HK1152951B - Novel tumor marker - Google Patents

Description

Novel tumor marker

Technical Field

The invention relates to the field of diagnosis and treatment of tumors, in particular to a novel tumor marker, a method and a kit for diagnosing occurrence and metastasis of tumors, a method and a medicament for treating tumors and tumor metastasis.

Background

Currently, about 1100 million people are diagnosed with tumors worldwide each year. And it is expected that there will be 1600 million new cases annually before 2020. In 2005, 760 million of the 5800 million deaths worldwide were caused by cancer (approximately 13% of total deaths), and this number has a tendency to increase year by year, with an expectation that 900 million will die of cancer by 2015 and 1140 million by 2030 (World Health Organization, 2006).

Tumor markers are antigens and other bioactive substances that are produced or reduced by tumor cells due to changes in the expression level of genes during carcinogenesis, and are useful for early diagnosis, staging, monitoring of tumor progression, and evaluation of therapeutic effects of drugs (ASCO, 1996). It can have a tremendous impact on the clinical treatment of tumors, especially if it can be detected before the clinical condition occurs or can be used for real-time detection of the therapeutic effect. At present, in order to meet the requirements of clinical diagnosis and treatment of tumors, the research and development of tumor markers needs to be accelerated urgently.

Most of the tumor markers for early diagnosis of tumors at present can not be widely used in physical examination due to lack of sensitivity and specificity. For example, for liver cancer, alpha-fetoprotein and ultrasound examination are commonly used as means for diagnosing high-risk patients, and indeed the survival rate of liver cancer patients is significantly improved, but the sensitivity is low; the tumor antigen CA-125 has higher sensitivity but lacks specificity. Similarly, the blood tumor marker CA15-3 for breast cancer detection is hardly used in early diagnosis due to its low sensitivity. Therefore, early diagnosis of tumors and differentiation between benign and malignant tumors remain a clinical problem, and new technologies and methods are needed to find new tumor markers and to improve the sensitivity and reliability of tumor marker detection.

The emergence of tumor proteomics brings new hopes for the discovery of new tumor markers, and the general investigation, early diagnosis and prognosis of tumors. Malignant transformation of tumors is accompanied by changes in the expression levels of certain proteins, which can be detected qualitatively and quantitatively at the protein level, which is the main research content of tumor proteomics. The protein information of the tumor obtained by the method can provide valuable help for more effective diagnosis, prognosis and evaluation of treatment effect.

Heat shock protein 90 α (Hsp90 α) is a molecular chaperone protein whose presence is essential for the stability and function of many tumor-associated proteins. Hsp90 a is one of the most abundant chaperones in the cytoplasm of eukaryotic cells and accounts for about 1-2% of total protein in cells. The major functions of intracellular Hsp90 a include stabilizing proteins (e.g., estrogen receptors) and helpingProtein maturation (e.g., certain kinases and signaling proteins) has also been implicated in several other physiological processes in the cell, including the evolutionary stabilization of muteins, cytoskeletal rearrangement, nuclear protein transport, cell proliferation and apoptosis, protein degradation, antigen presentation, and lipopolysaccharide recognition. Hsp90 a is also associated with a variety of diseases, including cancer, autoimmune diseases, and cardiovascular diseases. For example, monoclonal antibodies epitope the LKVIRK sequence of Hsp90 a are therapeutically active against fungal infections and are currently being tested by Neutec under the trade name "Neutec

There are papers that found Hsp90 a to be secreted under extreme conditions (Liao et al (2000) j. biol. chem.275, 189-96), and as a classical intracellular protein, there are few reports on how Hsp90 a appears and functions extracellularly. In previous reports, Hsp90 a was found to aid antigen presentation in antigen presenting cells, and was also found to be one of four proteins associated with lipid rafts on the cell surface, binding to lipopolysaccharide, causing a response by intracellular proteins (Triantafilou et al (2002) Trends in Immunology 23, 301-4).

Hsp90 a has also been found to be highly expressed on the surface of many tumor cells, including small cell carcinomas, melanomas, and liver cancer cell lines (Ferrarini et al (1992) int.j. cancer 51, 613-19). The high expression of Hsp90 a on the surface of these cell lines is presumed to be linked to antigen presentation, but there is currently no definitive evidence.

Hsp90 a has also been reported to aid transport of transmembrane proteins (Schlater et al (2002) biochem. J.362, 675-84) and has been implicated in Drug efflux (Drug efflux) in leukemias, lung and ovarian cancers (Rappa et al (2002) Oncol. Res.12, 113-9and Rappa et al (2000) Anticancer Drug Des 15, 127-34).

Intracellular Hsp90 a is also an important target for the development of antitumor drugs at present. Intracellular Hsp90 a is involved in the regulation of many signaling pathways leading to cellular carcinogenesis. Inhibition of Hsp90 a can cause selective degradation of some signaling proteins associated with cell proliferation, cell cycle regulation, and apoptosis. Many known antibiotics (e.g., Geldanamycin, Radicicol, and courromycin Al, etc.) have recently been found to be inhibitors of Hsp90 a, and WO 00/53169 describes this finding and suggests methods of inhibiting chaperonin activity by inhibiting the binding of chaperonin to its substrate, which is believed to function. However, the inhibitors mentioned in WO 00/53169 are directed against intracellular Hsp90 a.

The Geldanamycin analog 17-AAG, which is also an inhibitor of Hsp90 α, has been clinically tested, but 17-AAG is believed to be highly toxic due to nonspecific inhibition by binding to proteins of various cellular components (Dunn (2002) j. natl. cancer inst.94, 1194-5). In addition, direct inhibition of intracellular Hsp90 a activity is also at risk due to the lack of sufficient knowledge of the various cellular signaling processes involved in the binding of intracellular Hsp90 a to its substrate.

EP1457499a1 describes the function of extracellular Hsp90 a in tumor cell invasion, Hsp90 a promotes tumor invasion by promoting the secretion or activation of the matrix metalloproteinase MMP-2. Based on this finding, EP1457499a1 suggests that tumor invasion and metastasis can be inhibited by inhibiting the activity of extracellular Hsp90 a, and that the tumor cell invasiveness and its invasiveness correlation with Hsp90 a can be determined by examining the tumor cell response to inhibitors of Hsp90 a.

WO/2008/070472 proposes the detection and prognosis of the anti-tumor therapeutic effect against Hsp90 a by detecting Hsp90 a and its related factors in plasma, examples of which provide a correlation between the level of plasma Hsp90 a and the therapeutic effect of its inhibitors BA (including 17-AAG and 17-DMAG), and a correlation between the size of the tumor volume and the level of plasma Hsp90 a in a mouse tumor model. However, no form of Hsp90 a was identified in the blood of cancer patients, nor was Hsp90 a found to be associated with the malignancy, especially metastasis, of cancer patients; the use of Hsp90 a as an independent tumor marker in tumor diagnosis and prognosis has not been suggested either.

Another article reported that Hsp90 a in serum was associated with the clinical stage of non-small cell lung cancer (Xu et al (2007) j. cancer mol.3, 107-. Hsp90 a in the serum of patients with lung cancer is obviously improved compared with normal people and patients with benign tumors. However, the article also does not identify the presence of Hsp90 a in the blood of tumor patients, nor does it suggest its association with tumor metastasis; in addition, the article only researches on non-small cell lung cancer, and does not relate to the correlation and specificity of the Hsp90 alpha in the blood plasma of breast cancer, liver cancer and pancreatic cancer; and this article only qualitatively investigated the changes in serum Hsp90 a levels in neoplastic patients, and does not define the changes in absolute levels, and the normal and abnormal ranges of levels necessary for diagnosis and prognosis of tumors.

Disclosure of Invention

The present invention is based on the discovery that the level of Hsp90 a in the blood is associated with the development of a variety of tumors and their malignancy and metastasis. Therefore, Hsp90 a in blood can be used as a new tumor marker. The present inventors found that Hsp90 a in blood is different from intracellular Hsp90 a in that 4 amino acids are deleted from the C-terminus.

Thus, in one aspect, the invention provides an isolated polypeptide comprising or consisting of the amino acid sequence of SEQ ID No. 1.

The polypeptide of the invention may be phosphorylated, wherein one or more amino acid residues selected from the group consisting of: threonine at position 90, serine at position 231, serine at position 263, tyrosine at position 309, and combinations thereof. Preferably, the threonine at position 90 of the polypeptide is phosphorylated.

The polypeptide of the invention can be used as a tumor marker. By using specific conjugates of the polypeptides of the present invention to detect the levels of the polypeptides of the present invention in the blood, it is possible to help judge the occurrence of various tumors and their malignancy and metastasis.

Thus, in another aspect, the present invention relates to the use of a specific binding agent of a polypeptide of the present invention for the preparation of a kit, which can be used, for example, for aiding in the determination of the presence, stage and/or metastasis of a tumor by detecting the level of a polypeptide of the present invention in plasma, for screening tumors in a high risk group by detecting the level of a polypeptide of the present invention in plasma, for prognosis of a patient with a tumor by detecting the level of a polypeptide of the present invention in plasma, or for determining whether surgery, radiation therapy or drug therapy is effective and/or for deciding when to stop therapy in a patient with a tumor by detecting the level of a polypeptide of the present invention in plasma.

Preferably, the specific binding agent for the polypeptide of the invention is an antibody specific for the polypeptide of the invention. Preferably, the antibody is a monoclonal antibody or antigen binding fragment thereof, such as scFv, Fab ', and F (ab') 2. In a specific embodiment, the antibody is monoclonal antibody E9 or D10 produced by a cell line with the preservation number of CGMCC No.2903 or 2904.

According to the invention, the antibody specifically binds to a polypeptide of the invention, and preferably specifically binds to a polypeptide of the invention in plasma. Preferably, the antibody specifically binds to a phosphorylated polypeptide of the invention, wherein one or more amino acid residues of the polypeptide corresponding to SEQ ID No.1 selected from the group consisting of: threonine at position 90, serine at position 231, serine at position 263, tyrosine at position 309, and combinations thereof. More preferably, the antibody specifically binds to threonine at position 90 is a phosphorylated polypeptide of the invention.

In another aspect, the invention relates to the use of an inhibitor of a polypeptide of the invention for the preparation of a pharmaceutical composition for the prevention or treatment of tumor metastasis.

According to one embodiment of the invention, the inhibitor is an antibody specific for the polypeptide of the invention. Preferably, the antibody is a humanized antibody or an antigen-binding fragment thereof. In one embodiment, the antibody specifically binds to a phosphorylated polypeptide of the invention, wherein one or more amino acid residues of the polypeptide corresponding to SEQ ID No.1 selected from the group consisting of: threonine at position 90, serine at position 231, serine at position 263, tyrosine at position 309, and combinations thereof. In a preferred embodiment, the antibody specifically binds to a polypeptide of the invention in which threonine at position 90 is phosphorylated. In a specific embodiment, the antibody of the present invention is monoclonal antibody E9 or D10 produced by a cell line with the preservation number of CGMCC No.2903 or 2904.

In various uses of the invention, the tumor may be, for example, lung cancer, liver cancer, stomach cancer, esophageal cancer, osteosarcoma, pancreatic cancer, lymphoma, colon cancer, breast cancer, prostate cancer, oral cancer, nasopharyngeal cancer, cervical cancer, leukemia, malignant melanoma, sarcoma, renal cancer, biliary cancer.

The invention also relates to antibodies that specifically bind to a polypeptide of the invention, which antibodies specifically bind to a polypeptide of the invention in plasma. In a specific embodiment, the antibody is monoclonal antibody E9 or D10 produced by a cell line with the preservation number of CGMCC No.2903 or 2904. Preferably, the antibody is a humanized antibody or an antigen-binding fragment thereof. In one embodiment, the antibody specifically binds to a phosphorylated polypeptide of the invention, wherein one or more amino acid residues of the polypeptide corresponding to SEQ id No.1 selected from the group consisting of: threonine at position 90, serine at position 231, serine at position 263, tyrosine at position 309, and combinations thereof. In a preferred embodiment, the antibody specifically binds to a polypeptide of the invention in which threonine at position 90 is phosphorylated.

In another aspect, the invention relates to a method of inhibiting tumor invasion and metastasis, comprising the step of inhibiting phosphorylation of Hsp90 a in tumor cells. In one embodiment, the methods of the invention comprise inhibiting phosphorylation of threonine at position 90 of Hsp90 a in tumor cells. In a particular embodiment, the method of the invention comprises overexpressing a nucleic acid encoding protein phosphorylase 5(PP5) in tumor cells, and preferably PP5 by means of gene introduction.

Drawings

FIG. 1: the plasma level of Hsp90 a is elevated in tumor-bearing mice compared to normal mice.

FIG. 2: the level of Hsp90 a in plasma of malignant patients is elevated compared to normal persons.

FIG. 3: detection of the titers of murine mAbs E9 and D10

FIG. 4: standard curves for plasma Hsp90 a concentrations were determined using murine mab E9 and rabbit polyclonal antibody S2 (sandwich ELISA).

FIG. 5: the content of Hsp90 alpha in plasma of liver cancer patients, lung cancer patients, breast cancer patients, pancreatic cancer patients, benign breast cysts and hysteromyoma patients is quantitatively compared by using a sandwich ELISA method.

A: measuring the content of the plasma Hsp90 alpha of the liver cancer patient by using a sandwich ELISA method, wherein the content of the plasma Hsp90 alpha of the benign tumor patient is within the interval of 2-10ng/ml, and most of the content is concentrated at 2-5 ng/ml; 69% (20/29) of the liver cancer patients have the content of Hsp90 alpha in plasma of more than 50ng/ml, the mean value of the content is more than 10 times higher than that of benign tumor patients, the result is basically consistent with the result of immunoblotting, and the correlation between the content level of plasma Hsp90 alpha and the malignancy degree of the tumor is also preliminarily shown.

B: the content of plasma Hsp90 a in lung cancer patients is measured by a sandwich ELISA, 64 percent (9/14) of the content of plasma Hsp90 a in lung cancer patients is more than 50ng/ml compared with benign tumor patients, the mean value is increased by more than 10 times compared with benign tumor patients, and the correlation between the content level of plasma Hsp90 a and the malignancy degree of tumors is shown.

C: the determination of the plasma Hsp90 a content in breast cancer patients by sandwich ELISA can be more than 5 times higher than that in benign tumor patients, and the total level is significantly different from that in benign tumor patients.

D: the content of plasma Hsp90 a in pancreatic cancer patients is measured by using a sandwich ELISA, compared with benign tumor patients, the content of 100 percent (10/10) of plasma Hsp90 a in pancreatic cancer patients is more than 50ng/ml, the mean value is increased by more than 10 times compared with benign tumor patients, and the correlation between the content level of plasma Hsp90 a and the malignancy degree of tumors is shown.

FIG. 6: the content of Hsp90 alpha in blood plasma of patients with tumor metastasis and patients without tumor metastasis are quantitatively compared by using a sandwich ELISA method.

A: the liver cancer patients are grouped according to whether tumors have metastasis, and the comparison of two groups shows that the content of plasma Hsp90 alpha of the patients with tumor metastasis is more than 200ng/ml, and the content of plasma Hsp90 alpha of the patients without tumor metastasis is between 50ng/ml and 200 ng/ml.

B: the lung cancer patients are grouped according to whether tumors have metastasis, and the comparison of two groups shows that the content of plasma Hsp90 alpha of the patients with tumor metastasis is more than 200ng/ml, and the content of plasma Hsp90 alpha of the patients without tumor metastasis is between 50 and 200 ng/ml.

C: the breast cancer patients are grouped according to whether tumors have metastasis, and the comparison of the two groups shows that the content of plasma Hsp90 alpha of the patients with tumor metastasis is remarkably increased.

FIG. 7: the content of Hsp90 alpha in plasma of inflammatory patients (including pneumonia and hepatitis patients), normal patients and tumor patients is quantitatively compared by using a sandwich ELISA method.

A: to confirm that the elevation of plasma Hsp90 a levels in tumor patients is specific, we compared the plasma Hsp90 a levels in pneumonia patients, normal patients and tumor patients and found that the plasma Hsp90 a levels in pneumonia patients are between 2-10ng/ml, which is not significantly different from normal.

B: the content of Hsp90 alpha in the plasma of hepatitis patients (hepatitis A and hepatitis B) is between 2 and 10ng/ml, and compared with normal people, the content is not obviously different.

FIG. 8: hsp90 a in plasma is secreted by tumor cells.

FIG. 9: determination of the C-terminal deletion of Hsp90 a secreted by tumor cells.

FIG. 10: hsp90 a in plasma is a C-terminal deletion of 4 amino acids.

FIG. 11: detection of the phosphorylated form of Hsp90 a in plasma.

FIG. 12: the level of threonine phosphorylated Hsp90 a at position 90 in the plasma of neoplastic patients is elevated.

FIG. 13: the increase in threonine phosphorylated Hsp90 a at position 90 in plasma of neoplastic patients is consistent with the increase in total Hsp90 a in plasma of neoplastic patients.

FIG. 14: phosphorylation of threonine at position 90 is essential for the secretion of Hsp90 a.

FIG. 15: PP5 dephosphorylates threonine 90 of Hsp90 a.

A: the free phosphate groups released were detected by incubation of purified PP5 in combination with threonine-phosphorylated Hsp90 α at position 90 (pT90-Hsp90 α). Peptide was used as a positive control. The results show that PP5 can directly dephosphorylate threonine 90 of Hsp90 a.

B: in a human breast cancer cell line MCF-7, human PP5 is excessively expressed or the expression of human PP5 is inhibited by using an RNA interference technology, and the phosphorylation of threonine at position 90 of Hsp90 alpha is observed, so that the result shows that the threonine phosphorylation at position 90 of Hsp90 alpha (pT90-Hsp90 alpha) is obviously reduced (which is 0.55 of a control group) after the human PP5 is excessively expressed; threonine-phosphorylated Hsp90 a (pT90-Hsp90 a) at position 90 increased significantly (as compared to 1.58 of the control) when the expression of endogenous human PP5 was suppressed.

FIG. 16: intracellular PP5 regulates the secretion of Hsp90 a.

A: in the breast cancer cell line MCF-7, human PP5 is overexpressed, and the change of cell secretion of Hsp90 alpha is observed, so that the result shows that the cell secretion of Hsp90 alpha is obviously reduced after human PP5 is overexpressed.

B: in the breast cancer cell line MCF-7, the expression of human PP5 is inhibited by using an RNA interference technology, the change of the cell secretion Hsp90 alpha is observed, and the result shows that the cell secretion Hsp90 alpha is obviously increased when the expression of endogenous human PP5 is inhibited.

FIG. 17: the expression level of PP5 was correlated with the secretion of Hsp90 α by cells.

FIG. 18: the expression level of PP5 was correlated with the ability of tumor cells to migrate.

FIG. 19: antibodies specific for Hsp90 a are capable of inhibiting tumor cell migration.

FIG. 20: hsp90 a-specific antibodies inhibit the activity of tumor metastasis.

Microbiological material preservation information

The SP2/0-Ag14 mouse hybridoma cell line of the generated monoclonal antibody E9 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (CGMCC, Datun Luo of the sunward area of Beijing, China academy of sciences microorganism) at 24 months and 2 months in 2009 with the preservation number of CGMCC No. 2903.

The SP2/0-Ag14 mouse hybridoma cell line of the generated monoclonal antibody D10 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (CGMCC, Datun Luo of the sunward area of Beijing, China academy of sciences microorganism) at 24 months and 2 months in 2009 with the preservation number of CGMCC No. 2904.

Detailed Description

The canceration of cells is caused by changes in certain signal transduction pathways of cells, accompanied by changes in the expression level, modification type and spatial distribution of proteins, and the information on the changes in these proteins can be used to monitor the occurrence and progression of tumors, and these proteins are called tumor markers. With the progress and development of proteomics methods and technologies, qualitative and quantitative monitoring of changes of tumor proteome becomes possible, and a plurality of new tumor markers are discovered, so that more accurate and reliable bases are provided for clinical diagnosis and prognosis of tumors.

The present invention is based on the discovery of a novel plasma tumor marker, Hsp90 a present in plasma. Compared with the intracellular Hsp90 alpha (the amino acid sequence of which is SEQ ID No.3 and the coding nucleic acid sequence of which is SEQ ID No.4), the C terminal of the Hsp90 alpha in the blood plasma lacks 4 amino acids.

Thus, in one aspect, the invention provides an isolated polypeptide which is Hsp90 a in plasma or serum, said polypeptide comprising or consisting of the amino acid sequence of SEQ ID No. 1. In the present application, the term "polypeptide of the invention" refers to Hsp90 a in plasma or serum, which comprises or consists of the amino acid sequence of SEQ ID No. 1. Preferably, the term "polypeptide of the invention" refers in the present application to a polypeptide consisting of the amino acid sequence of SEQ id No. 1. In the present application, the term "Hsp 90a in plasma" or "Hsp 90a in serum" equivalently refers to the non-intracellular and cell-surface Hsp90 a protein present in blood, either alone or in combination with other extracellular proteins in blood. In the present application, the term "polypeptide" is used interchangeably with "protein".

The invention also relates to polynucleotides encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID No. 1. In a specific embodiment, the polynucleotide comprises or consists of the sequence of SEQ ID No. 2.

The present inventors have also found that the polypeptide of the invention is phosphorylated in the plasma form, wherein one or more amino acid residues selected from the group consisting of: threonine at position 90, serine at position 231, serine at position 263, tyrosine at position 309, and combinations thereof. Preferably, the threonine at position 90 of the polypeptide is phosphorylated.

No specific existing form of plasma Hsp90 alpha has been found, and the level of the Hsp90 alpha is not reported to be related to the occurrence and development of tumors. EP1457499a1 describes extracellular Hsp90 a and suggests that inhibitors thereof may be useful in the treatment of tumor metastasis, as well as in the diagnosis of the invasive capacity of tumor cells, and whether this capacity is dependent on Hsp90 a. However, the method described in EP1457499a1 detects Hsp90 a on the surface of cell membranes, does not relate to the level of Hsp90 a in plasma, and does not relate to the determination of the malignancy of tumors based on the level of Hsp90 a, and is useful for early diagnosis, staging, detection of therapeutic effects and prognosis of tumors. WO/2008/070472 proposes to judge the effect of anti-tumor therapy against Hsp90 a by detecting Hsp90 a and its related factors in plasma, but does not mention the use of Hsp90 a as an independent tumor marker in tumor diagnosis and prognosis.

The inventor finds that the level of Hsp90 alpha in plasma is related to the malignancy degree of tumors, particularly metastasis, by detecting blood from nearly hundreds of tumor patients (with 4 types of tumors such as breast cancer, liver cancer, pancreatic cancer, lung cancer) and the like, and inflammatory reaction has no influence on the level of Hsp90 alpha in plasma. Therefore, Hsp90 alpha in the plasma is a novel tumor marker and can be used for diagnosis and prognosis of tumors and metastasis thereof.

Thus, in another aspect, the invention relates to a kit for detecting the level of a polypeptide of the invention in plasma. The kit of the invention is directed to specific conjugates comprising, for example, a polypeptide of the invention. The kit can be used for detecting the content of Hsp90 alpha in blood plasma.

The invention also relates to the use of specific binders for the polypeptides of the invention in the preparation of a kit, which can be used, for example, to aid in the determination of the presence, malignancy or metastasis of a tumor by detecting Hsp90 a levels in the plasma; for tumor screening of high risk population by detecting Hsp90 a levels in plasma; for determining the prognosis of a patient with a tumor by detecting the level of Hsp90 a in the plasma; or for determining whether surgery, radiation therapy or drug treatment is effective and/or for deciding when to stop treatment in a patient with a tumor by detecting the level of Hsp90 a in the plasma.

In the present application, the term "specific binding agent" refers to a molecule that binds with high affinity to a polypeptide of the invention, which also includes molecules that bind with high affinity to Hsp90 a in or on the surface of cells. The specific binding substance is for example a protein, preferably the specific binding substance is an Hsp90 a specific antibody. In a preferred embodiment, the antibody is a monoclonal antibody or antigen binding fragment thereof, such as scFv, Fab ', and F (ab') 2. In a specific embodiment, the antibody is monoclonal antibody E9 or D10 produced by a cell line with the preservation number of CGMCC No.2903 or 2904.

Monoclonal antibodies are obtained by selecting cells that secrete the desired antibody and culturing in vitro. Methods for preparing monoclonal antibodies are well known in the art (K' hler G & Milstein C. (1975) Nature.256, 495-7). The specific process for preparing a monoclonal antibody specifically recognizing Hsp90 a is as follows: BALB/C mice were immunized with recombinant human Hsp90 a, and primary immunization was performed using 100 μ g of antigen plus Freund's complete adjuvant by back subcutaneous multiple injection; the second immunization is carried out after 3 weeks, the dosage is the same, and the intraperitoneal injection is carried out by adding Freund incomplete adjuvant; after 3 weeks, carrying out third immunization, wherein the dose is the same as the above, and no adjuvant is added for intraperitoneal injection (blood is collected after 5-7 days to measure the titer); the immunization was boosted after another 3 weeks at a dose of 200 μ g and injected intraperitoneally. After 3 days, splenocytes are taken and fused with SP2/0-Ag14(SP2/0) hybridoma (source: ATCC, number: CRL-1581), HAT is used for screening, the hybridoma is cloned by a limiting dilution method, and the hybridoma is identified by immunoblotting and ELISA methods, so that the hybridoma cell strain secreting the antibody specifically recognizing Hsp90 alpha is finally obtained.

According to the present invention, the antibodies useful for preparing the above-described kits specifically bind Hsp90 a, and preferably specifically bind Hsp90 a in plasma. In one embodiment, the antibody specifically binds phosphorylated Hsp90 a, wherein one or more amino acid residues of the Hsp90 a selected from the group consisting of: threonine at position 90, serine at position 231, serine at position 263, tyrosine at position 309, and combinations thereof. In a preferred embodiment, the antibody specifically binds threonine at position 90 is phosphorylated Hsp90 a.

The invention also relates to methods for detecting the level of a polypeptide of the invention in plasma. The level of Hsp90 a in the plasma can be determined by any suitable method. The method comprises directly or indirectly determining the level of the polypeptide of the invention in plasma, in order to facilitate the determination of the occurrence of a tumor and its malignancy and metastasis.

Direct assay methods include detection of the polypeptides of the invention using specific binders for the polypeptides, for example immunoblotting or ELISA detection using specific antibodies that recognize the polypeptides.

Indirect assays include, for example, reflecting the concentration of Hsp90 α by detecting the activity of Hsp90 α, such as luciferase heat denaturation assays based on the detection of Hsp90 α chaperone activity (Johnson et al (2000) j. biol. chem., 275, 32499-32507).

Preferably, the content of Hsp90 a in the plasma is detected by ELISA or immunoblotting method, which essentially comprises the following steps:

a) collecting whole blood of individual such as tumor patient, centrifuging according to conventional method to obtain blood plasma or blood serum;

b) detecting the level of Hsp90 a in the plasma or serum obtained in step a) by ELISA or immunoblotting, and using the plasma of healthy normal persons as negative control and the plasma of diagnosed malignant patients as positive control, optionally generating a standard curve of the Hsp90 a concentration;

c) according to the measured content of the plasma Hsp90 alpha, the occurrence, the malignancy degree and the stage of the tumor are judged. Thereby making a judgment on the diagnosis, prognosis and treatment effect of the tumor.

Other detection means based on antigen-antibody reaction can be used in step b), and other detection means based on other principles that can directly or indirectly reflect the concentration of Hsp90 a, such as reflecting the concentration of Hsp90 a by detecting the activity of Hsp90 a.

Standard curves for determining Hsp90 a concentration Hsp90 a standards were purified from the plasma of tumor patients and were obtained by recombinant expression of genes, including full-length, fragments of Hsp90 a, and recombinant proteins containing Hsp90 a sequence and complexes conjugated to other groups. "standard curve for Hsp90 a concentration" refers to the corresponding curve of concentration and absorbance measurements determined by ELISA using a standard sample of known concentration of Hsp90 a. By "Hsp 90a standard" is meant a sample of plasma Hsp90 a protein, recombinant Hsp90 a protein, fragments and derivatives that are greater than 95% pure.

The term "determining the malignancy degree of the tumor" refers to determining the level of the Hsp90 a in the plasma of a patient according to the concentration of the Hsp90 a in a test sample, and the values of a negative control and a positive control, so as to determine the tumor nature (i.e. benign or malignant) of the patient.

ELISA methods that can be used include both sandwich and competitive methods. Among them, the competitive method having high sensitivity is preferable.

The general steps of the sandwich method include: a) connecting the specific antibody with a solid phase carrier to form a solid phase antibody, and washing to remove the unbound antibody and impurities; b) adding a detected sample: contacting the solid phase antibody with the antigen in the sample for a period of time to allow the antigen in the sample to be combined with the antibody on the solid phase carrier to form a solid phase antigen-antibody immune complex, and washing to remove other unbound substances; c) adding an enzyme-labeled antibody: combining the antigen on the solid-phase immune complex with the enzyme-labeled antibody, and thoroughly washing the unbound enzyme-labeled antibody, wherein the enzyme amount on the solid-phase carrier is positively correlated with the amount of the detected substance in the specimen; d) the enzyme in the sandwich complex catalyzes the substrate to a colored product upon addition of the substrate. The antigen is either qualitative or quantitative depending on the degree of color reaction.

The general steps of the competition method include: a) connecting the specific antibody with a solid phase carrier to form a solid phase antibody, and washing; b) the mixed solution of the detected specimen and a certain amount of enzyme-labeled antigen is added into the tube to be detected, so that the mixed solution reacts with the solid-phase antibody. If the specimen to be tested contains no antigen, the enzyme-labeled antigen can be smoothly bound to the solid-phase antibody. If the specimen contains an antigen, the antigen binds to the solid-phase antibody at the same chance as the enzyme-labeled antigen, and competitively takes up the chance of binding the enzyme-labeled antigen to the solid-phase carrier, thereby reducing the amount of binding between the enzyme-labeled antigen and the solid-phase carrier. Only adding enzyme labeled antigen into the reference tube, after heat preservation, combining the enzyme labeled antigen and the solid phase antibody to the most sufficient amount, and washing. c) The substrate is added for color development, and the reference tube has the darkest color because the most enzyme-labeled antigen is combined. The difference between the color depth of the reference tube and the color depth of the tube to be detected represents the amount of the antigen in the sample to be detected. The lighter the color of the tube to be detected is, the more the antigen content in the sample is.

The method uses an ELISA sandwich method for detection, and the used antibodies are derived from specific antibodies of plasma Hsp90 alpha of two different species, wherein the plating antibody is preferably rabbit polyclonal antibody with strong binding capacity, and the other antibody is preferably mouse monoclonal antibody with good recognition specificity. Both antibodies must not cross-react.

If the method is used for detection by using an ELISA competition method, the used antibody is a specific antibody of plasma Hsp90 alpha, and the antibody has stronger recognition capability and recognition specificity on competitors and Hsp90 alpha.

If the method is used for detection by an ELISA competition method, the competitor is a marker of a plasma Hsp90 alpha standard. And the label does not interfere with the binding of Hsp90 a standard to its antibody.

If the method is used for detection by an immunoblotting method, the antibody used is a specific antibody of plasma Hsp90 alpha; the secondary antibody can be coupled with horseradish peroxidase or alkaline phosphatase; the reaction substrate includes DAB and a fluorescent substrate, and among them, a fluorescent substrate having high sensitivity is preferable.

The sensitivity of the above detection method should be 10ng/ml or less.

Antibodies specific for plasma Hsp90 a, as used in the present invention, include full length antibodies, fragments and derivatives thereof.

The antibodies of the present invention may also be replaced by other Hsp90 a specific conjugates, including small molecule compounds, polypeptides and derivatives thereof.

The invention relates to a marker of a plasma Hsp90 alpha standard substance, wherein the marker group comprises biotin and various fluorescent labeling reagents. Among them, biotin-labeled competitors are preferred.

Cancers that can be detected using the kit of the present invention include, but are not limited to, lung cancer, liver cancer, stomach cancer, esophageal cancer, osteosarcoma, pancreatic cancer, lymph cancer, colon cancer, breast cancer, prostate cancer, oral cancer, nasopharyngeal cancer, cervical cancer, leukemia, malignant melanoma, sarcoma, renal cancer, biliary cancer, and the like. "tumor and its malignancy" refers to whether a tumor is benign, malignant, or metastatic.

Preliminary studies by the present inventors have shown that normal human plasma Hsp90 a ranges from 2 to 50ng/ml, more concentrated at 2 to 10ng/ml, and that tumor-diagnosed patients have plasma Hsp90 a levels higher than normal, while plasma Hsp90 a levels are higher than 50ng/ml, and mostly higher than 200ng/ml levels in patients with metastases. This makes plasma Hsp90 a level a new tumor marker, which can be used to help determine whether a tumor exists, especially whether metastasis of the tumor exists.

Thus, in one embodiment, the kits or methods of the invention can be used to determine whether a tumor, particularly a malignant tumor, is present in an individual and whether the tumor has metastasized. For this purpose, the kit or method of the present invention can be used to determine the level of Hsp90 a in a plasma sample from a suspected patient having a tumor or a suspected patient having a tumor metastasis, optionally in comparison with a normal control, and then to determine the likelihood of the patient developing a tumor or tumor metastasis based on the level of Hsp90 a in the sample. Elevated levels of Hsp90 a indicate a greater likelihood that the patient will have a malignant tumor, whereas for patients known to have a tumor, significantly elevated levels of Hsp90 a indicate a greater likelihood that the tumor will metastasize.

In another embodiment, the kit or method of the present invention can be used for tumor screening of high risk population by detecting Hsp90 a level in plasma. To this end, the kit or method of the invention may be used to determine the level of Hsp90 a in a plasma sample from a high risk group, optionally compared to a normal control, and then to determine which individuals in the group may have developed a tumor based on the level of Hsp90 a in the sample. An elevated level of Hsp90 a suggests that the individual is more likely to develop a malignancy. It is known to the person skilled in the art that different types of tumours have their associated high risk population depending on the type of tumour, individual factors such as age, family history, lifestyle, working environment, history of exposure to harmful substances, etc. For example, patients with chronic hepatitis b or c are at high risk for hepatocellular carcinoma.

In another embodiment, the kit or method of the invention can be used to determine the prognosis of a patient with a tumor by detecting the level of Hsp90 a in the plasma. To this end, the kit or method of the invention may be used to determine the level of Hsp90 a in a plasma sample from a patient with a tumour, optionally compared to a normal control or previous Hsp90 a levels in the plasma of the patient, and the prognosis of the patient with a tumour is determined from the level of Hsp90 a in the sample. Maintenance of high levels of Hsp90 a or further elevation of Hsp90 a levels may be associated with adverse prognosis. Thus, the clinician may be prompted to make a closer look at the patient and change the current treatment regimen if necessary.

In another embodiment, the kit or method of the present invention can be used to determine whether surgery, radiation therapy or drug treatment is effective and/or to decide when to stop treatment for a patient with a tumor by detecting the level of Hsp90 a in the plasma. To this end, the kits or methods of the invention can be used to measure the level of Hsp90 a in a plasma sample from a tumor patient, optionally compared to a normal control or previous Hsp90 a levels in the plasma of the patient, and then based on the Hsp90 a levels in the sample, determine whether surgery, radiation therapy or drug treatment is effective and/or determine when to stop the treatment.

As will be appreciated by those skilled in the art, the method of determining whether a patient has a malignant tumor or a tumor metastasis by detecting the level of Hsp90 a in a plasma sample is an auxiliary detection method in the tumor diagnosis process, as is the case with all known tumor markers or methods of screening and diagnosing tumors using tumor markers. According to the results of this method, it is not possible to directly determine whether a patient has a malignant tumor, whether metastasis of the malignant tumor exists, and also to directly determine the tissue origin of the malignant tumor, the type of pathology thereof, or the location and number of metastatic lesions. Therefore, for oncologists, diagnostic determinations are still made by imaging, pathology, and other means, as well as by the judgment of the clinician. In this respect, the method of the invention provides only supplementary reference information for the clinician or the researcher. However, as mentioned in the specification and demonstrated in the examples, the detection of the level of Hsp90 a in plasma provides valuable reference information for determining the presence of a malignant tumor in a patient, and in particular, the presence of metastasis of a tumor. Similarly, the purpose of the methods for screening tumors of high risk groups, judging prognosis of tumor patients, evaluating treatment effect and the like is to provide auxiliary reference information for clinicians or scientific researchers.

The present inventors further confirmed that Hsp90 a in plasma is derived from tumor cells, but is different from Hsp90 a in tumor cells, and thus it is possible to inhibit tumor development and metastasis by inhibiting Hsp90 a in plasma. The inventor also proves that the specific antibody of the plasma Hsp90 alpha can inhibit the tumor metastasis of mice, so that the plasma Hsp90 alpha can be used as a new target to screen new anti-cancer drugs.

In another aspect, the invention relates to the use of an inhibitor of a polypeptide of the invention for the preparation of a pharmaceutical composition for the prevention or treatment of tumor metastasis.

According to one embodiment of the invention, the inhibitor is an Hsp90 a-specific antibody. Preferably, the antibody is a humanized antibody or an antigen-binding fragment thereof. In one embodiment, the antibody specifically binds phosphorylated Hsp90 a, wherein one or more amino acid residues of the Hsp90 a selected from the group consisting of: threonine at position 90, serine at position 231, serine at position 263, tyrosine at position 309, and combinations thereof. In a preferred embodiment, the antibody that specifically binds threonine at position 90 is phosphorylated Hsp90 a. In a specific embodiment, the antibody is monoclonal antibody E9 or D10 produced by a cell line with the preservation number of CGMCC No.2903 or 2904. As described in the examples below, the antibody can completely inhibit lymph node metastasis of mouse tumors, and the efficiency of inhibiting lung metastasis can reach 56%.

Thus, in another aspect the invention also relates to an antibody that specifically binds to a polypeptide of the invention, said antibody specifically binding to Hsp90 a in plasma. In a specific embodiment, the antibody is monoclonal antibody E9 or D10 produced by a cell line with the preservation number of CGMCC No.2903 or 2904. Preferably, the antibody is a humanized antibody or an antigen-binding fragment thereof. In one embodiment, the antibody specifically binds phosphorylated Hsp90 a, wherein one or more amino acid residues of the Hsp90 a selected from the group consisting of: threonine at position 90, serine at position 231, serine at position 263, tyrosine at position 309, and combinations thereof. In a preferred embodiment, the antibody that specifically binds threonine at position 90 is phosphorylated Hsp90 a. Preferably, the antibody inhibits the growth, in particular metastasis, of a tumor. The invention also relates to a conjugate comprising an antibody of the invention and a detection or therapeutic moiety. The detection moiety is, for example, a fluorophore and the therapeutic moiety is, for example, a tumor chemotherapeutic agent.

The inventor also finds that the level of secretion of Hsp90 alpha by tumor cells is in certain correlation with the expression level of intracellular Protein phosphatase 5(PP 5). In benign tumors, the secretion of Hsp90 a is low, while the expression level of PP5 is high; in malignant tumors, the secretion of Hsp90 a is high, while the expression level of PP5 is low. Therefore, the level of secretion of Hsp90 a by tumor cells and the level of intracellular PP5 are in negative correlation. Therefore, by detecting the expression level of PP5 in tumor tissues, the level of Hsp90 alpha secreted by tumor cells into plasma can be predicted, and the early diagnosis of tumors can be carried out.

The invention also proves that the secretion of Hsp90 alpha is inhibited and regulated by PP5 in cells through experiments. When the nucleic acid encoding PP5 is overexpressed in cells, the secretion of Hsp90 a is inhibited, the expression level of PP5 in the cells is reduced, and the secretion of Hsp90 a by the cells is obviously increased. It is therefore possible to inhibit tumor development and metastasis by overexpressing nucleic acids encoding PP5, thereby inhibiting the secretion of Hsp90 a. Through our experiments, we also found that over-expression of PP5 can inhibit migration of breast cancer MCF-7, so PP5 can be a new tumor treatment target.

Thus, in another aspect, the invention relates to a method of inhibiting tumor invasion and metastasis, comprising the step of inhibiting phosphorylation of Hsp90 a in tumor cells. In one embodiment, the methods of the invention comprise inhibiting phosphorylation of threonine at position 90 of Hsp90 a in tumor cells. In a specific embodiment, the method of the invention comprises overexpressing a nucleic acid encoding PP5 in tumor cells, and preferably PP5 by means of gene transfer. In one embodiment, the method of the invention comprises overexpressing in the tumor cells a nucleic acid encoding PP5 comprising the amino acid sequence of SEQ ID No.5, in a particular embodiment the nucleic acid comprises the nucleotide sequence shown in SEQ ID No. 6. The invention also relates to the use of a vector carrying the above nucleic acid encoding PP5 operably linked to a promoter for the preparation of a medicament for inhibiting the phosphorylation of Hsp90 a in tumor cells by over-expressing PP5 in the tumor cells. The medicine can be used for inhibiting invasion and metastasis of tumors.

The invention also provides a method and a model for screening antitumor drugs by using the plasma Hsp90 alpha and derivatives thereof, including but not limited to searching binding protein, small peptide and small molecule compound of the plasma Hsp90 alpha and screening inhibitors for inhibiting the activity of the plasma Hsp90 alpha.

Examples

Example 1: collection and preparation of mouse plasma sample and detection of plasma Hsp90 alpha

Selecting Balb/c mice (purchased from Beijing Wittingle laboratory animal technology Co., Ltd.) with average weight of about 20 g, randomly dividing into two groups, wherein each group contains 3 mice, one of the groups is inoculated with H22 mouse liver cancer cells (CCTCC, serial number: GDC091), and the number of each inoculated cell is 10⁶Control groups were not inoculated with tumors. When the tumor diameter of the mice grows to 2 cm on average (about 20 days), blood is taken from the fundus venous plexus, and anticoagulant is added into the blood to avoid hemolysis. If hemolysis occurs, the sample is collected again. The whole blood was collected and centrifuged twice at 6000g at 4 ℃ to obtain the supernatant, and the plasma was analyzed for the level of Hsp90 a by immunoblotting using Rabbit anti-human Hsp90 a pAb (Labversion). The total protein content of the samples was determined using the BCA method (Pierce) so that the loading amount (protein amount) of each sample remained the same. The results are shown in figure 1, and compared with normal mice, the content of Hsp90 alpha in the plasma of tumor-bearing mice is increased.

Example 2: collection and preparation of plasma samples of normal human and tumor patients, and detection of plasma Hsp90 alpha

Taking whole blood of normal people or cancer patients, sending the whole blood to a laboratory under the condition of low temperature (about 4 ℃) within 24 hours, avoiding hemolysis, and collecting samples again if the hemolysis occurs. Centrifuging twice at 4 ℃ and 6000g, taking supernatant, detecting the content of Hsp90 alpha in plasma by an immunoblotting method, and storing at-80 ℃ after subpackaging if the content cannot be detected immediately. The test results are compared with clinical diagnosis to verify the correlation between the content of Hsp90 alpha in plasma and the malignancy degree of tumor.

The specific operation method of the immunoblotting detection comprises the following steps: plasma samples were mixed 1: 1 with loading buffer, and loaded 1-2 microliters for SDS-PAGE, the primary antibody being a specific antibody recognizing plasma Hsp90 alpha (rat monoclonal antibody SPA-840, Stressgen) and the secondary antibody being a goat anti-rat antibody conjugated with horseradish peroxidase (purchased from China fir gold bridge). The results are shown in fig. 2, and the content of Hsp90 alpha in the plasma of the liver cancer patient is detected by immunoblotting, and the increase is about 10 times compared with the normal human (A); the plasma Hsp90 a content of benign breast cysts and uterine fibroids is about 2-fold higher than that of normal persons (B).

Example 3: preparation of Hsp90 alpha-specific rabbit polyclonal antibody and mouse monoclonal antibody

The composition was prepared using a mixture of Hsp90 α -Sal 1-Re: ACGCGTCGACTTAGTCTACTTCTTCCATGC (SEQ ID No.8) and Hsp90 α -Sph 1-For: ACATGCATGCATGCCTGAGGAAACCCAGACC (SEQ ID No.9) and Pfu DNA polymerase (origin: NEB) from a human liver cDNA library (origin: Stratagene) to obtain the full-length sequence of Hsp 90. alpha. was amplified, the fragment was digested simultaneously with the vector pQE80L (origin: Qiagen) using Sph1 and Sal1 (origin: NEB), and the resulting fragment was ligated with T4 ligase (origin: NEB). The ligation product is transformed into a Top10 escherichia coli competent cell (source: Transgen) for amplification and verification, and the verified plasmid is transformed into a BL21DE3 escherichia coli competent cell (source: Transgen) for expression to obtain the recombinant human Hsp90 alpha protein. The purification method of the recombinant human Hsp90 alpha protein comprises the following steps: ion exchange chromatography SP HP, pH6.8, collecting conductivity 10ms/ml elution peak; q HP, pH7.8, collected the conductance 19ms/ml elution peak.

Adult male New Zealand white rabbits were immunized with > 95% pure recombinant human Hsp90 a protein and injected intradermally in multiple sites into the back of the New Zealand white rabbits at an antigen dose of 100. mu.g/rabbit. After 2 weeks, the 2 nd immunization (halving the antigen dose) was performed in the same manner, followed by 1 booster immunization every 1 week for 2 boosts, and blood was collected from the ear vein 7 to 10 days after the booster immunization to measure the titer of the antibody in the serum. 8 days after the last 1 immunization, carotid artery was cannulated for exsanguination, serum was isolated, and stored at-20 ℃. Serum was purified using an affinity column coupled to antigen. The purified rabbit polyclonal antibody was named S2.

BALB/C mice were immunized with recombinant human Hsp90 a, and primary immunization was performed using 100 μ g of antigen plus Freund's complete adjuvant by back subcutaneous multiple injection; the second immunization is carried out after 3 weeks, the dosage is the same, and the intraperitoneal injection is carried out by adding Freund incomplete adjuvant; after 3 weeks, carrying out third immunization, wherein the dose is the same as the above, and no adjuvant is added for intraperitoneal injection (blood is collected after 5-7 days to measure the titer); the immunization was boosted after another 3 weeks at a dose of 200 μ g and injected intraperitoneally. After 3 days, splenocytes are taken and fused with SP2/0-Ag14(SP2/0) hybridoma (source: ATCC, number: CRL-1581), HAT is used for screening, the hybridoma is cloned by a limiting dilution method, and the hybridoma is identified by immunoblotting and ELISA methods, so that hybridoma cell strains E9 and D10 which secrete antibodies capable of specifically recognizing Hsp90 alpha are finally obtained, wherein the preservation numbers are respectively as follows: CGMCC No.2903 and 2904, which was deposited in CGMCC at 24 months 2 in 2009.

The titers of E9 and D10 were measured by indirect ELISA, and as a result, as shown in FIG. 3, the titers of E9 and D10 both reached 500,000, which was used to measure Hsp90 a in plasma. The indirect ELISA method comprises the following specific steps: plating overnight at 4 ℃ with recombinant human Hsp90 a at a coating concentration of 10 μ g/ml; blocking at 37 deg.C for one hour, adding E9 or D10 diluted in gradient according to 1: 400, 1: 1600, 1: 6400, 1: 25600, 1: 102400, 1: 509600, and incubating at room temperature for 2 hours; this was then incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (purchased from sequoia jessamine) for 1 hour at room temperature, developed using o-phenylenediamine and read at OD490 nm.

Example 4: determination of Standard difficult Curve for plasma Hsp90 alpha concentration Using murine monoclonal antibody E9 and Rabbit polyclonal antibody S2 (Sandwich ELISA)

In the method of determining plasma Hsp90 a concentration using sandwich ELISA, antibodies derived from two different species of antibodies specific for plasma Hsp90 a were used, wherein the plating antibody used was rabbit polyclonal antibody S2 with strong binding capacity (preparation described in example 3), and the other antibody preferably recognized murine mab E9 with good specificity (preparation described in example 3). The two antibodies have no cross reaction, good repeatability and high detection sensitivity. The detection level of the method can reach 5ng/ml as seen by the standard curve of FIG. 4.

Example 5: human plasma sample collection and preparation, and determination of plasma Hsp90 alpha and determination of tumor malignancy (sandwich ELISA)

Whole blood collected from normal, cancer or inflammatory patients within 24 hours is sent to a laboratory at low temperature to avoid hemolysis. If hemolysis occurs, the sample is collected again. Centrifuging twice at 4 ℃ and 6000g, taking supernatant, detecting the content of Hsp90 alpha in plasma by adopting an ELISA method, and storing at-80 ℃ after subpackaging if the content cannot be detected immediately. The test results are compared with clinical diagnosis to verify the correlation between the content of Hsp90 alpha in plasma and the malignancy degree of tumor.

Sandwich ELISA uses two different sources of antibodies against Hsp90 a, of which the home-made rabbit polyclonal antibody S2 (preparation described in example 3) was used for plating overnight at 4 ℃, after blocking for one hour at 37 ℃ the test plasma sample and the standard curve sample were added, the test plasma was diluted 10-fold and 100 μ l was added per well; samples of the standard curve were spiked with a known amount of standard Hsp90 a sample per well and 10 microliters of blank negative serum to exclude serum background; after incubation for 2 hours at 37 ℃, another antibody, homemade murine mab E9 (preparation described in example 3) was added and incubated for 2 hours at 37 ℃; this was followed by incubation with horseradish peroxidase-conjugated goat anti-mouse secondary antibody for 1 hour, developed using o-phenylenediamine and read at OD 450 nm. The results are shown in FIGS. 5, 6 and 7.

As shown in FIG. 5, the content of plasma Hsp90 a in benign tumor patients (including benign breast cyst and hysteromyoma patients, in 7 cases) was in the range of 2-10ng/ml, and most of them was concentrated in 2-5 ng/ml; 69% (20/29) of liver cancer patients (29 cases) had an Hsp90 α content of 50ng/ml or more in their plasma, with an average of > 10-fold increase compared to benign tumor patients, P0.00263, student's t-test (A); 64% (9/14) lung cancer patients (14 cases) had plasma Hsp90 α levels above 50ng/ml, with mean > 10-fold increase compared to benign tumor patients, P0.0497, student's t-test (B); 78% (25/32) of breast cancer patients (32 cases) had plasma Hsp90 α levels above 50ng/ml, with mean > 10-fold increase compared to benign tumor patients, P < 0.001, student's t-test (C); 100% (10/10) pancreatic cancer patients (10 cases) had plasma levels of Hsp90 α above 50ng/ml, with a mean > 10-fold increase, P < 0.05, compared to benign tumor patients, student's t-test (D).

As shown in fig. 6, in patients with liver cancer (17 cases, 7 cases in which metastasis occurred) (a), lung cancer (10 cases, 2 cases in which metastasis occurred) (B), and breast cancer (21 cases, 10 cases in which metastasis occurred) (C), the plasma Hsp90 α content in patients with metastasis was significantly increased compared to those without metastasis, wherein the P value of liver cancer was 0.003, the P value of breast cancer was 0.002, and the student t test.

As shown in figure 7, the content of Hsp90 alpha in the plasma of inflammatory patients, including 10 cases of pneumonia (A) and hepatitis patients (5 cases of hepatitis A and hepatitis B) (B), is between 2 ng/ml and 10ng/ml, and has no significant difference compared with normal people (3 cases), and the P values are 0.2988, 0.5177 and 0.138 respectively, and the student t test.

The above plasma samples, wherein normal human samples were from healthy volunteers, tumor patients and inflammatory patient samples were from Beijing tumor Hospital and Xiamen's first Hospital.

Example 6: hsp90 a in plasma is secreted by tumor cells

Selecting nude mice (purchased from Beijing Wittiaxle laboratory animal technology Co., Ltd.) with average body weight of about 20 g, dividing into two groups, each group containing 6 mice, and inoculating into oxterHela cervical cancer cells (source: ATCC, accession number: CCL-2) were added, and the number of cells per recipient was 10⁶And (4) respectively. The control group was normal mice without tumor inoculation. When mouse tumors grow to an average of 2 cm in diameter (about 20 days), the fundus venous plexus is bled and Hsp90 α in the plasma is detected using an antibody (rat mab) that specifically recognizes Hsp90 α of human origin but not of murine origin. As shown in fig. 8, Hsp90 a in the plasma of mice inoculated with a human tumor was recognized by an antibody that specifically recognizes Hsp90 a of human origin, but not of murine origin, indicating that Hsp90 a in the plasma is secreted by tumor cells.

Example 7: determination that Hsp90 alpha secreted by tumor cells is in C-terminal deletion condition

The composition was prepared using a method described by Hsp90 alpha-pc 3.1-Nhe 1-For-Myc: GCTAGCTAGCGCCACCATGGAACAAAAACTCATCTCAGAAGAGGATCTGCCTGAGGAAACCCAGACCCAAGAC (SEQ ID No.10) and Hsp90 α -pc3.1-Xho 1-Re-nosup: CCCGCTCGAGTGTCTACTTCTTCCATGCGTGATG (SEQ ID No.12), and Pfu DNA polymerase (origin: NEB), using pQE80L-Hsp90 alpha plasmid obtained in example 3 as template, amplifying to obtain Hsp90 alpha full-length sequence, constructing into pcDNA3.1/Myc-His (-) (origin of vector: Invitrogen) by enzyme digestion and connection, obtaining Hsp90 alpha with an additional Myc tag at N terminal, and naming as Myc-H; or using a mixture of Hsp90 alpha-pc 3.1-Nhe 1-For-His-Myc: GCTAGCTAGCGCCACCATGCATCATCATCATCATCATGAACAAAAACTCATCTCAGAAGAGGATCTGCCTGAGGAAACCCAGACCCAAGAC (SEQ ID No.11) and the nucleotide sequence of SEQ ID No.12 (synthesized from Invitrogen) was additionally added a continuous His-Myc tag at its N-terminus, named His-Myc-H. Both vectors were transiently transfected to over-express in the human breast cancer cell line MCF-7 and secretion of exogenous Hsp90 a (i.e., over-expressed Hsp90 a, as distinguished from endogenous, background Hsp90 a) was observed. Changes in Hsp90 a secreted into extracellular medium were detected with anti-Hsp 90a, anti-Myc tag, anti-His tag antibodies. The results showed that Hsp90 a secreted extracellularly was in the form of a C-terminal deletion (fig. 9A).

Mutation is carried out on the last four amino acids (EEVD) at the C terminal of the Myc-His-H by taking the Myc-His-H as a template, wherein EE- > AA indicates two EE mutations to two Ala (amplified using primers consisting of the nucleotide sequences of SEQ ID No.11 and Hsp90 alpha-EE-AA: GGCCGCTCGAGTGTCTACTGCTGCCATGCGTGATGTG (SEQ ID No. 13)), VD- > AA indicates two VD mutations to two Ala (amplified using primers consisting of the nucleotide sequences of SEQ ID No.11 and Hsp90 alpha-VD-AA: GGCCGCTCGAGTTGCTGCTTCTTCCATGCGTGATGTG (SEQ ID No. 14)), All Ala indicates EEVD mutations to Ala (amplified using primers consisting of the nucleotide sequences of SEQ ID No.11 and 90 alpha-VD EEAAAA: GGCCGCTCGAGTTGCTGCTGCTGCCATGCGTGATGTG (SEQ ID No. 15)), and C.DELTA.4 indicates the last four amino acid deletion of EEVD (amplified using primers consisting of the nucleotide sequences of SEQ ID No.11 and Hsp90 alpha-C.DELTA.4-Xho: CCGCTCGAGTCATGCGTGATGTGTCGTCATCTC (SEQ ID No. 16)). In the human breast cancer cell line MCF-7, several mutants were transiently transfected and over-expressed, and secretion of exogenous Hsp90 a (i.e., over-expressed Hsp90 a, as distinguished from endogenous, background Hsp90 a) was observed. Changes in Hsp90 a secreted into extracellular medium were detected with antibodies against Hsp90 a. The results show that the secretion of Hsp90 a is regulated by four amino acids at the C-terminal, and that any point mutation or deletion of the four amino acids can result in that Hsp90 a secreted to the outside is no longer present in the form of a C-terminal deletion, demonstrating that Hsp90 a secreted to the outside lacks the four amino acids at the C-terminal EEVD (fig. 9B).

Example 8: detection of the form of Hsp90 a in human plasma

Taking whole blood collected within 24 hours of a liver cancer patient, centrifuging twice to take plasma, and detecting the form of Hsp90 alpha in the plasma by adopting a co-immunoprecipitation and immunoblotting method. Namely, firstly, a rabbit polyclonal antibody (source: Labvision) which specifically recognizes Hsp90 alpha is used to carry out immunoprecipitation on Hsp90 alpha in plasma, and then a rabbit polyclonal antibody (Anti-C4) which specifically recognizes EEVD (4 amino acids at the C terminal of Hsp90 alpha) (self-made, an antigen used for immunization is a peptide segment which is synthesized from Siberian cockroaches and is coupled with a carrier protein) which is used for detecting the condition that the C terminal of Hsp90 alpha in plasma is deleted is used. As a result, as shown in fig. 10, the rabbit polyclonal antibody specifically recognizing the C-terminal 4 amino acids EEVD of Hsp90 α was able to recognize Hsp90 α in whole cells, but not Hsp90 α in plasma, indicating that Hsp90 α in plasma is a form of deletion of 4 amino acids from the C-terminal, unlike Hsp90 α in cells (fig. 10).

Example 9: detection of the phosphorylated form of Hsp90 a in human plasma

Taking whole blood collected within 24 hours of a liver cancer patient, centrifuging twice to take plasma, and detecting the form of Hsp90 alpha in the plasma by adopting a co-immunoprecipitation and immunoblotting method. Namely, Hsp90 a in plasma was first immunoprecipitated with rabbit polyclonal antibody (source: Labvision) specifically recognizing Hsp90 a, and threonine phosphorylation at the 90 th site of Hsp90 a in plasma was detected with antibody (Rabbitanti-phosphorus- (Ser/Thr) PKA substrate pAb, Cell signaling) specifically recognizing threonine phosphorylation at the 90 th site of Hsp90 a.

As a result, as shown in fig. 11, Hsp90 a in plasma was able to be recognized by an antibody that specifically recognizes threonine phosphorylation at position 90 of Hsp90 a, indicating that Hsp90 a in plasma is a form of threonine phosphorylation at position 90.

Example 10: determination of the threonine phosphorylated Hsp90 alpha content at position 90 in human plasma samples

Taking whole blood collected within 24 hours of normal people and liver cancer patients, centrifuging twice, taking blood plasma, and detecting the relative content of Hsp90 alpha in the blood plasma by adopting a sandwich ELISA method. The operation steps are that firstly, self-made rabbit source polyclonal antibody S2 (the preparation method is described in example 3) is used for plating at 4 ℃ overnight, the sample of the blood plasma to be detected is added after being sealed for one hour at 37 ℃, the blood plasma to be detected is diluted by 10 times, and 100 microliters of the blood plasma is added into each hole; after 2 hours of incubation at 37 ℃, an antibody (Cell signaling) specifically recognizing threonine phosphorylated Hsp90 a at the 90 th site was added, and incubation was performed for 2 hours at 37 ℃; the goat anti-rabbit secondary antibody conjugated with horseradish peroxidase was then incubated for 1 hour, developed using o-phenylenediamine and read at OD490 nm. The results show that the detection value of the plasma of the liver cancer patients is obviously higher than that of normal people, the P value is 0.003, and the student t test shows that the content of threonine phosphorylated Hsp90 alpha at the 90 th site in the plasma of the liver cancer patients is increased (figure 12).

Example 11: consistency test of threonine phosphorylated Hsp90 a content and total Hsp90 a content at position 90 in human plasma samples

The same plasma aliquots (8 total aliquots) of liver cancer patients were tested for threonine phosphorylated Hsp90 a content and total Hsp90 a content at site 90 simultaneously. The total content of Hsp90 a was determined as in example 5; the method for detecting the content of threonine phosphorylated Hsp90 a at the 90 th site is the same as that in example 10. The results showed that the threonine-phosphorylated Hsp90 a content at the 90 th site and the total Hsp90 a content in plasma of patients with liver cancer were consistent, and further demonstrated that Hsp90 a in plasma was phosphorylated at the 90 th site, and that the increase in the total Hsp90 a content represented the increase in the threonine-phosphorylated Hsp90 a content at the 90 th site (fig. 13).

Example 12: threonine phosphorylation at position 90 is essential for the secretion of Hsp90 a

Using the pcDNA3.1-Myc-His-Hsp90 a plasmid obtained in example 3 as a template (also known as wild-type Hsp90 a (WT Hsp90 a)), the plasmid was prepared using Hsp90 a-T89A-Sense: GATCGAACTCTTGCAATTGTGGATACTGGAATTGGAATG (SEQ ID No.17) and Hsp90 α -T89-AntiSense: CATTCCAATTCCAGTATCCACAATTGCAAGAGTTCGATC (SEQ ID No.18) to construct mutant Hsp90 alpha (T90A), namely threonine at position 90 is mutated to alanine, so that the threonine cannot be phosphorylated, and the primer is named as (T90AHsp90 alpha). In the human breast cancer cell line MCF-7 (purchased from ATCC, No. HTB-22), wild-type Hsp90 a (WT) and mutant and Hsp90 a (T90A) were overexpressed, and the medium was collected and observed for secretion of exogenous Hsp90 a (i.e., overexpressed Hsp90 a, as distinguished from endogenous, background Hsp90 a). Changes in Hsp90 a secreted into extracellular medium were detected with antibodies against Hsp90 a.

The results showed that exogenous wild-type Hsp90 a could be detected extracellularly, whereas T90A mutant could not be detected, confirming that phosphorylation of threonine at position 90 is essential for secretion of Hsp90 a (fig. 14).

Example 13: dephosphorylation of threonine 90 of Hsp90 alpha by PP5

Preparation of threonine-phosphorylated Hsp90 a at position 90 (pT90-Hsp90 a): recombinant human Hsp90 a protein and recombinant protein kinase a (Promega, usa) were incubated in reaction buffer (NEB, uk) for 1 hour at 30 ℃ with mixing, and then the pT90-Hsp90 a protein was purified and the free phosphate groups were removed by three dialysis. The purified pT90-Hsp90 alpha protein and recombinant human PP5 protein were mixed and incubated at 30 degrees, and the released free phosphate groups were detected using a non-radioactive serine/threonine phosphatase activity detection kit (Promega, USA). The polypeptide substrate is a component of the kit and serves as a positive control. The results are shown in fig. 15A, PP5 was incubated with the polypeptide substrate, the release of free phosphate groups was significantly increased, P value < 0.005, student t test, indicating that PP5 can directly dephosphorylate the polypeptide substrate (positive control); when the pT90-Hsp90 alpha protein and PP5 are incubated together, the release of free phosphate groups is also obviously increased, the P value is less than 0.005, and the student t test shows that PP5 can directly dephosphorize threonine at the 90 th position of Hsp90 alpha.

The full-length sequence of PP5 (SEQ ID No.6) was amplified from a human liver cDNA library and constructed to pcDNA3.1/Myc-His (-) (vector source: Invitrogen) after correct gene sequencing. The vector is transferred into a human breast cancer cell line MCF-7 for overexpression. Or 5'-ACTCGAACACCTCGCTAAAGAGCTC-3' (SEQ ID No.7) is used as a target sequence of siRNA of PP5 by utilizing an RNA interference technology, specific small RNA (synthesized by Invitrogen) aiming at human PP5 is transferred, the expression of human PP5 is inhibited, the phosphorylation of threonine at position 90 of Hsp90 alpha is observed, and as a result, as shown in figure 15B, after excessive expression of human PP5, threonine phosphorylated Hsp90 alpha (pT90-Hsp90 alpha) at position 90 is obviously reduced (which is 0.55 of a control group); threonine-phosphorylated Hsp90 a (pT90-Hsp90 a) at position 90 increased significantly (as compared to 1.58 of the control) when the expression of endogenous human PP5 was suppressed.

Example 14: modulating levels of secreted Hsp90 a by promoting or inhibiting expression of PP5

PP5 is capable of causing phosphorylation dephosphorylation of threonine 90 of Hsp90 a. PP5-NheI-For was used: CTAGCTAGCATGTACCCATACGACGTCCCAGACTACGCT (SEQ ID No.19) and PP 5-XhoI-Re: CCGCTCGAGTTAATGATGATGATGATGATGCACGTGTACC (SEQ ID No.20), and the full-length sequence of PP5 was obtained by amplifying the cDNA library of human liver with primers consisting of nucleotide sequence, and was constructed into pcDNA3.1/Myc-His (-) (vector source: Invitrogen) after the gene sequencing was correct. The vector is transferred into a human breast cancer cell line MCF-7, overexpression is carried out, the level of Hsp90 alpha secreted by the cells is observed, and the result shows that the Hsp90 alpha secreted by the cells is obviously reduced after human PP5 is overexpressed (FIG. 16A).

In the breast cancer cell line MCF-7, inhibition of expression of human PP5 using RNA interference technology (i.e. transfer of specific small RNA for human PP5, Invitrogen) modulated the level of cellular secretion of Hsp90 a, showing that cellular secretion of Hsp90 a was significantly increased when endogenous expression of human PP5 was inhibited (fig. 16B).

Example 15: detection of PP5 content and determination of tumor malignancy

In human breast cancer cell lines MCF-7, SKBR3, MDA-MB-453, 435s and 231(ATCC, numbered HTB-22, -30, -131, -129 and HTB-26, respectively), the relationship between the expression level of PP5 in cells and the secretion of Hsp90 alpha by cells was examined by immunoblotting. MCF-7 and SKBR3 are breast cancer cell lines with low malignancy, and in a nude mouse tumor formation model, the two cell lines can only form in-situ tumors but can not generate metastasis; MDA-MB-453, 435s and 231 are breast cancer cell lines with higher malignancy, and in a nude mouse tumor formation model, the two cell lines can form in-situ tumor and can generate metastasis, wherein the MDA-MB-435s and 231 are commonly used for establishing a tumor metastasis model. In FIG. 17, the five breast cancer cell lines are arranged in order of the degree of malignancy from low to high.

The results showed that when the expression level of PP5 in cells was high, the cells secreted less Hsp90 a, and when the expression level of PP5 in cells was low, the cells secreted more Hsp90 a (fig. 17); meanwhile, the level of secretory Hsp90 alpha is positively correlated with the malignancy of the tumor, PP5 is negatively correlated with the malignancy of the tumor (FIG. 17), and the levels of secretory Hsp90 alpha and its regulatory factor PP5 can be used for judging the malignancy of the tumor.

Example 16: relationship between PP5 expression level and tumor cell migration ability

The relationship between the expression level of PP5 and the migration capacity of tumor cells was examined by using a wound healing model (wound healing model).

In the human breast cancer cell line MCF-7, over-expressing human PP5 or reducing the expression of endogenous PP5 by using small RNA interference, then respectively inoculating 12-hole plates, scraping partial cells by using a gun tip when the cells are close to the bottom of a full culture dish to form a wound, sucking the scraped cells, replacing fresh DMEM (GIBCO) medium, and placing the cells in an incubator at 37 ℃ for continuous culture. Photographs were taken at 0h, 12h, 24h to record "wounds" (FIG. 18A). The effect of the expression level of PP5 on cell migration was examined by the rate of "wound" healing. The results show that over-expression of PP5 can inhibit the cell migration ability of MCF-7, while PP5 interference can promote the cell migration ability of MCF-7 (FIG. 18B).

Example 17: detection of tumor cell migration inhibitory activity by plasma Hsp90 a specific antibody.

The activity of plasma Hsp90 a-specific antibodies in inhibiting tumor cell migration was tested using a wound healing model (wound healing model).

MCF-7 and MDA-MB-231 cells (ATCC, accession numbers HTB-22 and HTB-26, respectively) were seeded into 12-well plates, and when the cells approached the bottom of the full-length dish, a portion of the cells were scraped off using a gun tip to form a "wound", the scraped cells were aspirated away, replaced with fresh DMEM medium (GIBCO), and a mouse monoclonal antibody specific for plasma Hsp90 α, E9(20 μ g/ml) or control IgG (20 μ g/ml), were added, and the culture was continued in an incubator at 37 ℃. Pictures were taken at 0h, 6h, 12h, 24h, 48h and 72h to record "wounds". The inhibition of cell migration by Hsp90 a antibodies was examined by the rate of "wound" healing. The results are shown in FIG. 19. Antibodies specific for plasma Hsp90 a inhibited MDA-MB-231 (fig. 19A) and MCF-7 (fig. 19B) cell migration by > 40%.

Example 18: antibodies specific for plasma Hsp90 a inhibit the detection of tumor metastasis activity.

Nude mice (purchased from Beijing Wittiaxle laboratory animals technology Co., Ltd.) with an average weight of about 20 g were selected, and B16/F10 malignant melanoma cells (ATCC, accession number: CRL-6475) were inoculated into the tail vein of each of the nude mice, the number of which was 2X 10⁵And (4) respectively. The following day, 8 animals were randomly assigned to each group, and a negative control group (IgG) and an administration group (Hsp 90. alpha. Ab) (mouse monoclonal antibody E9) were assigned, respectively, and administered once every other day at a dose of 40. mu.g/mouse, and after 15 days, metastasis was examined. The results are shown in fig. 20, and the specific antibody of plasma Hsp90 a can completely inhibit lymphometastasis of B16/F10 cells (a), and the inhibition rate of lung metastasis is 56% (B).

Sequence listing

<110> Qinghua university

PROTGEN Ltd.

<120> a novel tumor marker

<130>I200901930CB

<160>20

<170>PatentIn version 3.3

<210>1

<211>728

<212>PRT

<213>Homo sapiens

<400>1

Met Pro Glu Glu Thr Gln Thr Gln Asp Gln Pro Met Glu Glu Glu Glu

1 5 10 15

Val Glu Thr Phe Ala Phe Gln Ala Glu Ile Ala Gln Leu Met Ser Leu

20 25 30

Ile Ile Asn Thr Phe Tyr Ser Asn Lys Glu Ile Phe Leu Arg Glu Leu

35 40 45

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

50 55 60

Thr Asp Pro Ser Lys Leu Asp Ser Gly Lys Glu Leu His Ile Asn Leu

65 70 75 80

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

85 90 95

Gly Met Thr Lys Ala Asp Leu Ile Asn Asn Leu Gly Thr Ile Ala Lys

100 105 110

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

115 120 125

Ser Met Ile Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu Val

130 135 140

Ala Glu Lys Val Thr Val Ile Thr Lys His Asn Asp Asp Glu Gln Tyr

145 150 155 160

Ala Trp Glu Ser Ser Ala Gly Gly Ser Phe Thr Val Arg Thr Asp Thr

165 170 175

Gly Glu Pro Met Gly Arg Gly Thr Lys Val Ile Leu His Leu Lys Glu

180 185 190

Asp Gln Thr Glu Tyr Leu Glu Glu Arg Arg Ile Lys Glu Ile Val Lys

195 200 205

Lys His Ser Gln Phe Ile Gly Tyr Pro Ile Thr Leu Phe Val Glu Lys

210 215 220

Glu Arg Asp Lys Glu Val Ser Asp Asp Glu Ala Glu Glu Lys Glu Asp

225 230 235 240

Lys Glu Glu Glu Lys Glu Lys Glu Glu Lys Glu Ser Glu Asp Lys Pro

245 250 255

Glu Ile Glu Asp Val Gly Ser Asp Glu Glu Glu Glu Lys Lys Asp Gly

260 265 270

Asp Lys Lys Lys Lys Lys Lys Ile Lys Glu Lys Tyr Ile Asp Gln Glu

275 280 285

Glu Leu Asn Lys Thr Lys Pro Ile Trp Thr Arg Asn Pro Asp Asp Ile

290 295 300

Thr Asn Glu Glu Tyr Gly Glu Phe Tyr Lys Ser Leu Thr Asn Asp Trp

305 310 315 320

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

325 330 335

Phe Arg Ala Leu Leu Phe Val Pro Arg Arg Ala Pro Phe Asp Leu Phe

340 345 350

Glu Asn Arg Lys Lys Lys Asn Asn Ile Lys Leu Tyr Val Arg Arg Val

355 360 365

Phe Ile Met Asp Asn Cys Glu Glu Leu Ile Pro Glu Tyr Leu Asn Phe

370 375 380

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

385 390 395 400

Glu Met Leu Gln Gln Ser Lys Ile Leu Lys Val Ile Arg Lys Asn Leu

405 410 415

Val Lys Lys Cys Leu Glu Leu Phe Thr Glu Leu Ala Glu Asp Lys Glu

420 425 430

Asn Tyr Lys Lys Phe Tyr Glu Gln Phe Ser Lys Asn Ile Lys Leu Gly

435 440 445

Ile His Glu Asp Ser Gln Asn Arg Lys Lys Leu Ser Glu Leu Leu Arg

450 455 460

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

465 470 475 480

Cys Thr Arg Met Lys Glu Asn Gln Lys His Ile Tyr Tyr Ile Thr Gly

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Thr Lys Glu Gly Leu Glu Leu Pro Glu Asp Glu Glu Glu Lys Lys Lys

545 550 555 560

Gln Glu Glu Lys Lys Thr Lys Phe Glu Asn Leu Cys Lys Ile Met Lys

565 570 575

Asp Ile Leu Glu Lys Lys Val Glu Lys Val Val Val Ser Asn Arg Leu

580 585 590

Val Thr Ser Pro Cys Cys Ile Val Thr Ser Thr Tyr Gly Trp Thr Ala

595 600 605

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

610 615 620

Met Gly Tyr Met Ala Ala Lys Lys His Leu Glu Ile Asn Pro Asp His

625 630 635 640

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

645 650 655

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

660 665 670

Ser Ser Gly Phe Ser Leu Glu Asp Pro Gln Thr His Ala Asn Arg Ile

675 680 685

Tyr Arg Met Ile Lys Leu Gly Leu Gly Ile Asp Glu Asp Asp Pro Thr

690 695 700

Ala Asp Asp Thr Ser Ala Ala Val Thr Glu Glu Met Pro Pro Leu Glu

705 710 715 720

Gly Asp Asp Asp Thr Ser Arg Met

725

<210>2

<211>2184

<212>DNA

<213>Homo sapiens

<400>2

atgcctgagg aaacccagac ccaagaccaa ccgatggagg aggaggaggt tgagacgttc 60

gcctttcagg cagaaattgc ccagttgatg tcattgatca tcaatacttt ctactcgaac 120

aaagagatct ttctgagaga gctcatttca aattcatcag atgcattgga caaaatccgg 180

tatgaaactt tgacagatcc cagtaaatta gactctggga aagagctgca tattaacctt 240

ataccgaaca aacaagatcg aactctcact attgtggata ctggaattgg aatgaccaag 300

gctgacttga tcaataacct tggtactatc gccaagtctg ggaccaaagc gttcatggaa 360

gctttgcagg ctggtgcaga tatctctatg attggccagt tcggtgttgg tttttattct 420

gcttatttgg ttgctgagaa agtaactgtg atcaccaaac ataacgatga tgagcagtac 480

gcttgggagt cctcagcagg gggatcattc acagtgagga cagacacagg tgaacctatg 540

ggtcgtggaa caaaagttat cctacacctg aaagaagacc aaactgagta cttggaggaa 600

cgaagaataa aggagattgt gaagaaacat tctcagttta ttggatatcc cattactctt 660

tttgtggaga aggaacgtga taaagaagta agcgatgatg aggctgaaga aaaggaagac 720

aaagaagaag aaaaagaaaa agaagagaaa gagtcggaag acaaacctga aattgaagat 780

gttggttctg atgaggaaga agaaaagaag gatggtgaca agaagaagaa gaagaagatt 840

aaggaaaagt acatcgatca agaagagctc aacaaaacaa agcccatctg gaccagaaat 900

cccgacgata ttactaatga ggagtacgga gaattctata agagcttgac caatgactgg 960

gaagatcact tggcagtgaa gcatttttca gttgaaggac agttggaatt cagagccctt 1020

ctatttgtcc cacgacgtgc tccttttgat ctgtttgaaa acagaaagaa aaagaacaat 1080

atcaaattgt atgtacgcag agttttcatc atggataact gtgaggagct aatccctgaa 1140

tatctgaact tcattagagg ggtggtagac tcggaggatc tccctctaaa catatcccgt 1200

gagatgttgc aacaaagcaa aattttgaaa gttatcagga agaatttggt caaaaaatgc 1260

ttagaactct ttactgaact ggcggaagat aaagagaact acaagaaatt ctatgagcag 1320

ttctctaaaa acataaagct tggaatacac gaagactctc aaaatcggaa gaagctttca 1380

gagctgttaa ggtactacac atctgcctct ggtgatgaga tggtttctct caaggactac 1440

tgcaccagaa tgaaggagaa ccagaaacat atctattata tcacaggtga gaccaaggac 1500

caggtagcta actcagcctt tgtggaacgt cttcggaaac atggcttaga agtgatctat 1560

atgattgagc ccattgatga gtactgtgtc caacagctga aggaatttga ggggaagact 1620

ttagtgtcag tcaccaaaga aggcctggaa cttccagagg atgaagaaga gaaaaagaag 1680

caggaagaga aaaaaacaaa gtttgagaac ctctgcaaaa tcatgaaaga catattggag 1740

aaaaaagttg aaaaggtggt tgtgtcaaac cgattggtga catctccatg ctgtattgtc 1800

acaagcacat atggctggac agcaaacatg gagagaatca tgaaagctca agccctaaga 1860

gacaactcaa caatgggtta catggcagca aagaaacacc tggagataaa ccctgaccat 1920

tccattattg agaccttaag gcaaaaggca gaggctgata agaacgacaa gtctgtgaag 1980

gatctggtca tcttgcttta tgaaactgcg ctcctgtctt ctggcttcag tctggaagat 2040

ccccagacac atgctaacag gatctacagg atgatcaaac ttggtctggg tattgatgaa 2100

gatgacccta ctgctgatga taccagtgct gctgtaactg aagaaatgcc accccttgaa 2160

ggagatgacg acacatcacg catg 2184

<210>3

<211>732

<212>PRT

<213>Homo sapiens

<400>3

Met Pro Glu Glu Thr Gln Thr Gln Asp Gln Pro Met Glu Glu Glu Glu

1 5 10 15

Val Glu Thr Phe Ala Phe Gln Ala Glu Ile Ala Gln Leu Met Ser Leu

20 25 30

Ile Ile Asn Thr Phe Tyr Ser Asn Lys Glu Ile Phe Leu Arg Glu Leu

35 40 45

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

50 55 60

Thr Asp Pro Ser Lys Leu Asp Ser Gly Lys Glu Leu His Ile Asn Leu

65 70 75 80

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

85 90 95

Gly Met Thr Lys Ala Asp Leu Ile Asn Asn Leu Gly Thr Ile Ala Lys

100 105 110

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

115 120 125

Ser Met Ile Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu Val

130 135 140

Ala Glu Lys Val Thr Val Ile Thr Lys His Asn Asp Asp Glu Gln Tyr

145 150 155 160

Ala Trp Glu Ser Ser Ala Gly Gly Ser Phe Thr Val Arg Thr Asp Thr

165 170 175

Gly Glu Pro Met Gly Arg Gly Thr Lys Val Ile Leu His Leu Lys Glu

180 185 190

Asp Gln Thr Glu Tyr Leu Glu Glu Arg Arg Ile Lys Glu Ile Val Lys

195 200 205

Lys His Ser Gln Phe Ile Gly Tyr Pro Ile Thr Leu Phe Val Glu Lys

210 215 220

Glu Arg Asp Lys Glu Val Ser Asp Asp Glu Ala Glu Glu Lys Glu Asp

225 230 235 240

Lys Glu Glu Glu Lys Glu Lys Glu Glu Lys Glu Ser Glu Asp Lys Pro

245 250 255

Glu Ile Glu Asp Val Gly Ser Asp Glu Glu Glu Glu Lys Lys Asp Gly

260 265 270

Asp Lys Lys Lys Lys Lys Lys Ile Lys Glu Lys Tyr Ile Asp Gln Glu

275 280 285

Glu Leu Asn Lys Thr Lys Pro Ile Trp Thr Arg Asn Pro Asp Asp Ile

290 295 300

Thr Asn Glu Glu Tyr Gly Glu Phe Tyr Lys Ser Leu Thr Asn Asp Trp

305 310 315 320

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

325 330 335

Phe Arg Ala Leu Leu Phe Val Pro Arg Arg Ala Pro Phe Asp Leu Phe

340 345 350

Glu Asn Arg Lys Lys Lys Asn Asn Ile Lys Leu Tyr Val Arg Arg Val

355 360 365

Phe Ile Met Asp Asn Cys Glu Glu Leu Ile Pro Glu Tyr Leu Asn Phe

370 375 380

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

385 390 395 400

Glu Met Leu Gln Gln Ser Lys Ile Leu Lys Val Ile Arg Lys Asn Leu

405 410 415

Val Lys Lys Cys Leu Glu Leu Phe Thr Glu Leu Ala Glu Asp Lys Glu

420 425 430

Asn Tyr Lys Lys Phe Tyr Glu Gln Phe Ser Lys Asn Ile Lys Leu Gly

435 440 445

Ile His Glu Asp Ser Gln Asn Arg Lys Lys Leu Ser Glu Leu Leu Arg

450 455 460

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

465 470 475 480

Cys Thr Arg Met Lys Glu Asn Gln Lys His Ile Tyr Tyr Ile Thr Gly

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Thr Lys Glu Gly Leu Glu Leu Pro Glu Asp Glu Glu Glu Lys Lys Lys

545 550 555 560

Gln Glu Glu Lys Lys Thr Lys Phe Glu Asn Leu Cys Lys Ile Met Lys

565 570 575

Asp Ile Leu Glu Lys Lys Val Glu Lys Val Val Val Ser Asn Arg Leu

580 585 590

Val Thr Ser Pro Cys Cys Ile Val Thr Ser Thr Tyr Gly Trp Thr Ala

595 600 605

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

610 615 620

Met Gly Tyr Met Ala Ala Lys Lys His Leu Glu Ile Asn Pro Asp His

625 630 635 640

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

645 650 655

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

660 665 670

Ser Ser Gly Phe Ser Leu Glu Asp Pro Gln Thr His Ala Asn Arg Ile

675 680 685

Tyr Arg Met Ile Lys Leu Gly Leu Gly Ile Asp Glu Asp Asp Pro Thr

690 695 700

Ala Asp Asp Thr Ser Ala Ala Val Thr Glu Glu Met Pro Pro Leu Glu

705 710 715 720

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

725 730

<210>4

<211>2199

<212>DNA

<213>Homo sapiens

<400>4

atgcctgagg aaacccagac ccaagaccaa ccgatggagg aggaggaggt tgagacgttc 60

gcctttcagg cagaaattgc ccagttgatg tcattgatca tcaatacttt ctactcgaac 120

aaagagatct ttctgagaga gctcatttca aattcatcag atgcattgga caaaatccgg 180

tatgaaactt tgacagatcc cagtaaatta gactctggga aagagctgca tattaacctt 240

ataccgaaca aacaagatcg aactctcact attgtggata ctggaattgg aatgaccaag 300

gctgacttga tcaataacct tggtactatc gccaagtctg ggaccaaagc gttcatggaa 360

gctttgcagg ctggtgcaga tatctctatg attggccagt tcggtgttgg tttttattct 420

gcttatttgg ttgctgagaa agtaactgtg atcaccaaac ataacgatga tgagcagtac 480

gcttgggagt cctcagcagg gggatcattc acagtgagga cagacacagg tgaacctatg 540

ggtcgtggaa caaaagttat cctacacctg aaagaagacc aaactgagta cttggaggaa 600

cgaagaataa aggagattgt gaagaaacat tctcagttta ttggatatcc cattactctt 660

tttgtggaga aggaacgtga taaagaagta agcgatgatg aggctgaaga aaaggaagac 720

aaagaagaag aaaaagaaaa agaagagaaa gagtcggaag acaaacctga aattgaagat 780

gttggttctg atgaggaaga agaaaagaag gatggtgaca agaagaagaa gaagaagatt 840

aaggaaaagt acatcgatca agaagagctc aacaaaacaa agcccatctg gaccagaaat 900

cccgacgata ttactaatga ggagtacgga gaattctata agagcttgac caatgactgg 960

gaagatcact tggcagtgaa gcatttttca gttgaaggac agttggaatt cagagccctt 1020

ctatttgtcc cacgacgtgc tccttttgat ctgtttgaaa acagaaagaa aaagaacaat 1080

atcaaattgt atgtacgcag agttttcatc atggataact gtgaggagct aatccctgaa 1140

tatctgaact tcattagagg ggtggtagac tcggaggatc tccctctaaa catatcccgt 1200

gagatgttgc aacaaagcaa aattttgaaa gttatcagga agaatttggt caaaaaatgc 1260

ttagaactct ttactgaact ggcggaagat aaagagaact acaagaaatt ctatgagcag 1320

ttctctaaaa acataaagct tggaatacac gaagactctc aaaatcggaa gaagctttca 1380

gagctgttaa ggtactacac atctgcctct ggtgatgaga tggtttctct caaggactac 1440

tgcaccagaa tgaaggagaa ccagaaacat atctattata tcacaggtga gaccaaggac 1500

caggtagcta actcagcctt tgtggaacgt cttcggaaac atggcttaga agtgatctat 1560

atgattgagc ccattgatga gtactgtgtc caacagctga aggaatttga ggggaagact 1620

ttagtgtcag tcaccaaaga aggcctggaa cttccagagg atgaagaaga gaaaaagaag 1680

caggaagaga aaaaaacaaa gtttgagaac ctctgcaaaa tcatgaaaga catattggag 1740

aaaaaagttg aaaaggtggt tgtgtcaaac cgattggtga catctccatg ctgtattgtc 1800

acaagcacat atggctggac agcaaacatg gagagaatca tgaaagctca agccctaaga 1860

gacaactcaa caatgggtta catggcagca aagaaacacc tggagataaa ccctgaccat 1920

tccattattg agaccttaag gcaaaaggca gaggctgata agaacgacaa gtctgtgaag 1980

gatctggtca tcttgcttta tgaaactgcg ctcctgtctt ctggcttcag tctggaagat 2040

ccccagacac atgctaacag gatctacagg atgatcaaac ttggtctggg tattgatgaa 2100

gatgacccta ctgctgatga taccagtgct gctgtaactg aagaaatgcc accccttgaa 2160

ggagatgacg acacatcacg catggaagaa gtagactaa 2199

<210>5

<211>499

<212>PRT

<213>Homo sapiens

<400>5

Met Ala Met Ala Glu Gly Glu Arg Thr Glu Cys Ala Glu Pro Pro Arg

1 5 10 15

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

20 25 30

Thr Gln Ala Asn Asp Tyr Phe Lys Ala Lys Asp Tyr Glu Asn Ala Ile

35 40 45

Lys Phe Tyr Ser Gln Ala Ile Glu Leu Asn Pro Ser Asn Ala Ile Tyr

50 55 60

Tyr Gly Asn Arg Ser Leu Ala Tyr Leu Arg Thr Glu Cys Tyr Gly Tyr

65 70 75 80

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

85 90 95

Lys Gly Tyr Tyr Arg Arg Ala Ala Ser Asn Met Ala Leu Gly Lys Phe

100 105 110

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

115 120 125

Asp Lys Asp Ala Lys Met Lys Tyr Gln Glu Cys Asn Lys Ile Val Lys

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Glu Leu Met Gln Trp Tyr Lys Asp Gln Lys Lys Leu His Arg Lys Cys

195 200 205

Ala Tyr Gln Ile Leu Val Gln Val Lys Glu Val Leu Ser Lys Leu Ser

210 215 220

Thr Leu Val Glu Thr Thr Leu Lys Glu Thr Glu Lys Ile Thr Val Cys

225 230 235 240

Glv Asp Thr His Glv Gln Phe Tyr Asp Leu Leu Asn Ile Phe Glu Leu

245 250 255

Asn Gly Leu Pro Ser Glu Thr Asn Pro Tyr Ile Phe Asn Gly Asp Phe

260 265 270

Val Asp Arg Gly Ser Phe Ser Val Glu Val Ile Leu Thr Leu Phe Gly

275 280 285

Phe Lys Leu Leu Tyr Pro Asp His Phe His Leu Leu Arg Gly Asn His

290 295 300

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

305 310 315 320

Ala Lys Tyr Thr Ala Gln Met Tyr Glu Leu Phe Ser Glu Val Phe Glu

325 330 335

Trp Leu Pro Leu Ala Gln Cys Ile Asn Gly Lys Val Leu Ile Met His

340 345 350

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

355 360 365

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

370 375 380

Leu Trp Ser Asp Pro Gln Pro Gln Asn Gly Arg Ser Ile Ser Lys Arg

385 390 395 400

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

405 410 415

Glu Asn Asn Leu Asp Tyr Ile Ile Arg Ser His Glu Val Lys Ala Glu

420 425 430

Gly Tyr Gln Val Ala His Gly Gly Arg Cys Val Thr Val Phe Ser Ala

435 440 445

Pro Asn Tyr Cys Asp Gln Met Gly Asn Lys Ala Ser Tyr Ile His Leu

450 455 460

Gln Gly Ser Asp Leu Arg Pro Gln Phe His Gln Phe Thr Ala Val Pro

465 470 475 480

His Pro Asn Val Lys Pro Met Ala Tyr Ala Asn Thr Leu Leu Gln Leu

485 490 495

Gly Met Met

<210>6

<211>1500

<212>DNA

<213>Homo sapiens

<400>6

atggcgatgg cggagggcga gaggactgag tgtgctgagc ccccccggga cgaacccccg 60

gctgatggag ctctgaagcg ggcagaggag ctcaagactc aggccaatga ctacttcaaa 120

gccaaggact acgagaacgc catcaagttc tacagccagg ccatcgagct gaaccccagc 180

aatgccatct actatggcaa ccgcagcctg gcctacctgc gcactgagtg ctatggctac 240

gcgctgggag acgccacgcg ggccattgag ctggacaaga agtacatcaa gggttattac 300

cgccgggctg ccagcaacat ggcactgggc aagttccggg ccgcgctgcg agactacgag 360

acggtggtca aggtgaagcc ccatgacaag gatgccaaaa tgaaatacca ggagtgcaac 420

aagatcgtga agcagaaggc ctttgagcgg gccatcgcgg gcgacgagca caagcgctcc 480

gtggtggact cgctggacat cgagagcatg accattgagg atgagtacag cggacccaag 540

cttgaagacg gcaaagtgac aatcagtttc atgaaggagc tcatgcagtg gtacaaggac 600

cagaagaaac tgcaccggaa atgtgcctac cagattctgg tacaggtcaa agaggtcctc 660

tccaagctga gcacgctcgt ggaaaccaca ctcaaagaga cagagaagat tacagtatgt 720

ggggacaccc atggccagtt ctatgacctc ctcaacatat tcgagctcaa cggtttaccc 780

tcggagacca acccctatat atttaatggt gactttgtgg accgaggctc cttctctgta 840

gaagtgatcc tcaccctttt cggcttcaag ctcctgtacc cagatcactt tcacctcctt 900

cgaggcaacc acgagacaga caacatgaac cagatctacg gtttcgaggg tgaggtgaag 960

gccaagtaca cagcccagat gtacgagctc tttagcgagg tgttcgagtg gctcccgttg 1020

gcccagtgca tcaacggcaa agtgctgatc atgcacggag gcctgttcag tgaagacggt 1080

gtcaccctgg atgacatccg gaaaattgag cggaatcgac aacccccaga ttcagggccc 1140

atgtgtgacc tgctctggtc agatccacag ccacagaacg ggcgctcgat cagcaagcgg 1200

ggcgtgagct gtcagtttgg gcctgacgtc accaaggcct tcttggaaga gaacaacctg 1260

gactatatca tccgcagcca cgaagtcaag gccgagggct acgaggtggc tcacggaggc 1320

cgctgtgtca ccgtcttctc tgcccccaac tactgcgacc agatggggaa caaagcctcc 1380

tacatccacc tccagggctc tgacctacgg cctcagttcc accagttcac agcagtgcct 1440

catcccaacg tcaagcccat ggcctatgcc aacacgctgc tgcagctagg aatgatgtga 1500

<210>7

<211>25

<212>DNA

<213>Artificial

<220>

<223>Target sequence of PP5 siRNA

<400>7

actcgaacac ctcgctaaag agctc 25

<210>8

<211>30

<212>DNA

<213>Artificial

<220>

<223>Hsp90α-Sal1-Re

<400>8

acgcgtcgac ttagtctact tcttccatgc 30

<210>9

<211>31

<212>DNA

<213>Artificial

<220>

<223>Hsp90α-Sph1-For

<400>9

acatgcatgc atgcctgagg aaacccagac c 31

<210>10

<211>73

<212>DNA

<213>Artificial

<220>

<223>Hsp90α-pc3.1-Nhe1-For-Myc：

<400>10

gctagctagc gccaccatgg aacaaaaact catctcagaa gaggatctgc ctgaggaaac 60

ccagacccaa gac 73

<210>11

<211>91

<212>DNA

<213>Artificial

<220>

<223>Hsp90α-pc3.1-Nhe1-For-His-Myc：

<400>11

gctagctagc gccaccatgc atcatcatca tcatcatgaa caaaaactca tctcagaaga 60

ggatctgcct gaggaaaccc agacccaaga c 91

<210>12

<211>34

<212>DNA

<213>Artificial

<220>

<223>Hsp90α-pc3.1-Xho1-Re-nostop

<400>12

cccgctcgag tgtctacttc ttccatgcgt gatg 34

<210>13

<211>37

<212>DNA

<213>Artificial

<220>

<223>Hsp90α-EE-AA

<400>13

ggccgctcga gtgtctactg ctgccatgcg tgatgtg 37

<210>14

<211>37

<212>DNA

<213>Artificial

<220>

<223>Hsp90α-VD-AA

<400>14

ggccgctcga gttgctgctt cttccatgcg tgatgtg 37

<210>15

<211>37

<212>DNA

<213>Artificial

<220>

<223>Hsp90α-EEVD-AAAA

<400>15

ggccgctcga gttgctgctg ctgccatgcg tgatgtg 37

<210>16

<211>33

<212>DNA

<213>Artificial

<220>

<223>Hsp90α-CΔ4-Xho

<400>16

ccgctcgagt catgcgtgat gtgtcgtcat ctc 33

<210>17

<211>39

<212>DNA

<213>Artificial

<220>

<223>Hsp90α-T89A-Sense

<400>17

gatcgaactc ttgcaattgt ggatactgga attggaatg 39

<210>18

<211>39

<212>DNA

<213>Artificial

<220>

<223>Hsp90α-T89-AntiSense

<400>18

cattccaatt ccagtatcca caattgcaag agttcgatc 39

<210>19

<211>39

<212>DNA

<213>Artificial

<220>

<223>PP5-NheI-For

<400>19

ctagctagca tgtacccata cgacgtccca gactacgct 39

<210>20

<211>40

<212>DNA

<213>Artificial

<220>

<223>PP5-XhoI-Re

<400>20

ccgctcgagt taatgatgat gatgatgatg cacgtgtacc 40

Claims (17)

1. An isolated polypeptide consisting of the amino acid sequence of SEQ ID No. 1.

2. The polypeptide of claim 1, wherein one or more amino acid residues in the amino acid sequence of SEQ ID No.1 selected from the group consisting of: threonine at position 90, serine at position 231, serine at position 263, tyrosine at position 309, and combinations thereof.

3. The polypeptide of claim 2, wherein the threonine at position 90 is phosphorylated.

4. Use of a specific binder for a polypeptide according to any one of claims 1-3, wherein said specific binder is an antibody that specifically binds to a phosphorylated said polypeptide, for the preparation of a kit for determining the presence, stage and/or metastasis of a tumor by detecting the level of the polypeptide consisting of the amino acid sequence of SEQ ID No.1 in plasma.

5. Use of a specific binder for a polypeptide according to any one of claims 1-3, wherein said specific binder is an antibody that specifically binds to a phosphorylated said polypeptide, for the preparation of a kit for tumor screening of high risk groups by detecting the level of the polypeptide consisting of the amino acid sequence of SEQ ID No.1 in plasma.

6. Use of a specific binder for a polypeptide according to any one of claims 1-3, wherein said specific binder is an antibody that specifically binds to a phosphorylated said polypeptide, for the preparation of a kit for the determination of the prognosis of a patient with a tumor by detecting the level of the polypeptide consisting of the amino acid sequence of SEQ ID No.1 in the plasma.

7. Use of a specific binder for a polypeptide according to any of claims 1-3, wherein said specific binder is an antibody that specifically binds to a phosphorylated said polypeptide, for the preparation of a kit for determining whether surgery, radiotherapy or drug treatment is effective and/or for deciding when to stop the treatment in a patient with a tumor by detecting the level of the polypeptide consisting of the amino acid sequence of SEQ ID No.1 in plasma.

8. The use of any one of claims 4-7, wherein the tumor is selected from the group consisting of lung cancer, liver cancer, stomach cancer, esophageal cancer, osteosarcoma, pancreatic cancer, lymphoma, colon cancer, breast cancer, prostate cancer, oral cancer, nasopharyngeal cancer, cervical cancer, leukemia, malignant melanoma, sarcoma, renal cancer, biliary cancer.

9. The use of claim 8, wherein the antibody is a monoclonal antibody or an antigen-binding fragment thereof.

10. The use of claim 9, wherein the antigen binding fragment is selected from the group consisting of scFv, Fab ', and F (ab') 2.

11. Use according to any one of claims 4 to 7, wherein the polypeptide is phosphorylated at one or more amino acid residues corresponding to SEQ ID No.1 selected from the group consisting of: threonine at position 90, serine at position 231, serine at position 263, tyrosine at position 309, and combinations thereof.

12. The use of claim 11, wherein the antibody specifically binds threonine at position 90 is a phosphorylated said polypeptide.

13. Use of an antibody specific for a polypeptide according to any one of claims 1-3, wherein said antibody specifically binds to a phosphorylated said polypeptide, wherein said polypeptide is phosphorylated at one or more amino acid residues corresponding to SEQ ID No.1 selected from the group consisting of: threonine at position 90, serine at position 231, serine at position 263, tyrosine at position 309, and combinations thereof.

14. The use of claim 13, wherein the antibody is a humanized antibody or an antigen-binding fragment thereof.

15. The use of claim 13, wherein the antibody specifically binds threonine at position 90 is a phosphorylated said polypeptide.

16. The use of claim 13, wherein the antibody is monoclonal antibody E9 or D10 produced by a cell line with a accession number of CGMCC No.2903 or 2904.

17. The use of any one of claims 13-16, wherein the tumor is selected from the group consisting of lung cancer, liver cancer, stomach cancer, esophageal cancer, osteosarcoma, pancreatic cancer, lymphatic cancer, colon cancer, breast cancer, prostate cancer, oral cancer, nasopharyngeal cancer, cervical cancer, leukemia, malignant melanoma, sarcoma, renal cancer, biliary cancer.