gln Ser Phe Glu Lys

340 345 350

Pro Asn Val Lys Ala Gln Met Pro Thr Val Glu Arg Leu Ser Met Thr

355 360 365

Arg Tyr Phe Tyr Leu Phe Pro Gly Asn

370 375

<210> 31

<211> 242

<212> PRT

<213>People（Homo sapiens）

<400> 31

Met Ser Asn Pro Arg Ser Leu Glu Glu Glu Lys Tyr Asp Met Ser Gly

1 5 10 15

Ala Arg Leu Ala Leu Ile Leu Cys Val Thr Lys Ala Arg Glu Gly Ser

20 25 30

Glu Glu Asp Leu Asp Ala Leu Glu His Met Phe Arg Gln Leu Arg Phe

35 40 45

Glu Ser Thr Met Lys Arg Asp Pro Thr Ala Glu Gln Phe Gln Glu Glu

50 55 60

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

65 70 75 80

Cys Ala Phe Val Val Leu Met Ala His Gly Arg Glu Gly Phe Leu Lys

85 90 95

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

100 105 110

Asn Asn Lys Asn Cys Gln Ala Leu Arg Ala Lys Pro Lys Val Tyr Ile

115 120 125

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

130 135 140

Gly Asp Glu Ile Val Met Val Ile Lys Asp Ser Pro Gln Thr Ile Pro

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Thr Arg Arg Met Ala Glu Ala Glu Leu Val Gln Glu Gly Lys Ala

210 215 220

Arg Lys Thr Asn Pro Glu Ile Gln Ser Thr Leu Arg Lys Arg Leu Tyr

225 230 235 240

Leu Gln

<210> 32

<211> 480

<212> PRT

<213>People（Homo sapiens）

<400> 32

Met Ile Arg Ala Ala Pro Pro Pro Leu Phe Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Val Ser Trp Ala Ser Arg Gly Glu Ala Ala Pro Asp Gln

20 25 30

Asp Glu Ile Gln Arg Leu Pro Gly Leu Ala Lys Gln Pro Ser Phe Arg

35 40 45

Gln Tyr Ser Gly Tyr Leu Lys Gly Ser Gly Ser Lys His Leu His Tyr

50 55 60

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

65 70 75 80

Trp Leu Asn Gly Gly Pro Gly Cys Ser Ser Leu Asp Gly Leu Leu Thr

85 90 95

Glu His Gly Pro Phe Leu Val Gln Pro Asp Gly Val Thr Leu Glu Tyr

100 105 110

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

115 120 125

Pro Ala Gly Val Gly Phe Ser Tyr Ser Asp Asp Lys Phe Tyr Ala Thr

130 135 140

Asn Asp Thr Glu Val Ala Gln Ser Asn Phe Glu Ala Leu Gln Asp Phe

145 150 155 160

Phe Arg Leu Phe Pro Glu Tyr Lys Asn Asn Lys Leu Phe Leu Thr Gly

165 170 175

Glu Ser Tyr Ala Gly Ile Tyr Ile Pro Thr Leu Ala Val Leu Val Met

180 185 190

Gln Asp Pro Ser Met Asn Leu Gln Gly Leu Ala Val Gly Asn Gly Leu

195 200 205

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

210 215 220

His Gly Leu Leu Gly Asn Arg Leu Trp Ser Ser Leu Gln Thr His Cys

225 230 235 240

Cys Ser Gln Asn Lys Cys Asn Phe Tyr Asp Asn Lys Asp Leu Glu Cys

245 250 255

Val Thr Asn Leu Gln Glu Val Ala Arg Ile Val Gly Asn Ser Gly Leu

260 265 270

Asn Ile Tyr Asn Leu Tyr Ala Pro Cys Ala Gly Gly Val Pro Ser His

275 280 285

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

290 295 300

Phe Thr Arg Leu Pro Leu Lys Arg Met Trp His Gln Ala Leu Leu Arg

305 310 315 320

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

325 330 335

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

340 345 350

Pro Glu Gln Leu Pro Gln Trp Asp Met Cys Asn Phe Leu Val Asn Leu

355 360 365

Gln Tyr Arg Arg Leu Tyr Arg Ser Met Asn Ser Gln Tyr Leu Lys Leu

370 375 380

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

385 390 395 400

Met Ala Cys Asn Phe Met Gly Asp Glu Trp Phe Val Asp Ser Leu Asn

405 410 415

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

420 425 430

Ser Gly Glu Gln Ile Ala Gly Phe Val Lys Glu Phe Ser His Ile Ala

435 440 445

Phe Leu Thr Ile Lys Gly Ala Gly His Met Val Pro Thr Asp Lys Pro

450 455 460

Leu Ala Ala Phe Thr Met Phe Ser Arg Phe Leu Asn Lys Gln Pro Tyr

465 470 475 480

<210> 33

<211> 339

<212> PRT

<213>People（Homo sapiens）

<400> 33

Met Trp Gln Leu Trp Ala Ser Leu Cys Cys Leu Leu Val Leu Ala Asn

1 5 10 15

Ala Arg Ser Arg Pro Ser Phe His Pro Leu Ser Asp Glu Leu Val Asn

20 25 30

Tyr Val Asn Lys Arg Asn Thr Thr Trp Gln Ala Gly His Asn Phe Tyr

35 40 45

Asn Val Asp Met Ser Tyr Leu Lys Arg Leu Cys Gly Thr Phe Leu Gly

50 55 60

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

65 70 75 80

Pro Ala Ser Phe Asp Ala Arg Glu Gln Trp Pro Gln Cys Pro Thr Ile

85 90 95

Lys Glu Ile Arg Asp Gln Gly Ser Cys Gly Ser Cys Trp Ala Phe Gly

100 105 110

Ala Val Glu Ala Ile Ser Asp Arg Ile Cys Ile His Thr Asn Ala His

115 120 125

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

130 135 140

Met Cys Gly Asp Gly Cys Asn Gly Gly Tyr Pro Ala Glu Ala Trp Asn

145 150 155 160

Phe Trp Thr Arg Lys Gly Leu Val Ser Gly Gly Leu Tyr Glu Ser His

165 170 175

Val Gly Cys Arg Pro Tyr Ser Ile Pro Pro Cys Glu His His Val Asn

180 185 190

Gly Ser Arg Pro Pro Cys Thr Gly Glu Gly Asp Thr Pro Lys Cys Ser

195 200 205

Lys Ile Cys Glu Pro Gly Tyr Ser Pro Thr Tyr Lys Gln Asp Lys His

210 215 220

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

225 230 235 240

Ala Glu Ile Tyr Lys Asn Gly Pro Val Glu Gly Ala Phe Ser Val Tyr

245 250 255

Ser Asp Phe Leu Leu Tyr Lys Ser Gly Val Tyr Gln His Val Thr Gly

260 265 270

Glu Met Met Gly Gly His Ala Ile Arg Ile Leu Gly Trp Gly Val Glu

275 280 285

Asn Gly Thr Pro Tyr Trp Leu Val Ala Asn Ser Trp Asn Thr Asp Trp

290 295 300

Gly Asp Asn Gly Phe Phe Lys Ile Leu Arg Gly Gln Asp His Cys Gly

305 310 315 320

Ile Glu Ser Glu Val Val Ala Gly Ile Pro Arg Thr Asp Gln Tyr Trp

325 330 335

Glu Lys Ile

<210> 34

<211> 463

<212> PRT

<213>People（Homo sapiens）

<400> 34

Met Gly Ala Gly Pro Ser Leu Leu Leu Ala Ala Leu Leu Leu Leu Leu

1 5 10 15

Ser Gly Asp Gly Ala Val Arg Cys Asp Thr Pro Ala Asn Cys Thr Tyr

20 25 30

Leu Asp Leu Leu Gly Thr Trp Val Phe Gln Val Gly Ser Ser Gly Ser

35 40 45

Gln Arg Asp Val Asn Cys Ser Val Met Gly Pro Gln Glu Lys Lys Val

50 55 60

Val Val Tyr Leu Gln Lys Leu Asp Thr Ala Tyr Asp Asp Leu Gly Asn

65 70 75 80

Ser Gly His Phe Thr Ile Ile Tyr Asn Gln Gly Phe Glu Ile Val Leu

85 90 95

Asn Asp Tyr Lys Trp Phe Ala Phe Phe Lys Tyr Lys Glu Glu Gly Ser

100 105 110

Lys Val Thr Thr Tyr Cys Asn Glu Thr Met Thr Gly Trp Val His Asp

115 120 125

Val Leu Gly Arg Asn Trp Ala Cys Phe Thr Gly Lys Lys Val Gly Thr

130 135 140

Ala Ser Glu Asn Val Tyr Val Asn Ile Ala His Leu Lys Asn Ser Gln

145 150 155 160

Glu Lys Tyr Ser Asn Arg Leu Tyr Lys Tyr Asp His Asn Phe Val Lys

165 170 175

Ala Ile Asn Ala Ile Gln Lys Ser Trp Thr Ala Thr Thr Tyr Met Glu

180 185 190

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

195 200 205

Ser Arg Lys Ile Pro Arg Pro Lys Pro Ala Pro Leu Thr Ala Glu Ile

210 215 220

Gln Gln Lys Ile Leu His Leu Pro Thr Ser Trp Asp Trp Arg Asn Val

225 230 235 240

His Gly Ile Asn Phe Val Ser Pro Val Arg Asn Gln Ala Ser Cys Gly

245 250 255

Ser Cys Tyr Ser Phe Ala Ser Met Gly Met Leu Glu Ala Arg Ile Arg

260 265 270

Ile Leu Thr Asn Asn Ser Gln Thr Pro Ile Leu Ser Pro Gln Glu Val

275 280 285

Val Ser Cys Ser Gln Tyr Ala Gln Gly Cys Glu Gly Gly Phe Pro Tyr

290 295 300

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

305 310 315 320

Cys Phe Pro Tyr Thr Gly Thr Asp Ser Pro Cys Lys Met Lys Glu Asp

325 330 335

Cys Phe Arg Tyr Tyr Ser Ser Glu Tyr His Tyr Val Gly Gly Phe Tyr

340 345 350

Gly Gly Cys Asn Glu Ala Leu Met Lys Leu Glu Leu Val His His Gly

355 360 365

Pro Met Ala Val Ala Phe Glu Val Tyr Asp Asp Phe Leu His Tyr Lys

370 375 380

Lys Gly Ile Tyr His His Thr Gly Leu Arg Asp Pro Phe Asn Pro Phe

385 390 395 400

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

405 410 415

Ala Ser Gly Met Asp Tyr Trp Ile Val Lys Asn Ser Trp Gly Thr Gly

420 425 430

Trp Gly Glu Asn Gly Tyr Phe Arg Ile Arg Arg Gly Thr Asp Glu Cys

435 440 445

Ala Ile Glu Ser Ile Ala Val Ala Ala Thr Pro Ile Pro Lys Leu

450 455 460

<210> 35

<211> 412

<212> PRT

<213>People（Homo sapiens）

<400> 35

Met Gln Pro Ser Ser Leu Leu Pro Leu Ala Leu Cys Leu Leu Ala Ala

1 5 10 15

Pro Ala Ser Ala Leu Val Arg Ile Pro Leu His Lys Phe Thr Ser Ile

20 25 30

Arg Arg Thr Met Ser Glu Val Gly Gly Ser Val Glu Asp Leu Ile Ala

35 40 45

Lys Gly Pro Val Ser Lys Tyr Ser Gln Ala Val Pro Ala Val Thr Glu

50 55 60

Gly Pro Ile Pro Glu Val Leu Lys Asn Tyr Met Asp Ala Gln Tyr Tyr

65 70 75 80

Gly Glu Ile Gly Ile Gly Thr Pro Pro Gln Cys Phe Thr Val Val Phe

85 90 95

Asp Thr Gly Ser Ser Asn Leu Trp Val Pro Ser Ile His Cys Lys Leu

100 105 110

Leu Asp Ile Ala Cys Trp Ile His His Lys Tyr Asn Ser Asp Lys Ser

115 120 125

Ser Thr Tyr Val Lys Asn Gly Thr Ser Phe Asp Ile His Tyr Gly Ser

130 135 140

Gly Ser Leu Ser Gly Tyr Leu Ser Gln Asp Thr Val Ser Val Pro Cys

145 150 155 160

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

165 170 175

Gln Val Phe Gly Glu Ala Thr Lys Gln Pro Gly Ile Thr Phe Ile Ala

180 185 190

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

195 200 205

Asn Asn Val Leu Pro Val Phe Asp Asn Leu Met Gln Gln Lys Leu Val

210 215 220

Asp Gln Asn Ile Phe Ser Phe Tyr Leu Ser Arg Asp Pro Asp Ala Gln

225 230 235 240

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

245 250 255

Gly Ser Leu Ser Tyr Leu Asn Val Thr Arg Lys Ala Tyr Trp Gln Val

260 265 270

His Leu Asp Gln Val Glu Val Ala Ser Gly Leu Thr Leu Cys Lys Glu

275 280 285

Gly Cys Glu Ala Ile Val Asp Thr Gly Thr Ser Leu Met Val Gly Pro

290 295 300

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

305 310 315 320

Ile Gln Gly Glu Tyr Met Ile Pro Cys Glu Lys Val Ser Thr Leu Pro

325 330 335

Ala Ile Thr Leu Lys Leu Gly Gly Lys Gly Tyr Lys Leu Ser Pro Glu

340 345 350

Asp Tyr Thr Leu Lys Val Ser Gln Ala Gly Lys Thr Leu Cys Leu Ser

355 360 365

Gly Phe Met Gly Met Asp Ile Pro Pro Pro Ser Gly Pro Leu Trp Ile

370 375 380

Leu Gly Asp Val Phe Ile Gly Arg Tyr Tyr Thr Val Phe Asp Arg Asp

385 390 395 400

Asn Asn Arg Val Gly Phe Ala Glu Ala Ala Arg Leu

405 410

<210> 36

<211> 401

<212> PRT

<213>People（Homo sapiens）

<400> 36

Met Lys Thr Leu Leu Leu Leu Leu Leu Val Leu Leu Glu Leu Gly Glu

1 5 10 15

Ala Gln Gly Ser Leu His Arg Val Pro Leu Arg Arg His Pro Ser Leu

20 25 30

Lys Lys Lys Leu Arg Ala Arg Ser Gln Leu Ser Glu Phe Trp Lys Ser

35 40 45

His Asn Leu Asp Met Ile Gln Phe Thr Glu Ser Cys Ser Met Asp Gln

50 55 60

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

65 70 75 80

Thr Ile Ser Ile Gly Ser Pro Pro Gln Asn Phe Thr Val Ile Phe Asp

85 90 95

Thr Gly Ser Ser Asn Leu Trp Val Pro Ser Val Tyr Cys Thr Ser Pro

100 105 110

Ala Cys Lys Thr His Ser Arg Phe Gln Pro Ser Gln Ser Ser Thr Tyr

115 120 125

Ser Gln Pro Gly Gln Ser Phe Ser Ile Gln Tyr Gly Thr Gly Ser Leu

130 135 140

Ser Gly Ile Ile Gly Ala Asp Gln Val Ser Ala Phe Ala Thr Gln Val

145 150 155 160

Glu Gly Leu Thr Val Val Gly Gln Gln Phe Gly Glu Ser Val Thr Glu

165 170 175

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

180 185 190

Gly Tyr Pro Ser Leu Ala Val Gly Gly Val Thr Pro Val Phe Asp Asn

195 200 205

Met Met Ala Gln Asn Leu Val Asp Leu Pro Met Phe Ser Val Tyr Met

210 215 220

Ser Ser Asn Pro Glu Gly Gly Ala Gly Ser Glu Leu Ile Phe Gly Gly

225 230 235 240

Tyr Asp His Ser His Phe Ser Gly Ser Leu Asn Trp Val Pro Val Thr

245 250 255

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

260 265 270

Thr Val Met Phe Cys Ser Glu Gly Cys Gln Ala Ile Val Asp Thr Gly

275 280 285

Thr Ser Leu Ile Thr Gly Pro Ser Asp Lys Ile Lys Gln Leu Gln Asn

290 295 300

Ala Ile Gly Ala Ala Pro Val Asp Gly Glu Tyr Ala Val Glu Cys Ala

305 310 315 320

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

325 330 335

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

340 345 350

Met Gln Phe Cys Ser Ser Gly Phe Gln Gly Leu Asp Ile His Pro Pro

355 360 365

Ala Gly Pro Leu Trp Ile Leu Gly Asp Val Phe Ile Arg Gln Phe Tyr

370 375 380

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

385 390 395 400

Pro

<210> 37

<211> 484

<212> PRT

<213>People（Homo sapiens）

<400> 37

Met Ala Pro Trp Leu Gln Leu Leu Ser Leu Leu Gly Leu Leu Pro Gly

1 5 10 15

Ala Val Ala Ala Pro Ala Gln Pro Arg Ala Ala Ser Phe Gln Ala Trp

20 25 30

Gly Pro Pro Ser Pro Glu Leu Leu Ala Pro Thr Arg Phe Ala Leu Glu

35 40 45

Met Phe Asn Arg Gly Arg Ala Ala Gly Thr Arg Ala Val Leu Gly Leu

50 55 60

Val Arg Gly Arg Val Arg Arg Ala Gly Gln Gly Ser Leu Tyr Ser Leu

65 70 75 80

Glu Ala Thr Leu Glu Glu Pro Pro Cys Asn Asp Pro Met Val Cys Arg

85 90 95

Leu Pro Val Ser Lys Lys Thr Leu Leu Cys Ser Phe Gln Val Leu Asp

100 105 110

Glu Leu Gly Arg His Val Leu Leu Arg Lys Asp Cys Gly Pro Val Asp

115 120 125

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

130 135 140

Ser Ala Met Ile Ser Ser Leu Ser Gln Asn His Pro Asp Asn Arg Asn

145 150 155 160

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

165 170 175

Gln Asp Leu Pro Val Lys Met Ala Ser Ile Phe Lys Asn Phe Val Ile

180 185 190

Thr Tyr Asn Arg Thr Tyr Glu Ser Lys Glu Glu Ala Arg Trp Arg Leu

195 200 205

Ser Val Phe Val Asn Asn Met Val Arg Ala Gln Lys Ile Gln Ala Leu

210 215 220

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

225 230 235 240

Glu Glu Glu Phe Arg Thr Ile Tyr Leu Asn Thr Leu Leu Arg Lys Glu

245 250 255

Pro Gly Asn Lys Met Lys Gln Ala Lys Ser Val Gly Asp Leu Ala Pro

260 265 270

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

275 280 285

Gln Gly Met Cys Gly Ser Cys Trp Ala Phe Ser Val Thr Gly Asn Val

290 295 300

Glu Gly Gln Trp Phe Leu Asn Gln Gly Thr Leu Leu Ser Leu Ser Glu

305 310 315 320

Gln Glu Leu Leu Asp Cys Asp Lys Met Asp Lys Ala Cys Met Gly Gly

325 330 335

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

340 345 350

Thr Glu Asp Asp Tyr Ser Tyr Gln Gly His Met Gln Ser Cys Asn Phe

355 360 365

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

370 375 380

Gln Asn Glu Gln Lys Leu Ala Ala Trp Leu Ala Lys Arg Gly Pro Ile

385 390 395 400

Ser Val Ala Ile Asn Ala Phe Gly Met Gln Phe Tyr Arg His Gly Ile

405 410 415

Ser Arg Pro Leu Arg Pro Leu Cys Ser Pro Trp Leu Ile Asp His Ala

420 425 430

Val Leu Leu Val Gly Tyr Gly Asn Arg Ser Asp Val Pro Phe Trp Ala

435 440 445

Ile Lys Asn Ser Trp Gly Thr Asp Trp Gly Glu Lys Gly Tyr Tyr Tyr

450 455 460

Leu His Arg Gly Ser Gly Ala Cys Gly Val Asn Thr Met Ala Ser Ser

465 470 475 480

Ala Val Val Asp

<210> 38

<211> 255

<212> PRT

<213>People（Homo sapiens）

<400> 38

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

1 5 10 15

Glu Ala Gly Glu Ile Ile Gly Gly Arg Glu Ser Arg Pro His Ser Arg

20 25 30

Pro Tyr Met Ala Tyr Leu Gln Ile Gln Ser Pro Ala Gly Gln Ser Arg

35 40 45

Cys Gly Gly Phe Leu Val Arg Glu Asp Phe Val Leu Thr Ala Ala His

50 55 60

Cys Trp Gly Ser Asn Ile Asn Val Thr Leu Gly Ala His Asn Ile Gln

65 70 75 80

Arg Arg Glu Asn Thr Gln Gln His Ile Thr Ala Arg Arg Ala Ile Arg

85 90 95

His Pro Gln Tyr Asn Gln Arg Thr Ile Gln Asn Asp Ile Met Leu Leu

100 105 110

Gln Leu Ser Arg Arg Val Arg Arg Asn Arg Asn Val Asn Pro Val Ala

115 120 125

Leu Pro Arg Ala Gln Glu Gly Leu Arg Pro Gly Thr Leu Cys Thr Val

130 135 140

Ala Gly Trp Gly Arg Val Ser Met Arg Arg Gly Thr Asp Thr Leu Arg

145 150 155 160

Glu Val Gln Leu Arg Val Gln Arg Asp Arg Gln Cys Leu Arg Ile Phe

165 170 175

Gly Ser Tyr Asp Pro Arg Arg Gln Ile Cys Val Gly Asp Arg Arg Glu

180 185 190

Arg Lys Ala Ala Phe Lys Gly Asp Ser Gly Gly Pro Leu Leu Cys Asn

195 200 205

Asn Val Ala His Gly Ile Val Ser Tyr Gly Lys Ser Ser Gly Val Pro

210 215 220

Pro Glu Val Phe Thr Arg Val Ser Ser Phe Leu Pro Trp Ile Arg Thr

225 230 235 240

Thr Met Arg Ser Phe Lys Leu Leu Asp Gln Met Glu Thr Pro Leu

245 250 255

<210> 39

<211> 335

<212> PRT

<213>People（Homo sapiens）

<400> 39

Met Trp Ala Thr Leu Pro Leu Leu Cys Ala Gly Ala Trp Leu Leu Gly

1 5 10 15

Val Pro Val Cys Gly Ala Ala Glu Leu Cys Val Asn Ser Leu Glu Lys

20 25 30

Phe His Phe Lys Ser Trp Met Ser Lys His Arg Lys Thr Tyr Ser Thr

35 40 45

Glu Glu Tyr His His Arg Leu Gln Thr Phe Ala Ser Asn Trp Arg Lys

50 55 60

Ile Asn Ala His Asn Asn Gly Asn His Thr Phe Lys Met Ala Leu Asn

65 70 75 80

Gln Phe Ser Asp Met Ser Phe Ala Glu Ile Lys His Lys Tyr Leu Trp

85 90 95

Ser Glu Pro Gln Asn Cys Ser Ala Thr Lys Ser Asn Tyr Leu Arg Gly

100 105 110

Thr Gly Pro Tyr Pro Pro Ser Val Asp Trp Arg Lys Lys Gly Asn Phe

115 120 125

Val Ser Pro Val Lys Asn Gln Gly Ala Cys Gly Ser Cys Trp Thr Phe

130 135 140

Ser Thr Thr Gly Ala Leu Glu Ser Ala Ile Ala Ile Ala Thr Gly Lys

145 150 155 160

Met Leu Ser Leu Ala Glu Gln Gln Leu Val Asp Cys Ala Gln Asp Phe

165 170 175

Asn Asn His Gly Cys Gln Gly Gly Leu Pro Ser Gln Ala Phe Glu Tyr

180 185 190

Ile Leu Tyr Asn Lys Gly Ile Met Gly Glu Asp Thr Tyr Pro Tyr Gln

195 200 205

Gly Lys Asp Gly Tyr Cys Lys Phe Gln Pro Gly Lys Ala Ile Gly Phe

210 215 220

Val Lys Asp Val Ala Asn Ile Thr Ile Tyr Asp Glu Glu Ala Met Val

225 230 235 240

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

245 250 255

Gln Asp Phe Met Met Tyr Arg Thr Gly Ile Tyr Ser Ser Thr Ser Cys

260 265 270

His Lys Thr Pro Asp Lys Val Asn His Ala Val Leu Ala Val Gly Tyr

275 280 285

Gly Glu Lys Asn Gly Ile Pro Tyr Trp Ile Val Lys Asn Ser Trp Gly

290 295 300

Pro Gln Trp Gly Met Asn Gly Tyr Phe Leu Ile Glu Arg Gly Lys Asn

305 310 315 320

Met Cys Gly Leu Ala Ala Cys Ala Ser Tyr Pro Ile Pro Leu Val

325 330 335

<210> 40

<211> 329

<212> PRT

<213>People（Homo sapiens）

<400> 40

Met Trp Gly Leu Lys Val Leu Leu Leu Pro Val Val Ser Phe Ala Leu

1 5 10 15

Tyr Pro Glu Glu Ile Leu Asp Thr His Trp Glu Leu Trp Lys Lys Thr

20 25 30

His Arg Lys Gln Tyr Asn Asn Lys Val Asp Glu Ile Ser Arg Arg Leu

35 40 45

Ile Trp Glu Lys Asn Leu Lys Tyr Ile Ser Ile His Asn Leu Glu Ala

50 55 60

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

65 70 75 80

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

85 90 95

Leu Ser His Ser Arg Ser Asn Asp Thr Leu Tyr Ile Pro Glu Trp Glu

100 105 110

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

115 120 125

Pro Val Lys Asn Gln Gly Gln Cys Gly Ser Cys Trp Ala Phe Ser Ser

130 135 140

Val Gly Ala Leu Glu Gly Gln Leu Lys Lys Lys Thr Gly Lys Leu Leu

145 150 155 160

Asn Leu Ser Pro Gln Asn Leu Val Asp Cys Val Ser Glu Asn Asp Gly

165 170 175

Cys Gly Gly Gly Tyr Met Thr Asn Ala Phe Gln Tyr Val Gln Lys Asn

180 185 190

Arg Gly Ile Asp Ser Glu Asp Ala Tyr Pro Tyr Val Gly Gln Glu Glu

195 200 205

Ser Cys Met Tyr Asn Pro Thr Gly Lys Ala Ala Lys Cys Arg Gly Tyr

210 215 220

Arg Glu Ile Pro Glu Gly Asn Glu Lys Ala Leu Lys Arg Ala Val Ala

225 230 235 240

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

245 250 255

Gln Phe Tyr Ser Lys Gly Val Tyr Tyr Asp Glu Ser Cys Asn Ser Asp

260 265 270

Asn Leu Asn His Ala Val Leu Ala Val Gly Tyr Gly Ile Gln Lys Gly

275 280 285

Asn Lys His Trp Ile Ile Lys Asn Ser Trp Gly Glu Asn Trp Gly Asn

290 295 300

Lys Gly Tyr Ile Leu Met Ala Arg Asn Lys Asn Asn Ala Cys Gly Ile

305 310 315 320

Ala Asn Leu Ala Ser Phe Pro Lys Met

325

<210> 41

<211> 333

<212> PRT

<213>People（Homo sapiens）

<400> 41

Met Asn Pro Thr Leu Ile Leu Ala Ala Phe Cys Leu Gly Ile Ala Ser

1 5 10 15

Ala Thr Leu Thr Phe Asp His Ser Leu Glu Ala Gln Trp Thr Lys Trp

20 25 30

Lys Ala Met His Asn Arg Leu Tyr Gly Met Asn Glu Glu Gly Trp Arg

35 40 45

Arg Ala Val Trp Glu Lys Asn Met Lys Met Ile Glu Leu His Asn Gln

50 55 60

Glu Tyr Arg Glu Gly Lys His Ser Phe Thr Met Ala Met Asn Ala Phe

65 70 75 80

Gly Asp Met Thr Ser Glu Glu Phe Arg Gln Val Met Asn Gly Phe Gln

85 90 95

Asn Arg Lys Pro Arg Lys Gly Lys Val Phe Gln Glu Pro Leu Phe Tyr

100 105 110

Glu Ala Pro Arg Ser Val Asp Trp Arg Glu Lys Gly Tyr Val Thr Pro

115 120 125

Val Lys Asn Gln Gly Gln Cys Gly Ser Cys Trp Ala Phe Ser Ala Thr

130 135 140

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

145 150 155 160

Leu Ser Glu Gln Asn Leu Val Asp Cys Ser Gly Pro Gln Gly Asn Glu

165 170 175

Gly Cys Asn Gly Gly Leu Met Asp Tyr Ala Phe Gln Tyr Val Gln Asp

180 185 190

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

195 200 205

Glu Ser Cys Lys Tyr Asn Pro Lys Tyr Ser Val Ala Asn Asp Thr Gly

210 215 220

Phe Val Asp Ile Pro Lys Gln Glu Lys Ala Leu Met Lys Ala Val Ala

225 230 235 240

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

245 250 255

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

260 265 270

Asp Met Asp His Gly Val Leu Val Val Gly Tyr Gly Phe Glu Ser Thr

275 280 285

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

290 295 300

Glu Trp Gly Met Gly Gly Tyr Val Lys Met Ala Lys Asp Arg Arg Asn

305 310 315 320

His Cys Gly Ile Ala Ser Ala Ala Ser Tyr Pro Thr Val

325 330

<210> 42

<211> 334

<212> PRT

<213>People（Homo sapiens）

<400> 42

Met Asn Leu Ser Leu Val Leu Ala Ala Phe Cys Leu Gly Ile Ala Ser

1 5 10 15

Ala Val Pro Lys Phe Asp Gln Asn Leu Asp Thr Lys Trp Tyr Gln Trp

20 25 30

Lys Ala Thr His Arg Arg Leu Tyr Gly Ala Asn Glu Glu Gly Trp Arg

35 40 45

Arg Ala Val Trp Glu Lys Asn Met Lys Met Ile Glu Leu His Asn Gly

50 55 60

Glu Tyr Ser Gln Gly Lys His Gly Phe Thr Met Ala Met Asn Ala Phe

65 70 75 80

Gly Asp Met Thr Asn Glu Glu Phe Arg Gln Met Met Gly Cys Phe Arg

85 90 95

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

100 105 110

Asp Leu Pro Lys Ser Val Asp Trp Arg Lys Lys Gly Tyr Val Thr Pro

115 120 125

Val Lys Asn Gln Lys Gln Cys Gly Ser Cys Trp Ala Phe Ser Ala Thr

130 135 140

Gly Ala Leu Glu Gly Gln Met Phe Arg Lys Thr Gly Lys Leu Val Ser

145 150 155 160

Leu Ser Glu Gln Asn Leu Val Asp Cys Ser Arg Pro Gln Gly Asn Gln

165 170 175

Gly Cys Asn Gly Gly Phe Met Ala Arg Ala Phe Gln Tyr Val Lys Glu

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Ala Thr Val Gly Pro Ile Ser Val Ala Met Asp Ala Gly His Ser Ser

245 250 255

Phe Gln Phe Tyr Lys Ser Gly Ile Tyr Phe Glu Pro Asp Cys Ser Ser

260 265 270

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

275 280 285

Ala Asn Ser Asn Asn Ser Lys Tyr Trp Leu Val Lys Asn Ser Trp Gly

290 295 300

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

305 310 315 320

Asn His Cys Gly Ile Ala Thr Ala Ala Ser Tyr Pro Asn Val

325 330

<210> 43

<211> 321

<212> PRT

<213>People（Homo sapiens）

<400> 43

Met Asp Val Arg Ala Leu Pro Trp Leu Pro Trp Leu Leu Trp Leu Leu

1 5 10 15

Cys Arg Gly Gly Gly Asp Ala Asp Ser Arg Ala Pro Phe Thr Pro Thr

20 25 30

Trp Pro Arg Ser Arg Glu Arg Glu Ala Ala Ala Phe Arg Glu Ser Leu

35 40 45

Asn Arg His Arg Tyr Leu Asn Ser Leu Phe Pro Ser Glu Asn Ser Thr

50 55 60

Ala Phe Tyr Gly Ile Asn Gln Phe Ser Tyr Leu Phe Pro Glu Glu Phe

65 70 75 80

Lys Ala Ile Tyr Leu Arg Ser Lys Pro Ser Lys Phe Pro Arg Tyr Ser

85 90 95

Ala Glu Val His Met Ser Ile Pro Asn Val Ser Leu Pro Leu Arg Phe

100 105 110

Asp Trp Arg Asp Lys Gln Val Val Thr Gln Val Arg Asn Gln Gln Met

115 120 125

Cys Gly Gly Cys Trp Ala Phe Ser Val Val Gly Ala Val Glu Ser Ala

130 135 140

Tyr Ala Ile Lys Gly Lys Pro Leu Glu Asp Leu Ser Val Gln Gln Val

145 150 155 160

Ile Asp Cys Ser Tyr Asn Asn Tyr Gly Cys Asn Gly Gly Ser Thr Leu

165 170 175

Asn Ala Leu Asn Trp Leu Asn Lys Met Gln Val Lys Leu Val Lys Asp

180 185 190

Ser Glu Tyr Pro Phe Lys Ala Gln Asn Gly Leu Cys His Tyr Phe Ser

195 200 205

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

210 215 220

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

225 230 235 240

Leu Val Val Ile Val Asp Ala Val Ser Trp Gln Asp Tyr Leu Gly Gly

245 250 255

Ile Ile Gln His His Cys Ser Ser Gly Glu Ala Asn His Ala Val Leu

260 265 270

Ile Thr Gly Phe Asp Lys Thr Gly Ser Thr Pro Tyr Trp Ile Val Arg

275 280 285

Asn Ser Trp Gly Ser Ser Trp Gly Val Asp Gly Tyr Ala His Val Lys

290 295 300

Met Gly Ser Asn Val Cys Gly Ile Ala Asp Ser Val Ser Ser Ile Phe

305 310 315 320

Val

<210> 44

<211> 331

<212> PRT

<213>People（Homo sapiens）

<400> 44

Met Lys Arg Leu Val Cys Val Leu Leu Val Cys Ser Ser Ala Val Ala

1 5 10 15

Gln Leu His Lys Asp Pro Thr Leu Asp His His Trp His Leu Trp Lys

20 25 30

Lys Thr Tyr Gly Lys Gln Tyr Lys Glu Lys Asn Glu Glu Ala Val Arg

35 40 45

Arg Leu Ile Trp Glu Lys Asn Leu Lys Phe Val Met Leu His Asn Leu

50 55 60

Glu His Ser Met Gly Met His Ser Tyr Asp Leu Gly Met Asn His Leu

65 70 75 80

Gly Asp Met Thr Ser Glu Glu Val Met Ser Leu Met Ser Ser Leu Arg

85 90 95

Val Pro Ser Gln Trp Gln Arg Asn Ile Thr Tyr Lys Ser Asn Pro Asn

100 105 110

Arg Ile Leu Pro Asp Ser Val Asp Trp Arg Glu Lys Gly Cys Val Thr

115 120 125

Glu Val Lys Tyr Gln Gly Ser Cys Gly Ala Cys Trp Ala Phe Ser Ala

130 135 140

Val Gly Ala Leu Glu Ala Gln Leu Lys Leu Lys Thr Gly Lys Leu Val

145 150 155 160

Ser Leu Ser Ala Gln Asn Leu Val Asp Cys Ser Thr Glu Lys Tyr Gly

165 170 175

Asn Lys Gly Cys Asn Gly Gly Phe Met Thr Thr Ala Phe Gln Tyr Ile

180 185 190

Ile Asp Asn Lys Gly Ile Asp Ser Asp Ala Ser Tyr Pro Tyr Lys Ala

195 200 205

Met Asp Gln Lys Cys Gln Tyr Asp Ser Lys Tyr Arg Ala Ala Thr Cys

210 215 220

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

225 230 235 240

Ala Val Ala Asn Lys Gly Pro Val Ser Val Gly Val Asp Ala Arg His

245 250 255

Pro Ser Phe Phe Leu Tyr Arg Ser Gly Val Tyr Tyr Glu Pro Ser Cys

260 265 270

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

275 280 285

Asn Gly Lys Glu Tyr Trp Leu Val Lys Asn Ser Trp Gly His Asn Phe

290 295 300

Gly Glu Glu Gly Tyr Ile Arg Met Ala Arg Asn Lys Gly Asn His Cys

305 310 315 320

Gly Ile Ala Ser Phe Pro Ser Tyr Pro Glu Ile

325 330

<210> 45

<211> 376

<212> PRT

<213>People（Homo sapiens）

<400> 45

Met Ala Leu Thr Ala His Pro Ser Cys Leu Leu Ala Leu Leu Val Ala

1 5 10 15

Gly Leu Ala Gln Gly Ile Arg Gly Pro Leu Arg Ala Gln Asp Leu Gly

20 25 30

Pro Gln Pro Leu Glu Leu Lys Glu Ala Phe Lys Leu Phe Gln Ile Gln

35 40 45

Phe Asn Arg Ser Tyr Leu Ser Pro Glu Glu His Ala His Arg Leu Asp

50 55 60

Ile Phe Ala His Asn Leu Ala Gln Ala Gln Arg Leu Gln Glu Glu Asp

65 70 75 80

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

85 90 95

Glu Glu Phe Gly Gln Leu Tyr Gly Tyr Arg Arg Ala Ala Gly Gly Val

100 105 110

Pro Ser Met Gly Arg Glu Ile Arg Ser Glu Glu Pro Glu Glu Ser Val

115 120 125

Pro Phe Ser Cys Asp Trp Arg Lys Val Ala Ser Ala Ile Ser Pro Ile

130 135 140

Lys Asp Gln Lys Asn Cys Asn Cys Cys Trp Ala Met Ala Ala Ala Gly

145 150 155 160

Asn Ile Glu Thr Leu Trp Arg Ile Ser Phe Trp Asp Phe Val Asp Val

165 170 175

Ser Val Gln Glu Leu Leu Asp Cys Gly Arg Cys Gly Asp Gly Cys His

180 185 190

Gly Gly Phe Val Trp Asp Ala Phe Ile Thr Val Leu Asn Asn Ser Gly

195 200 205

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

210 215 220

Arg Cys His Pro Lys Lys Tyr Gln Lys Val Ala Trp Ile Gln Asp Phe

225 230 235 240

Ile Met Leu Gln Asn Asn Glu His Arg Ile Ala Gln Tyr Leu Ala Thr

245 250 255

Tyr Gly Pro Ile Thr Val Thr Ile Asn Met Lys Pro Leu Gln Leu Tyr

260 265 270

Arg Lys Gly Val Ile Lys Ala Thr Pro Thr Thr Cys Asp Pro Gln Leu

275 280 285

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

290 295 300

Glu Gly Ile Trp Ala Glu Thr Val Ser Ser Gln Ser Gln Pro Gln Pro

305 310 315 320

Pro His Pro Thr Pro Tyr Trp Ile Leu Lys Asn Ser Trp Gly Ala Gln

325 330 335

Trp Gly Glu Lys Gly Tyr Phe Arg Leu His Arg Gly Ser Asn Thr Cys

340 345 350

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

355 360 365

Lys Pro Arg Val Ser Cys Pro Pro

370 375

<210> 46

<211> 303

<212> PRT

<213>People（Homo sapiens）

<400> 46

Met Ala Arg Arg Gly Pro Gly Trp Arg Pro Leu Leu Leu Leu Val Leu

1 5 10 15

Leu Ala Gly Ala Ala Gln Gly Gly Leu Tyr Phe Arg Arg Gly Gln Thr

20 25 30

Cys Tyr Arg Pro Leu Arg Gly Asp Gly Leu Ala Pro Leu Gly Arg Ser

35 40 45

Thr Tyr Pro Arg Pro His Glu Tyr Leu Ser Pro Ala Asp Leu Pro Lys

50 55 60

Ser Trp Asp Trp Arg Asn Val Asp Gly Val Asn Tyr Ala Ser Ile Thr

65 70 75 80

Arg Asn Gln His Ile Pro Gln Tyr Cys Gly Ser Cys Trp Ala His Ala

85 90 95

Ser Thr Ser Ala Met Ala Asp Arg Ile Asn Ile Lys Arg Lys Gly Ala

100 105 110

Trp Pro Ser Thr Leu Leu Ser Val Gln Asn Val Ile Asp Cys Gly Asn

115 120 125

Ala Gly Ser Cys Glu Gly Gly Asn Asp Leu Ser Val Trp Asp Tyr Ala

130 135 140

His Gln His Gly Ile Pro Asp Glu Thr Cys Asn Asn Tyr Gln Ala Lys

145 150 155 160

Asp Gln Glu Cys Asp Lys Phe Asn Gln Cys Gly Thr Cys Asn Glu Phe

165 170 175

Lys Glu Cys His Ala Ile Arg Asn Tyr Thr Leu Trp Arg Val Gly Asp

180 185 190

Tyr Gly Ser Leu Ser Gly Arg Glu Lys Met Met Ala Glu Ile Tyr Ala

195 200 205

Asn Gly Pro Ile Ser Cys Gly Ile Met Ala Thr Glu Arg Leu Ala Asn

210 215 220

Tyr Thr Gly Gly Ile Tyr Ala Glu Tyr Gln Asp Thr Thr Tyr Ile Asn

225 230 235 240

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

245 250 255

Trp Ile Val Arg Asn Ser Trp Gly Glu Pro Trp Gly Glu Arg Gly Trp

260 265 270

Leu Arg Ile Val Thr Ser Thr Tyr Lys Asp Gly Lys Gly Ala Arg Tyr

275 280 285

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

290 295 300

<210> 47

<211> 338

<212> PRT

<213>People（Homo sapiens）

<400> 47

Met Leu Gln Lys Pro Lys Ser Val Lys Leu Arg Ala Leu Arg Ser Pro

1 5 10 15

Arg Lys Phe Gly Val Ala Gly Arg Ser Cys Gln Glu Val Leu Arg Lys

20 25 30

Gly Cys Leu Arg Phe Gln Leu Pro Glu Arg Gly Ser Arg Leu Cys Leu

35 40 45

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

50 55 60

Asp Asn Ala Glu Leu Val Leu Leu Thr Leu Gly Gln Ala Trp Gln Gly

65 70 75 80

Tyr Val Ser Asp Ile Arg Arg Phe Leu Ser Ala Phe His Glu Pro Gln

85 90 95

Val Gly Leu Ile Gln Ala Ala Gln Gln Leu Leu Cys Asp Glu Gln Ala

100 105 110

Pro Gln Arg Gln Arg Leu Leu Ala Asp Leu Leu His Asn Val Ser Gln

115 120 125

Asn Ile Ala Ala Glu Thr Arg Ala Glu Asp Pro Pro Trp Phe Glu Gly

130 135 140

Leu Glu Ser Arg Phe Gln Ser Lys Ser Gly Tyr Leu Arg Tyr Ser Cys

145 150 155 160

Glu Ser Arg Ile Arg Ser Tyr Leu Arg Glu Val Ser Ser Tyr Pro Ser

165 170 175

Thr Val Gly Ala Glu Ala Gln Glu Glu Phe Leu Arg Val Leu Gly Ser

180 185 190

Met Cys Gln Arg Leu Arg Ser Met Gln Tyr Asn Gly Ser Tyr Phe Asp

195 200 205

Arg Gly Ala Lys Gly Gly Ser Arg Leu Cys Thr Pro Glu Gly Trp Phe

210 215 220

Ser Cys Gln Gly Pro Phe Asp Met Asp Ser Cys Leu Ser Arg His Ser

225 230 235 240

Ile Asn Pro Tyr Ser Asn Arg Glu Ser Arg Ile Leu Phe Ser Thr Trp

245 250 255

Asn Leu Asp His Ile Ile Glu Lys Lys Arg Thr Ile Ile Pro Thr Leu

260 265 270

Val Glu Ala Ile Lys Glu Gln Asp Gly Arg Glu Val Asp Trp Glu Tyr

275 280 285

Phe Tyr Gly Leu Leu Phe Thr Ser Glu Asn Leu Lys Leu Val His Ile

290 295 300

Val Cys His Lys Lys Thr Thr His Lys Leu Asn Cys Asp Pro Ser Arg

305 310 315 320

Ile Tyr Lys Pro Gln Thr Arg Leu Lys Arg Lys Gln Pro Val Arg Lys

325 330 335

Arg Gln

<210> 48

<211> 360

<212> PRT

<213>People（Homo sapiens）

<400> 48

Met Ser Gln Glu Arg Pro Thr Phe Tyr Arg Gln Glu Leu Asn Lys Thr

1 5 10 15

Ile Trp Glu Val Pro Glu Arg Tyr Gln Asn Leu Ser Pro Val Gly Ser

20 25 30

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

35 40 45

Arg Val Ala Val Lys Lys Leu Ser Arg Pro Phe Gln Ser Ile Ile His

50 55 60

Ala Lys Arg Thr Tyr Arg Glu Leu Arg Leu Leu Lys His Met Lys His

65 70 75 80

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

85 90 95

Glu Glu Phe Asn Asp Val Tyr Leu Val Thr His Leu Met Gly Ala Asp

100 105 110

Leu Asn Asn Ile Val Lys Cys Gln Lys Leu Thr Asp Asp His Val Gln

115 120 125

Phe Leu Ile Tyr Gln Ile Leu Arg Gly Leu Lys Tyr Ile His Ser Ala

130 135 140

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

145 150 155 160

Asp Cys Glu Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg His Thr Asp

165 170 175

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

180 185 190

Ile Met Leu Asn Trp Met His Tyr Asn Gln Thr Val Asp Ile Trp Ser

195 200 205

Val Gly Cys Ile Met Ala Glu Leu Leu Thr Gly Arg Thr Leu Phe Pro

210 215 220

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

225 230 235 240

Thr Pro Gly Ala Glu Leu Leu Lys Lys Ile Ser Ser Glu Ser Ala Arg

245 250 255

Asn Tyr Ile Gln Ser Leu Thr Gln Met Pro Lys Met Asn Phe Ala Asn

260 265 270

Val Phe Ile Gly Ala Asn Pro Leu Ala Val Asp Leu Leu Glu Lys Met

275 280 285

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

290 295 300

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

305 310 315 320

Asp Pro Tyr Asp Gln Ser Phe Glu Ser Arg Asp Leu Leu Ile Asp Glu

325 330 335

Trp Lys Ser Leu Thr Tyr Asp Glu Val Ile Ser Phe Val Pro Pro Pro

340 345 350

Leu Asp Gln Glu Glu Met Glu Ser

355 360

<210> 49

<211> 364

<212> PRT

<213>People（Homo sapiens）

<400> 49

Met Ser Gly Pro Arg Ala Gly Phe Tyr Arg Gln Glu Leu Asn Lys Thr

1 5 10 15

Val Trp Glu Val Pro Gln Arg Leu Gln Gly Leu Arg Pro Val Gly Ser

20 25 30

Gly Ala Tyr Gly Ser Val Cys Ser Ala Tyr Asp Ala Arg Leu Arg Gln

35 40 45

Lys Val Ala Val Lys Lys Leu Ser Arg Pro Phe Gln Ser Leu Ile His

50 55 60

Ala Arg Arg Thr Tyr Arg Glu Leu Arg Leu Leu Lys His Leu Lys His

65 70 75 80

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

85 90 95

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

100 105 110

Leu Asn Asn Ile Val Lys Cys Gln Ala Leu Ser Asp Glu His Val Gln

115 120 125

Phe Leu Val Tyr Gln Leu Leu Arg Gly Leu Lys Tyr Ile His Ser Ala

130 135 140

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

145 150 155 160

Asp Cys Glu Leu Arg Ile Leu Asp Phe Gly Leu Ala Arg Gln Ala Asp

165 170 175

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

180 185 190

Ile Met Leu Asn Trp Met His Tyr Asn Gln Thr Val Asp Ile Trp Ser

195 200 205

Val Gly Cys Ile Met Ala Glu Leu Leu Gln Gly Lys Ala Leu Phe Pro

210 215 220

Gly Ser Asp Tyr Ile Asp Gln Leu Lys Arg Ile Met Glu Val Val Gly

225 230 235 240

Thr Pro Ser Pro Glu Val Leu Ala Lys Ile Ser Ser Glu His Ala Arg

245 250 255

Thr Tyr Ile Gln Ser Leu Pro Pro Met Pro Gln Lys Asp Leu Ser Ser

260 265 270

Ile Phe Arg Gly Ala Asn Pro Leu Ala Ile Asp Leu Leu Gly Arg Met

275 280 285

Leu Val Leu Asp Ser Asp Gln Arg Val Ser Ala Ala Glu Ala Leu Ala

290 295 300

His Ala Tyr Phe Ser Gln Tyr His Asp Pro Glu Asp Glu Pro Glu Ala

305 310 315 320

Glu Pro Tyr Asp Glu Ser Val Glu Ala Lys Glu Arg Thr Leu Glu Glu

325 330 335

Trp Lys Glu Leu Thr Tyr Gln Glu Val Leu Ser Phe Lys Pro Pro Glu

340 345 350

Pro Pro Lys Pro Pro Gly Ser Leu Glu Ile Glu Gln

355 360

<210> 50

<211> 367

<212> PRT

<213>People（Homo sapiens）

<400> 50

Met Ser Ser Pro Pro Pro Ala Arg Ser Gly Phe Tyr Arg Gln Glu Val

1 5 10 15

Thr Lys Thr Ala Trp Glu Val Arg Ala Val Tyr Arg Asp Leu Gln Pro

20 25 30

Val Gly Ser Gly Ala Tyr Gly Ala Val Cys Ser Ala Val Asp Gly Arg

35 40 45

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

50 55 60

Glu Leu Phe Ala Lys Arg Ala Tyr Arg Glu Leu Arg Leu Leu Lys His

65 70 75 80

Met Arg His Glu Asn Val Ile Gly Leu Leu Asp Val Phe Thr Pro Asp

85 90 95

Glu Thr Leu Asp Asp Phe Thr Asp Phe Tyr Leu Val Met Pro Phe Met

100 105 110

Gly Thr Asp Leu Gly Lys Leu Met Lys His Glu Lys Leu Gly Glu Asp

115 120 125

Arg Ile Gln Phe Leu Val Tyr Gln Met Leu Lys Gly Leu Arg Tyr Ile

130 135 140

His Ala Ala Gly Ile Ile His Arg Asp Leu Lys Pro Gly Asn Leu Ala

145 150 155 160

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

165 170 175

Gln Ala Asp Ser Glu Met Thr Gly Tyr Val Val Thr Arg Trp Tyr Arg

180 185 190

Ala Pro Glu Val Ile Leu Asn Trp Met Arg Tyr Thr Gln Thr Val Asp

195 200 205

Ile Trp Ser Val Gly Cys Ile Met Ala Glu Met Ile Thr Gly Lys Thr

210 215 220

Leu Phe Lys Gly Ser Asp His Leu Asp Gln Leu Lys Glu Ile Met Lys

225 230 235 240

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

245 250 255

Glu Ala Lys Asn Tyr Met Lys Gly Leu Pro Glu Leu Glu Lys Lys Asp

260 265 270

Phe Ala Ser Ile Leu Thr Asn Ala Ser Pro Leu Ala Val Asn Leu Leu

275 280 285

Glu Lys Met Leu Val Leu Asp Ala Glu Gln Arg Val Thr Ala Gly Glu

290 295 300

Ala Leu Ala His Pro Tyr Phe Glu Ser Leu His Asp Thr Glu Asp Glu

305 310 315 320

Pro Gln Val Gln Lys Tyr Asp Asp Ser Phe Asp Asp Val Asp Arg Thr

325 330 335

Leu Asp Glu Trp Lys Arg Val Thr Tyr Lys Glu Val Leu Ser Phe Lys

340 345 350

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

355 360 365

<210> 51

<211> 365

<212> PRT

<213>People（Homo sapiens）

<400> 51

Met Ser Leu Ile Arg Lys Lys Gly Phe Tyr Lys Gln Asp Val Asn Lys

1 5 10 15

Thr Ala Trp Glu Leu Pro Lys Thr Tyr Val Ser Pro Thr His Val Gly

20 25 30

Ser Gly Ala Tyr Gly Ser Val Cys Ser Ala Ile Asp Lys Arg Ser Gly

35 40 45

Glu Lys Val Ala Ile Lys Lys Leu Ser Arg Pro Phe Gln Ser Glu Ile

50 55 60

Phe Ala Lys Arg Ala Tyr Arg Glu Leu Leu Leu Leu Lys His Met Gln

65 70 75 80

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

85 90 95

Leu Arg Asn Phe Tyr Asp Phe Tyr Leu Val Met Pro Phe Met Gln Thr

100 105 110

Asp Leu Gln Lys Ile Met Gly Met Glu Phe Ser Glu Glu Lys Ile Gln

115 120 125

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

130 135 140

Gly Val Val His Arg Asp Leu Lys Pro Gly Asn Leu Ala Val Asn Glu

145 150 155 160

Asp Cys Glu Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg His Ala Asp

165 170 175

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

180 185 190

Val Ile Leu Ser Trp Met His Tyr Asn Gln Thr Val Asp Ile Trp Ser

195 200 205

Val Gly Cys Ile Met Ala Glu Met Leu Thr Gly Lys Thr Leu Phe Lys

210 215 220

Gly Lys Asp Tyr Leu Asp Gln Leu Thr Gln Ile Leu Lys Val Thr Gly

225 230 235 240

Val Pro Gly Thr Glu Phe Val Gln Lys Leu Asn Asp Lys Ala Ala Lys

245 250 255

Ser Tyr Ile Gln Ser Leu Pro Gln Thr Pro Arg Lys Asp Phe Thr Gln

260 265 270

Leu Phe Pro Arg Ala Ser Pro Gln Ala Ala Asp Leu Leu Glu Lys Met

275 280 285

Leu Glu Leu Asp Val Asp Lys Arg Leu Thr Ala Ala Gln Ala Leu Thr

290 295 300

His Pro Phe Phe Glu Pro Phe Arg Asp Pro Glu Glu Glu Thr Glu Ala

305 310 315 320

Gln Gln Pro Phe Asp Asp Ser Leu Glu His Glu Lys Leu Thr Val Asp

325 330 335

Glu Trp Lys Gln His Ile Tyr Lys Glu Ile Val Asn Phe Ser Pro Ile

340 345 350

Ala Arg Lys Asp Ser Arg Arg Arg Ser Gly Met Lys Leu

355 360 365

<210> 52

<211> 403

<212> PRT

<213>People（Homo sapiens）

<400> 52

Met Thr Ala Ile Ile Lys Glu Ile Val Ser Arg Asn Lys Arg Arg Tyr

1 5 10 15

Gln Glu Asp Gly Phe Asp Leu Asp Leu Thr Tyr Ile Tyr Pro Asn Ile

20 25 30

Ile Ala Met Gly Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn

35 40 45

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

50 55 60

Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala Lys

65 70 75 80

Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe Glu Asp His Asn Pro Pro

85 90 95

Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp Leu Asp Gln Trp Leu

100 105 110

Ser Glu Asp Asp Asn His Val Ala Ala Ile His Cys Lys Ala Gly Lys

115 120 125

Gly Arg Thr Gly Val Met Ile Cys Ala Tyr Leu Leu His Arg Gly Lys

130 135 140

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

145 150 155 160

Arg Asp Lys Lys Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr Val Tyr

165 170 175

Tyr Tyr Ser Tyr Leu Leu Lys Asn His Leu Asp Tyr Arg Pro Val Ala

180 185 190

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

195 200 205

Gly Thr Cys Asn Pro Gln Phe Val Val Cys Gln Leu Lys Val Lys Ile

210 215 220

Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp Lys Phe Met Tyr

225 230 235 240

Phe Glu Phe Pro Gln Pro Leu Pro Val Cys Gly Asp Ile Lys Val Glu

245 250 255

Phe Phe His Lys Gln Asn Lys Met Leu Lys Lys Asp Lys Met Phe His

260 265 270

Phe Trp Val Asn Thr Phe Phe Ile Pro Gly Pro Glu Glu Thr Ser Glu

275 280 285

Lys Val Glu Asn Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser Ile Cys

290 295 300

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

305 310 315 320

Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg Tyr

325 330 335

Phe Ser Pro Asn Phe Lys Val Lys Leu Tyr Phe Thr Lys Thr Val Glu

340 345 350

Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr Ser Val Thr Pro Asp

355 360 365

Val Ser Asp Asn Glu Pro Asp His Tyr Arg Tyr Ser Asp Thr Thr Asp

370 375 380

Ser Asp Pro Glu Asn Glu Pro Phe Asp Glu Asp Gln His Thr Gln Ile

385 390 395 400

Thr Lys Val

<210> 53

<211> 1154

<212> PRT

<213>People（Homo sapiens）

<400> 53

Met Gln Tyr Leu Asn Ile Lys Glu Asp Cys Asn Ala Met Ala Phe Cys

1 5 10 15

Ala Lys Met Arg Ser Ser Lys Lys Thr Glu Val Asn Leu Glu Ala Pro

20 25 30

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

35 40 45

Arg Leu Gly Ser Gly Glu Tyr Thr Ala Glu Glu Leu Cys Ile Arg Ala

50 55 60

Ala Gln Ala Cys Arg Ile Ser Pro Leu Cys His Asn Leu Phe Ala Leu

65 70 75 80

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

85 90 95

Val Asp Asp Lys Met Ser Leu Arg Leu His Tyr Arg Met Arg Phe Tyr

100 105 110

Phe Thr Asn Trp His Gly Thr Asn Asp Asn Glu Gln Ser Val Trp Arg

115 120 125

His Ser Pro Lys Lys Gln Lys Asn Gly Tyr Glu Lys Lys Lys Ile Pro

130 135 140

Asp Ala Thr Pro Leu Leu Asp Ala Ser Ser Leu Glu Tyr Leu Phe Ala

145 150 155 160

Gln Gly Gln Tyr Asp Leu Val Lys Cys Leu Ala Pro Ile Arg Asp Pro

165 170 175

Lys Thr Glu Gln Asp Gly His Asp Ile Glu Asn Glu Cys Leu Gly Met

180 185 190

Ala Val Leu Ala Ile Ser His Tyr Ala Met Met Lys Lys Met Gln Leu

195 200 205

Pro Glu Leu Pro Lys Asp Ile Ser Tyr Lys Arg Tyr Ile Pro Glu Thr

210 215 220

Leu Asn Lys Ser Ile Arg Gln Arg Asn Leu Leu Thr Arg Met Arg Ile

225 230 235 240

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

245 250 255

Cys Asp Ser Ser Val Ser Thr His Asp Leu Lys Val Lys Tyr Leu Ala

260 265 270

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

275 280 285

Ser Met Leu Leu Ile Ser Ser Glu Asn Glu Met Asn Trp Phe His Ser

290 295 300

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

305 310 315 320

Leu Gly Ile Gln Trp Arg His Lys Pro Asn Val Val Ser Val Glu Lys

325 330 335

Glu Lys Asn Lys Leu Lys Arg Lys Lys Leu Glu Asn Lys His Lys Lys

340 345 350

Asp Glu Glu Lys Asn Lys Ile Arg Glu Glu Trp Asn Asn Phe Ser Tyr

355 360 365

Phe Pro Glu Ile Thr His Ile Val Ile Lys Glu Ser Val Val Ser Ile

370 375 380

Asn Lys Gln Asp Asn Lys Lys Met Glu Leu Lys Leu Ser Ser His Glu

385 390 395 400

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

405 410 415

Ala Asp Ala His His Tyr Leu Cys Thr Asp Val Ala Pro Pro Leu Ile

420 425 430

Val His Asn Ile Gln Asn Gly Cys His Gly Pro Ile Cys Thr Glu Tyr

435 440 445

Ala Ile Asn Lys Leu Arg Gln Glu Gly Ser Glu Glu Gly Met Tyr Val

450 455 460

Leu Arg Trp Ser Cys Thr Asp Phe Asp Asn Ile Leu Met Thr Val Thr

465 470 475 480

Cys Phe Glu Lys Ser Glu Gln Val Gln Gly Ala Gln Lys Gln Phe Lys

485 490 495

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

500 505 510

Asp Arg Ser Phe Pro Ser Leu Gly Asp Leu Met Ser His Leu Lys Lys

515 520 525

Gln Ile Leu Arg Thr Asp Asn Ile Ser Phe Met Leu Lys Arg Cys Cys

530 535 540

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

545 550 555 560

Ala Gln Glu Trp Gln Pro Val Tyr Pro Met Ser Gln Leu Ser Phe Asp

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Asp Pro Ser His Arg Asp Ile Ser Leu Ala Phe Phe Glu Ala Ala Ser

625 630 635 640

Met Met Arg Gln Val Ser His Lys His Ile Val Tyr Leu Tyr Gly Val

645 650 655

Cys Val Arg Asp Val Glu Asn Ile Met Val Glu Glu Phe Val Glu Gly

660 665 670

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

675 680 685

Pro Trp Lys Phe Lys Val Ala Lys Gln Leu Ala Ser Ala Leu Ser Tyr

690 695 700

Leu Glu Asp Lys Asp Leu Val His Gly Asn Val Cys Thr Lys Asn Leu

705 710 715 720

Leu Leu Ala Arg Glu Gly Ile Asp Ser Glu Cys Gly Pro Phe Ile Lys

725 730 735

Leu Ser Asp Pro Gly Ile Pro Ile Thr Val Leu Ser Arg Gln Glu Cys

740 745 750

Ile Glu Arg Ile Pro Trp Ile Ala Pro Glu Cys Val Glu Asp Ser Lys

755 760 765

Asn Leu Ser Val Ala Ala Asp Lys Trp Ser Phe Gly Thr Thr Leu Trp

770 775 780

Glu Ile Cys Tyr Asn Gly Glu Ile Pro Leu Lys Asp Lys Thr Leu Ile

785 790 795 800

Glu Lys Glu Arg Phe Tyr Glu Ser Arg Cys Arg Pro Val Thr Pro Ser

805 810 815

Cys Lys Glu Leu Ala Asp Leu Met Thr Arg Cys Met Asn Tyr Asp Pro

820 825 830

Asn Gln Arg Pro Phe Phe Arg Ala Ile Met Arg Asp Ile Asn Lys Leu

835 840 845

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

850 855 860

Val Asp Pro Thr His Phe Glu Lys Arg Phe Leu Lys Arg Ile Arg Asp

865 870 875 880

Leu Gly Glu Gly His Phe Gly Lys Val Glu Leu Cys Arg Tyr Asp Pro

885 890 895

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

900 905 910

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

915 920 925

Leu Arg Asn Leu Tyr His Glu Asn Ile Val Lys Tyr Lys Gly Ile Cys

930 935 940

Thr Glu Asp Gly Gly Asn Gly Ile Lys Leu Ile Met Glu Phe Leu Pro

945 950 955 960

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

965 970 975

Leu Lys Gln Gln Leu Lys Tyr Ala Val Gln Ile Cys Lys Gly Met Asp

980 985 990

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

995 1000 1005

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

1010 1015 1020

Thr Lys Ala Ile Glu Thr Asp Lys Glu Tyr Tyr Thr Val Lys Asp Asp

1025 1030 1035 1040

Arg Asp Ser Pro Val Phe Trp Tyr Ala Pro Glu Cys Leu Met Gln Ser

1045 1050 1055

Lys Phe Tyr Ile Ala Ser Asp Val Trp Ser Phe Gly Val Thr Leu His

1060 1065 1070

Glu Leu Leu Thr Tyr Cys Asp Ser Asp Ser Ser Pro Met Ala Leu Phe

1075 1080 1085

Leu Lys Met Ile Gly Pro Thr His Gly Gln Met Thr Val Thr Arg Leu

1090 1095 1100

Val Asn Thr Leu Lys Glu Gly Lys Arg Leu Pro Cys Pro Pro Asn Cys

1105 1110 1115 1120

Pro Asp Glu Val Tyr Gln Leu Met Arg Lys Cys Trp Glu Phe Gln Pro

1125 1130 1135

Ser Asn Arg Thr Ser Phe Gln Asn Leu Ile Glu Gly Phe Glu Ala Leu

1140 1145 1150

Leu Lys

<210> 54

<211> 1132

<212> PRT

<213>People（Homo sapiens）

<400> 54

Met Gly Met Ala Cys Leu Thr Met Thr Glu Met Glu Gly Thr Ser Thr

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Glu Glu Ile Cys Ile Ala Ala Ser Lys Ala Cys Gly Ile Thr Pro Val

65 70 75 80

Tyr His Asn Met Phe Ala Leu Met Ser Glu Thr Glu Arg Ile Trp Tyr

85 90 95

Pro Pro Asn His Val Phe His Ile Asp Glu Ser Thr Arg His Asn Val

100 105 110

Leu Tyr Arg Ile Arg Phe Tyr Phe Pro Arg Trp Tyr Cys Ser Gly Ser

115 120 125

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

130 135 140

Leu Asp Asp Phe Val Met Ser Tyr Leu Phe Ala Gln Trp Arg His Asp

145 150 155 160

Phe Val His Gly Trp Ile Lys Val Pro Val Thr His Glu Thr Gln Glu

165 170 175

Glu Cys Leu Gly Met Ala Val Leu Asp Met Met Arg Ile Ala Lys Glu

180 185 190

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

195 200 205

Phe Leu Pro Lys Cys Ile Arg Ala Lys Ile Gln Asp Tyr His Ile Leu

210 215 220

Thr Arg Lys Arg Ile Arg Tyr Arg Phe Arg Arg Phe Ile Gln Gln Phe

225 230 235 240

Ser Gln Cys Lys Ala Thr Ala Arg Asn Leu Lys Leu Lys Tyr Leu Ile

245 250 255

Asn Leu Glu Thr Leu Gln Ser Ala Phe Tyr Thr Glu Lys Phe Glu Val

260 265 270

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

275 280 285

Ile Ile Thr Gly Asn Gly Gly Ile Gln Trp Ser Arg Gly Lys His Lys

290 295 300

Glu Ser Glu Thr Leu Thr Glu Gln Asp Leu Gln Leu Tyr Cys Asp Phe

305 310 315 320

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

325 330 335

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

340 345 350

Glu Ile Glu Leu Ser Ser Leu Arg Glu Ala Leu Ser Phe Val Ser Leu

355 360 365

Ile Asp Gly Tyr Tyr Arg Leu Thr Ala Asp Ala His His Tyr Leu Cys

370 375 380

Lys Glu Val Ala Pro Pro Ala Val Leu Glu Asn Ile Gln Ser Asn Cys

385 390 395 400

His Gly Pro Ile Ser Met Asp Phe Ala Ile Ser Lys Leu Lys Lys Ala

405 410 415

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

420 425 430

Asn Lys Tyr Phe Leu Thr Phe Ala Val Glu Arg Glu Asn Val Ile Glu

435 440 445

Tyr Lys His Cys Leu Ile Thr Lys Asn Glu Asn Glu Glu Tyr Asn Leu

450 455 460

Ser Gly Thr Lys Lys Asn Phe Ser Ser Leu Lys Asp Leu Leu Asn Cys

465 470 475 480

Tyr Gln Met Glu Thr Val Arg Ser Asp Asn Ile Ile Phe Gln Phe Thr

485 490 495

Lys Cys Cys Pro Pro Lys Pro Lys Asp Lys Ser Asn Leu Leu Val Phe

500 505 510

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

515 520 525

Pro Thr His Met Asn Gln Met Val Phe His Lys Ile Arg Asn Glu Asp

530 535 540

Leu Ile Phe Asn Glu Ser Leu Gly Gln Gly Thr Phe Thr Lys Ile Phe

545 550 555 560

Lys Gly Val Arg Arg Glu Val Gly Asp Tyr Gly Gln Leu His Glu Thr

565 570 575

Glu Val Leu Leu Lys Val Leu Asp Lys Ala His Arg Asn Tyr Ser Glu

580 585 590

Ser Phe Phe Glu Ala Ala Ser Met Met Ser Lys Leu Ser His Lys His

595 600 605

Leu Val Leu Asn Tyr Gly Val Cys Val Cys Gly Asp Glu Asn Ile Leu

610 615 620

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

625 630 635 640

Asn Lys Asn Cys Ile Asn Ile Leu Trp Lys Leu Glu Val Ala Lys Gln

645 650 655

Leu Ala Trp Ala Met His Phe Leu Glu Glu Asn Thr Leu Ile His Gly

660 665 670

Asn Val Cys Ala Lys Asn Ile Leu Leu Ile Arg Glu Glu Asp Arg Lys

675 680 685

Thr Gly Asn Pro Pro Phe Ile Lys Leu Ser Asp Pro Gly Ile Ser Ile

690 695 700

Thr Val Leu Pro Lys Asp Ile Leu Gln Glu Arg Ile Pro Trp Val Pro

705 710 715 720

Pro Glu Cys Ile Glu Asn Pro Lys Asn Leu Asn Leu Ala Thr Asp Lys

725 730 735

Trp Ser Phe Gly Thr Thr Leu Trp Glu Ile Cys Ser Gly Gly Asp Lys

740 745 750

Pro Leu Ser Ala Leu Asp Ser Gln Arg Lys Leu Gln Phe Tyr Glu Asp

755 760 765

Arg His Gln Leu Pro Ala Pro Lys Trp Ala Glu Leu Ala Asn Leu Ile

770 775 780

Asn Asn Cys Met Asp Tyr Glu Pro Asp Phe Arg Pro Ser Phe Arg Ala

785 790 795 800

Ile Ile Arg Asp Leu Asn Ser Leu Phe Thr Pro Asp Tyr Glu Leu Leu

805 810 815

Thr Glu Asn Asp Met Leu Pro Asn Met Arg Ile Gly Ala Leu Gly Phe

820 825 830

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

835 840 845

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

850 855 860

Met Cys Arg Tyr Asp Pro Leu Gln Asp Asn Thr Gly Glu Val Val Ala

865 870 875 880

Val Lys Lys Leu Gln His Ser Thr Glu Glu His Leu Arg Asp Phe Glu

885 890 895

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

900 905 910

Tyr Lys Gly Val Cys Tyr Ser Ala Gly Arg Arg Asn Leu Lys Leu Ile

915 920 925

Met Glu Tyr Leu Pro Tyr Gly Ser Leu Arg Asp Tyr Leu Gln Lys His

930 935 940

Lys Glu Arg Ile Asp His Ile Lys Leu Leu Gln Tyr Thr Ser Gln Ile

945 950 955 960

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

965 970 975

Leu Ala Thr Arg Asn Ile Leu Val Glu Asn Glu Asn Arg Val Lys Ile

980 985 990

Gly Asp Phe Gly Leu Thr Lys Val Leu Pro Gln Asp Lys Glu Tyr Tyr

995 1000 1005

Lys Val Lys Glu Pro Gly Glu Ser Pro Ile Phe Trp Tyr Ala Pro Glu

1010 1015 1020

Ser Leu Thr Glu Ser Lys Phe Ser Val Ala Ser Asp Val Trp Ser Phe

1025 1030 1035 1040

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

1045 1050 1055

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

1060 1065 1070

Met Ile Val Phe His Leu Ile Glu Leu Leu Lys Asn Asn Gly Arg Leu

1075 1080 1085

Pro Arg Pro Asp Gly Cys Pro Asp Glu Ile Tyr Met Ile Met Thr Glu

1090 1095 1100

Cys Trp Asn Asn Asn Val Asn Gln Arg Pro Ser Phe Arg Asp Leu Ala

1105 1110 1115 1120

Leu Arg Val Asp Gln Ile Arg Asp Asn Met Ala Gly

1125 1130

<210> 55

<211> 1124

<212> PRT

<213>People（Homo sapiens）

<400> 55

Met Ala Pro Pro Ser Glu Glu Thr Pro Leu Ile Pro Gln Arg Ser Cys

1 5 10 15

Ser Leu Leu Ser Thr Glu Ala Gly Ala Leu His Val Leu Leu Pro Ala

20 25 30

Arg Gly Pro Gly Pro Pro Gln Arg Leu Ser Phe Ser Phe Gly Asp His

35 40 45

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

50 55 60

Pro Val Tyr His Ser Leu Phe Ala Leu Ala Thr Glu Asp Leu Ser Cys

65 70 75 80

Trp Phe Pro Pro Ser His Ile Phe Ser Val Glu Asp Ala Ser Thr Gln

85 90 95

Val Leu Leu Tyr Arg Ile Arg Phe Tyr Phe Pro Asn Trp Phe Gly Leu

100 105 110

Glu Lys Cys His Arg Phe Gly Leu Arg Lys Asp Leu Ala Ser Ala Ile

115 120 125

Leu Asp Leu Pro Val Leu Glu His Leu Phe Ala Gln His Arg Ser Asp

130 135 140

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

145 150 155 160

Glu Cys Leu Ser Leu Ala Val Leu Asp Leu Ala Arg Met Ala Arg Glu

165 170 175

Gln Ala Gln Arg Pro Gly Glu Leu Leu Lys Thr Val Ser Tyr Lys Ala

180 185 190

Cys Leu Pro Pro Ser Leu Arg Asp Leu Ile Gln Gly Leu Ser Phe Val

195 200 205

Thr Arg Arg Arg Ile Arg Arg Thr Val Arg Arg Ala Leu Arg Arg Val

210 215 220

Ala Ala Cys Gln Ala Asp Arg His Ser Leu Met Ala Lys Tyr Ile Met

225 230 235 240

Asp Leu Glu Arg Leu Asp Pro Ala Gly Ala Ala Glu Thr Phe His Val

245 250 255

Gly Leu Pro Gly Ala Leu Gly Gly His Asp Gly Leu Gly Leu Leu Arg

260 265 270

Val Ala Gly Asp Gly Gly Ile Ala Trp Thr Gln Gly Glu Gln Glu Val

275 280 285

Leu Gln Pro Phe Cys Asp Phe Pro Glu Ile Val Asp Ile Ser Ile Lys

290 295 300

Gln Ala Pro Arg Val Gly Pro Ala Gly Glu His Arg Leu Val Thr Val

305 310 315 320

Thr Arg Thr Asp Asn Gln Ile Leu Glu Ala Glu Phe Pro Gly Leu Pro

325 330 335

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

340 345 350

Thr Asp Ser Gln His Phe Phe Cys Lys Glu Val Ala Pro Pro Arg Leu

355 360 365

Leu Glu Glu Val Ala Glu Gln Cys His Gly Pro Ile Thr Leu Asp Phe

370 375 380

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

385 390 395 400

Leu Arg Arg Ser Pro Gln Asp Phe Asp Ser Phe Leu Leu Thr Val Cys

405 410 415

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

420 425 430

Ser Pro Thr Gly Thr Phe Leu Leu Val Gly Leu Ser Arg Pro His Ser

435 440 445

Ser Leu Arg Glu Leu Leu Ala Thr Cys Trp Asp Gly Gly Leu His Val

450 455 460

Asp Gly Val Ala Val Thr Leu Thr Ser Cys Cys Ile Pro Arg Pro Lys

465 470 475 480

Glu Lys Ser Asn Leu Ile Val Val Gln Arg Gly His Ser Pro Pro Thr

485 490 495

Ser Ser Leu Val Gln Pro Gln Ser Gln Tyr Gln Leu Ser Gln Met Thr

500 505 510

Phe His Lys Ile Pro Ala Asp Ser Leu Glu Trp His Glu Asn Leu Gly

515 520 525

His Gly Ser Phe Thr Lys Ile Tyr Arg Gly Cys Arg His Glu Val Val

530 535 540

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

545 550 555 560

Lys His Lys Asn Cys Met Glu Ser Phe Leu Glu Ala Ala Ser Leu Met

565 570 575

Ser Gln Val Ser Tyr Arg His Leu Val Leu Leu His Gly Val Cys Met

580 585 590

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

595 600 605

Asp Met Tyr Leu Arg Lys Arg Gly His Leu Val Pro Ala Ser Trp Lys

610 615 620

Leu Gln Val Val Lys Gln Leu Ala Tyr Ala Leu Asn Tyr Leu Glu Asp

625 630 635 640

Lys Gly Leu Pro His Gly Asn Val Ser Ala Arg Lys Val Leu Leu Ala

645 650 655

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

660 665 670

Gly Val Ser Pro Ala Val Leu Ser Leu Glu Met Leu Thr Asp Arg Ile

675 680 685

Pro Trp Val Ala Pro Glu Cys Leu Arg Glu Ala Gln Thr Leu Ser Leu

690 695 700

Glu Ala Asp Lys Trp Gly Phe Gly Ala Thr Val Trp Glu Val Phe Ser

705 710 715 720

Gly Val Thr Met Pro Ile Ser Ala Leu Asp Pro Ala Lys Lys Leu Gln

725 730 735

Phe Tyr Glu Asp Arg Gln Gln Leu Pro Ala Pro Lys Trp Thr Glu Leu

740 745 750

Ala Leu Leu Ile Gln Gln Cys Met Ala Tyr Glu Pro Val Gln Arg Pro

755 760 765

Ser Phe Arg Ala Val Ile Arg Asp Leu Asn Ser Leu Ile Ser Ser Asp

770 775 780

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

785 790 795 800

Gly Leu Trp Asn Gly Ala Gln Leu Tyr Ala Cys Gln Asp Pro Thr Ile

805 810 815

Phe Glu Glu Arg His Leu Lys Tyr Ile Ser Gln Leu Gly Lys Gly Asn

820 825 830

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

835 840 845

Gly Ala Leu Val Ala Val Lys Gln Leu Gln His Ser Gly Pro Asp Gln

850 855 860

Gln Arg Asp Phe Gln Arg Glu Ile Gln Ile Leu Lys Ala Leu His Ser

865 870 875 880

Asp Phe Ile Val Lys Tyr Arg Gly Val Ser Tyr Gly Pro Gly Arg Gln

885 890 895

Ser Leu Arg Leu Val Met Glu Tyr Leu Pro Ser Gly Cys Leu Arg Asp

900 905 910

Phe Leu Gln Arg His Arg Ala Arg Leu Asp Ala Ser Arg Leu Leu Leu

915 920 925

Tyr Ser Ser Gln Ile Cys Lys Gly Met Glu Tyr Leu Gly Ser Arg Arg

930 935 940

Cys Val His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Glu Ser Glu

945 950 955 960

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

965 970 975

Asp Lys Asp Tyr Tyr Val Val Arg Glu Pro Gly Gln Ser Pro Ile Phe

980 985 990

Trp Tyr Ala Pro Glu Ser Leu Ser Asp Asn Ile Phe Ser Arg Gln Ser

995 1000 1005

Asp Val Trp Ser Phe Gly Val Val Leu Tyr Glu Leu Phe Thr Tyr Cys

1010 1015 1020

Asp Lys Ser Cys Ser Pro Ser Ala Glu Phe Leu Arg Met Met Gly Cys

1025 1030 1035 1040

Glu Arg Asp Val Pro Ala Leu Cys Arg Leu Leu Glu Leu Leu Glu Glu

1045 1050 1055

Gly Gln Arg Leu Pro Ala Pro Pro Ala Cys Pro Ala Glu Val His Glu

1060 1065 1070

Leu Met Lys Leu Cys Trp Ala Pro Ser Pro Gln Asp Arg Pro Ser Phe

1075 1080 1085

Ser Ala Leu Gly Pro Gln Leu Asp Met Leu Trp Ser Gly Ser Arg Gly

1090 1095 1100

Cys Glu Thr His Ala Phe Thr Ala His Pro Glu Gly Lys His His Ser

1105 1110 1115 1120

Leu Ser Phe Ser

<210> 56

<211> 1187

<212> PRT

<213>People（Homo sapiens）

<400> 56

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

1 5 10 15

Asp Gly Ala Gln Pro Met Ala Ala Met Gly Gly Leu Lys Val Leu Leu

20 25 30

His Trp Ala Gly Pro Gly Gly Gly Glu Pro Trp Val Thr Phe Ser Glu

35 40 45

Ser Ser Leu Thr Ala Glu Glu Val Cys Ile His Ile Ala His Lys Val

50 55 60

Gly Ile Thr Pro Pro Cys Phe Asn Leu Phe Ala Leu Phe Asp Ala Gln

65 70 75 80

Ala Gln Val Trp Leu Pro Pro Asn His Ile Leu Glu Ile Pro Arg Asp

85 90 95

Ala Ser Leu Met Leu Tyr Phe Arg Ile Arg Phe Tyr Phe Arg Asn Trp

100 105 110

His Gly Met Asn Pro Arg Glu Pro Ala Val Tyr Arg Cys Gly Pro Pro

115 120 125

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

130 135 140

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

145 150 155 160

Val Asn Asp Val Ala Ser Leu Trp Glu Leu Ser Thr Glu Glu Glu Ile

165 170 175

His His Phe Lys Asn Glu Ser Leu Gly Met Ala Phe Leu His Leu Cys

180 185 190

His Leu Ala Leu Arg His Gly Ile Pro Leu Glu Glu Val Ala Lys Lys

195 200 205

Thr Ser Phe Lys Asp Cys Ile Pro Arg Ser Phe Arg Arg His Ile Arg

210 215 220

Gln His Ser Ala Leu Thr Arg Leu Arg Leu Arg Asn Val Phe Arg Arg

225 230 235 240

Phe Leu Arg Asp Phe Gln Pro Gly Arg Leu Ser Gln Gln Met Val Met

245 250 255

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

260 265 270

Glu Arg Val Pro Val Cys His Leu Arg Leu Leu Ala Gln Ala Glu Gly

275 280 285

Glu Pro Cys Tyr Ile Arg Asp Ser Gly Val Ala Pro Thr Asp Pro Gly

290 295 300

Pro Glu Ser Ala Ala Gly Pro Pro Thr His Glu Val Leu Val Thr Gly

305 310 315 320

Thr Gly Gly Ile Gln Trp Trp Pro Val Glu Glu Glu Val Asn Lys Glu

325 330 335

Glu Gly Ser Ser Gly Ser Ser Gly Arg Asn Pro Gln Ala Ser Leu Phe

340 345 350

Gly Lys Lys Ala Lys Ala His Lys Ala Val Gly Gln Pro Ala Asp Arg

355 360 365

Pro Arg Glu Pro Leu Trp Ala Tyr Phe Cys Asp Phe Arg Asp Ile Thr

370 375 380

His Val Val Leu Lys Glu His Cys Val Ser Ile His Arg Gln Asp Asn

385 390 395 400

Lys Cys Leu Glu Leu Ser Leu Pro Ser Arg Ala Ala Ala Leu Ser Phe

405 410 415

Val Ser Leu Val Asp Gly Tyr Phe Arg Leu Thr Ala Asp Ser Ser His

420 425 430

Tyr Leu Cys His Glu Val Ala Pro Pro Arg Leu Val Met Ser Ile Arg

435 440 445

Asp Gly Ile His Gly Pro Leu Leu Glu Pro Phe Val Gln Ala Lys Leu

450 455 460

Arg Pro Glu Asp Gly Leu Tyr Leu Ile His Trp Ser Thr Ser His Pro

465 470 475 480

Tyr Arg Leu Ile Leu Thr Val Ala Gln Arg Ser Gln Ala Pro Asp Gly

485 490 495

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

500 505 510

Ala Phe Val Leu Glu Gly Trp Gly Arg Ser Phe Pro Ser Val Arg Glu

515 520 525

Leu Gly Ala Ala Leu Gln Gly Cys Leu Leu Arg Ala Gly Asp Asp Cys

530 535 540

Phe Ser Leu Arg Arg Cys Cys Leu Pro Gln Pro Gly Glu Thr Ser Asn

545 550 555 560

Leu Ile Ile Met Arg Gly Ala Arg Ala Ser Pro Arg Thr Leu Asn Leu

565 570 575

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

580 585 590

Ser His Leu Gly Gln Gly Thr Arg Thr Asn Val Tyr Glu Gly Arg Leu

595 600 605

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

610 615 620

Asp Pro Leu Val Pro Gly Arg Asp Arg Gly Gln Glu Leu Arg Val Val

625 630 635 640

Leu Lys Val Leu Asp Pro Ser His His Asp Ile Ala Leu Ala Phe Tyr

645 650 655

Glu Thr Ala Ser Leu Met Ser Gln Val Ser His Thr His Leu Ala Phe

660 665 670

Val His Gly Val Cys Val Arg Gly Pro Glu Asn Ile Met Val Thr Glu

675 680 685

Tyr Val Glu His Gly Pro Leu Asp Val Trp Leu Arg Arg Glu Arg Gly

690 695 700

His Val Pro Met Ala Trp Lys Met Val Val Ala Gln Gln Leu Ala Ser

705 710 715 720

Ala Leu Ser Tyr Leu Glu Asn Lys Asn Leu Val His Gly Asn Val Cys

725 730 735

Gly Arg Asn Ile Leu Leu Ala Arg Leu Gly Leu Ala Glu Gly Thr Ser

740 745 750

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

755 760 765

Arg Glu Glu Arg Val Glu Arg Ile Pro Trp Leu Ala Pro Glu Cys Leu

770 775 780

Pro Gly Gly Ala Asn Ser Leu Ser Thr Ala Met Asp Lys Trp Gly Phe

785 790 795 800

Gly Ala Thr Leu Leu Glu Ile Cys Phe Asp Gly Glu Ala Pro Leu Gln

805 810 815

Ser Arg Ser Pro Ser Glu Lys Glu His Phe Tyr Gln Arg Gln His Arg

820 825 830

Leu Pro Glu Pro Ser Cys Pro Gln Leu Ala Thr Leu Thr Ser Gln Cys

835 840 845

Leu Thr Tyr Glu Pro Thr Gln Arg Pro Ser Phe Arg Thr Ile Leu Arg

850 855 860

Asp Leu Thr Arg Leu Gln Pro His Asn Leu Ala Asp Val Leu Thr Val

865 870 875 880

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

885 890 895

Leu Lys Lys Ile Arg Asp Leu Gly Glu Gly His Phe Gly Lys Val Ser

900 905 910

Leu Tyr Cys Tyr Asp Pro Thr Asn Asp Gly Thr Gly Glu Met Val Ala

915 920 925

Val Lys Ala Leu Lys Ala Asp Cys Gly Pro Gln His Arg Ser Gly Trp

930 935 940

Lys Gln Glu Ile Asp Ile Leu Arg Thr Leu Tyr His Glu His Ile Ile

945 950 955 960

Lys Tyr Lys Gly Cys Cys Glu Asp Gln Gly Glu Lys Ser Leu Gln Leu

965 970 975

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

980 985 990

His Ser Ile Gly Leu Ala Gln Leu Leu Leu Phe Ala Gln Gln Ile Cys

995 1000 1005

Glu Gly Met Ala Tyr Leu His Ala Gln His Tyr Ile His Arg Asp Leu

1010 1015 1020

Ala Ala Arg Asn Val Leu Leu Asp Asn Asp Arg Leu Val Lys Ile Gly

1025 1030 1035 1040

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

1045 1050 1055

Val Arg Glu Asp Gly Asp Ser Pro Val Phe Trp Tyr Ala Pro Glu Cys

1060 1065 1070

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

1075 1080 1085

Val Thr Leu Tyr Glu Leu Leu Thr His Cys Asp Ser Ser Gln Ser Pro

1090 1095 1100

Pro Thr Lys Phe Leu Glu Leu Ile Gly Ile Ala Gln Gly Gln Met Thr

1105 1110 1115 1120

Val Leu Arg Leu Thr Glu Leu Leu Glu Arg Gly Glu Arg Leu Pro Arg

1125 1130 1135

Pro Asp Lys Cys Pro Cys Glu Val Tyr His Leu Met Lys Asn Cys Trp

1140 1145 1150

Glu Thr Glu Ala Ser Phe Arg Pro Thr Phe Glu Asn Leu Ile Pro Ile

1155 1160 1165

Leu Lys Thr Val His Glu Lys Tyr Gln Gly Gln Ala Pro Ser Val Phe

1170 1175 1180

Ser Val Cys

1185

<210> 57

<211> 906

<212> PRT

<213>People（Homo sapiens）

<400> 57

Met Ala Gly Asn Val Lys Lys Ser Ser Gly Ala Gly Gly Gly Ser Gly

1 5 10 15

Ser Gly Gly Ser Gly Ser Gly Gly Leu Ile Gly Leu Met Lys Asp Ala

20 25 30

Phe Gln Pro His His His His His His His Leu Ser Pro His Pro Pro

35 40 45

Gly Thr Val Asp Lys Lys Met Val Glu Lys Cys Trp Lys Leu Met Asp

50 55 60

Lys Val Val Arg Leu Cys Gln Asn Pro Lys Leu Ala Leu Lys Asn Ser

65 70 75 80

Pro Pro Tyr Ile Leu Asp Leu Leu Pro Asp Thr Tyr Gln His Leu Arg

85 90 95

Thr Ile Leu Ser Arg Tyr Glu Gly Lys Met Glu Thr Leu Gly Glu Asn

100 105 110

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

115 120 125

Thr Ile Ser Leu Phe Lys Glu Gly Lys Glu Arg Met Tyr Glu Glu Asn

130 135 140

Ser Gln Pro Arg Arg Asn Leu Thr Lys Leu Ser Leu Ile Phe Ser His

145 150 155 160

Met Leu Ala Glu Leu Lys Gly Ile Phe Pro Ser Gly Leu Phe Gln Gly

165 170 175

Asp Thr Phe Arg Ile Thr Lys Ala Asp Ala Ala Glu Phe Trp Arg Lys

180 185 190

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

195 200 205

Leu His Glu Val His Pro Ile Ser Ser Gly Leu Glu Ala Met Ala Leu

210 215 220

Lys Ser Thr Ile Asp Leu Thr Cys Asn Asp Tyr Ile Ser Val Phe Glu

225 230 235 240

Phe Asp Ile Phe Thr Arg Leu Phe Gln Pro Trp Ser Ser Leu Leu Arg

245 250 255

Asn Trp Asn Ser Leu Ala Val Thr His Pro Gly Tyr Met Ala Phe Leu

260 265 270

Thr Tyr Asp Glu Val Lys Ala Arg Leu Gln Lys Phe Ile His Lys Pro

275 280 285

Gly Ser Tyr Ile Phe Arg Leu Ser Cys Thr Arg Leu Gly Gln Trp Ala

290 295 300

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

305 310 315 320

Asn Lys Pro Leu Phe Gln Ala Leu Ile Asp Gly Phe Arg Glu Gly Phe

325 330 335

Tyr Leu Phe Pro Asp Gly Arg Asn Gln Asn Pro Asp Leu Thr Gly Leu

340 345 350

Cys Glu Pro Thr Pro Gln Asp His Ile Lys Val Thr Gln Glu Gln Tyr

355 360 365

Glu Leu Tyr Cys Glu Met Gly Ser Thr Phe Gln Leu Cys Lys Ile Cys

370 375 380

Ala Glu Asn Asp Lys Asp Val Lys Ile Glu Pro Cys Gly His Leu Met

385 390 395 400

Cys Thr Ser Cys Leu Thr Ser Trp Gln Glu Ser Glu Gly Gln Gly Cys

405 410 415

Pro Phe Cys Arg Cys Glu Ile Lys Gly Thr Glu Pro Ile Val Val Asp

420 425 430

Pro Phe Asp Pro Arg Gly Ser Gly Ser Leu Leu Arg Gln Gly Ala Glu

435 440 445

Gly Ala Pro Ser Pro Asn Tyr Asp Asp Asp Asp Asp Glu Arg Ala Asp

450 455 460

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

465 470 475 480

Pro Pro Ser Pro Phe Ser Met Ala Pro Gln Ala Ser Leu Pro Pro Val

485 490 495

Pro Pro Arg Leu Asp Leu Leu Pro Gln Arg Val Cys Val Pro Ser Ser

500 505 510

Ala Ser Ala Leu Gly Thr Ala Ser Lys Ala Ala Ser Gly Ser Leu His

515 520 525

Lys Asp Lys Pro Leu Pro Val Pro Pro Thr Leu Arg Asp Leu Pro Pro

530 535 540

Pro Pro Pro Pro Asp Arg Pro Tyr Ser Val Gly Ala Glu Ser Arg Pro

545 550 555 560

Gln Arg Arg Pro Leu Pro Cys Thr Pro Gly Asp Cys Pro Ser Arg Asp

565 570 575

Lys Leu Pro Pro Val Pro Ser Ser Arg Leu Gly Asp Ser Trp Leu Pro

580 585 590

Arg Pro Ile Pro Lys Val Pro Val Ser Ala Pro Ser Ser Ser Asp Pro

595 600 605

Trp Thr Gly Arg Glu Leu Thr Asn Arg His Ser Leu Pro Phe Ser Leu

610 615 620

Pro Ser Gln Met Glu Pro Arg Pro Asp Val Pro Arg Leu Gly Ser Thr

625 630 635 640

Phe Ser Leu Asp Thr Ser Met Ser Met Asn Ser Ser Pro Leu Val Gly

645 650 655

Pro Glu Cys Asp His Pro Lys Ile Lys Pro Ser Ser Ser Ala Asn Ala

660 665 670

Ile Tyr Ser Leu Ala Ala Arg Pro Leu Pro Val Pro Lys Leu Pro Pro

675 680 685

Gly Glu Gln Cys Glu Gly Glu Glu Asp Thr Glu Tyr Met Thr Pro Ser

690 695 700

Ser Arg Pro Leu Arg Pro Leu Asp Thr Ser Gln Ser Ser Arg Ala Cys

705 710 715 720

Asp Cys Asp Gln Gln Ile Asp Ser Cys Thr Tyr Glu Ala Met Tyr Asn

725 730 735

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

740 745 750

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

755 760 765

Asn Glu Asp Asp Gly Tyr Asp Val Pro Lys Pro Pro Val Pro Ala Val

770 775 780

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

785 790 795 800

Gly Trp Leu Ser Leu Asp Gly Asp Pro Thr Thr Asn Val Thr Glu Gly

805 810 815

Ser Gln Val Pro Glu Arg Pro Pro Lys Pro Phe Pro Arg Arg Ile Asn

820 825 830

Ser Glu Arg Lys Ala Gly Ser Cys Gln Gln Gly Ser Gly Pro Ala Ala

835 840 845

Ser Ala Ala Thr Ala Ser Pro Gln Leu Ser Ser Glu Ile Glu Asn Leu

850 855 860

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

865 870 875 880

Ala Gln Asn Asn Ile Glu Met Ala Lys Asn Ile Leu Arg Glu Phe Val

885 890 895

Ser Ile Ser Ser Pro Ala His Val Ala Thr

900 905

<210> 58

<211> 465

<212> PRT

<213>People（Homo sapiens）

<400> 58

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

1 5 10 15

Val Asp Ser Asp Thr Ser Ile Phe Gln Leu Lys Glu Val Val Ala Lys

20 25 30

Arg Gln Gly Val Pro Ala Asp Gln Leu Arg Val Ile Phe Ala Gly Lys

35 40 45

Glu Leu Arg Asn Asp Trp Thr Val Gln Asn Cys Asp Leu Asp Gln Gln

50 55 60

Ser Ile Val His Ile Val Gln Arg Pro Trp Arg Lys Gly Gln Glu Met

65 70 75 80

Asn Ala Thr Gly Gly Asp Asp Pro Arg Asn Ala Ala Gly Gly Cys Glu

85 90 95

Arg Glu Pro Gln Ser Leu Thr Arg Val Asp Leu Ser Ser Ser Val Leu

100 105 110

Pro Gly Asp Ser Val Gly Leu Ala Val Ile Leu His Thr Asp Ser Arg

115 120 125

Lys Asp Ser Pro Pro Ala Gly Ser Pro Ala Gly Arg Ser Ile Tyr Asn

130 135 140

Ser Phe Tyr Val Tyr Cys Lys Gly Pro Cys Gln Arg Val Gln Pro Gly

145 150 155 160

Lys Leu Arg Val Gln Cys Ser Thr Cys Arg Gln Ala Thr Leu Thr Leu

165 170 175

Thr Gln Gly Pro Ser Cys Trp Asp Asp Val Leu Ile Pro Asn Arg Met

180 185 190

Ser Gly Glu Cys Gln Ser Pro His Cys Pro Gly Thr Ser Ala Glu Phe

195 200 205

Phe Phe Lys Cys Gly Ala His Pro Thr Ser Asp Lys Glu Thr Ser Val

210 215 220

Ala Leu His Leu Ile Ala Thr Asn Ser Arg Asn Ile Thr Cys Ile Thr

225 230 235 240

Cys Thr Asp Val Arg Ser Pro Val Leu Val Phe Gln Cys Asn Ser Arg

245 250 255

His Val Ile Cys Leu Asp Cys Phe His Leu Tyr Cys Val Thr Arg Leu

260 265 270

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

275 280 285

Cys Val Ala Gly Cys Pro Asn Ser Leu Ile Lys Glu Leu His His Phe

290 295 300

Arg Ile Leu Gly Glu Glu Gln Tyr Asn Arg Tyr Gln Gln Tyr Gly Ala

305 310 315 320

Glu Glu Cys Val Leu Gln Met Gly Gly Val Leu Cys Pro Arg Pro Gly

325 330 335

Cys Gly Ala Gly Leu Leu Pro Glu Pro Asp Gln Arg Lys Val Thr Cys

340 345 350

Glu Gly Gly Asn Gly Leu Gly Cys Gly Phe Ala Phe Cys Arg Glu Cys

355 360 365

Lys Glu Ala Tyr His Glu Gly Glu Cys Ser Ala Val Phe Glu Ala Ser

370 375 380

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

385 390 395 400

Ala Arg Trp Glu Ala Ala Ser Lys Glu Thr Ile Lys Lys Thr Thr Lys

405 410 415

Pro Cys Pro Arg Cys His Val Pro Val Glu Lys Asn Gly Gly Cys Met

420 425 430

His Met Lys Cys Pro Gln Pro Gln Cys Arg Leu Glu Trp Cys Trp Asn

435 440 445

Cys Gly Cys Glu Trp Asn Arg Val Cys Met Gly Asp His Trp Phe Asp

450 455 460

Val

465

<210> 59

<211> 108

<212> PRT

<213>People（Homo sapiens）

<400> 59

Met Ala Ala Ala Met Asp Val Asp Thr Pro Ser Gly Thr Asn Ser Gly

1 5 10 15

Ala Gly Lys Lys Arg Phe Glu Val Lys Lys Trp Asn Ala Val Ala Leu

20 25 30

Trp Ala Trp Asp Ile Val Val Asp Asn Cys Ala Ile Cys Arg Asn His

35 40 45

Ile Met Asp Leu Cys Ile Glu Cys Gln Ala Asn Gln Ala Ser Ala Thr

50 55 60

Ser Glu Glu Cys Thr Val Ala Trp Gly Val Cys Asn His Ala Phe His

65 70 75 80

Phe His Cys Ile Ser Arg Trp Leu Lys Thr Arg Gln Val Cys Pro Leu

85 90 95

Asp Asn Arg Glu Trp Glu Phe Gln Lys Tyr Gly His

100 105

<210> 60

<211> 501

<212> PRT

<213>People（Homo sapiens）

<400> 60

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

1 5 10 15

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

20 25 30

Leu Cys Ser Ala Cys Arg Asn Val Leu Arg Arg Pro Phe Gln Ala Gln

35 40 45

Cys Gly His Arg Tyr Cys Ser Phe Cys Leu Ala Ser Ile Leu Ser Ser

50 55 60

Gly Pro Gln Asn Cys Ala Ala Cys Val His Glu Gly Ile Tyr Glu Glu

65 70 75 80

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

85 90 95

Arg Arg Glu Val Glu Ser Leu Pro Ala Val Cys Pro Ser Asp Gly Cys

100 105 110

Thr Trp Lys Gly Thr Leu Lys Glu Tyr Glu Ser Cys His Glu Gly Arg

115 120 125

Cys Pro Leu Met Leu Thr Glu Cys Pro Ala Cys Lys Gly Leu Val Arg

130 135 140

Leu Gly Glu Lys Glu Arg His Leu Glu His Glu Cys Pro Glu Arg Ser

145 150 155 160

Leu Ser Cys Arg His Cys Arg Ala Pro Cys Cys Gly Ala Asp Val Lys

165 170 175

Ala His His Glu Val Cys Pro Lys Phe Pro Leu Thr Cys Asp Gly Cys

180 185 190

Gly Lys Lys Lys Ile Pro Arg Glu Lys Phe Gln Asp His Val Lys Thr

195 200 205

Cys Gly Lys Cys Arg Val Pro Cys Arg Phe His Ala Ile Gly Cys Leu

210 215 220

Glu Thr Val Glu Gly Glu Lys Gln Gln Glu His Glu Val Gln Trp Leu

225 230 235 240

Arg Glu His Leu Ala Met Leu Leu Ser Ser Val Leu Glu Ala Lys Pro

245 250 255

Leu Leu Gly Asp Gln Ser His Ala Gly Ser Glu Leu Leu Gln Arg Cys

260 265 270

Glu Ser Leu Glu Lys Lys Thr Ala Thr Phe Glu Asn Ile Val Cys Val

275 280 285

Leu Asn Arg Glu Val Glu Arg Val Ala Met Thr Ala Glu Ala Cys Ser

290 295 300

Arg Gln His Arg Leu Asp Gln Asp Lys Ile Glu Ala Leu Ser Ser Lys

305 310 315 320

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

325 330 335

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

340 345 350

Val Phe Ile Trp Lys Ile Ser Asp Phe Ala Arg Lys Arg Gln Glu Ala

355 360 365

Val Ala Gly Arg Ile Pro Ala Ile Phe Ser Pro Ala Phe Tyr Thr Ser

370 375 380

Arg Tyr Gly Tyr Lys Met Cys Leu Arg Ile Tyr Leu Asn Gly Asp Gly

385 390 395 400

Thr Gly Arg Gly Thr His Leu Ser Leu Phe Phe Val Val Met Lys Gly

405 410 415

Pro Asn Asp Ala Leu Leu Arg Trp Pro Phe Asn Gln Lys Val Thr Leu

420 425 430

Met Leu Leu Asp Gln Asn Asn Arg Glu His Val Ile Asp Ala Phe Arg

435 440 445

Pro Asp Val Thr Ser Ser Ser Phe Gln Arg Pro Val Asn Asp Met Asn

450 455 460

Ile Ala Ser Gly Cys Pro Leu Phe Cys Pro Val Ser Lys Met Glu Ala

465 470 475 480

Lys Asn Ser Tyr Val Arg Asp Asp Ala Ile Phe Ile Lys Ala Ile Val

485 490 495

Asp Leu Thr Gly Leu

500

<210> 61

<211> 491

<212> PRT

<213>People（Homo sapiens）

<400> 61

Met Cys Asn Thr Asn Met Ser Val Pro Thr Asp Gly Ala Val Thr Thr

1 5 10 15

Ser Gln Ile Pro Ala Ser Glu Gln Glu Thr Leu Val Arg Pro Lys Pro

20 25 30

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

35 40 45

Thr Met Lys Glu Val Leu Phe Tyr Leu Gly Gln Tyr Ile Met Thr Lys

50 55 60

Arg Leu Tyr Asp Glu Lys Gln Gln His Ile Val Tyr Cys Ser Asn Asp

65 70 75 80

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

85 90 95

Arg Lys Ile Tyr Thr Met Ile Tyr Arg Asn Leu Val Val Val Asn Gln

100 105 110

Gln Glu Ser Ser Asp Ser Gly Thr Ser Val Ser Glu Asn Arg Cys His

115 120 125

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

130 135 140

Glu Lys Pro Ser Ser Ser His Leu Val Ser Arg Pro Ser Thr Ser Ser

145 150 155 160

Arg Arg Arg Ala Ile Ser Glu Thr Glu Glu Asn Ser Asp Glu Leu Ser

165 170 175

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

180 185 190

Phe Asp Glu Ser Leu Ala Leu Cys Val Ile Arg Glu Ile Cys Cys Glu

195 200 205

Arg Ser Ser Ser Ser Glu Ser Thr Gly Thr Pro Ser Asn Pro Asp Leu

210 215 220

Asp Ala Gly Val Ser Glu His Ser Gly Asp Trp Leu Asp Gln Asp Ser

225 230 235 240

Val Ser Asp Gln Phe Ser Val Glu Phe Glu Val Glu Ser Leu Asp Ser

245 250 255

Glu Asp Tyr Ser Leu Ser Glu Glu Gly Gln Glu Leu Ser Asp Glu Asp

260 265 270

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

275 280 285

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

290 295 300

Cys Thr Ser Cys Asn Glu Met Asn Pro Pro Leu Pro Ser His Cys Asn

305 310 315 320

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

325 330 335

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

340 345 350

Ala Glu Glu Gly Phe Asp Val Pro Asp Cys Lys Lys Thr Ile Val Asn

355 360 365

Asp Ser Arg Glu Ser Cys Val Glu Glu Asn Asp Asp Lys Ile Thr Gln

370 375 380

Ala Ser Gln Ser Gln Glu Ser Glu Asp Tyr Ser Gln Pro Ser Thr Ser

385 390 395 400

Ser Ser Ile Ile Tyr Ser Ser Gln Glu Asp Val Lys Glu Phe Glu Arg

405 410 415

Glu Glu Thr Gln Asp Lys Glu Glu Ser Val Glu Ser Ser Leu Pro Leu

420 425 430

Asn Ala Ile Glu Pro Cys Val Ile Cys Gln Gly Arg Pro Lys Asn Gly

435 440 445

Cys Ile Val His Gly Lys Thr Gly His Leu Met Ala Cys Phe Thr Cys

450 455 460

Ala Lys Lys Leu Lys Lys Arg Asn Lys Pro Cys Pro Val Cys Arg Gln

465 470 475 480

Pro Ile Gln Met Ile Val Leu Thr Tyr Phe Pro

485 490

<210> 62

<211> 240

<212> PRT

<213>Sea pansy

<400> 62

Met Thr Ser Lys Val Tyr Asp Pro Glu Gln Arg Lys Arg Met Ile Thr

1 5 10 15

Gly Pro Gln Trp Trp Ala Arg Cys Lys Gln Met Asn Val Leu Asp Ser

20 25 30

Phe Ile Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val Ile

35 40 45

Phe Leu His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val

50 55 60

Pro His Ile Glu Pro Val Ala Arg Cys Ile Ile Pro Asp Leu Ile Gly

65 70 75 80

Met Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp

85 90 95

His Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys

100 105 110

Lys Ile Ile Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His

115 120 125

Tyr Ser Tyr Glu His Gln Asp Lys Ile Lys Ala Ile Val His Ala Glu

130 135 140

Ser Val Val Asp Val Ile Glu Ser Trp Asp Glu Trp Pro Asp Ile Glu

145 150 155 160

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

165 170 175

Glu Asn Asn Phe Phe Val Glu Thr Met Leu Pro Ser Lys Ile Met Arg

180 185 190

Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu

195 200 205

Lys Gly Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu Ile Pro

210 215 220

Leu Val Lys Gly Gly Lys Pro Asp Val Val Gln Ile Val Arg Asn Tyr

225 230 235 240

<210> 63

<211> 320

<212> PRT

<213>People（Homo sapiens）

<400> 63

Met Glu Leu Leu Ser Pro Pro Leu Arg Asp Val Asp Leu Thr Ala Pro

1 5 10 15

Asp Gly Ser Leu Cys Ser Phe Ala Thr Thr Asp Asp Phe Tyr Asp Asp

20 25 30

Pro Cys Phe Asp Ser Pro Asp Leu Arg Phe Phe Glu Asp Leu Asp Pro

35 40 45

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

50 55 60

Phe Pro Ala Ala Val His Pro Ala Pro Gly Ala Arg Glu Asp Glu His

65 70 75 80

Val Arg Ala Pro Ser Gly His His Gln Ala Gly Arg Cys Leu Leu Trp

85 90 95

Ala Cys Lys Ala Cys Lys Arg Lys Thr Thr Asn Ala Asp Arg Arg Lys

100 105 110

Ala Ala Thr Met Arg Glu Arg Arg Arg Leu Ser Lys Val Asn Glu Ala

115 120 125

Phe Glu Thr Leu Lys Arg Cys Thr Ser Ser Asn Pro Asn Gln Arg Leu

130 135 140

Pro Lys Val Glu Ile Leu Arg Asn Ala Ile Arg Tyr Ile Glu Gly Leu

145 150 155 160

Gln Ala Leu Leu Arg Asp Gln Asp Ala Ala Pro Pro Gly Ala Ala Ala

165 170 175

Ala Phe Tyr Ala Pro Gly Pro Leu Pro Pro Gly Arg Gly Gly Glu His

180 185 190

Tyr Ser Gly Asp Ser Asp Ala Ser Ser Pro Arg Ser Asn Cys Ser Asp

195 200 205

Gly Met Met Asp Tyr Ser Gly Pro Pro Ser Gly Ala Arg Arg Arg Asn

210 215 220

Cys Tyr Glu Gly Ala Tyr Tyr Asn Glu Ala Pro Ser Glu Pro Arg Pro

225 230 235 240

Gly Lys Ser Ala Ala Val Ser Ser Leu Asp Cys Leu Ser Ser Ile Val

245 250 255

Glu Arg Ile Ser Thr Glu Ser Pro Ala Ala Pro Ala Leu Leu Leu Ala

260 265 270

Asp Val Pro Ser Glu Ser Pro Pro Arg Arg Gln Glu Ala Ala Ala Pro

275 280 285

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

290 295 300

Pro Gln Cys Pro Ala Gly Ala Asn Pro Asn Pro Ile Tyr Gln Val Leu

305 310 315 320

<210> 64

<211> 607

<212> PRT

<213>People（Homo sapiens）

<400> 64

Met Ala Glu Lys Arg Arg Gly Ser Pro Cys Ser Met Leu Ser Leu Lys

1 5 10 15

Ala His Ala Phe Ser Val Glu Ala Leu Ile Gly Ala Glu Lys Gln Gln

20 25 30

Gln Leu Gln Lys Lys Arg Arg Lys Leu Gly Ala Glu Glu Ala Ala Gly

35 40 45

Ala Val Asp Asp Gly Gly Cys Ser Arg Gly Gly Gly Ala Gly Glu Lys

50 55 60

Gly Ser Ser Glu Gly Asp Glu Gly Ala Ala Leu Pro Pro Pro Ala Gly

65 70 75 80

Ala Thr Ser Gly Pro Ala Arg Ser Gly Ala Asp Leu Glu Arg Gly Ala

85 90 95

Ala Gly Gly Cys Glu Asp Gly Phe Gln Gln Gly Ala Ser Pro Leu Ala

100 105 110

Ser Pro Gly Gly Ser Pro Lys Gly Ser Pro Ala Arg Ser Leu Ala Arg

115 120 125

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

130 135 140

Gly Ala Glu Leu Trp Lys Arg Phe His Glu Ile Gly Thr Glu Met Ile

145 150 155 160

Ile Thr Lys Ala Gly Arg Arg Met Phe Pro Ala Met Arg Val Lys Ile

165 170 175

Ser Gly Leu Asp Pro His Gln Gln Tyr Tyr Ile Ala Met Asp Ile Val

180 185 190

Pro Val Asp Asn Lys Arg Tyr Arg Tyr Val Tyr His Ser Ser Lys Trp

195 200 205

Met Val Ala Gly Asn Ala Asp Ser Pro Val Pro Pro Arg Val Tyr Ile

210 215 220

His Pro Asp Ser Pro Ala Ser Gly Glu Thr Trp Met Arg Gln Val Ile

225 230 235 240

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

245 250 255

His Ile Ile Leu His Ser Met His Lys Tyr Gln Pro Arg Val His Val

260 265 270

Ile Arg Lys Asp Cys Gly Asp Asp Leu Ser Pro Ile Lys Pro Val Pro

275 280 285

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

290 295 300

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

305 310 315 320

Arg Asn Pro Phe Ala Lys Gly Phe Arg Asp Ser Gly Arg Asn Arg Met

325 330 335

Gly Leu Glu Ala Leu Val Glu Ser Tyr Ala Phe Trp Arg Pro Ser Leu

340 345 350

Arg Thr Leu Thr Phe Glu Asp Ile Pro Gly Ile Pro Lys Gln Gly Asn

355 360 365

Ala Ser Ser Ser Thr Leu Leu Gln Gly Thr Gly Asn Gly Val Pro Ala

370 375 380

Thr His Pro His Leu Leu Ser Gly Ser Ser Cys Ser Ser Pro Ala Phe

385 390 395 400

His Leu Gly Pro Asn Thr Ser Gln Leu Cys Ser Leu Ala Pro Ala Asp

405 410 415

Tyr Ser Ala Cys Ala Arg Ser Gly Leu Thr Leu Asn Arg Tyr Ser Thr

420 425 430

Ser Leu Ala Glu Thr Tyr Asn Arg Leu Thr Asn Gln Ala Gly Glu Thr

435 440 445

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

450 455 460

Ser Val Asn Met Ser Met Gly Gly Thr Asp Gly Asp Thr Phe Ser Cys

465 470 475 480

Pro Gln Thr Ser Leu Ser Met Gln Ile Ser Gly Met Ser Pro Gln Leu

485 490 495

Gln Tyr Ile Met Pro Ser Pro Ser Ser Asn Ala Phe Ala Thr Asn Gln

500 505 510

Thr His Gln Gly Ser Tyr Asn Thr Phe Arg Leu His Ser Pro Cys Ala

515 520 525

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

530 535 540

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

545 550 555 560

Gly Thr Met Thr Asp Arg Gln Met Leu Pro Pro Val Glu Gly Val His

565 570 575

Leu Leu Ser Ser Gly Gly Gln Gln Ser Phe Phe Asp Ser Arg Thr Leu

580 585 590

Gly Ser Leu Thr Leu Ser Ser Ser Gln Val Ser Ala His Met Val

595 600 605

<210> 65

<211> 393

<212> PRT

<213>People（Homo sapiens）

<400> 65

Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln

1 5 10 15

Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu

20 25 30

Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp

35 40 45

Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro

50 55 60

Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala Pro Ala Ala Pro

65 70 75 80

Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser

85 90 95

Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly

100 105 110

Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro

115 120 125

Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln

130 135 140

Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met

145 150 155 160

Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys

165 170 175

Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln

180 185 190

His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp

195 200 205

Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu

210 215 220

Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser

225 230 235 240

Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr

245 250 255

Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val

260 265 270

Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn

275 280 285

Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr

290 295 300

Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys

305 310 315 320

Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu

325 330 335

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

340 345 350

Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His

355 360 365

Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met

370 375 380

Phe Lys Thr Glu Gly Pro Asp Ser Asp

385 390

<210> 66

<211> 215

<212> PRT

<213>People（Homo sapiens）

<400> 66

Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr

1 5 10 15

Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro

20 25 30

Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg

35 40 45

Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala

50 55 60

Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro

65 70 75 80

Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys

85 90 95

Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys

100 105 110

Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys

115 120 125

Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr

130 135 140

Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala

145 150 155 160

Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val

165 170 175

Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu

180 185 190

Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu

195 200 205

Glu Glu Asp Asp Asp Asp Glu

210 215

<210> 67

<211> 356

<212> PRT

<213>People（Homo sapiens）

<400> 67

Met Thr Lys Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu Pro Gln Pro

1 5 10 15

Gln Gly Pro Pro Ser Trp Thr Asp Glu Cys Leu Ser Ser Gln Asp Glu

20 25 30

Glu His Glu Ala Asp Lys Lys Glu Asp Asp Leu Glu Thr Met Asn Ala

35 40 45

Glu Glu Asp Ser Leu Arg Asn Gly Gly Glu Glu Glu Asp Glu Asp Glu

50 55 60

Asp Leu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Gln Lys

65 70 75 80

Pro Lys Arg Arg Gly Pro Lys Lys Lys Lys Met Thr Lys Ala Arg Leu

85 90 95

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

100 105 110

Arg Met His Gly Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val

115 120 125

Pro Cys Tyr Ser Lys Thr Gln Lys Leu Ser Lys Ile Glu Thr Leu Arg

130 135 140

Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile Leu Arg Ser Gly

145 150 155 160

Lys Ser Pro Asp Leu Val Ser Phe Val Gln Thr Leu Cys Lys Gly Leu

165 170 175

Ser Gln Pro Thr Thr Asn Leu Val Ala Gly Cys Leu Gln Leu Asn Pro

180 185 190

Arg Thr Phe Leu Pro Glu Gln Asn Gln Asp Met Pro Pro His Leu Pro

195 200 205

Thr Ala Ser Ala Ser Phe Pro Val His Pro Tyr Ser Tyr Gln Ser Pro

210 215 220

Gly Leu Pro Ser Pro Pro Tyr Gly Thr Met Asp Ser Ser His Val Phe

225 230 235 240

His Val Lys Pro Pro Pro His Ala Tyr Ser Ala Ala Leu Glu Pro Phe

245 250 255

Phe Glu Ser Pro Leu Thr Asp Cys Thr Ser Pro Ser Phe Asp Gly Pro

260 265 270

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

275 280 285

Pro Ser Ala Glu Phe Glu Lys Asn Tyr Ala Phe Thr Met His Tyr Pro

290 295 300

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

305 310 315 320

Thr Ala Ala Pro Arg Cys Glu Ile Pro Ile Asp Asn Ile Met Ser Phe

325 330 335

Asp Ser His Ser His His Glu Arg Val Met Ser Ala Gln Leu Asn Ala

340 345 350

Ile Phe His Asp

355

<210> 68

<211> 498

<212> PRT

<213>People（Homo sapiens）

<400> 68

Met Asn Gln Ser Ile Pro Val Ala Pro Thr Pro Pro Arg Arg Val Arg

1 5 10 15

Leu Lys Pro Trp Leu Val Ala Gln Val Asn Ser Cys Gln Tyr Pro Gly

20 25 30

Leu Gln Trp Val Asn Gly Glu Lys Lys Leu Phe Cys Ile Pro Trp Arg

35 40 45

His Ala Thr Arg His Gly Pro Ser Gln Asp Gly Asp Asn Thr Ile Phe

50 55 60

Lys Ala Trp Ala Lys Glu Thr Gly Lys Tyr Thr Glu Gly Val Asp Glu

65 70 75 80

Ala Asp Pro Ala Lys Trp Lys Ala Asn Leu Arg Cys Ala Leu Asn Lys

85 90 95

Ser Arg Asp Phe Arg Leu Ile Tyr Asp Gly Pro Arg Asp Met Pro Pro

100 105 110

Gln Pro Tyr Lys Ile Tyr Glu Val Cys Ser Asn Gly Pro Ala Pro Thr

115 120 125

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

130 135 140

Glu Glu Glu Glu Glu Leu Gln Arg Met Leu Pro Ser Leu Ser Leu Thr

145 150 155 160

Glu Asp Val Lys Trp Pro Pro Thr Leu Gln Pro Pro Thr Leu Arg Pro

165 170 175

Pro Thr Leu Gln Pro Pro Thr Leu Gln Pro Pro Val Val Leu Gly Pro

180 185 190

Pro Ala Pro Asp Pro Ser Pro Leu Ala Pro Pro Pro Gly Asn Pro Ala

195 200 205

Gly Phe Arg Glu Leu Leu Ser Glu Val Leu Glu Pro Gly Pro Leu Pro

210 215 220

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

225 230 235 240

Ser Pro His Met Leu Pro Leu Thr Asp Leu Glu Ile Lys Phe Gln Tyr

245 250 255

Arg Gly Arg Pro Pro Arg Ala Leu Thr Ile Ser Asn Pro His Gly Cys

260 265 270

Arg Leu Phe Tyr Ser Gln Leu Glu Ala Thr Gln Glu Gln Val Glu Leu

275 280 285

Phe Gly Pro Ile Ser Leu Glu Gln Val Arg Phe Pro Ser Pro Glu Asp

290 295 300

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

305 310 315 320

Leu Asp Arg Gly Leu Ile Leu Gln Leu Gln Gly Gln Asp Leu Tyr Ala

325 330 335

Ile Arg Leu Cys Gln Cys Lys Val Phe Trp Ser Gly Pro Cys Ala Ser

340 345 350

Ala His Asp Ser Cys Pro Asn Pro Ile Gln Arg Glu Val Lys Thr Lys

355 360 365

Leu Phe Ser Leu Glu His Phe Leu Asn Glu Leu Ile Leu Phe Gln Lys

370 375 380

Gly Gln Thr Asn Thr Pro Pro Pro Phe Glu Ile Phe Phe Cys Phe Gly

385 390 395 400

Glu Glu Trp Pro Asp Arg Lys Pro Arg Glu Lys Lys Leu Ile Thr Val

405 410 415

Gln Val Val Pro Val Ala Ala Arg Leu Leu Leu Glu Met Phe Ser Gly

420 425 430

Glu Leu Ser Trp Ser Ala Asp Ser Ile Arg Leu Gln Ile Ser Asn Pro

435 440 445

Asp Leu Lys Asp Arg Met Val Glu Gln Phe Lys Glu Leu His His Ile

450 455 460

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

465 470 475 480

Ala Gly Leu Gly Val Gly Gln Gly Pro Trp Pro Met His Pro Ala Gly

485 490 495

Met Gln

<210> 69

<211> 427

<212> PRT

<213>People（Homo sapiens）

<400> 69

Met Gly Thr Pro Lys Pro Arg Ile Leu Pro Trp Leu Val Ser Gln Leu

1 5 10 15

Asp Leu Gly Gln Leu Glu Gly Val Ala Trp Val Asn Lys Ser Arg Thr

20 25 30

Arg Phe Arg Ile Pro Trp Lys His Gly Leu Arg Gln Asp Ala Gln Gln

35 40 45

Glu Asp Phe Gly Ile Phe Gln Ala Trp Ala Glu Ala Thr Gly Ala Tyr

50 55 60

Val Pro Gly Arg Asp Lys Pro Asp Leu Pro Thr Trp Lys Arg Asn Phe

65 70 75 80

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

85 90 95

Ser Lys Asp Pro His Asp Pro His Lys Ile Tyr Glu Phe Val Asn Ser

100 105 110

Gly Val Gly Asp Phe Ser Gln Pro Asp Thr Ser Pro Asp Thr Asn Gly

115 120 125

Gly Gly Ser Thr Ser Asp Thr Gln Glu Asp Ile Leu Asp Glu Leu Leu

130 135 140

Gly Asn Met Val Leu Ala Pro Leu Pro Asp Pro Gly Pro Pro Ser Leu

145 150 155 160

Ala Val Ala Pro Glu Pro Cys Pro Gln Pro Leu Arg Ser Pro Ser Leu

165 170 175

Asp Asn Pro Thr Pro Phe Pro Asn Leu Gly Pro Ser Glu Asn Pro Leu

180 185 190

Lys Arg Leu Leu Val Pro Gly Glu Glu Trp Glu Phe Glu Val Thr Ala

195 200 205

Phe Tyr Arg Gly Arg Gln Val Phe Gln Gln Thr Ile Ser Cys Pro Glu

210 215 220

Gly Leu Arg Leu Val Gly Ser Glu Val Gly Asp Arg Thr Leu Pro Gly

225 230 235 240

Trp Pro Val Thr Leu Pro Asp Pro Gly Met Ser Leu Thr Asp Arg Gly

245 250 255

Val Met Ser Tyr Val Arg His Val Leu Ser Cys Leu Gly Gly Gly Leu

260 265 270

Ala Leu Trp Arg Ala Gly Gln Trp Leu Trp Ala Gln Arg Leu Gly His

275 280 285

Cys His Thr Tyr Trp Ala Val Ser Glu Glu Leu Leu Pro Asn Ser Gly

290 295 300

His Gly Pro Asp Gly Glu Val Pro Lys Asp Lys Glu Gly Gly Val Phe

305 310 315 320

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

325 330 335

Gly Arg Ser Pro Arg Tyr Ala Leu Trp Phe Cys Val Gly Glu Ser Trp

340 345 350

Pro Gln Asp Gln Pro Trp Thr Lys Arg Leu Val Met Val Lys Val Val

355 360 365

Pro Thr Cys Leu Arg Ala Leu Val Glu Met Ala Arg Val Gly Gly Ala

370 375 380

Ser Ser Leu Glu Asn Thr Val Asp Leu His Ile Ser Asn Ser His Pro

385 390 395 400

Leu Ser Leu Thr Ser Asp Gln Tyr Lys Ala Tyr Leu Gln Asp Leu Val

405 410 415

Glu Gly Met Asp Phe Gln Gly Pro Gly Glu Ser

420 425

<210> 70

<211> 750

<212> PRT

<213>People（Homo sapiens）

<400> 70

Met Ser Gln Trp Tyr Glu Leu Gln Gln Leu Asp Ser Lys Phe Leu Glu

1 5 10 15

Gln Val His Gln Leu Tyr Asp Asp Ser Phe Pro Met Glu Ile Arg Gln

20 25 30

Tyr Leu Ala Gln Trp Leu Glu Lys Gln Asp Trp Glu His Ala Ala Asn

35 40 45

Asp Val Ser Phe Ala Thr Ile Arg Phe His Asp Leu Leu Ser Gln Leu

50 55 60

Asp Asp Gln Tyr Ser Arg Phe Ser Leu Glu Asn Asn Phe Leu Leu Gln

65 70 75 80

His Asn Ile Arg Lys Ser Lys Arg Asn Leu Gln Asp Asn Phe Gln Glu

85 90 95

Asp Pro Ile Gln Met Ser Met Ile Ile Tyr Ser Cys Leu Lys Glu Glu

100 105 110

Arg Lys Ile Leu Glu Asn Ala Gln Arg Phe Asn Gln Ala Gln Ser Gly

115 120 125

Asn Ile Gln Ser Thr Val Met Leu Asp Lys Gln Lys Glu Leu Asp Ser

130 135 140

Lys Val Arg Asn Val Lys Asp Lys Val Met Cys Ile Glu His Glu Ile

145 150 155 160

Lys Ser Leu Glu Asp Leu Gln Asp Glu Tyr Asp Phe Lys Cys Lys Thr

165 170 175

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

180 185 190

Lys Gln Glu Gln Leu Leu Leu Lys Lys Met Tyr Leu Met Leu Asp Asn

195 200 205

Lys Arg Lys Glu Val Val His Lys Ile Ile Glu Leu Leu Asn Val Thr

210 215 220

Glu Leu Thr Gln Asn Ala Leu Ile Asn Asp Glu Leu Val Glu Trp Lys

225 230 235 240

Arg Arg Gln Gln Ser Ala Cys Ile Gly Gly Pro Pro Asn Ala Cys Leu

245 250 255

Asp Gln Leu Gln Asn Trp Phe Thr Ile Val Ala Glu Ser Leu Gln Gln

260 265 270

Val Arg Gln Gln Leu Lys Lys Leu Glu Glu Leu Glu Gln Lys Tyr Thr

275 280 285

Tyr Glu His Asp Pro Ile Thr Lys Asn Lys Gln Val Leu Trp Asp Arg

290 295 300

Thr Phe Ser Leu Phe Gln Gln Leu Ile Gln Ser Ser Phe Val Val Glu

305 310 315 320

Arg Gln Pro Cys Met Pro Thr His Pro Gln Arg Pro Leu Val Leu Lys

325 330 335

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

340 345 350

Glu Leu Asn Tyr Asn Leu Lys Val Lys Val Leu Phe Asp Lys Asp Val

355 360 365

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

370 375 380

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

385 390 395 400

Ala Ala Glu Phe Arg His Leu Gln Leu Lys Glu Gln Lys Asn Ala Gly

405 410 415

Thr Arg Thr Asn Glu Gly Pro Leu Ile Val Thr Glu Glu Leu His Ser

420 425 430

Leu Ser Phe Glu Thr Gln Leu Cys Gln Pro Gly Leu Val Ile Asp Leu

435 440 445

Glu Thr Thr Ser Leu Pro Val Val Val Ile Ser Asn Val Ser Gln Leu

450 455 460

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

465 470 475 480

Pro Arg Asn Leu Ser Phe Phe Leu Thr Pro Pro Cys Ala Arg Trp Ala

485 490 495

Gln Leu Ser Glu Val Leu Ser Trp Gln Phe Ser Ser Val Thr Lys Arg

500 505 510

Gly Leu Asn Val Asp Gln Leu Asn Met Leu Gly Glu Lys Leu Leu Gly

515 520 525

Pro Asn Ala Ser Pro Asp Gly Leu Ile Pro Trp Thr Arg Phe Cys Lys

530 535 540

Glu Asn Ile Asn Asp Lys Asn Phe Pro Phe Trp Leu Trp Ile Glu Ser

545 550 555 560

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

565 570 575

Cys Ile Met Gly Phe Ile Ser Lys Glu Arg Glu Arg Ala Leu Leu Lys

580 585 590

Asp Gln Gln Pro Gly Thr Phe Leu Leu Arg Phe Ser Glu Ser Ser Arg

595 600 605

Glu Gly Ala Ile Thr Phe Thr Trp Val Glu Arg Ser Gln Asn Gly Gly

610 615 620

Glu Pro Asp Phe His Ala Val Glu Pro Tyr Thr Lys Lys Glu Leu Ser

625 630 635 640

Ala Val Thr Phe Pro Asp Ile Ile Arg Asn Tyr Lys Val Met Ala Ala

645 650 655

Glu Asn Ile Pro Glu Asn Pro Leu Lys Tyr Leu Tyr Pro Asn Ile Asp

660 665 670

Lys Asp His Ala Phe Gly Lys Tyr Tyr Ser Arg Pro Lys Glu Ala Pro

675 680 685

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

690 695 700

Glu Leu Ile Ser Val Ser Glu Val His Pro Ser Arg Leu Gln Thr Thr

705 710 715 720

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

725 730 735

Ile Val Gly Ser Val Glu Phe Asp Ser Met Met Asn Thr Val

740 745 750

<210> 71

<211> 225

<212> PRT

<213>People（Homo sapiens）

<400> 71

Met Val Thr His Ser Lys Phe Pro Ala Ala Gly Met Ser Arg Pro Leu

1 5 10 15

Asp Thr Ser Leu Arg Leu Lys Thr Phe Ser Ser Lys Ser Glu Tyr Gln

20 25 30

Leu Val Val Asn Ala Val Arg Lys Leu Gln Glu Ser Gly Phe Tyr Trp

35 40 45

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

50 55 60

Ala Gly Thr Phe Leu Ile Arg Asp Ser Ser Asp Gln Arg His Phe Phe

65 70 75 80

Thr Leu Ser Val Lys Thr Gln Ser Gly Thr Lys Asn Leu Arg Ile Gln

85 90 95

Cys Glu Gly Gly Ser Phe Ser Leu Gln Ser Asp Pro Arg Ser Thr Gln

100 105 110

Pro Val Pro Arg Phe Asp Cys Val Leu Lys Leu Val His His Tyr Met

115 120 125

Pro Pro Pro Gly Ala Pro Ser Phe Pro Ser Pro Pro Thr Glu Pro Ser

130 135 140

Ser Glu Val Pro Glu Gln Pro Ser Ala Gln Pro Leu Pro Gly Ser Pro

145 150 155 160

Pro Arg Arg Ala Tyr Tyr Ile Tyr Ser Gly Gly Glu Lys Ile Pro Leu

165 170 175

Val Leu Ser Arg Pro Leu Ser Ser Asn Val Ala Thr Leu Gln His Leu

180 185 190

Cys Arg Lys Thr Val Asn Gly His Leu Asp Ser Tyr Glu Lys Val Thr

195 200 205

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

210 215 220

Leu

225

<210> 72

<211> 770

<212> PRT

<213>People（Homo sapiens）

<400> 72

Met Ala Gln Trp Asn Gln Leu Gln Gln Leu Asp Thr Arg Tyr Leu Glu

1 5 10 15

Gln Leu His Gln Leu Tyr Ser Asp Ser Phe Pro Met Glu Leu Arg Gln

20 25 30

Phe Leu Ala Pro Trp Ile Glu Ser Gln Asp Trp Ala Tyr Ala Ala Ser

35 40 45

Lys Glu Ser His Ala Thr Leu Val Phe His Asn Leu Leu Gly Glu Ile

50 55 60

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

65 70 75 80

His Asn Leu Arg Arg Ile Lys Gln Phe Leu Gln Ser Arg Tyr Leu Glu

85 90 95

Lys Pro Met Glu Ile Ala Arg Ile Val Ala Arg Cys Leu Trp Glu Glu

100 105 110

Ser Arg Leu Leu Gln Thr Ala Ala Thr Ala Ala Gln Gln Gly Gly Gln

115 120 125

Ala Asn His Pro Thr Ala Ala Val Val Thr Glu Lys Gln Gln Met Leu

130 135 140

Glu Gln His Leu Gln Asp Val Arg Lys Arg Val Gln Asp Leu Glu Gln

145 150 155 160

Lys Met Lys Val Val Glu Asn Leu Gln Asp Asp Phe Asp Phe Asn Tyr

165 170 175

Lys Thr Leu Lys Ser Gln Gly Asp Met Gln Asp Leu Asn Gly Asn Asn

180 185 190

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

195 200 205

Ala Leu Asp Gln Met Arg Arg Ser Ile Val Ser Glu Leu Ala Gly Leu

210 215 220

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

225 230 235 240

Ala Asp Trp Lys Arg Arg Gln Gln Ile Ala Cys Ile Gly Gly Pro Pro

245 250 255

Asn Ile Cys Leu Asp Arg Leu Glu Asn Trp Ile Thr Ser Leu Ala Glu

260 265 270

Ser Gln Leu Gln Thr Arg Gln Gln Ile Lys Lys Leu Glu Glu Leu Gln

275 280 285

Gln Lys Val Ser Tyr Lys Gly Asp Pro Ile Val Gln His Arg Pro Met

290 295 300

Leu Glu Glu Arg Ile Val Glu Leu Phe Arg Asn Leu Met Lys Ser Ala

305 310 315 320

Phe Val Val Glu Arg Gln Pro Cys Met Pro Met His Pro Asp Arg Pro

325 330 335

Leu Val Ile Lys Thr Gly Val Gln Phe Thr Thr Lys Val Arg Leu Leu

340 345 350

Val Lys Phe Pro Glu Leu Asn Tyr Gln Leu Lys Ile Lys Val Cys Ile

355 360 365

Asp Lys Asp Ser Gly Asp Val Ala Ala Leu Arg Gly Ser Arg Lys Phe

370 375 380

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

385 390 395 400

Asn Gly Ser Leu Ser Ala Glu Phe Lys His Leu Thr Leu Arg Glu Gln

405 410 415

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

420 425 430

Thr Glu Glu Leu His Leu Ile Thr Phe Glu Thr Glu Val Tyr His Gln

435 440 445

Gly Leu Lys Ile Asp Leu Glu Thr His Ser Leu Pro Val Val Val Ile

450 455 460

Ser Asn Ile Cys Gln Met Pro Asn Ala Trp Ala Ser Ile Leu Trp Tyr

465 470 475 480

Asn Met Leu Thr Asn Asn Pro Lys Asn Val Asn Phe Phe Thr Lys Pro

485 490 495

Pro Ile Gly Thr Trp Asp Gln Val Ala Glu Val Leu Ser Trp Gln Phe

500 505 510

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

515 520 525

Ala Glu Lys Leu Leu Gly Pro Gly Val Asn Tyr Ser Gly Cys Gln Ile

530 535 540

Thr Trp Ala Lys Phe Cys Lys Glu Asn Met Ala Gly Lys Gly Phe Ser

545 550 555 560

Phe Trp Val Trp Leu Asp Asn Ile Ile Asp Leu Val Lys Lys Tyr Ile

565 570 575

Leu Ala Leu Trp Asn Glu Gly Tyr Ile Met Gly Phe Ile Ser Lys Glu

580 585 590

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

595 600 605

Arg Phe Ser Glu Ser Ser Lys Glu Gly Gly Val Thr Phe Thr Trp Val

610 615 620

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

625 630 635 640

Thr Lys Gln Gln Leu Asn Asn Met Ser Phe Ala Glu Ile Ile Met Gly

645 650 655

Tyr Lys Ile Met Asp Ala Thr Asn Ile Leu Val Ser Pro Leu Val Tyr

660 665 670

Leu Tyr Pro Asp Ile Pro Lys Glu Glu Ala Phe Gly Lys Tyr Cys Arg

675 680 685

Pro Glu Ser Gln Glu His Pro Glu Ala Asp Pro Gly Ser Ala Ala Pro

690 695 700

Tyr Leu Lys Thr Lys Phe Ile Cys Val Thr Pro Thr Thr Cys Ser Asn

705 710 715 720

Thr Ile Asp Leu Pro Met Ser Pro Arg Thr Leu Asp Ser Leu Met Gln

725 730 735

Phe Gly Asn Asn Gly Glu Gly Ala Glu Pro Ser Ala Gly Gly Gln Phe

740 745 750

Glu Ser Leu Thr Phe Asp Met Glu Leu Thr Ser Glu Cys Ala Thr Ser

755 760 765

Pro Met

770

<210> 73

<211> 246

<212> PRT

<213>People（Homo sapiens）

<400> 73

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

1 5 10 15

Gly Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Trp Gly

20 25 30

Asn Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg

35 40 45

Leu Leu Met Tyr Tyr Ala Ser Asn Leu Ala Ser Gly Val Ser Ser Arg

50 55 60

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

65 70 75 80

Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Gly Phe Glu

85 90 95

Cys Ser Gly Gly Asp Cys Val Gly Phe Gly Gly Gly Thr Glu Leu Glu

100 105 110

Ile Leu Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Ala

130 135 140

Pro Gly Gly Ser Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu

145 150 155 160

Ser Ser Asp Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

165 170 175

Glu Trp Ile Gly Thr Ile Tyr Gly Ser Ala Gly Thr Tyr Tyr Ala Thr

180 185 190

Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp

195 200 205

Leu Lys Met Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys

210 215 220

Thr Arg Ala Phe Ser Asn Thr Arg Leu Asp Leu Trp Gly Gln Gly Thr

225 230 235 240

Leu Val Thr Ile Ser Ser

245

<210> 74

<211> 233

<212> PRT

<213>People（Homo sapiens）

<400> 74

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

1 5 10 15

Leu Ser Gln Lys Gly Tyr Ser Trp Ser Gln Phe Ser Asp Val Glu Glu

20 25 30

Asn Arg Thr Glu Ala Pro Glu Gly Thr Glu Ser Glu Met Glu Thr Pro

35 40 45

Ser Ala Ile Asn Gly Asn Pro Ser Trp His Leu Ala Asp Ser Pro Ala

50 55 60

Val Asn Gly Ala Thr Gly His Ser Ser Ser Leu Asp Ala Arg Glu Val

65 70 75 80

Ile Pro Met Ala Ala Val Lys Gln Ala Leu Arg Glu Ala Gly Asp Glu

85 90 95

Phe Glu Leu Arg Tyr Arg Arg Ala Phe Ser Asp Leu Thr Ser Gln Leu

100 105 110

His Ile Thr Pro Gly Thr Ala Tyr Gln Ser Phe Glu Gln Val Val Asn

115 120 125

Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg Ile Val Ala Phe Phe

130 135 140

Ser Phe Gly Gly Ala Leu Cys Val Glu Ser Val Asp Lys Glu Met Gln

145 150 155 160

Val Leu Val Ser Arg Ile Ala Ala Trp Met Ala Thr Tyr Leu Asn Asp

165 170 175

His Leu Glu Pro Trp Ile Gln Glu Asn Gly Gly Trp Asp Thr Phe Val

180 185 190

Glu Leu Tyr Gly Asn Asn Ala Ala Ala Glu Ser Arg Lys Gly Gln Glu

195 200 205

Arg Phe Asn Arg Trp Phe Leu Thr Gly Met Thr Val Ala Gly Val Val

210 215 220

Leu Leu Gly Ser Leu Phe Ser Arg Lys

225 230

<210> 75

<211> 312

<212> PRT

<213>People（Homo sapiens）

<400> 75

Met Ala Asn Asn Asp Ala Val Leu Lys Arg Leu Glu Gln Lys Gly Ala

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Pro Ser Gly Thr Pro Leu His Ala Asn Ser Met Val Ser Glu Asn Val

85 90 95

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

100 105 110

Ile Lys Gly Gly Thr Gly Asp Glu Lys Lys Ala Lys Glu Lys Ile Glu

115 120 125

Lys Lys Gly Glu Lys Lys Glu Lys Lys Gln Gln Ser Ile Ala Gly Ser

130 135 140

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

145 150 155 160

Cys Ile Ile Thr Ala Arg Lys His Pro Asp Ala Asp Ser Leu Tyr Val

165 170 175

Glu Glu Val Asp Val Gly Glu Ile Ala Pro Arg Thr Val Val Ser Gly

180 185 190

Leu Val Asn His Val Pro Leu Glu Gln Met Gln Asn Arg Met Val Ile

195 200 205

Leu Leu Cys Asn Leu Lys Pro Ala Lys Met Arg Gly Val Leu Ser Gln

210 215 220

Ala Met Val Met Cys Ala Ser Ser Pro Glu Lys Ile Glu Ile Leu Ala

225 230 235 240

Pro Pro Asn Gly Ser Val Pro Gly Asp Arg Ile Thr Phe Asp Ala Phe

245 250 255

Pro Gly Glu Pro Asp Lys Glu Leu Asn Pro Lys Lys Lys Ile Trp Glu

260 265 270

Gln Ile Gln Pro Asp Leu His Thr Asn Asp Glu Cys Val Ala Thr Tyr

275 280 285

Lys Gly Val Pro Phe Glu Val Lys Gly Lys Gly Val Cys Arg Ala Gln

290 295 300

Thr Met Ser Asn Ser Gly Ile Lys

305 310

<210> 76

<211> 320

<212> PRT

<213>People（Homo sapiens）

<400> 76

Met Pro Met Tyr Gln Val Lys Pro Tyr His Gly Gly Gly Ala Pro Leu

1 5 10 15

Arg Val Glu Leu Pro Thr Cys Met Tyr Arg Leu Pro Asn Val His Gly

20 25 30

Arg Ser Tyr Gly Pro Ala Pro Gly Ala Gly His Val Gln Glu Glu Ser

35 40 45

Asn Leu Ser Leu Gln Ala Leu Glu Ser Arg Gln Asp Asp Ile Leu Lys

50 55 60

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

65 70 75 80

Gln Thr Pro Asp Ala Asp Leu Asp Val Thr Asn Ile Ile Gln Ala Asp

85 90 95

Glu Pro Thr Thr Leu Thr Thr Asn Ala Leu Asp Leu Asn Ser Val Leu

100 105 110

Gly Lys Asp Tyr Gly Ala Leu Lys Asp Ile Val Ile Asn Ala Asn Pro

115 120 125

Ala Ser Pro Pro Leu Ser Leu Leu Val Leu His Arg Leu Leu Cys Glu

130 135 140

His Phe Arg Val Leu Ser Thr Val His Thr His Ser Ser Val Lys Ser

145 150 155 160

Val Pro Glu Asn Leu Leu Lys Cys Phe Gly Glu Gln Asn Lys Lys Gln

165 170 175

Pro Arg Gln Asp Tyr Gln Leu Gly Phe Thr Leu Ile Trp Lys Asn Val

180 185 190

Pro Lys Thr Gln Met Lys Phe Ser Ile Gln Thr Met Cys Pro Ile Glu

195 200 205

Gly Glu Gly Asn Ile Ala Arg Phe Leu Phe Ser Leu Phe Gly Gln Lys

210 215 220

His Asn Ala Val Asn Ala Thr Leu Ile Asp Ser Trp Val Asp Ile Ala

225 230 235 240

Ile Phe Gln Leu Lys Glu Gly Ser Ser Lys Glu Lys Ala Ala Val Phe

245 250 255

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

260 265 270

Glu Leu Thr Val Ala Asp Val Val Leu Trp Ser Val Leu Gln Gln Ile

275 280 285

Gly Gly Cys Ser Val Thr Val Pro Ala Asn Val Gln Arg Trp Met Arg

290 295 300

Ser Cys Glu Asn Leu Ala Pro Phe Asn Thr Ala Leu Lys Leu Leu Lys

305 310 315 320

<210> 77

<211> 233

<212> PRT

<213>People（Homo sapiens）

<400> 77

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

225 230

<210> 78

<211> 267

<212> PRT

<213>People（Homo sapiens）

<400> 78

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Ser Gly Ser Gly Leu Arg Ser Arg Ala Gln Ala Ser Asn Ser Ala Val

245 250 255

Asp Gly Thr Ala Gly Pro Gly Ser Thr Gly Ser

260 265

<210> 79

<211> 425

<212> PRT

<213>People（Homo sapiens）

<400> 79

Met Ala Asp Lys Glu Ala Ala Phe Asp Asp Ala Val Glu Glu Arg Val

1 5 10 15

Ile Asn Glu Glu Tyr Lys Ile Trp Lys Lys Asn Thr Pro Phe Leu Tyr

20 25 30

Asp Leu Val Met Thr His Ala Leu Glu Trp Pro Ser Leu Thr Ala Gln

35 40 45

Trp Leu Pro Asp Val Thr Arg Pro Glu Gly Lys Asp Phe Ser Ile His

50 55 60

Arg Leu Val Leu Gly Thr His Thr Ser Asp Glu Gln Asn His Leu Val

65 70 75 80

Ile Ala Ser Val Gln Leu Pro Asn Asp Asp Ala Gln Phe Asp Ala Ser

85 90 95

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

100 105 110

Gly Lys Ile Glu Ile Glu Ile Lys Ile Asn His Glu Gly Glu Val Asn

115 120 125

Arg Ala Arg Tyr Met Pro Gln Asn Pro Cys Ile Ile Ala Thr Lys Thr

130 135 140

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

145 150 155 160

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

165 170 175

Gln Lys Glu Gly Tyr Gly Leu Ser Trp Asn Pro Asn Leu Ser Gly His

180 185 190

Leu Leu Ser Ala Ser Asp Asp His Thr Ile Cys Leu Trp Asp Ile Ser

195 200 205

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

210 215 220

Gly His Thr Ala Val Val Glu Asp Val Ser Trp His Leu Leu His Glu

225 230 235 240

Ser Leu Phe Gly Ser Val Ala Asp Asp Gln Lys Leu Met Ile Trp Asp

245 250 255

Thr Arg Ser Asn Asn Thr Ser Lys Pro Ser His Ser Val Asp Ala His

260 265 270

Thr Ala Glu Val Asn Cys Leu Ser Phe Asn Pro Tyr Ser Glu Phe Ile

275 280 285

Leu Ala Thr Gly Ser Ala Asp Lys Thr Val Ala Leu Trp Asp Leu Arg

290 295 300

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

305 310 315 320

Phe Gln Val Gln Trp Ser Pro His Asn Glu Thr Ile Leu Ala Ser Ser

325 330 335

Gly Thr Asp Arg Arg Leu Asn Val Trp Asp Leu Ser Lys Ile Gly Glu

340 345 350

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

355 360 365

Ile His Gly Gly His Thr Ala Lys Ile Ser Asp Phe Ser Trp Asn Pro

370 375 380

Asn Glu Pro Trp Val Ile Cys Ser Val Ser Glu Asp Asn Ile Met Gln

385 390 395 400

Val Trp Gln Met Ala Glu Asn Ile Tyr Asn Asp Glu Asp Pro Glu Gly

405 410 415

Ser Val Asp Pro Glu Gly Gln Gly Ser

420 425

<210> 80

<211> 375

<212> PRT

<213>People（Homo sapiens）

<400> 80

Met Ala Asp His Ser Phe Ser Asp Gly Val Pro Ser Asp Ser Val Glu

1 5 10 15

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

20 25 30

Gln Asn Pro Arg Val Leu Ala Ala Leu Gln Glu Arg Leu Asp Asn Val

35 40 45

Pro His Thr Pro Ser Ser Tyr Ile Glu Thr Leu Pro Lys Ala Val Lys

50 55 60

Arg Arg Ile Asn Ala Leu Lys Gln Leu Gln Val Arg Cys Ala His Ile

65 70 75 80

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

85 90 95

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

100 105 110

Asp Val Glu Pro Thr Asp Ala Glu Ser Glu Trp His Ser Glu Asn Glu

115 120 125

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

130 135 140

Glu Lys Ala Ala Ala Thr Ala Glu Glu Pro Asp Pro Lys Gly Ile Pro

145 150 155 160

Glu Phe Trp Phe Thr Ile Phe Arg Asn Val Asp Met Leu Ser Glu Leu

165 170 175

Val Gln Glu Tyr Asp Glu Pro Ile Leu Lys His Leu Gln Asp Ile Lys

180 185 190

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

195 200 205

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

210 215 220

Tyr Lys Met Lys Ser Glu Pro Asp Lys Ala Asp Pro Phe Ser Phe Glu

225 230 235 240

Gly Pro Glu Ile Val Asp Cys Asp Gly Cys Thr Ile Asp Trp Lys Lys

245 250 255

Gly Lys Asn Val Thr Val Lys Thr Ile Lys Lys Lys Gln Lys His Lys

260 265 270

Gly Arg Gly Thr Val Arg Thr Ile Thr Lys Gln Val Pro Asn Glu Ser

275 280 285

Phe Phe Asn Phe Phe Asn Pro Leu Lys Ala Ser Gly Asp Gly Glu Ser

290 295 300

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

305 310 315 320

His Phe Phe Arg Glu Arg Ile Val Pro Arg Ala Val Leu Tyr Phe Thr

325 330 335

Gly Glu Ala Ile Glu Asp Asp Asp Asn Phe Glu Glu Gly Glu Glu Gly

340 345 350

Glu Glu Glu Glu Leu Glu Gly Asp Glu Glu Gly Glu Asp Glu Asp Asp

355 360 365

Ala Glu Ile Asn Pro Lys Val

370 375

<210> 81

<211> 1132

<212> PRT

<213>People（Homo sapiens）

<400> 81

Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser

1 5 10 15

His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly

20 25 30

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

35 40 45

Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro

50 55 60

Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu

65 70 75 80

Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val

85 90 95

Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro

100 105 110

Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr

115 120 125

Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val

130 135 140

Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val

145 150 155 160

Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr

165 170 175

Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly

180 185 190

Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg

195 200 205

Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg

210 215 220

Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg

225 230 235 240

Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp

245 250 255

Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val

260 265 270

Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala

275 280 285

Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His

290 295 300

Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro

305 310 315 320

Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly

325 330 335

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

340 345 350

Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser

355 360 365

Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln

370 375 380

Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His

385 390 395 400

Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg

405 410 415

Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln

420 425 430

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

435 440 445

Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe

450 455 460

Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser

465 470 475 480

Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser

485 490 495

Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met

500 505 510

Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys

515 520 525

Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe

530 535 540

Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe

545 550 555 560

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

565 570 575

Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His

580 585 590

Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln

595 600 605

His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile

610 615 620

Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val

625 630 635 640

Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser

645 650 655

Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg

660 665 670

Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg

675 680 685

Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro

690 695 700

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

705 710 715 720

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

725 730 735

Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His

740 745 750

Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp

755 760 765

Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser

770 775 780

Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu

785 790 795 800

Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His

805 810 815

Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro

820 825 830

Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp

835 840 845

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

850 855 860

Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser

930 935 940

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

945 950 955 960

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

965 970 975

Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn

980 985 990

Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln

995 1000 1005

Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln Gln

1010 1015 1020

Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr Ala

1025 1030 1035 1040

Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser Leu

1045 1050 1055

Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu Ala Val Gln Trp

1060 1065 1070

Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg Val Thr

1075 1080 1085

Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr Gln Leu Ser

1090 1095 1100

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

1105 1110 1115 1120

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

1125 1130

<210> 82

<211> 462

<212> PRT

<213>People（Homo sapiens）

<400> 82

Met Glu Thr Glu Gln Pro Glu Glu Thr Phe Pro Asn Thr Glu Thr Asn

1 5 10 15

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

20 25 30

Phe Lys Arg Ser Arg Asn Thr Asp Glu Met Val Glu Leu Arg Ile Leu

35 40 45

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

50 55 60

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

65 70 75 80

Ser Ser Gly Pro Glu Arg Ile Leu Ser Ile Ser Ala Asp Ile Glu Thr

85 90 95

Ile Gly Glu Ile Leu Lys Lys Ile Ile Pro Thr Leu Glu Glu Gly Leu

100 105 110

Gln Leu Pro Ser Pro Thr Ala Thr Ser Gln Leu Pro Leu Glu Ser Asp

115 120 125

Ala Val Glu Cys Leu Asn Tyr Gln His Tyr Lys Gly Ser Asp Phe Asp

130 135 140

Cys Glu Leu Arg Leu Leu Ile His Gln Ser Leu Ala Gly Gly Ile Ile

145 150 155 160

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

165 170 175

Thr Ile Lys Leu Phe Gln Glu Cys Cys Pro His Ser Thr Asp Arg Val

180 185 190

Val Leu Ile Gly Gly Lys Pro Asp Arg Val Val Glu Cys Ile Lys Ile

195 200 205

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

210 215 220

Tyr Asp Pro Asn Phe Tyr Asp Glu Thr Tyr Asp Tyr Gly Gly Phe Thr

225 230 235 240

Met Met Phe Asp Asp Arg Arg Gly Arg Pro Val Gly Phe Pro Met Arg

245 250 255

Gly Arg Gly Gly Phe Asp Arg Met Pro Pro Gly Arg Gly Gly Arg Pro

260 265 270

Met Pro Pro Ser Arg Arg Asp Tyr Asp Asp Met Ser Pro Arg Arg Gly

275 280 285

Pro Pro Pro Pro Pro Pro Gly Arg Gly Gly Arg Gly Gly Ser Arg Ala

290 295 300

Arg Asn Leu Pro Leu Pro Pro Pro Pro Pro Pro Arg Gly Gly Asp Leu

305 310 315 320

Met Ala Tyr Asp Arg Arg Gly Arg Pro Gly Asp Arg Tyr Asp Gly Met

325 330 335

Val Gly Phe Ser Ala Asp Glu Thr Trp Asp Ser Ala Ile Asp Thr Trp

340 345 350

Ser Pro Ser Glu Trp Gln Met Ala Tyr Glu Pro Gln Gly Gly Ser Gly

355 360 365

Tyr Asp Tyr Ser Tyr Ala Gly Gly Arg Gly Ser Tyr Gly Asp Leu Gly

370 375 380

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

385 390 395 400

Ser Ile Ile Gly Lys Gly Gly Gln Arg Ile Lys Gln Ile Arg His Glu

405 410 415

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

420 425 430

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

435 440 445

Tyr Leu Leu Gln Asn Ser Val Lys Gln Tyr Ser Gly Lys Phe

450 455 460

<210> 83

<211> 745

<212> PRT

<213>People（Homo sapiens）

<400> 83

Met Glu Arg Pro Pro Gly Leu Arg Pro Gly Ala Gly Gly Pro Trp Glu

1 5 10 15

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

20 25 30

Gln His Arg Glu Leu Asp Leu Lys Ile Ala Ile Lys Ser Cys Arg Leu

35 40 45

Glu Leu Ser Thr Lys Asn Arg Glu Arg Trp Cys His Glu Ile Gln Ile

50 55 60

Met Lys Lys Leu Asn His Ala Asn Val Val Lys Ala Cys Asp Val Pro

65 70 75 80

Glu Glu Leu Asn Ile Leu Ile His Asp Val Pro Leu Leu Ala Met Glu

85 90 95

Tyr Cys Ser Gly Gly Asp Leu Arg Lys Leu Leu Asn Lys Pro Glu Asn

100 105 110

Cys Cys Gly Leu Lys Glu Ser Gln Ile Leu Ser Leu Leu Ser Asp Ile

115 120 125

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

130 135 140

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

145 150 155 160

His Lys Ile Ile Asp Leu Gly Tyr Ala Lys Asp Val Asp Gln Gly Ser

165 170 175

Leu Cys Thr Ser Phe Val Gly Thr Leu Gln Tyr Leu Ala Pro Glu Leu

180 185 190

Phe Glu Asn Lys Pro Tyr Thr Ala Thr Val Asp Tyr Trp Ser Phe Gly

195 200 205

Thr Met Val Phe Glu Cys Ile Ala Gly Tyr Arg Pro Phe Leu His His

210 215 220

Leu Gln Pro Phe Thr Trp His Glu Lys Ile Lys Lys Lys Asp Pro Lys

225 230 235 240

Cys Ile Phe Ala Cys Glu Glu Met Ser Gly Glu Val Arg Phe Ser Ser

245 250 255

His Leu Pro Gln Pro Asn Ser Leu Cys Ser Leu Val Val Glu Pro Met

260 265 270

Glu Asn Trp Leu Gln Leu Met Leu Asn Trp Asp Pro Gln Gln Arg Gly

275 280 285

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

290 295 300

Asp His Ile Leu Asn Leu Lys Ile Val His Ile Leu Asn Met Thr Ser

305 310 315 320

Ala Lys Ile Ile Ser Phe Leu Leu Pro Pro Asp Glu Ser Leu His Ser

325 330 335

Leu Gln Ser Arg Ile Glu Arg Glu Thr Gly Ile Asn Thr Gly Ser Gln

340 345 350

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

355 360 365

Ser Gln Cys Val Leu Asp Gly Val Arg Gly Cys Asp Ser Tyr Met Val

370 375 380

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

385 390 395 400

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

405 410 415

Gln Leu Pro Ile Ile Gln Leu Arg Lys Val Trp Ala Glu Ala Val His

420 425 430

Tyr Val Ser Gly Leu Lys Glu Asp Tyr Ser Arg Leu Phe Gln Gly Gln

435 440 445

Arg Ala Ala Met Leu Ser Leu Leu Arg Tyr Asn Ala Asn Leu Thr Lys

450 455 460

Met Lys Asn Thr Leu Ile Ser Ala Ser Gln Gln Leu Lys Ala Lys Leu

465 470 475 480

Glu Phe Phe His Lys Ser Ile Gln Leu Asp Leu Glu Arg Tyr Ser Glu

485 490 495

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

500 505 510

Glu Met Glu Glu Lys Ala Ile His Tyr Ala Glu Val Gly Val Ile Gly

515 520 525

Tyr Leu Glu Asp Gln Ile Met Ser Leu His Ala Glu Ile Met Glu Leu

530 535 540

Gln Lys Ser Pro Tyr Gly Arg Arg Gln Gly Asp Leu Met Glu Ser Leu

545 550 555 560

Glu Gln Arg Ala Ile Asp Leu Tyr Lys Gln Leu Lys His Arg Pro Ser

565 570 575

Asp His Ser Tyr Ser Asp Ser Thr Glu Met Val Lys Ile Ile Val His

580 585 590

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

595 600 605

Ser Lys Leu Leu Gly Cys Lys Gln Lys Ile Ile Asp Leu Leu Pro Lys

610 615 620

Val Glu Val Ala Leu Ser Asn Ile Lys Glu Ala Asp Asn Thr Val Met

625 630 635 640

Phe Met Gln Gly Lys Arg Gln Lys Glu Ile Trp His Leu Leu Lys Ile

645 650 655

Ala Cys Thr Gln Ser Ser Ala Arg Ser Leu Val Gly Ser Ser Leu Glu

660 665 670

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

675 680 685

Glu His Asp His Ser Leu Ser Cys Val Val Thr Pro Gln Asp Gly Glu

690 695 700

Thr Ser Ala Gln Met Ile Glu Glu Asn Leu Asn Cys Leu Gly His Leu

705 710 715 720

Ser Thr Ile Ile His Glu Ala Asn Glu Glu Gln Gly Asn Ser Met Met

725 730 735

Asn Leu Asp Trp Ser Trp Leu Thr Glu

740 745

<210> 84

<211> 968

<212> PRT

<213>People（Homo sapiens）

<400> 84

Met Ala Glu Asp Asp Pro Tyr Leu Gly Arg Pro Glu Gln Met Phe His

1 5 10 15

Leu Asp Pro Ser Leu Thr His Thr Ile Phe Asn Pro Glu Val Phe Gln

20 25 30

Pro Gln Met Ala Leu Pro Thr Asp Gly Pro Tyr Leu Gln Ile Leu Glu

35 40 45

Gln Pro Lys Gln Arg Gly Phe Arg Phe Arg Tyr Val Cys Glu Gly Pro

50 55 60

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

65 70 75 80

Tyr Pro Gln Val Lys Ile Cys Asn Tyr Val Gly Pro Ala Lys Val Ile

85 90 95

Val Gln Leu Val Thr Asn Gly Lys Asn Ile His Leu His Ala His Ser

100 105 110

Leu Val Gly Lys His Cys Glu Asp Gly Ile Cys Thr Val Thr Ala Gly

115 120 125

Pro Lys Asp Met Val Val Gly Phe Ala Asn Leu Gly Ile Leu His Val

130 135 140

Thr Lys Lys Lys Val Phe Glu Thr Leu Glu Ala Arg Met Thr Glu Ala

145 150 155 160

Cys Ile Arg Gly Tyr Asn Pro Gly Leu Leu Val His Pro Asp Leu Ala

165 170 175

Tyr Leu Gln Ala Glu Gly Gly Gly Asp Arg Gln Leu Gly Asp Arg Glu

180 185 190

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

195 200 205

Leu Ser Val Val Arg Leu Met Phe Thr Ala Phe Leu Pro Asp Ser Thr

210 215 220

Gly Ser Phe Thr Arg Arg Leu Glu Pro Val Val Ser Asp Ala Ile Tyr

225 230 235 240

Asp Ser Lys Ala Pro Asn Ala Ser Asn Leu Lys Ile Val Arg Met Asp

245 250 255

Arg Thr Ala Gly Cys Val Thr Gly Gly Glu Glu Ile Tyr Leu Leu Cys

260 265 270

Asp Lys Val Gln Lys Asp Asp Ile Gln Ile Arg Phe Tyr Glu Glu Glu

275 280 285

Glu Asn Gly Gly Val Trp Glu Gly Phe Gly Asp Phe Ser Pro Thr Asp

290 295 300

Val His Arg Gln Phe Ala Ile Val Phe Lys Thr Pro Lys Tyr Lys Asp

305 310 315 320

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

325 330 335

Ser Asp Leu Glu Thr Ser Glu Pro Lys Pro Phe Leu Tyr Tyr Pro Glu

340 345 350

Ile Lys Asp Lys Glu Glu Val Gln Arg Lys Arg Gln Lys Leu Met Pro

355 360 365

Asn Phe Ser Asp Ser Phe Gly Gly Gly Ser Gly Ala Gly Ala Gly Gly

370 375 380

Gly Gly Met Phe Gly Ser Gly Gly Gly Gly Gly Gly Thr Gly Ser Thr

385 390 395 400

Gly Pro Gly Tyr Ser Phe Pro His Tyr Gly Phe Pro Thr Tyr Gly Gly

405 410 415

Ile Thr Phe His Pro Gly Thr Thr Lys Ser Asn Ala Gly Met Lys His

420 425 430

Gly Thr Met Asp Thr Glu Ser Lys Lys Asp Pro Glu Gly Cys Asp Lys

435 440 445

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

450 455 460

Thr Glu Gln Asp Gln Glu Pro Ser Glu Ala Thr Val Gly Asn Gly Glu

465 470 475 480

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

485 490 495

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

500 505 510

Ala Asn Ala Leu Phe Asp Tyr Ala Val Thr Gly Asp Val Lys Met Leu

515 520 525

Leu Ala Val Gln Arg His Leu Thr Ala Val Gln Asp Glu Asn Gly Asp

530 535 540

Ser Val Leu His Leu Ala Ile Ile His Leu His Ser Gln Leu Val Arg

545 550 555 560

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

565 570 575

Met Arg Asn Asp Leu Tyr Gln Thr Pro Leu His Leu Ala Val Ile Thr

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Ala Leu Leu Leu Asp His Pro Asn Gly Asp Gly Leu Asn Ala Ile His

645 650 655

Leu Ala Met Met Ser Asn Ser Leu Pro Cys Leu Leu Leu Leu Val Ala

660 665 670

Ala Gly Ala Asp Val Asn Ala Gln Glu Gln Lys Ser Gly Arg Thr Ala

675 680 685

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

690 695 700

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

705 710 715 720

Thr Pro Leu His Ile Ala Ala Gly Arg Gly Ser Thr Arg Leu Ala Ala

725 730 735

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

740 745 750

Leu Tyr Asp Leu Asp Asp Ser Trp Glu Asn Ala Gly Glu Asp Glu Gly

755 760 765

Val Val Pro Gly Thr Thr Pro Leu Asp Met Ala Thr Ser Trp Gln Val

770 775 780

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

785 790 795 800

Asp Leu Leu Ala Gln Gly Asp Met Lys Gln Leu Ala Glu Asp Val Lys

805 810 815

Leu Gln Leu Tyr Lys Leu Leu Glu Ile Pro Asp Pro Asp Lys Asn Trp

820 825 830

Ala Thr Leu Ala Gln Lys Leu Gly Leu Gly Ile Leu Asn Asn Ala Phe

835 840 845

Arg Leu Ser Pro Ala Pro Ser Lys Thr Leu Met Asp Asn Tyr Glu Val

850 855 860

Ser Gly Gly Thr Val Arg Glu Leu Val Glu Ala Leu Arg Gln Met Gly

865 870 875 880

Tyr Thr Glu Ala Ile Glu Val Ile Gln Ala Ala Ser Ser Pro Val Lys

885 890 895

Thr Thr Ser Gln Ala His Ser Leu Pro Leu Ser Pro Ala Ser Thr Arg

900 905 910

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

915 920 925

Val Glu Thr Ser Phe Arg Lys Leu Ser Phe Thr Glu Ser Leu Thr Ser

930 935 940

Gly Ala Ser Leu Leu Thr Leu Asn Lys Met Pro His Asp Tyr Gly Gln

945 950 955 960

Glu Gly Pro Leu Glu Gly Lys Ile

965

Claims (21)

1. a kind of method for largely generating the excretion body comprising cargo protein on film includes the following steps：

A) fusion being made of excretion body specific marker object and cargo protein by importing coding in generating cell in excretion body The polynucleotides of albumen prepare transformed cells；

B) transformed cells of incubation step a)；And

C) cargo protein is separated on the inside of excretion body film.

2. the method according to claim 1 for largely generating the excretion body comprising cargo protein, wherein the cargo Albumen is super repressor I kB proteins, Bax (Bcl-2 correlations X protein), Peroxiredoxin I, the peroxide for inhibiting NF- κ B Compound oxidoreducing enzyme II, cre recombinase, Cas9 (CRISPR associated protein 9s), Cpf1 (irrigate Pseudomonas and Fu Langxi from Prey The CRISPR 1 of this Bordetella) or GBA (β-glucocerebrosidase).

3. the method according to claim 1 for largely generating excretion body, wherein it is choosing that the excretion body, which generates cell, The group that free bone-marrow-derived lymphocyte, T lymphocytes, Dendritic Cells, megacaryocyte, macrophage, stem cell and tumour cell form One or more cells.

4. the method according to claim 1 for largely generating excretion body, wherein the excretion body specific marker object It is CD9, CD63, CD81 or CD82.

5. a kind of method for largely generating the excretion body comprising cargo protein includes the following steps：

(a) polynucleotides and encoding fusion protein of encoding fusion protein (fusion protein I) are imported in excretion body generates cell The polynucleotides of (fusion protein II), the fusion protein I specifically bind egg by excretion body specific marker object and the first light White composition, the fusion protein II are special by cargo protein and the second light that can be connect with the described first smooth binding proteins specific Specific binding protein forms；

(b) with can cause between the described first smooth binding proteins specific and the second smooth binding proteins specific be conjugated Light irradiates the excretion body and generates cell；And

(c) irradiation is terminated after completing production excretion body during the excretion body generates cell.

6. the method according to claim 5 for largely generating the excretion body comprising cargo protein, wherein described first Light binding proteins specific is selected from by CIB (cryptochrome interaction bHLH protein), the CIBN (N of CIB Terminal domains), PhyB (phytochrome B), PIF (phytochrome interaction factor), FKF1 (flavine combine, Kelch weight Multiple, Fbox 1), the group that forms of GIGANTEA, CRY (cryptochrome) and PHR (photolyase homology region) it is one or more Albumen.

7. the methods according to claim 5 for largely generating the excretion bodies comprising cargo protein, wherein when described the When one smooth binding proteins specific is CIB or CIBN, the second smooth binding proteins specific is CRY or PHR, and described the The combination of one smooth binding proteins specific and the second smooth binding proteins specific passes through the light with wavelength for 460~490nm It irradiates to realize.

8. the methods according to claim 5 for largely generating the excretion bodies comprising cargo protein, wherein when described the When one smooth binding proteins specific is PhyB, the second smooth binding proteins specific is PIF, and first light is specific The combination of binding protein and the second smooth binding proteins specific is realized by being irradiated for the light of 600~650nm with wavelength.

9. the methods according to claim 5 for largely generating the excretion bodies comprising cargo protein, wherein when described the When one smooth binding proteins specific is GIGANTEA, the second smooth binding proteins specific is FKF1, and first light The combination of binding proteins specific and the second smooth binding proteins specific with the light that wavelength is 460~490nm by being irradiated To realize.

10. the method according to claim 5 for largely generating the excretion body comprising cargo protein, wherein the goods Object albumen be the super repressor I kB proteins for inhibiting NF- κ B, Bax (the relevant X proteins of Bcl-2), Peroxiredoxin I, Peroxiredoxin II, cre recombinase, Cas9 (CRISPR associated protein 9s), Cpf1 are (from the fertile Pseudomonas of Prey and not The CRISPR 1 of Lang Xisi Bordetellas) or GBA (β-glucocerebrosidase).

11. protein drug is delivered to born of the same parents using the excretion body prepared by the method according to claim 1 or claim 5 The method of matter colloidal sol.

12. for albumen will to be included by the method preparation according to claim 1 or claim 5 as active constituent The excretion body of drug is delivered to the pharmaceutical composition of cytosol.

13. a kind of carrier for generating excretion body, including：

(a) the first expression vector includes the fusion egg of coding excretion body specific marker object and the first smooth binding proteins specific The polynucleotides of (the first fusion protein) in vain；And

(b) the second expression vector, including multiple cloning sites and coding will be combined with the described above first smooth binding proteins specific The second smooth binding proteins specific polynucleotides, encode cargo protein polynucleotides can be fed to it is described polyclonal Site.

14. the carrier according to claim 13 for generating excretion body, wherein second expression vector will be in excretion (second melts the fusion protein that expression is made of the described second smooth binding proteins specific and the cargo protein in body production cell Hop protein).

15. the excretion body comprising cargo protein prepared by the method according to claim 1 or claim 5.

16. a kind of condition for generating target gene knocks out the composition of allele, including by claim 1 or claim Excretion body comprising Cre recombinases prepared by the method described in 5 is as active constituent.

17. a kind of composition manipulating DNA sequence dna, including prepared by the method according to claim 1 or claim 5 Including the excretion body of Cas9 and Cpf1 albumen and the guiding RNA special to target DNA sequence.

18. a kind of method for treating or preventing Gaucher disease includes by pharmacy effective dose by according to claim 1 or power Profit requires the step of excretion body comprising β-glucocerebrosidase prepared by the method described in 5 is as delivery of active ingredients.

19. a kind of method for treating or preventing active oxygen relevant disease includes by pharmacy effective dose by according to right It is required that 1 or claim 5 described in method prepare include Peroxiredoxin I or Peroxiredoxin II Excretion body as delivery of active ingredients the step of.

20. a kind of method for treating or preventing diseases associated with inflammation includes by pharmacy effective dose by according to claim 1 Or the excretion body comprising super repressor I kB proteins for preparing of method described in claim 5 as delivery of active ingredients the step of.

21. a kind of method for treating or preventing cancer includes by pharmacy effective dose by according to claim 1 or right It is required that the excretion body comprising Bax (the relevant X proteins of Bcl-2) albumen prepared by the method described in 5 is as delivery of active ingredients Step.