WO2012152222A1 - 一种双功能肿瘤诊断试剂及方法 - Google Patents

一种双功能肿瘤诊断试剂及方法 Download PDF

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WO2012152222A1
WO2012152222A1 PCT/CN2012/075291 CN2012075291W WO2012152222A1 WO 2012152222 A1 WO2012152222 A1 WO 2012152222A1 CN 2012075291 W CN2012075291 W CN 2012075291W WO 2012152222 A1 WO2012152222 A1 WO 2012152222A1
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ferritin
tissue
protein
tumor
bifunctional
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French (fr)
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阎锡蕴
梁敏敏
范克龙
杨东玲
卢迪
冯静
段德民
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中国科学院生物物理研究所
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Priority to US14/117,229 priority Critical patent/US20150037817A1/en
Priority to EP12782401.9A priority patent/EP2733490B1/en
Priority to JP2014509596A priority patent/JP6117185B2/ja
Publication of WO2012152222A1 publication Critical patent/WO2012152222A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7023—(Hyper)proliferation
    • G01N2800/7028—Cancer

Definitions

  • the invention belongs to the intersection of nanotechnology, biomimetic, immunology and biomedicine.
  • the present invention provides a tumor diagnostic test which combines the specific recognition of cancer and the dual function of color development, and a method for the diagnosis of cancer tissues and cancer cells using the tumor diagnostic reagent. Background technique
  • Pathological biopsy is the most accurate and reliable, and is recognized as the "gold standard" for tumor diagnosis at home and abroad (Shi, et al, (2008) Am. J. Clin. Pathol, 129:358-366; Taylor et al. (2006) Biotech Histochem., 81:3-12.; Larsson, et al, (1988) Immunocytochemistry: Theory and Practice. Boca Raton, FL: CRC Press 41-73).
  • the main staining methods for pathological sections are hematoxylin-eosin staining (HE:), immunohistochemistry, and immunofluorescence.
  • HE staining mainly uses hematoxylin to make the chromatin in the nucleus purple-blue.
  • the common eosin makes the cytoplasm and the extracellular matrix are red, thus providing information on cell morphology.
  • the pathologists based on these cell morphology. Change, roughly identify the cancerous changes of tissue cells.
  • the staining method is convenient and rapid, but it can only provide changes in histomorphology, and the amount of information is small. For more complex and polymorphic tumors, correct judgment cannot be made.
  • Immunohistochemistry or immunofluorescence uses a primary antibody to bind to the detected tissue antigen, a fluorescent signal molecule or an enzyme-labeled secondary antibody, a triple antibody, and a primary antibody, and then reacts with a fluorescent signal or an enzyme substrate to provide a desired antigen to be detected. Localization and semi-quantitative information to identify pathological changes in tissue cells such as carcinogenesis, necrosis, and inflammatory cell infiltration. Immunohistochemistry or immunofluorescence can provide detailed information on the distribution, content and cell morphology of a particular antigen. It is very meaningful to study pathology in depth, but immunostaining requires the incubation of primary, secondary or even tertiary antibodies.
  • Natural human ferritin is a globular ferritin that is free from 24 heavy or light chain subunits. The ratios are complexed, and the heavy and light chain subunits have high homology with molecular weights of 21 kDa and 19 kDa (Theil, ( ⁇ 9S7) A nu. Rev. Biochem" 56:289-315 ). The subunit composition of ferritin is different in different tissues. The heavy chain subunit is mainly in the heart, while the light chain subunit is more in the liver. The two subunits also change due to environmental requirements, providing elastic iron for the organism. Ion supply.
  • iron ions form crystals together with phosphate and hydroxide ions, which have similar chemical properties to hydrated iron oxide in minerals (Harrison) , et al, (1996) Biochim. Biophys. Acta 1275: 161-203; Levi, et al. (1988) J. Biol. Chem. 263: 18086-18092; Ford, et al. ( ⁇ 9S4) Philos Trans. R. Soc. LondB Biol. Sci. 304:551-565).
  • heavy chain subunit ferritin can specifically bind to activated lymphocytes, leukemia cells, cervical cancer cells, lymphoma cells, etc.
  • the transferrin receptor is a key molecule for obtaining iron in cells, and is required for the rapid growth of proliferating cells and tumor cells.
  • Many tumors such as hepatocellular carcinoma, breast cancer, pancreatic cancer, gastric cancer, colon cancer, etc., highly express Tf l . Therefore, Tf l is a specific target for tumor recognition and treatment (Larrick, et al. (1979) J. Supramol. Struct ⁇ ⁇ 5 ⁇ 9 -5?>6; Daniels, et al. (2006) Clin. Immunol. , ⁇ 2 ⁇ : ⁇ 44- ⁇ 5S; Ryschich, et al. (2004) Eur. J. Caner 40: 1418-1422.
  • TfRl is a co-receptor of heavy chain subunit ferritin, and heavy chain subunit ferritin can be used for tumor specific recognition.
  • the natural protein shells with nano-cavity structures of different sizes mainly include deferoxin, heat shock protein (HSP, diameter 12 nm), DNA binding protein (DPs, diameter 9 nm), acridine synthase (LS, diameter 15 Nm) and viral protein shells, such as: cowpea green mottle virus (CCMV, diameter 28nm) and cowpea mosaic virus protein (CMV, diameter 31nm), these nanocavities provide a nanoparticle growth and can effectively prevent nanoparticles Good aggregation during the synthesis process
  • the spatial structure using this special structure, can synthesize various sizes of inorganic nanoparticles in the cavity to realize the controllable synthesis of nanomaterials in size, shape and crystal structure (Turyanska L., et al.
  • iron oxide nanoparticles have catalytic activity similar to peroxidase.
  • iron oxide nanoparticles can react with substrates of horseradish peroxidase such as DAB and TMB. , producing the same reaction product as peroxidase, thereby producing a catalytic similar to peroxidase.
  • horseradish peroxidase such as DAB and TMB.
  • the invention provides a novel tumor diagnostic reagent and method by utilizing the peroxidase activity of the nano material and combining the tumor recognition protein.
  • the tumor diagnostic reagent provided by the invention consists of a protein shell and a nanoparticle core, and the protein shell can specifically recognize cancer tissues and cells, and the nanoparticle core has peroxidase catalytic activity and can react with the substrate of horseradish peroxidase. color.
  • the tumor diagnostic reagent provided by the invention can realize tissue-specific recognition and complete color development, and does not require primary antibody, secondary antibody, and third antibody to be incubated, and the antibody does not require enzyme or signal molecule labeling, and operates. Simple, convenient and fast.
  • an aspect of the present invention provides a bifunctional tumor diagnostic reagent characterized in that it is composed of a protein shell that specifically recognizes cancer tissues and/or cancer cells and an inorganic nanocore having peroxidase catalytic activity.
  • the protein shell of the present invention which specifically recognizes cancerous tissues and/or cancer cells can be selected from a number of proteins specifically recognizing cancerous tissues and/or cancer cells disclosed in the art.
  • the protein shell of the invention is a genetically recombinant or native ferritin.
  • the deferoxin can be self-assembled from any of 12 or 24 heavy chain subunits and light chain subunits.
  • natural ferritin may be derived from eukaryotes, prokaryotes, preferably from mammals.
  • the protein shell of the invention is a genetically recombined full heavy chain subunit ferritin.
  • the protein shell of the invention is also a heat shock protein (HSP, 12 nm in diameter), a DNA binding protein (DPs, 9 nm in diameter), acridine synthase (LS, 15 nm in diameter) or a viral protein shell.
  • the viral protein shell includes cowpea green mottle virus (CCMV, diameter 28 nm) and cowpea mosaic virus protein (CMV, diameter 31 nm).
  • the protein shells of the invention are chemically coupled or genetically fused to target molecules and/or signal molecules.
  • the targeting molecules of the invention comprise a polypeptide or a nucleic acid aptamer.
  • the targeting molecule of the invention is an antibody.
  • the signal molecules of the invention comprise fluorescent, radionuclides.
  • the inorganic nanocore of the present invention has peroxidase catalytic activity and is capable of reacting with a substrate to develop color, including but not limited to: magnetic iron oxide nanoparticles, iron sulfide nanoparticles, noble metal doped iron oxide. Nanoparticles, bimetallic alloy nanoparticles or cerium oxide nanoparticles.
  • the protein shell of the invention is a genetically recombined full heavy chain subunit ferritin.
  • the inorganic core of the present invention is a magnetic iron oxide nanoparticle.
  • kits comprising the bifunctional tumor diagnostic reagent of the present invention.
  • the kit further comprises a peroxidase substrate.
  • the peroxidase substrate comprises DAB, TMB.
  • the kit of the invention further includes instructions.
  • the use of the dual-function tumor diagnostic reagent of the present invention for preparing a tumor diagnostic reagent is provided.
  • the bifunctional tumor diagnostic reagent of the present invention is composed of a protein shell and a nanoparticle core, and the protein shell can specifically recognize cancer tissues and cells, and the nanoparticle core has peroxidase catalytic activity and can be combined with horseradish peroxidase.
  • the substrate reacts to develop color and can therefore be used to diagnose tumors.
  • a further aspect of the invention provides a biomimetic protein consisting of a protein shell that specifically recognizes cancerous tissue and/or cancer cells and an inorganic nanocore having peroxidase catalytic activity.
  • the biomimetic protein of the invention is a biomimetic ferritin.
  • the protein shell of the invention is a genetically recombinant or native ferritin.
  • the deferoxin can be self-assembled from any of 12 or 24 heavy chain subunits and light chain subunits.
  • the native ferritin can be derived from eukaryotes, prokaryotes, preferably from a mammal.
  • the protein shell of the invention is a genetically recombined full heavy chain subunit ferritin. The invention also provides the use of the biomimetic protein for preparing a tumor detecting reagent.
  • the biomimetic protein of the present invention is composed of a protein shell and a nanoparticle core, and the protein shell can specifically recognize cancer tissues and cells, and the nanoparticle core has peroxidase catalytic activity and can react with a substrate of horseradish peroxidase. Color development, so it can be used to diagnose tumors.
  • a further aspect of the invention provides a method of detecting a tumor in an individual, comprising: obtaining a body tissue or a cell sample from the individual, contacting the sample with the bifunctional tumor diagnostic reagent of the invention or the biomimetic protein of the invention, The substrate is directly developed, thereby determining the presence of the tumor in the individual.
  • the tissue is a perforated biopsy specimen, a postoperative pathological section, or a necropsy tissue of a clinical patient or animal model.
  • the cell is exfoliated cells, tissue lysed cells, or cultured cells in vitro in the body's circulatory system.
  • the reagents and methods of the present invention are useful for clinical cancer screening, early diagnosis, cancer cell metastasis diagnosis, therapeutic monitoring, tumor imaging, necropsy histopathological analysis, or postoperative cancer recurrence evaluation.
  • the tumor diagnostic reagent provided by the invention can realize tissue-specific recognition and color development, and does not require primary antibody, secondary antibody, and third antibody to be incubated, and the antibody does not need enzyme or signal molecule labeling, and operates. Simple, convenient and fast.
  • Figure 1 Among them, A: TEM electron micrograph of bionic ferritin; B: bionic ferritin has peroxidase activity, catalyzes the color reaction of HRP substrate TMB; C: bionic ferritin has peroxidase activity, catalyzes HRP The substrate DAB produces a color reaction.
  • the antibody of (Transferrin receptor 1, Tf l) competes with the bionic ferritin receptor site, a: PBS control, b: Cy5.5-labeled bionic ferritin, c: Excessive anti-TfR1 antibody blocks the receptor site; C : Cell-binding saturation curve of biomimetic ferritin.
  • Figure 5 Staining of various human cancer tissues and normal tissues by bionic ferritin.
  • A1 colon cancer grade II
  • ⁇ 2 colonic mucosa
  • B1 non-specific invasive ductal carcinoma grade I
  • ⁇ 2 mammary fibrosis
  • C1 ovarian serous papillary adenocarcinoma grade III
  • C2 ovarian tissue
  • D1 Prostate cancer grade 4
  • D2 Prostatic smooth muscle tissue
  • E1 Lung squamous cell carcinoma grade III
  • ⁇ 2 Lung tissue with congestion
  • F1 Esophageal cancer grade III
  • F2 Esophageal chronic inflammation with epithelial hyperplasia
  • G1 Thymus Cancer
  • G2 Thymus tissue
  • HI Cervical squamous cell carcinoma grade III
  • H2 Chronic cervicitis.
  • FIG. 6 Fluorescently labeled bionic ferritin shells specifically recognize a variety of human cancer tissues.
  • A1 Colon cancer grade III
  • A2 colonic mucosal chronic inflammation with epithelial hyperplasia
  • B1 non-specific invasive ductal carcinoma of the breast grade I
  • B2 breast tissue
  • C1 ovarian serous papillary adenocarcinoma grade III
  • C2 ovary Tissue
  • D1 colon adenocarcinoma grade II
  • D2 colonic mucosal tissue
  • E1 prostate cancer grade 4
  • E2 benign prostatic hyperplasia with PIN grade I
  • F1 lung squamous cell carcinoma grade III
  • F2 lung tissue
  • G1 Ovarian serous papillary adenocarcinoma grade III
  • G2 ovarian tissue
  • HI lung squamous cell carcinoma grade III
  • H2 lung tissue with congestion
  • II esophageal squamous cell carcinoma grade III
  • Example 1 Preparation of Biomimetic Ferritin
  • bionic ferritin The iron-nanocore full-heavy chain human ferritin, named bionic ferritin, is used for tumor detection.
  • the specific operation is as follows: First, construct the recombinant plasmid of human heavy chain subunit ferritin gene sequence
  • PCR target gene product and pET-12b plasmid (Novagen, Inc., Madison, WI, USA) were digested with restriction endonucleases Ndel and BamHI, and the target gene was ligated to the vector pET-12b, and the obtained gene was obtained.
  • the recombinant plasmid pET12b-HFn of the full-heavy chain subunit ferritin gene sequence is shown in the following figure, wherein the restriction site is indicated by a red arrow and the antibiotic resistance is ampicillin.
  • the formation principle of the pseudo-magnetic ferritin is shown in the following formula (1).
  • the collected product was purified by exclusion chromatography to remove the denatured heteroprotein to obtain a magnetized full-chain subunit of the bionic ferritin.
  • Fig. 1A is a TEM electron micrograph of the synthetic bionic ferritin. It can be seen that the bionic iron egg is uniformly dispersed. The outer diameter of the protein shell is about 12 nm and the inner diameter is about 8 nm.
  • Example 2 Peroxidase activity of bionic ferritin
  • biomimetic ferritin In order to study the binding of biomimetic ferritin to human tumor cells, common human tumor cells were selected. Fluorescent molecularly labeled biomimetic ferritin was incubated, and cell-streaming was used to detect the binding of biomimetic ferritin to each tumor cell.
  • the experimental procedure was as follows: NHS-activated Cy5.5 (Cy5.5-NHS, purchased from GE Healthcare) was labeled onto the bionic ferritin according to the labeling method provided in the instructions. Each strain of cells was cultured to about 1 ⁇ 10 5 , trypsinized, washed three times with 0.3% BSA/PBS, and added with 50 g/ml of Cy5.5-labeled biomimetic ferritin, and incubated at 4 ° C for 45 minutes. The cells were then washed three times with 0.3% BSA/PBS, finally resuspended in PBS, and the sample was fluorescently detected.
  • the binding of the labeled protoplastin to the cells was mostly able to be competed by an excess of unlabeled proto-fetoprotein, and further, the binding of the bionic ferritin to the cancer cells was specific, and the cell surface was only A small amount of non-competitive non-specific binding molecules.
  • mice The paraffin sections of the mouse xenografts were prepared as follows: Cultured HT-29, SMMC-7721 and SKOV-3 tumor cells (purchased from ATCC) were collected, washed in serum-free medium, and resuspended in PBS buffer. Female BALB/c mice (purchased from Beijing Experimental Animal Center) were injected subcutaneously with 5 ⁇ 10 6 HT-29, SMMC-7721 or SKOV-3 cells in the scapular region, and the mice were treated with a tumor diameter of 0.4-0.6 cm.
  • the tumor was removed and fixed with 4% paraformaldehyde for 12-24 hours, rinsed with 70% ethanol solution, and then dehydrated by 70-80-90-95-95-100% ethanol for 35 minutes, immersed in 1:1 ethanol. / xylene solution for 5 minutes, taken out and then immersed in xylene for 20 minutes, transparent 2 times, put into the hot melt paraffin (melting point 56-58 ° C) for 4 hours, remove the waxing tissue and transfer it into the wax-containing carton. The cut surface is facing down, and the paraffin wax surface is condensed and then embedded, and immersed in cold water to solidify to obtain a paraffin block.
  • the paraffin-embedded tissues were cut into 5 ⁇ sections using a Le 1C a paraffin slicer, and spread on a glass slide at 42 ° C. The samples were naturally dried and baked at 50 ° C for 6 hours to obtain HT-29, SMMC-7721 and SKOV. -3 mouse transplanted tumor paraffin sections.
  • the anti-Tf l antibody CD71 stained tumor tissue method is as follows: The paraffin section of the transplanted tumor is dewaxed to water, washed 3 times with PBS, and added to a 0.3% 3 ⁇ 40 2 methanol solution for 30 minutes to eliminate tissue endogenous peroxidation.
  • Enzyme Add 1 mM citrate antigen repair solution, heat in a microwave oven for 30 minutes, antigen retrieval; rinse with deionized water, 5% normal sheep serum blocked at 37 °C for 1 hour, force B Anti-TfRl antibody CD71 (BD Pharmingen ), overnight at 4°C; after rinsing with PBS, add 1000-fold diluted secondary antibody, incubate at 37 °C for 1 hour, rinse with PBS; add HRP-labeled 2000 diluted primary antibody, incubate at 37 °C for 1 hour, rinse with PBS, 1 Add 1 ml of Buffer, 3 ⁇ 40 2 and DAB in DAB deionized water to the deionized water, mix and add to the slide tissue specimen for 5 mm, rinse with deionized water, counterstain with hematoxylin, dehydrated and sealed. , microscopic imaging observation.
  • bionic ferritin-implanted tumor tissue is as follows: dewaxing the paraffin sections of the transplanted tumor tissue, eliminating endogenous peroxidase, antigen repair, and blocking the serum, adding bionic ferritin, incubating for 45 minutes at room temperature, rinsing with PBS, DAB staining, hematoxylin counterstaining, conventional dehydration sealing, microscopic observation.
  • FITC-labeled bionic ferritin-infected mouse xenografts Fluorescein isothiocyanate FITC (purchased from Sigma-Aldrich) was labeled with a bionic ferritin shell according to the method provided in the instructions. After the paraffin section of the transplanted tumor tissue was blocked, add the FITC-labeled bionic ferritin shell and incubate for 45 minutes at room temperature. Clock, PBS rinse 2 times DAPI stained for 10 minutes. The quencher was sealed and observed by fluorescence confocal. As shown in Fig.
  • the bionic ferritin can specifically recognize the tumor cells in the tissue, and the staining region and the antibody CD71 are similar, and the tumor staining effect and the antibody are comparable, and the cell morphology can be clearly distinguished.
  • the fluorescence confocal results of the biomimetic ferritin shell to identify tumor tissue have further verified that the bionic ferritin shell can achieve specific recognition of tumors.
  • bionic ferritin can be integrated with antigen-specific recognition and staining, without the need for traditional immunohistochemical primary antibody, secondary antibody, triple antibody, etc., and does not require HRP enzymes. The modification of signal molecules is therefore simpler, more convenient and faster than traditional immunohistochemistry methods.
  • liver tissue samples came from Beijing Anzhen Hospital and Beijing Cancer Hospital. All liver tissue samples were large samples of surgically resected tumors and adjacent tissues. All tissues were embedded in paraffin.
  • biomimetic ferritin staining tissue sectioning method is briefly described as follows: paraffin-embedded liver cancer tissue and normal liver tissue are dewaxed, endogenous peroxidase is eliminated, antigen-repaired, serum-blocked, and bionic ferritin is added, and incubation is carried out at room temperature. Minutes, PBS rinse, DAB staining, hematoxylin counterstaining, conventional dehydration mounting, microscopic observation.
  • biomimetic ferritin fluorescent staining tissue section is briefly described as follows: paraffin-embedded liver cancer tissue and normal liver tissue are dewaxed, serum is blocked, FITC-labeled bionic ferritin shell is added, and incubation is performed for 45 minutes at room temperature, and DAPI staining is performed twice in PBS. 10 minutes. The quencher was sealed and observed by fluorescence confocal.
  • the results are shown in Fig. 4.
  • the bionic ferritin specifically recognizes cancerous cells in the liver tissue and does not stain the normal tissues surrounding the tumor.
  • the liver tissue chronic inflammation, nodular cirrhosis, biliary cirrhosis and other tissues are also Not recognized.
  • the positive rate was 54/55 in all detected hepatocellular carcinoma tissues, while the positive rate of normal liver tissue, hepatitis, and cirrhosis was 7/45. It was confirmed that the biomimetic ferritin specifically recognizes liver cancer tissues.
  • Example 6 Biomimetic ferritin specifically recognizes multiple human cancer tissues
  • biomimetic iron protein staining tissue section is briefly described as follows: Deacetylation of paraffin-embedded human tumor tissue, normal tissue or corresponding inflammatory tissue, elimination of endogenous peroxidase, addition of bionic ferritin after antigen retrieval and serum blocking, Incubate for 45 minutes at room temperature, rinse with PBS, DAB stain, hematoxylin counterstaining, conventional dehydrated mounting, microscopic observation, tissue recognition results are shown in Figure 5 and Table 2.
  • the biomimetic ferritin fluorescent staining tissue section is briefly described as follows: Paraffin-embedded tumor tissue or normal, inflamed tissue is dewaxed, serum is blocked, FITC-labeled bionic ferritin shell is added, incubation at room temperature for 45 minutes, PBS rinse 2 times, DAPI Dye the core for 10 minutes. The quenching agent was sealed and observed by fluorescence confocal observation. The results are shown in Fig. 6.
  • bionic ferritin is useful for colon cancer, breast cancer, ovarian cancer, colon adenocarcinoma, lung squamous cell carcinoma, prostate cancer, esophageal squamous cell carcinoma, thymic carcinoma, and cervical squamous cell carcinoma. It shows a strong combination, and there is no corresponding combination of its corresponding chronic inflammation of the colon, breast tissue, ovarian tissue, colonic mucosa, lung tissue, benign prostatic hyperplasia, chronic esophagus, thymus tissue, cervical tissue and chronic inflammation of the cervix. .
  • the biomimetic ferritin-based cancer tissue detection and recognition method can be integrated with tumor-specific recognition and staining, without the need for primary antibody, secondary antibody, triple antibody, etc. Or the labeling of the signal molecule, the operation is simple and convenient, and the staining effect on the tumor tissue and the antibody immunohistochemistry method are comparable.
  • Table 2 Specific recognition analysis of clinical tumor tissue and corresponding normal tissues by bionic ferritin
  • Transferrin receptor is a marker of malignant phenotype in human pancreatic cancer and in neuroendocrine carcinoma of the pancreas, Eur. J. Caner, 40: 1418-1422.

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一种双功能肿瘤诊断试剂及方法 技术领域
本发明属于纳米技术、 生物仿生、 免疫学和生物医药的交叉领域。特 别地，本发明提供一种集癌灶特异识别与显色双功能于一体的肿瘤诊断试 剂， 以及所述肿瘤诊断试剂用于癌组织及癌细胞诊断的方法。 背景技术
恶性肿瘤已经成为日益常见且严重威胁人类生命和生活质量的主要 疾病之一。病理切片检查最准确、可靠， 是国内外公认的肿瘤诊断的 "金 标准" ( Shi, et al, (2008) Am. J. Clin. Pathol, 129:358-366; Taylor et al. (2006) Biotech Histochem., 81 :3-12.; Larsson, et al, (1988) Immunocytochemistry: Theory and Practice. Boca Raton, FL: CRC Press 41-73 )。 目前， 病理切片的主要染色方法有苏木精-伊红染色 (HE：)、 免疫 组化、免疫荧光。 HE染色主要通过苏木精使细胞核内的染色质着紫蓝色， 通用伊红使细胞质和细胞外基质中的成分着红色，以此提供细胞形态学的 信息，病理医生根据这些细胞形态学的改变，粗略的鉴定组织细胞的癌变， 该染色方法操作方便、 快速， 但是仅能够提供组织形态学的变化， 信息量 少， 对于较为复杂、 多型性的肿瘤， 就不能作出正确的判断。 免疫组化或 免疫荧光利用一抗与被检测组织抗原结合， 荧光信号分子或酶标记的二 抗、 三抗与一抗结合， 然后通过荧光信号或酶底物反应显色， 提供所要检 测抗原的定位和半定量信息， 进而鉴定组织细胞的癌变、 坏死、 炎性细胞 浸润等病理改变。免疫组化或免疫荧光能够提供某特定抗原的分布、含量 及细胞形态学的详细信息， 对病理学的深入研究十分有意义， 但是免疫染 色需要一抗、 二抗甚至三抗的多歩孵育， PBS反复冲洗， 及酶或是荧光分 子的标记， 歩骤复杂， 操作时间长。 因此， 有必要研究简单、 快速且能够 提供丰富信息量的癌组织、癌细胞检测试剂和方法， 以提高临床病理诊断 效率， 为病人的治疗赢得时间。
天然的人铁蛋白是一种球状储铁蛋白， 由 24个重链或轻链亚基任意 比例复合而成， 重链和轻链亚基具有高度同源性， 分子量分别为 21 kDa 和 19 kDa ( Theil, (\9S7) A nu. Rev. Biochem" 56:289-315 )。 人体不同器官 不同组织中铁蛋白的亚基组成不同， 心脏中以重链亚基为主， 而肝脏则以 轻链亚基较多， 两种亚基也会因环境需求作改变， 为生物体提供弹性的铁 离子供应。然而只有重链亚基才能够利用氧气将二价铁离子转为三价铁离 子， 使铁离子能顺利进入铁蛋白， 所以增加重链亚基， 能增加该细胞利用 铁的能力；而增加轻链亚基，则能够增加铁储存的效率。铁蛋白球壳之中， 铁离子和磷酸盐、氢氧根离子一同形成结晶， 与矿物中的水合氧化铁具有 类似的化学性质（Harrison, et al, (1996) Biochim. Biophys. Acta 1275: 161-203; Levi, et al. (1988) J. Biol. Chem. 263: 18086-18092; Ford, et al. (\9S4) Philos Trans. R. Soc. LondB Biol. Sci. 304:551-565)。
除了在细胞内储存铁，重链亚基铁蛋白还能够特异地结合激活的淋巴 细胞、白血病细胞、宫颈癌细胞、淋巴瘤细胞等（Fargion, et al. (1988) Blood 71 :753-757; Moss, et al.(1992) J. Lab Clin. Med. 119:273; Bretscher, et al. (1983) EMBO J. 2:599-603; Takami, et al. (1986) Biochim. Biophys Acta 884:31-38; Fargion, et al. (1991) Blood, 78: 1056-1061 )。 Li等报道细胞表面 的转铁蛋白受体 1 (transferrin receptor l, Tf l )作为重链亚基铁蛋白的共 受体，调节铁蛋白与细胞的相互作用（Li, et al., (2010) Proc. Natl. Acad. Sci.
[/ S 107: 3505-3510)。 转铁蛋白受体是细胞获得铁元素的关键分子， 是 增殖细胞、 肿瘤细胞快速生长所必需的， 许多肿瘤如肝细胞癌、 乳腺癌、 胰腺癌、 胃癌、 结肠癌等均高表达 Tf l , 因此 Tf l成为肿瘤识别和治疗 的特异靶点（Larrick, et al. (1979) J. Supramol. Struct Λ \ 5Ί 9 -5?>6; Daniels, et al. (2006) Clin. Immunol. , \2\ : \44-\5S; Ryschich, et al. (2004 ) Eur. J. Caner 40: 1418-1422. )。利用 TfRl是重链亚基铁蛋白的共受体， 重链亚基铁蛋白 可用于肿瘤特异识别。
具有不同尺寸大小的纳米空腔结构的天然蛋白壳主要包括去铁蛋白、 热休克蛋白 （HSP, 直径 12 nm)、 DNA结合蛋白 （DPs, 直径 9 nm)、 喋 啶合酶（LS , 直径 15 nm)和病毒蛋白壳，如：豇豆退绿斑驳病毒（CCMV, 直径 28nm) 和豇豆花叶病毒蛋白 （CMV, 直径 31nm)， 这些纳米空腔提 供了一个纳米颗粒生长，并能有效阻止纳米颗粒在合成过程中聚集的良好 空间结构,利用这一特殊结构可以在其空腔内部合成各种尺度的无机纳米 颗粒，实现纳米材料在大小、形状、晶体结构上的可控合成（Turyanska L., et al. (2009) Small, 5: 1738-1741 ; Meldrum FC, et al. (2009) Science, 257:522-523;Galvez N.， et al. (2006) J. Mater. Chem., 16:2757-2761 ; Hennequin B.， et al. (2008) Adv. Mater., 20:3592-3593; Yamashita L, et al. (2004) Chem. Lett, 33: 1158-1159)。此外，这些不同结构的蛋白壳容易化学 偶联或是基因融合上靶向分子和信号分子， 实现癌症的靶向识别和检测， 比如去铁蛋白通过基因融合 RGD能够实现癌细胞靶向识别（Uchida M., et al. (2006) J. Am. Chem. Soc" 128: 16626-16633 )， 通过化学标记信号分子 Cy5.5或纳米空腔装载同位素 64Cu、 钆壳实现肿瘤活体成像（Lm X., et al. (2011) Nano Lett., 11 : 814—819; Geninatti CS.， et al. (2006) Cancer Res., 66: 9196-201. )。
我们课题组于 2007年首次发现氧化铁纳米颗粒具有类似过氧化物酶 的催化活性， 即在过氧化氢存在下， 氧化铁纳米颗粒能够与 DAB , TMB 等辣根过氧化物酶的底物反应， 生成与过氧化物酶相同的反应产物， 从而 产生与过氧化物酶相类似的催化作用。基于这一发现我们提出了氧化铁纳 米颗粒模拟酶的概念 ( Gao L, et al., (2007) Nature Nanotech., 2:577-583； 中 国专利 200610057413.9)。 发明内容
本发明利用纳米材料的过氧化物酶活性， 结合肿瘤识别蛋白， 提供了 一种新型肿瘤诊断试剂及方法。
本发明所提供的肿瘤诊断试剂由蛋白壳及纳米颗粒核组成，蛋白壳能 够特异识别癌组织及细胞， 纳米颗粒核具有过氧化物酶催化活性， 能够同 辣根过氧化酶的底物反应显色。本发明所提供的肿瘤诊断试剂能够实现组 织特异识别、 显色一歩完成， 和传统免疫方法相比， 无需一抗、 二抗、 三 抗的多歩孵育， 抗体无需酶或是信号分子标记， 操作简单、 方便、 快速。
因此， 本发明一方面提供双功能肿瘤诊断试剂， 其特征在于它是由特 异识别癌组织和 /或癌细胞的蛋白壳及具有过氧化物酶催化活性的无机纳 米核组成。 本发明的特异识别癌组织和 /或癌细胞的蛋白壳可从本领域中公开的 众多特异识别癌组织和 /或癌细胞的蛋白中选择。 在一些实施方案中， 本 发明所述蛋白壳是基因重组的或是天然的去铁蛋白。 在一些实施方案中， 去铁蛋白可以由 12个或是 24个重链亚基和轻链亚基任意比例自组装而 成。 本发明中， 天然去铁蛋白可以来源于真核生物、 原核生物， 优选地来 源于哺乳动物。在一些实施方案中， 本发明的蛋白壳是基因重组的全重链 亚基铁蛋白。
在一些实施方案中， 本发明所述蛋白壳还是热休克蛋白 （HSP, 直径 12 nm)、 DNA结合蛋白（DPs,直径 9 nm)、喋啶合酶（LS, 直径 15 nm) 或病毒蛋白壳， 病毒蛋白壳包括豇豆退绿斑驳病毒（CCMV, 直径 28nm) 和豇豆花叶病毒蛋白 （CMV, 直径 31nm)，
在一些实施方案中，本发明所述蛋白壳化学偶联或是基因融合靶向分 子和 /或信号分子。 在一些所述方案中， 本发明所述靶向分子包括多肽或 核酸适配体。在一些所述方案中， 本发明所述靶向分子是抗体。在一些实 施方案中， 本发明所述信号分子包括荧光、 放射核素。
在一些实施方案中， 本发明所述无机纳米核具有过氧化物酶催化活 性， 能够与底物反应显色， 包括但不限于： 磁性氧化铁纳米颗粒、 硫化铁 纳米颗粒、贵金属掺杂氧化铁纳米颗粒、双金属合金纳米颗粒或氧化铈纳 米颗粒。
在一些实施方案中，本发明所述蛋白壳是基因重组的全重链亚基铁蛋 白。 在一些实施方案中， 本发明所述的无机内核为磁性氧化铁纳米颗粒。
本发明另一方面提供肿瘤诊断试剂盒，其包括本发明所述的双功能肿 瘤诊断试剂。在一些实施方案中， 所述试剂盒还包括过氧化物酶底物。在 一些实施方案中， 所述过氧化物酶的底物包括 DAB、 TMB。 在一些实施 方案中， 本发明的试剂盒还包括说明书。
本发明又一方面提供本发明所述双功能肿瘤诊断试剂在制备肿瘤诊 断试剂中的用途。如上所述， 本发明的双功能肿瘤诊断试剂由蛋白壳及纳 米颗粒核组成， 蛋白壳能够特异识别癌组织及细胞， 纳米颗粒核具有过氧 化物酶催化活性， 能够同辣根过氧化酶的底物反应显色， 因此可用于诊断 肿瘤。 本发明又一方面提供仿生蛋白， 其由特异识别癌组织和 /或癌细胞的 蛋白壳及具有过氧化物酶催化活性的无机纳米核组成。 在一些实施方案 中， 本发明的仿生蛋白是仿生铁蛋白。在一些实施方案中， 本发明所述蛋 白壳是基因重组的或是天然的去铁蛋白。在一些实施方案中， 去铁蛋白可 以由 12个或是 24个重链亚基和轻链亚基任意比例自组装而成。在一些实 施方案中， 天然去铁蛋白可以来源于真核生物、 原核生物， 优选地来源于 哺乳动物。在一些实施方案中， 本发明的蛋白壳是基因重组的全重链亚基 铁蛋白。本发明还提供该仿生蛋白在制备肿瘤检测试剂中的用途。如上所 述， 本发明的仿生蛋白由蛋白壳及纳米颗粒核组成， 蛋白壳能够特异识别 癌组织及细胞， 纳米颗粒核具有过氧化物酶催化活性， 能够同辣根过氧化 酶的底物反应显色， 因此可用于诊断肿瘤。
本发明又一方面提供检测个体中肿瘤的方法， 其包括： 从所述个体获 得机体组织或细胞样品，将该样品与本发明所述的双功能肿瘤诊断试剂或 本发明的仿生蛋白接触， 加入底物直接显色， 由此测定所述个体中肿瘤的 存在。在一些实施方案中， 所述组织为临床病人或是动物模型的穿剌活组 织标本、 术后病理切片或尸体剖检组织。在一些实施方案中， 所述细胞为 机体循环系统中脱落细胞、 组织溶解细胞或体外培养细胞。
本发明所述试剂和方法可应用于临床癌症筛查、早期诊断、癌细胞转 移诊断、 治疗监控、 肿瘤成像、 尸体剖检组织病理分析或术后癌症复发评 价。本发明所提供的肿瘤诊断试剂能够实现组织特异识别、显色一歩完成， 和传统免疫方法相比， 无需一抗、 二抗、 三抗的多歩孵育， 抗体无需酶或 是信号分子标记， 操作简单、 方便、 快速。 附图说明
结合下面的附图对本发明的优选实施方案进行说明。
图 1 : 其中， A: 仿生铁蛋白的 TEM电镜图； B: 仿生铁蛋白具有过 氧化物酶活性， 催化 HRP底物 TMB产生颜色反应； C: 仿生铁蛋白具有 过氧化物酶活性， 催化 HRP底物 DAB产生颜色反应。
图 2: 流式分析结直肠癌细胞 HT29和仿生铁蛋白的相互作用。 A: 细胞表面受体竞争结合； a: PBS对照， b: Cy5.5标记的仿生铁蛋白染色， c: 过量未标记的仿生铁蛋白封闭细胞表面受体； B: 过量抗转铁蛋白受体
(Transferrin receptor 1, Tf l) 的抗体竞争结合仿生铁蛋白受体位点， a: PBS对照， b: Cy5.5标记的仿生铁蛋白， c: 过量抗 TfRl 的抗体封闭受 体位点； C: 仿生铁蛋白的细胞结合饱和曲线。
图 3: 仿生铁蛋白特异识别小鼠移植肿瘤， 对肿瘤组织的着色区及染 色效果和抗体具有可比性， （标尺为 100μπι)。
图 4: 仿生铁蛋白特异识别人肝癌组织切片， 不识别正常肝组织或是 肝炎肝、 硬化等组织。 A-D: 4 例肝细胞癌； Ε: 正常肝组织 F: 肝组织 慢性炎 G: 结节性肝硬化 Η: 胆汁性肝硬变 I： 肝细胞癌 J: 正常肝组 织 Κ: 肝细胞癌 L: 肝组织慢性炎， （标尺为 100μπι)。
图 5: 仿生铁蛋白对多种人癌组织及正常组织的染色。 A1: 结肠癌 II 级， Α2: 结肠粘膜组织， B1: 乳腺非特殊性浸润性导管癌 I级， Β2: 乳 腺纤维腺病， C1: 卵巢浆液性乳头状腺癌 III级， C2: 卵巢组织， D1: 前 列腺癌 4级， D2: 前列腺平滑肌组织， E1: 肺鳞状细胞癌 III级， Ε2: 肺 组织伴淤血， F1: 食道细胞癌 III级， F2: 食道慢性炎伴上皮增生， G1: 胸腺癌， G2: 胸腺组织， HI: 宫颈鳞状细胞癌 III级， H2: 宫颈慢性炎。
图 6: 荧光标记的仿生铁蛋白壳对多种人癌组织的特异识别。 A1: 结 肠癌 III级， A2: 结肠粘膜慢性炎伴上皮增生， B1: 乳腺非特殊性浸润性 导管癌 I级， B2: 乳腺组织， C1: 卵巢浆液性乳头状腺癌 III级， C2: 卵 巢组织， D1: 结肠腺癌 II级， D2: 结肠粘膜组织， E1: 前列腺癌 4级， E2: 前列腺增生症伴 PIN I级， F1: 肺鳞状细胞癌 III级， F2: 肺组织， G1: 卵巢浆液性乳头状腺癌 III级， G2: 卵巢组织， HI: 肺鳞状细胞癌 III 级， H2: 肺组织伴淤血， II： 食道鳞状细胞癌 III级， 12: 食道慢性炎伴上 皮增生， J1: 胸腺癌， J2: 胸腺组织， K1: 宫颈鳞状细胞癌 II级， K2: 宫颈组织， L1: 宫颈鳞状细胞癌 III级， L2: 宫颈慢性炎， （标尺为 50μπι)。
图 7: 本发明所用质粒的图谱。 具体实施方式
下列实施例的描述是为了说明目的， 而不意于限制本发明的范围。 实施例 1仿生铁蛋白的制备 通过基因重组全重链人铁蛋白壳， 利用人体生物矿化原理， 将铁离子 转载进入蛋白壳， 再氧化成氧化铁纳米颗粒， 我们仿生合成了包裹有磁性 氧化铁纳米内核的全重链人铁蛋白， 命名为仿生铁蛋白， 用于肿瘤检测。 具体操作如下： 首先构建人全重链亚基铁蛋白基因序列的重组质粒
pET12b-HFn ( Santambrogio P. et al.,(2000), Protein Expr. Purif. , 19：
212-218)。 人骨骼肌总 cDNA文库购自 Invitrogen公司 （D8090-01 , Carlsbad, CA, USA), 利用 PCR技术将全重链人铁蛋白基因从 cDNA中 分离并扩增， 设计的两个引物如下： 正向 PCR引物： 5?-A GTC GCC CAT ATG ACG ACC GCG TCC-3 ' , 酶切位点为 Ndel (如下划线所示）， 加有 7 个酶切位点保护碱基； 反向 PCR引物： 5' -GCC GGA TCC TTA GCT TTC ATT ATC AC -3' , 酶切位点为 BamHI (如下划线所示）， 加有 3个酶切位 点保护碱基。 552 bp的基因产物为全重链人铁蛋白目的基因产物， 序列如 下：
Figure imgf000009_0001
CTTTGACAAGCACACCCTGGGAGACAGTGATAATGAAAGCTAG。 然后将获 得的 PCR目的基因产物和 pET-12b plasmid (Novagen, Inc., Madison, WI, USA)用限制性内切酶 Ndel和 BamHI酶切， 目的基因连接到载体 pET-12b 上， 获得包含有人全重链亚基铁蛋白基因序列的重组质粒 pET12b-HFn， 下图为质粒图谱， 其中酶切位点用红色箭头标出， 抗生素抗性为氨苄。 获得的质粒 pET12b-HFn转染到表达菌株 BL21DE3 (购自北京全式 金生物技术有限公司）中表达重链亚基铁蛋白。将培养的表达目的蛋白菌 液超声裂解， 离心去除大肠杆菌残片， 上清液 65 °C加热 10分钟， 沉淀 杂蛋白， 离心去除， 上清液在排阻色谱 Sepharose 4B柱子上分离纯化， 获 得重链亚基铁蛋白， 电泳鉴定纯度， BCA测定蛋白浓度。
仿生铁蛋白生物矿化简述如下： 将浓度 100 mM的氯化钠溶液除气， 同纯化后的铁蛋白壳加入氮气环境下的密闭反应器中， 反应温度维持在 65 °C， pH值稳定在 8.5， 按照每个铁蛋白壳 1000-5000个 Fe的比率加入 N¾;)2Fe(S04:)2 · 6¾0,双氧水作为氧化剂和 Fe 同时加入， 加入的量按照 ¾02:Fe2+=l :3， 双氧水和 Fe加完后体系继续反应 5分钟， 加热 200 浓 度 300 mM的柠檬酸钠络合剩余的 Fe， 仿生磁性铁蛋白形成原理见下面 公式（1 )。 收集产品经排阻色谱纯化， 去除变性的杂蛋白， 得到磁化的全 重链亚基的仿生铁蛋白。
3Fe^ + + 2 0 : ~ Fc ¾04 + 6 H" ( 1 )
图 1A为合成的仿生铁蛋白的 TEM电镜表征图， 由图可见仿生铁蛋 白分散均匀， 蛋白壳外直径约约为 12 nm， 内径约为 8 nm。 实施例 2 仿生铁蛋白的过氧化物酶活性
基于我们课题组的报道，磁性氧化铁纳米颗粒具有类似过氧化物酶的 催化活性（Gao L, et al. (2007) Nature Nanotech., 2: 577-583. )， 由于仿生铁 蛋白内包裹有氧化铁的内核， 因此应当具有类氧化物酶的活性。我们使用 辣根过氧化物酶的底物检测仿生铁蛋白的酶活性， 具体方法如下： 在仿生 铁蛋白中加入 30% ¾02和 TMB或是 DAB , 观察颜色变化。 如图 1B所 示， 当 TMB中加入仿生铁蛋白和 ¾02后， 变成深蓝色， DAB中加入仿 生铁蛋白和 ¾02后， 变为深棕色， 和辣根过氧化物酶出现同样的颜色反 应， 说明仿生铁蛋白具有类过氧化物酶的活性。 实施例 3 仿生铁蛋白结合肿瘤细胞
为了研究仿生铁蛋白与人肿瘤细胞的结合，选取常见的人肿瘤细胞与 荧光分子标记的仿生铁蛋白进行孵育，应用细胞流式检测仿生铁蛋白与各 肿瘤细胞的结合。
实验方法如下： 按照说明书提供的标记方法， 将 NHS活化的 Cy5.5 (Cy5.5-NHS, 购自 GE Healthcare )标记到仿生铁蛋白上。 培养各株细胞 至 1 X 105左右， 胰酶消化， 0.3%的 BSA/PBS洗细胞三次， 加入 50 g/ml 的 Cy5.5标记的仿生铁蛋白， 4°C孵育 45分钟。然后再用 0.3%的 BSA/PBS 洗细胞三次， 最后重悬于 PBS中， 流式检测样品荧光。
结果如表 1所示， 在所检验的 12株肿瘤细胞中， 仿生铁蛋白与其中 的 11株有反应活性， 分别属于肝细胞癌、 结直肠癌、 乳腺癌、 黑色素瘤、 单核细胞淋巴瘤、 宫颈癌、 白血病和前列腺癌。 证明仿生铁蛋白能够和大 部分癌细胞特异结合。 表 -1
流式分析仿生铁蛋白和人肿瘤细胞的反应活性
Figure imgf000011_0001
* SMMC-7721和 U251购自中国科学院典型培养物保藏委员会细胞库， MX-1购自凯基生物， 其余购自 ATCC。 实施例 4癌细胞和仿生铁蛋白的相互作用
为研究仿生铁蛋白与肿瘤细胞表面受体结合情况， 将不同浓度 Cy5.5 标记的仿生铁蛋白与结直肠癌细胞 HT29孵育， 得出仿生铁蛋白结合饱和 曲线， 并用过量抗 Tf l 的抗体封闭受体位点， 证明 TfRl介导仿生铁蛋 白与癌细胞的相互作用。
实验方法如下： 将培养癌细胞 HT29至 1 X 105左右， 胰酶消化， 0.3% 的 BSA/PBS洗细胞三次， 加入不同浓度的 Cy5.5标记的仿生铁蛋白， 4°C 孵育 45分钟。 然后再用 0.3%的 BSA/PBS洗细胞三次， 最后重悬于 PBS 中，流式检测样品荧光。根据荧光强度值与仿生铁蛋白的浓度作饱和曲线。 结果如图 2C所示， 仿生铁蛋白与肿瘤细胞的结合具有可饱和性， 说明这 种结合是特异的， 具有高亲和力。
接下来根据饱和曲线， 选择合适浓度的 Cy5.5标记的仿生铁蛋白， 作 竞争结合抑制实验。 同样当细胞至 1 X 105左右， 胰酶消化， 缓冲液清洗 后， 同时加入一定浓度的 Cy5.5标记仿生铁蛋白和过量的未标记仿生铁蛋 白， 4°C孵育 45分钟， 洗细胞三次， 最后重悬于 PBS中， 流式检测样品 荧光。 结果如图 2A所示， 标记仿生铁蛋白和细胞的结合， 大部分能够被 过量的未标记仿生铁蛋白给竞争下来，进一歩说明仿生铁蛋白与癌细胞的 结合是特异的， 细胞表面仅有少量的未竞争下来的非特异结合分子。
文献（Li L. et al, (2010) Proc. Natl. Acad. Set 107: 3505-3510 )报道人 铁蛋白和转铁蛋白共用受体 TfRl ,为证实 TfRl介导仿生铁蛋白与癌细胞 的相互作用， 我们用过量的抗 TfRl的抗体与 Cy5.5标记的仿生铁蛋白竞 争细胞表面受体。 具体实验方法如下： 当细胞 HT29培养细胞数至 1 X 105 左右， 胰酶消化， 清洗三次， 共同加入过量的鼠抗人 TfRl 的抗体 CD71 (购自 BD Pharmingen)和 Cy5.5标记的仿生铁蛋白， 4°C孵育 45分钟，用 0.3%的 BSA/PBS洗细胞三次， 最后重悬于 PBS中， 流式检测样品荧光。 结果见图 2B ,约有 50%被竞争下来，说明 TfRl介导仿生铁蛋白与癌细胞 的相互作用。
实施例 5 仿生铁蛋白特异检测小鼠移植肿瘤
为检测仿生铁蛋白对肿瘤组织的识别及染色能力，我们将其与裸鼠移 植瘤组织进行孵育， 并与传统的抗体免疫组化结果相比较。具体实验方法 如下：
小鼠移植瘤石蜡切片的制备方法如下： 收集培养的 HT-29 , SMMC-7721和 SKOV-3肿瘤细胞（购自 ATCC) , 无血清培养基洗涤， 重 悬于 PBS缓冲液中。 雌性 BALB/c小鼠 （购自北京实验动物中心） 在肩 胛部位皮下注射 5 X 106个 HT-29, SMMC-7721或 SKOV-3细胞， 待肿瘤 直径达到 0.4-0.6厘米时， 将小鼠处死， 取出肿瘤， 并用 4%多聚甲醛固定 12-24小时， 用 70%乙醇溶液冲洗， 再由 70-80-90-95-95-100%乙醇逐级脱 水 35分钟，浸入 1 :1乙醇 /二甲苯溶液 5分钟，取出再浸入二甲苯 20分钟 透明 2次， 投入已热熔的石蜡 （溶点 56-58 ° C) 中 4小时， 取出浸蜡组 织移入含熔蜡的纸盒中， 切面朝下， 待石蜡表面凝结后包埋， 浸入冷水中 凝固， 得石蜡块。 用 Le1Ca石蜡切片机将石蜡包埋组织切成 5 μπι切片， 在载玻片上 42 °C展片， 自然干燥后 50 ° C烤片 6小时， 制得 HT-29, SMMC-7721和 SKOV-3的鼠移植肿瘤石蜡切片。
Anti-Tf l 抗体 CD71染移植瘤组织方法简述如下： 将移植瘤石蜡切 片脱蜡至水， PBS冲洗 3次，加入含 0.3 % ¾02的甲醇溶液孵育 30分钟， 消除组织内源性过氧化物酶； 再加 I mM柠檬酸抗原修复液， 微波炉中加 热 30分钟， 抗原修复； 去离子水漂洗后， 5 %普通羊血清 37 °C封闭 1小 时， 力 B Anti-TfRl 抗体 CD71 (BD Pharmingen) , 4°C过夜； PBS漂洗后， 加 1000倍稀释的二抗， 37 °C孵育 1小时， PBS漂洗；加 HRP标记的 2000 稀释的三抗， 37°C孵育 1小时， PBS漂洗， 1 ml去离子水中加 DAB试剂 盒中的 Buffer, ¾02， DAB各 1滴， 混匀后滴加到玻片组织标本上显色 5 mm, 去离子水冲洗， 苏木精复染， 脱水封片， 显微成像观察。
仿生铁蛋白染鼠移植瘤组织方法简述如下：将移植瘤组织石蜡切片脱 蜡、 消除内源性过氧化物酶、 抗原修复、 血清封闭后加入仿生铁蛋白， 室 温孵育 45分钟， PBS漂洗， DAB染色， 苏木精复染， 常规脱水封片， 显 微镜观察。
FITC标记的仿生铁蛋白染鼠移植瘤： 参照说明书提供的方法将异硫 氰酸荧光素 FITC (购自 Sigma- Aldrich) 标记仿生铁蛋白壳。 移植瘤组织 石蜡切片血清封闭后， 加入 FITC标记的仿生铁蛋白壳， 室温孵育 45分 钟， PBS冲洗 2次 DAPI染核 10分钟。 放淬灭剂封片， 荧光共聚焦观察。 结果如图 3所示， 仿生铁蛋白能够特异识别组织中的肿瘤细胞， 着色 区域和抗体 CD71—致， 肿瘤染色效果和抗体具有可比性， 能够清晰的分 辨出细胞形态的变化。仿生铁蛋白壳识别肿瘤组织的荧光共聚焦结果进一 歩验证了仿生铁蛋白壳能够实现对肿瘤的特异识别。和传统的免疫组化方 法相比较， 仿生铁蛋白能够集抗原特异识别和染色一歩实现， 无需传统免 疫组化的一抗、 二抗、 三抗等的多歩孵育过程， 也不需要 HRP酶等信号 分子的修饰， 因此， 比传统免疫组化方法更加简单、 方便、 快速。 实施例 6仿生铁蛋白染识别临床人肝癌组织
肝组织样本来来自北京安贞医院和北京肿瘤医院。所有肝组织样本均 是手术切除的肿瘤及邻近组织的大块样本。 所有组织用石蜡包埋。
仿生铁蛋白染组织切片方法简述如下：将石蜡包埋的肝癌组织和正常 肝组织脱蜡处理， 消除内源性过氧化物酶， 经抗原修复、 血清封闭后加入 仿生铁蛋白， 室温孵育 45分钟， PBS漂洗， DAB染色， 苏木精复染， 常 规脱水封片， 显微镜观察。
仿生铁蛋白荧光染组织切片简述如下：将石蜡包埋的肝癌组织和正常 肝组织脱蜡处理， 血清封闭后， 加入 FITC标记仿生铁蛋白壳， 室温孵育 45分钟， PBS冲洗 2次 DAPI染核 10分钟。 放淬灭剂封片， 荧光共聚焦 观察。
结果如图 4所示， 仿生铁蛋白特异识别肝组织中癌化的细胞， 对肿瘤 周围的正常组织不染色识别； 对肝组织慢性炎、 结节性肝硬化、胆汁性肝 硬变等组织也不识别。 在所有检测的肝细胞癌组织中阳性率为 54/55， 而 肝正常组织、 肝炎、 肝硬化的阳性率为 7/45。证实仿生铁蛋白能特异性地 识别肝癌组织。 实施例 6 仿生铁蛋白特异识别多种人癌组织
为了系统研究仿生铁蛋白对癌变组织和人体正常组织的特异识别能 力， 我们选取例人肿瘤组织和 48例人正常组织或炎症组织的石蜡切片标 本， 进行仿生铁蛋白染色。病人组织样本来来自北京安贞医院、 北京肿瘤 医院和西安奥美生物科技有限公司。所有组织样本均是手术切除的肿瘤及 邻近组织的大块样本。 所有组织用石蜡包埋。
仿生铁蛋白染组织切片简述如下： 将石蜡包埋的人肿瘤组织、正常组 织或相应炎症组织脱蜡处理， 消除内源性过氧化物酶， 经抗原修复、 血清 封闭后加入仿生铁蛋白， 室温孵育 45分钟， PBS漂洗， DAB染色， 苏木 精复染， 常规脱水封片， 显微镜观察， 组织识别结果见图 5和表 2。
仿生铁蛋白荧光染组织切片简述如下： 将石蜡包埋的肿瘤组织或正 常、 炎症组织脱蜡处理， 血清封闭后， 加入 FITC标记仿生铁蛋白壳， 室 温孵育 45分钟， PBS冲洗 2次， DAPI染核 10分钟。 放淬灭剂封片， 荧 光共聚焦观察， 结果见图 6。
如图 5， 6所示， 仿生铁蛋白对结肠癌、 乳腺癌、 卵巢癌、 结肠腺癌、 肺鳞状细胞癌、 前列腺癌、 食道鳞状细胞癌、 胸腺癌及宫颈鳞状细胞癌等 都表现出强烈的结合， 而对其相应的结肠慢性炎、 乳腺组织、 卵巢组织、 结肠粘膜组织、 肺组织、 前列腺增生、 食道慢性炎、 胸腺组织、 宫颈组织 及宫颈慢性炎等都没有相应的结合。表 2的分析结果表明， 仿生铁蛋白对 肝细胞癌 (54/55)、 肺鳞状细胞癌 (50/52)、 胸腺癌 (4/4)、 宫颈鳞状细胞癌 (4/4), 食道癌 （3/3 )、 结肠癌 （6/6)及卵巢癌 (4/4)等都表现出非常好的染 色灵敏性和特异性。 图 5、 图 6的组织识别效果证明仿生铁蛋白能够特异 识别多种人癌组织， 尤其对肝细胞癌、 肺癌、 胸腺癌、 宫颈癌、 卵巢癌和 结肠癌能够强烈地染色识别， 而对相应的正常或是炎症组织没有识别。和 传统的抗体免疫组化方法相比较，基于仿生铁蛋白的癌组织检测识别方法 能够集肿瘤特异识别和染色一歩实现， 无需一抗、 二抗、 三抗等的多歩孵 育或是 HRP等酶或信号分子的标记， 操作简单方便， 且对肿瘤组织的染 色效果和抗体免疫组化方法具有可比性。 表 2 仿生铁蛋白对临床肿瘤组织及相应的正常组织的特异识别分析
Figure imgf000015_0001
肺鳞状细胞癌 50/52 肺组织 1/56 胸腺癌 4/4 胸腺组织 0/4 宫颈鳞状细胞癌 28/28 宫颈组织 /慢性炎 0/52 前列腺腺癌 22/22 前列腺平滑肌 /前列腺增生 2/16 乳腺癌非特殊性 乳腺组织 /乳腺纤维病 /增生
4/5 1/4 浸润性导管癌 纤维组织 /乳腺脂肪组织 卵巢癌 55/56 卵巢组织 /卵巢上皮增生 1/16 食道平滑肌 /慢性炎伴上皮
食道鳞状细胞癌 3/3 0/5
增生 结肠粘膜组织 /结肠粘膜慢
结肠腺癌 23/23 1/41
性炎
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中国专利 200610057413.9
Theil EC, (1987), Ferritin: structure, gene regulation, and cellular function in animals, plants, and microorganisms. Annu. Rev. Biochem., 56:289-315. Harrison PM., et al. (1996) The ferritins: molecular properties, iron storage function and cellular regulation, Biochim. Biophys. Acta, 1275: 161-203. Levi S.， et al. (1988), Mechanism of ferritin iron uptake— Activity of the H-chain and deletion mapping of the ferro-oxidase site— A study of iron uptake and ferro-oxidase activity of human-liver, recombinant H-chain ferritins, and of 2H-chain deletion mutants, J. Biol. Chem., 263 : 18086-18092.
Ford GC, et al. (1984), Ferritin: Design and formation of an iron-storage molecule, Philos Trans. R. Soc. Lond B Biol. Sci., 304:551-565.
Fargion S, et al. (1988) Characteristics and expression of binding sites specific for ferritin H-chain on human cell lines. Blood 71 :753-757. Moss D, et al.(1992) Characterization of the ferritin receptors of human T lymphoid (MOLT-4) cells, J. Lab Clin. Med. 119:273-279.
Bretscher MS, et al. (1983) Distribution of ferritin receptors and coated pits on giant HeLa cells, EMBO J. 2:599-603.
Takami M, et al. (1986) Human placental ferritin receptor. Biochim. Biophys Acta 884:31-38.
Fargion S, et al. (1991) Specific binding sites for H-ferritin on human lymphocytes: Modulation during cellular proliferation and potential implication in cell growth control, Blood 78: 1056-1061.
Moss D.， et al. (1992) Functional roles of the ferritin receptors of human liver, hepatoma, lymphoid and erythroid cells, J. Inorg. Biochem. 47:219-227.
Li L., et al. (2010), Binding and uptake of H-ferritin are mediated by human transferrin receptor- 1, Proc. Natl. Acad. Sci. U S A, 107: 3505-3510.
Larrick JW., et al. (1979), Modulation of cell surface iron transferrin receptors by cellular density and state of activation, J. Supramol. Struct., 11 :579-586.
Daniels TR., et al. (2006), The transferrin receptor part I: biology and targeting with cytotoxic antibodies for the treatment of cancer, Clin. Immunol, U\ : 144-158.
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Claims

权 利 要 求
1. 双功能肿瘤诊断试剂， 其特征在于它是由特异识别癌组织和 /或癌 细胞的蛋白壳及具有过氧化物酶催化活性的无机纳米核组成。
2. 根据权利要求 1 所述的双功能肿瘤诊断试剂， 其特征在于： 所述 蛋白壳包括去铁蛋白、 热休克蛋白、 DNA结合蛋白、 喋啶合酶、 和具有 纳米空腔结构的病毒蛋白壳，所述纳米空腔结构的病毒蛋白壳包括豇豆退 绿斑驳病毒和豇豆花叶病毒蛋白。
3. 根据权利要求 2所述的双功能肿瘤诊断试剂， 其特征在于： 所述 去铁蛋白由 12个或是 24个重链亚基和轻链亚基任意比例自组装而成。
4. 根据权利要求 3所述的双功能肿瘤诊断试剂， 其特征在于： 所述 去铁蛋白是天然去铁蛋白或是基因重组的去铁蛋白，其中天然去铁蛋白来 源于真核生物或原核生物， 优选来源于哺乳动物。
5. 根据权利要求 1-4中任意一项所述的双功能肿瘤诊断试剂，其特征 在于： 所述蛋白壳包括化学偶联或是基因融合靶向分子和 /或信号分子的 蛋白壳， 所述靶向分子包括多肽或核酸适配体， 所述多肽包括抗体， 所述 信号分子包括荧光分子、 放射核素和酶。
6. 根据权利要求 1-4中任意一项所述的双功能肿瘤诊断试剂，其特征 在于： 所述无机纳米核包括磁性氧化铁纳米颗粒、硫化铁纳米颗粒、 贵金 属掺杂氧化铁纳米颗粒、 双金属合金纳米颗粒和氧化铈纳米颗粒。
7. 根据权利要求 1-4中任意一项所述的双功能肿瘤诊断试剂，其特征 在于： 所述蛋白壳是基因重组的全重链亚基铁蛋白， 所述的无机内核为磁 性氧化铁纳米颗粒。
8. 肿瘤诊断试剂盒，其包括权利要求 1-7中任意一项所述的双功能肿 瘤诊断试剂和过氧化物酶底物。
9. 仿生铁蛋白及其在制备肿瘤检测试剂中的用途， 该仿生铁蛋白由 蛋白壳及无机纳米核组成， 所述蛋白壳是基因重组的全重链亚基铁蛋白， 所述的无机内核为磁性氧化铁纳米颗粒。
10. 检测个体中肿瘤的方法， 包括： 从所述个体获得机体组织或细胞 样品， 将该样品与权利要求 1-7中任意一项所述的双功能肿瘤诊断试剂接 触， 加入底物直接显色， 由此测定所述个体中肿瘤的存在， 优选地所述组 织为临床病人或是动物模型的穿剌活组织标本、术后病理切片或尸体剖检 组织； 所述细胞为机体循环系统中脱落细胞、组织溶解细胞或体外培养细 胞。
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