CN109852666B - Specific target spot and diagnostic reagent for liver cancer diagnosis - Google Patents

Specific target spot and diagnostic reagent for liver cancer diagnosis Download PDF

Info

Publication number
CN109852666B
CN109852666B CN201910044299.3A CN201910044299A CN109852666B CN 109852666 B CN109852666 B CN 109852666B CN 201910044299 A CN201910044299 A CN 201910044299A CN 109852666 B CN109852666 B CN 109852666B
Authority
CN
China
Prior art keywords
liver cancer
glu
hccfn
leu
lys
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910044299.3A
Other languages
Chinese (zh)
Other versions
CN109852666A (en
Inventor
阎锡蕴
江冰
范克龙
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Biophysics of CAS
Original Assignee
Institute of Biophysics of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Biophysics of CAS filed Critical Institute of Biophysics of CAS
Priority to CN201910044299.3A priority Critical patent/CN109852666B/en
Publication of CN109852666A publication Critical patent/CN109852666A/en
Application granted granted Critical
Publication of CN109852666B publication Critical patent/CN109852666B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to a specific target spot and a diagnostic reagent for liver cancer diagnosis, wherein the specific target spot is GRP78 protein, and the diagnostic reagent is as follows: a nanoparticle loaded with a substance having peroxidase catalytic activity and fused to the surface thereof the targeting polypeptide SP94, said nanoparticle being a ferritin particle.

Description

Specific target spot and diagnostic reagent for liver cancer diagnosis
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to a specific target spot and a diagnostic reagent for liver cancer diagnosis.
Background
Liver cancer has become one of the major diseases that are increasingly common and seriously threatening human life and quality of life. The diagnosis of liver cancer is the subject and problem of greatest concern for current tumor researchers.
The pathological section examination is the most accurate and reliable and is the gold standard recognized at home and abroad for tumor diagnosis (Shi, et al., (2008) am.J. Clin.Pathol.,129: 358-. At present, the main staining methods of pathological sections include hematoxylin-eosin (HE) staining, immunohistochemistry and immunofluorescence. The HE staining method is convenient and rapid to operate, can only provide the change of the tissue morphology, has little information, and cannot make correct judgment on more complex and various tumors. Immunohistochemistry or immunofluorescence utilizes the combination of a primary antibody and a tissue antigen to be detected, the combination of a fluorescent signal molecule or enzyme-labeled secondary antibody and tertiary antibody, and the primary antibody, and then the chromogenic reaction is carried out through a fluorescent signal or an enzyme substrate, so as to provide positioning and semi-quantitative information of the antigen to be detected, and further identify pathological changes of tissue cells, such as canceration, necrosis, inflammatory cell infiltration and the like. Immunohistochemistry or immunofluorescence can provide detailed information on the distribution, content and cell morphology of a specific antigen, and is significant for deep study of pathology, but immunostaining requires multi-step incubation of primary antibody, secondary antibody and even tertiary antibody, repeated washing of PBS, and labeling of enzyme or fluorescent molecules, and has complex steps and long operation time. Therefore, there is a need for the research of cancer tissues, cancer cell detection reagents and methods that are simple, rapid and capable of providing a rich amount of information to improve the efficiency of clinical pathological diagnosis and to win time for the treatment of patients.
The subject group firstly discovers in 2007 that the iron oxide nanoparticles have catalytic activity similar to peroxidase, namely in the presence of hydrogen peroxide, the iron oxide nanoparticles can react with a substrate of horseradish peroxidase such as DAB, TMB and the like to generate a reaction product similar to peroxidase, so that catalytic action similar to peroxidase is generated. Based on this discovery, we propose the concept of nanoenzyme (Gao L, Zhuang J, Yan X, et al., (2007) Nature Nanotech.,2: 577-. And the ferroferric oxide nanoenzyme is biomimetically synthesized in the inner cavity of the human heavy chain ferritin, and a new tumor diagnosis reagent M-HFn (Fan K, et al., (2012) Nature Nanotech.,7(7):459-464) aiming at a target TFR1 (transferrin receptor 1) is developed.
The Pyrococcus furiosus ferritin is a protein shell with an inner diameter of 8nm and an outer diameter of 12nm formed by 24 subunits. The Pyrococcus furiosus ferritin has excellent thermal stability, is very suitable for long-term storage and adaptation to external condition changes, and has natural advantages when being used as a diagnostic reagent. From the perspective of the nanometer material, the internal space of the natural protein shell can be filled with drugs and fluorescent small molecules. The metal ions can be loaded into the protein shell cavity by utilizing the catalytic oxidation of the ferritin subunit of the pyrococcus furiosus, so that the metal oxide nanoparticles which are wrapped by the protein shell and have uniform particle size are formed. And the protein shell can be fused with small molecular polypeptide or antibody by means of genetic engineering, and the small molecular polypeptide or antibody is used as a polypeptide, antibody or vaccine display platform.
It has been reported that the liver Cancer targeting peptide SP94 can specifically target liver Cancer cells, but the receptor of SP94 on liver Cancer cells is unknown (Lo, a.et al, (2008) Mol Cancer ther, 7(3): 579-89). Here, we identified SP94 as glucose regulatory protein 78(GRP78) as the receptor on hepatoma cells by co-immunoprecipitation and mass spectrometry.
SP94 polypeptide is displayed on the outer surface of the Pyrococcus furiosus ferritin in a fusion expression mode, and the ferritin shell with liver cancer targeting property, HccFn for short, is synthesized. By utilizing the bionics principle, the synthesized bionic ferritin nanoenzyme (HccFn (Co)3O4) The outer shell can specifically target malignant liver cancer tissues, and the inner core has peroxidase activity. The application of the bifunctional bionic nano ferritin to liver cancer diagnosis brings new reagents, new technologies and new ideas to liver cancer diagnosis.
Disclosure of Invention
The invention firstly relates to a specific molecular target for tumor diagnosis, wherein the molecular target is GRP78 protein, preferably, the tumor is liver cancer, and the amino acid sequence of the GRP78 protein is shown as SEQ ID NO. 4.
SEQ ID NO.4：
MKLSLVAAMLLLLSAARAEEEDKKEDVGTVVGIDLGTTYSCVGVFKNGRVEIIANDQGNRITPSYVAFTPEGERLIGDAAKNQLTSNPENTVFDAKRLIGRTWNDPSVQQDIKFLPFKVVEKKTKPYIQVDIGGGQTKTFAPEEISAMVLTKMKETAEAYLGKKVTHAVVTVPAYFNDAQRQATKDAGTIAGLNVMRIINEPTAAAIAYGLDKREGEKNILVFDLGGGTFDVSLLTIDNGVFEVVATNGDTHLGGEDFDQRVMEHFIKLYKKKTGKDVRKDNRAVQKLRREVEKAKRALSSQHQARIEIESFYEGEDFSETLTRAKFEELNMDLFRSTMKPVQKVLEDSDLKKSDIDEIVLVGGSTRIPKIQQLVKEFFNGKEPSRGINPDEAVAYGAAVQAGVLSGDQDTGDLVLLDVCPLTLGIETVGGVMTKLIPRNTVVPTKKSQIFSTASDNQPTVTIKVYEGERPLTKDNHLLGTFDLTGIPPAPRGVPQIEVTFEIDVNGILRVTAEDKGTGNKNKITITNDQNRLTPEEIERMVNDAEKFAEEDKKLKERIDTRNELESYAYSLKNQIGDKEKLGGKLSSEDKETMEKAVEEKIEWLESHQDADIEDFKAKKKELEEIVQPIISKLYGSAGP PPTGEEDTAEKDEL。
The invention also relates to a targeting polypeptide SP94 for marking the molecular target GRP78, wherein the amino acid sequence of the SP94 polypeptide is shown as SEQ ID NO. 1.
SEQ ID NO.1：SFSIIHTPILPL。
The invention also relates to a tumor diagnostic reagent which can react with a substrate of horseradish peroxidase and develop color, wherein the diagnostic reagent is as follows: the nanoparticle is loaded with a substance with peroxidase catalytic activity and fused with the targeting polypeptide SP94 on the surface, the nanoparticle is a ferritin particle, and preferably, the tumor is liver cancer.
The ferritin particles are preferably human heavy chain ferritin 24-mer particles or furiosaelis ferritin 24-mer particles, and most preferably, the ferritin particles are furiosaelis ferritin 24-mer particles.
The amino acid sequence of the Pyrococcus furiosus ferritin is shown in SEQ ID NO. 2.
SEQ ID NO.2：
MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE。
Preferably, the targeting polypeptide SP94 is connected with the protein of the nanoparticle into a fusion protein through a connecting sequence, and the connecting sequence is shown as SEQ ID NO. 3.
SEQ ID NO.3：GGGSGGGGSGGGS。
The material with peroxidase catalytic activity is metal oxide, preferably, the material with peroxidase catalytic activity is Fe3O4Or Co3O4Most preferably, the substance having peroxidase catalytic activity is Co3O4。
The invention also relates to application of the molecular target, the targeting polypeptide SP94 or a diagnostic reagent in preparing a diagnostic kit for diagnosing liver cancer or judging the differentiation degree and the tumor invasion degree of the liver cancer.
Preferably, the detection sample of the diagnostic kit is a puncture biopsy specimen, a postoperative pathological section, a necropsy tissue or exfoliated cells, tissue lysed cells or in-vitro cultured cells in a body circulatory system.
The invention also relates to the application of the molecular target, the targeting polypeptide SP94, the diagnostic reagent or the diagnostic kit in diagnosing liver cancer or judging the differentiation degree of the liver cancer and the invasion degree of tumor.
Drawings
FIG. 1, SP94 targeting peptide specifically binds to GRP78 on the surface of hepatoma cells. FIG. 1A is a schematic diagram of the identification of SP94 receptor by co-immunoprecipitation and mass spectrometry; FIG. 1B shows the surface protein of hepatoma carcinoma cell HepG2 specifically bound to SP94 target peptide analyzed by gel electrophoresis; FIG. 1C is the result of mass spectrometry analysis of SP94 targeting peptide receptor protein; FIG. 1D shows that SP94 targeting peptide receptor protein can be specifically recognized by GRP78 protein antibody; FIG. 1E shows that after the GRP78 gene is knocked out on liver cancer cell HepG2, the affinity of SP94 and HepG2 is reduced; FIG. 1F shows that SP94 has increased affinity for 3T3-L1 cells following overexpression of the GRP78 gene on murine 3T3-L1 cells that do not express GRP 78.
FIG. 2, preparation and characterization of liver cancer targeting ferritin HccFn protein shell. FIG. 2A is a structural schematic of a natural Pyrococcus furiosus ferritin (pfFn); FIG. 2D is a structural schematic of Pyrococcus furiosus ferritin (HccFn) surface-fused to express SP94 targeting peptide; FIG. 2B is a transmission electron microscope and dynamic light scattering analysis of pfFn; fig. 2E is transmission electron microscopy and dynamic light scattering analysis of HccFn; FIG. 2C is a confocal laser microscopy image of FITC-labeled pfFn incubated with hepatoma cell HepG 2; fig. 2F is confocal laser microscopy imaging of FITC fluorescently labeled HccFn co-incubated with liver cancer cells HepG 2.
FIG. 3 shows a diagnostic reagent HccFn (Co) for liver cancer3O4) Preparation and characterization of (1). FIGS. 3A and 3D are Pyrococcus furiosus ferritin (pfFn (Co) with cobaltosic oxide nanoparticles loaded in the inner cavity3O4) FIG. 3A) and HccFn (Co)3O4) (FIG. 3D) structural schematic; FIG. 3B shows pfFn (Co)3O4) Transmission electron microscopy and dynamic light scattering analysis; FIG. 3E is HccFn (Co)3O4) Transmission electron microscopy and dynamic light scattering analysis; FIG. 3C shows pfFn (Co)3O4) The transmission electron microscope and the particle size distribution of the cobaltosic oxide core are statistically analyzed; FIG. 3F is HccFn (Co)3O4) The transmission electron microscope and the particle size distribution statistical analysis of the cobaltosic oxide core are carried out.
FIG. 4 shows the loading of Co3O4And loading of Fe3O4The peroxidase activity of HccFn (a) was compared. FIG. 4A shows HccFn (Co) under the same conditions3O4) And HccFn (Fe)3O4) Carrying out catalytic TMB color reaction comparison; FIG. 4B is HccFn (Co)3O4) And HccFn (Fe)3O4) OD562nm values versus time for catalyzed TMB color development; FIG. 4C is HccFn (Co)3O4) And HccFn (Fe)3O4) Catalytic substrate hydrogen peroxide (H)2O2) The saturation curve of (c); FIG. 4D shows HccFn (Co)3O4) And HccFn (Fe)3O4) Saturation curve of catalytic substrate TMB.
FIG. 5 shows a diagnostic reagent HccFn (Co) for liver cancer3O4) Can specifically identify clinical liver cancer tissues. FIG. 5A shows HccFn (Co)3O4) A schematic diagram of catalyzing the DAB color development of a peroxidase substrate; FIG. 5B is based on HccFn (Co)3O4) The novel method for diagnosing the immunohistochemical liver cancer is shown; FIG. 5C is a graph based on HccFn (Co)3O4) The immunohistochemical liver cancer diagnosis method can specifically identify clinical liver cancer tissues.
FIG. 6 shows a diagnostic reagent HccFn (Co) for liver cancer3O4) Can be used for prognosis of liver cancer. FIG. 6A shows HccFn (Co)3O4) Can distinguish liver cancer from non-cancer tissues and can present differential staining; FIG. 6B shows HccFn (Co)3O4) Statistical analysis of staining in liver cancer and tissues adjacent to the cancer; FIG. 6C shows HccFn (Co)3O4) Analyzing the difference of the liver cancer tissue staining at different differentiation stages; FIG. 6D shows HccFn (Co)3O4) Analyzing the difference of the staining of liver cancer tissues at different tumor invasion stages; FIG. 6E shows HccFn (Co)3O4) And (3) analyzing the correlation between the dyeing intensity and the life cycle of the liver cancer patient.
Detailed Description
Example 1 identification of specific receptors of SP94 targeting peptides on the surface of hepatoma cells
In order to identify specific receptors of SP94 targeting peptides on the surface of liver cancer cells, SP94 labeled by Biotin (Biotin) is firstly incubated with liver cancer cells HepG2, and then a chemical cross-linking agent DTSSP is added to fix the interaction between SP94 and the receptors. HepG2 cells were subsequently lysed, co-incubated with streptavidin-labeled resin, and centrifuged to obtain the receptor protein-SP 94-biotin-streptavidin-resin complex (FIG. 1A). Through gel electrophoresis analysis, a 78kDa receptor protein was found to bind specifically to SP94 (FIG. 1B). Subsequent mass spectrometry showed that the receptor protein was glucose regulatory protein 78(GRP78) (fig. 1C). The protein interacting with SP94 was further confirmed to be GRP78 by GRP78 protein antibody binding verification (fig. 1D). After knockout of GRP78 by HepG2 cells, the affinity of SP94 to HepG2 decreased; after 3T3-L1 over-expressed GRP78, SP94 had increased affinity for 3T 3-L1. All the results prove that GRP78 is a specific receptor of SP94 on the surface of hepatoma cells.
Example 2 preparation and characterization of liver cancer targeting ferritin HccFn protein Shell
SP94 liver cancer targeting peptide (SEQ ID NO:2) was expressed by fusion of linker protein (SEQ ID NO:3) at the N-terminus of Pyrococcus furiosus ferritin (SEQ ID NO:1) (FIG. 2A) by means of gene fusion, and we named this fusion protein HccFn (FIG. 2D). The fusion protein HccFn is a globular shell-like structural protein formed by self-assembly of 24 identical subunits. The results of transmission electron microscopy and dynamic light scattering analysis also indicate that both Pyrococcus furiosus ferritin (pfFn) (FIG. 2B) and HccFn (FIG. 2E) retain good homogeneity and a spherical shell-like structure. We fluorescently labeled pfFn and HccFn with FITC, and after incubation with liver cancer cell HepG2 for 30 min, HccFn specifically bound to HepG2 cell surface as observed by confocal laser scanning microscopy (fig. 2C, 3F).
Example 3 diagnostic reagent for liver cancer HccFn (Co)3O4) Preparation and characterization of
Based on the principle of natural mineralization of ferritin inner core, metal cobalt ions are loaded into protein shells of pfFn (figure 3A) and HccFn (figure 3D) and oxidized in inner cavities of the protein shells to form cobaltosic oxide nanoparticles, so that the pfFn (Co) wrapped with the cobaltosic oxide inner core is biomimetically synthesized3O4) And HccFn (Co)3O4). The specific operation is as follows:
(1) degassing 0.1M sodium chloride solution, adding purified ferritin shell into a closed reactor under nitrogen environment, maintaining reaction temperature at 65 deg.C and pH value at 8.5,
(2) 4000 Co per ferritin shell2+Adding Co (NO) in a ratio of3)2Hydrogen peroxide as oxidant and Co are added simultaneously in the amount of H2O2:Co2+＝1:3，
(3) After the addition of hydrogen peroxide and Co was completed, the reaction was continued for 5 minutes, and 200. mu.L of 300mM sodium citrate was added to complex the remaining Co.
The formation principle of cobaltosic oxide nanoparticles is shown in the following formula (1).
Figure BDA0001948641350000041
Collecting the product, purifying by exclusion chromatography, removing denatured impurity protein to obtain pfFn (Co) wrapped with cobaltosic oxide nanometer core3O4) And HccFn (Co)3O4)。
Transmission electron microscopy and dynamic light scattering analysis showed that the pfFn (fig. 3B) and HccFn (fig. 3E) protein shells loaded into the cobaltosic oxide nanocore retained good homogeneity and a complete globular shell-like structure. Meanwhile, transmission electron microscope analysis on the cobaltosic oxide nano-core shows that cobaltosic oxide nano-particles with uniform particle sizes are formed inside protein shells of pfFn (figure 3C) and HccFn (figure 3F).
Example 4 diagnostic reagent for liver cancer HccFn (Co)3O4) Has a peroxidase activity higher than that of Fe3O4HccFn of
We have previously reported that the inner cavity is loaded with ferroferric oxide (Fe)3O4) The recombinant human heavy chain ferritin protein shell of nanoparticle (cloning Fan et al, Magnetotherin nanoparticles for targeting and visualizing tissue tissues, NATURE NAOTECHNOLOGY, VOL 7, JULY 2012), M-HFn for short, has peroxidase activity. Here, we synthesized a chamber loaded tricobalt tetraoxide (Co)3O4) Nanoparticles and ferroferric oxide(Fe3O4) Nanoparticle HccFn ferritin protein shell, abbreviated HccFn (Co)3O4) And HccFn (Fe)3O4). We used TMB and hydrogen peroxide (H)2O2) Detection of HccFn (Co) as a reaction substrate3O4) And HccFn (Fe)3O4) The peroxidase activity of (a). The activity experiment result shows that HccFn (Co)3O4) The peroxidase activity of the protease is obviously higher than that of HccFn (Fe)3O4) (FIGS. 4A, 4B).
The activity detection system is as follows: 800mM H2O20.2mg/mL TMB, and 0.2M sodium acetate solution (pH 4.5) were added to the solution, and the same concentration (0.25. mu.M) of HccFn (Co)3O4),HccFn(Fe3O4) And HccFn. The OD652nm light absorption value change was detected.
Meanwhile, the result of the enzyme activity kinetic analysis experiment of the peroxidase shows that HccFn (Co)3O4) For substrates TMB and H2O2The affinity of the compounds is remarkably higher than that of HccFn (Fe)3O4). Detection substrate H2O2The enzyme activity profile of (1) was such that the concentration of substrate TMB was 800. mu.M (FIG. 4C); substrate H when the enzyme activity mechanical curve of substrate TMB is detected2O2Was 2400mM (FIG. 4D). The above experimental results show that HccFn (Co)3O4) The peroxidase activity of the protease is obviously higher than that of HccFn (Fe)3O4)。
Example 5 diagnostic reagent for liver cancer HccFn (Co)3O4) Can be used for identifying and visualizing the liver cancer tissue
HccFn(Co3O4) In a substrate H2O2And the peroxidase substrate DAB, the DAB can be catalyzed to generate oxidation color reaction, and yellow brown precipitate is generated (figure 5A). Using HccFn (Co)3O4) By this property we will refer to HccFn (Co)3O4) Can be used for clinical liver cancer tissue staining. Due to HccFn (Co)3O4) The protein shell can specifically recognize and combine liver cancer tissues (the recognition target point is GRP78) in the presence of peroxidase substrates DAB and H2O2Specific to liver cancer tissue in the presence ofHeterosexual staining (fig. 5B). Our experimental results showed that there was no pfFn (Co) displayed targeting peptide SP943O4) Not recognizing liver cancer tissue, HccFn (Co) targeting peptide SP94 was shown3O4) Specifically recognized liver cancer tissue, but not paracarcinoma tissue (fig. 5C).
Example 6 diagnostic reagent for liver cancer HccFn (Co)3O4) Can be used for prognosis of liver cancer
Mixing HccFn (Co)3O4) Used for clinical liver cancer tissue staining, we find HccFn (Co)3O4) Normal liver tissues are not stained, and obvious staining intensity differences appear in different clinical liver cancer tissue staining, and the staining degree is divided into: four types of strong staining, moderate staining, weak staining and no staining (fig. 6A). In 345 cases of non-cancer liver tissues and 424 cases of liver cancer tissues, we found HccFn (Co)3O4) The recognition sensitivity of the liver cancer tissue reaches 63.4 percent (269/424), and the recognition specificity reaches 79.3 percent (273/345) (FIG. 6B). And HccFn (Co)3O4) The staining degree of the liver cancer tissue is positively correlated with the differentiation degree and invasion degree of the liver cancer tumor. HccFn (Co) with reduced degree of differentiation of liver cancer (high differentiation, medium differentiation, low differentiation)3O4) The staining positive rate was increased (50% (24/48), 65.7% (159/242), 65.1% (84/129)), and HccFn (Co)3O4) The strong staining rate increased (14.5% (7/48), 16.9% (41/242), 30.2% (39/129)) (fig. 6C). HccFn (Co) with increasing degree of liver cancer invasion (T1, T2, T3, T4)3O4) The staining positive rate increased (32.4% (11/34), 59.5% (78/131), 64.5% (58/90), 100% (3/3)) (fig. 6D). Further analysis found that 5 years survival time and HccFn (Co) of liver cancer patients3O4) The staining degree of the liver cancer tissue is in negative correlation. HccFn (Co)3O4) The survival time of strongly stained liver cancer patients is significantly lower than that of non-strongly stained liver cancer patients. The prognosis information related to the liver cancer patient can be obtained through HccFn (Co)3O4) The liver cancer tissue is judged by staining analysis.
The above laboratory results show HccFn (Co)3O4) Can be used as a prognostic diagnostic reagent for liver cancer.
Finally, it should be noted that the above examples are only used to help those skilled in the art understand the essence of the present invention, and should not be construed as limiting the scope of protection.
SEQUENCE LISTING
<110> institute of biophysics of Chinese academy of sciences
<120> specific target spot and diagnostic reagent for liver cancer diagnosis
<130> CP11902025C
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 12
<212> PRT
<213> SP94 polypeptide
<400> 1
Ser Phe Ser Ile Ile His Thr Pro Ile Leu Pro Leu
1 5 10
<210> 2
<211> 174
<212> PRT
<213> Pyrococcus furiosus ferritin
<400> 2
Met Leu Ser Glu Arg Met Leu Lys Ala Leu Asn Asp Gln Leu Asn Arg
1 5 10 15
Glu Leu Tyr Ser Ala Tyr Leu Tyr Phe Ala Met Ala Ala Tyr Phe Glu
20 25 30
Asp Leu Gly Leu Glu Gly Phe Ala Asn Trp Met Lys Ala Gln Ala Glu
35 40 45
Glu Glu Ile Gly His Ala Leu Arg Phe Tyr Asn Tyr Ile Tyr Asp Arg
50 55 60
Asn Gly Arg Val Glu Leu Asp Glu Ile Pro Lys Pro Pro Lys Glu Trp
65 70 75 80
Glu Ser Pro Leu Lys Ala Phe Glu Ala Ala Tyr Glu His Glu Lys Phe
85 90 95
Ile Ser Lys Ser Ile Tyr Glu Leu Ala Ala Leu Ala Glu Glu Glu Lys
100 105 110
Asp Tyr Ser Thr Arg Ala Phe Leu Glu Trp Phe Ile Asn Glu Gln Val
115 120 125
Glu Glu Glu Ala Ser Val Lys Lys Ile Leu Asp Lys Leu Lys Phe Ala
130 135 140
Lys Asp Ser Pro Gln Ile Leu Phe Met Leu Asp Lys Glu Leu Ser Ala
145 150 155 160
Arg Ala Pro Lys Leu Pro Gly Leu Leu Met Gln Gly Gly Glu
165 170
<210> 3
<211> 13
<212> PRT
<213> ligation sequence
<400> 3
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser
1 5 10
<210> 4
<211> 654
<212> PRT
<213> GRP78 protein
<400> 4
Met Lys Leu Ser Leu Val Ala Ala Met Leu Leu Leu Leu Ser Ala Ala
1 5 10 15
Arg Ala Glu Glu Glu Asp Lys Lys Glu Asp Val Gly Thr Val Val Gly
20 25 30
Ile Asp Leu Gly Thr Thr Tyr Ser Cys Val Gly Val Phe Lys Asn Gly
35 40 45
Arg Val Glu Ile Ile Ala Asn Asp Gln Gly Asn Arg Ile Thr Pro Ser
50 55 60
Tyr Val Ala Phe Thr Pro Glu Gly Glu Arg Leu Ile Gly Asp Ala Ala
65 70 75 80
Lys Asn Gln Leu Thr Ser Asn Pro Glu Asn Thr Val Phe Asp Ala Lys
85 90 95
Arg Leu Ile Gly Arg Thr Trp Asn Asp Pro Ser Val Gln Gln Asp Ile
100 105 110
Lys Phe Leu Pro Phe Lys Val Val Glu Lys Lys Thr Lys Pro Tyr Ile
115 120 125
Gln Val Asp Ile Gly Gly Gly Gln Thr Lys Thr Phe Ala Pro Glu Glu
130 135 140
Ile Ser Ala Met Val Leu Thr Lys Met Lys Glu Thr Ala Glu Ala Tyr
145 150 155 160
Leu Gly Lys Lys Val Thr His Ala Val Val Thr Val Pro Ala Tyr Phe
165 170 175
Asn Asp Ala Gln Arg Gln Ala Thr Lys Asp Ala Gly Thr Ile Ala Gly
180 185 190
Leu Asn Val Met Arg Ile Ile Asn Glu Pro Thr Ala Ala Ala Ile Ala
195 200 205
Tyr Gly Leu Asp Lys Arg Glu Gly Glu Lys Asn Ile Leu Val Phe Asp
210 215 220
Leu Gly Gly Gly Thr Phe Asp Val Ser Leu Leu Thr Ile Asp Asn Gly
225 230 235 240
Val Phe Glu Val Val Ala Thr Asn Gly Asp Thr His Leu Gly Gly Glu
245 250 255
Asp Phe Asp Gln Arg Val Met Glu His Phe Ile Lys Leu Tyr Lys Lys
260 265 270
Lys Thr Gly Lys Asp Val Arg Lys Asp Asn Arg Ala Val Gln Lys Leu
275 280 285
Arg Arg Glu Val Glu Lys Ala Lys Arg Ala Leu Ser Ser Gln His Gln
290 295 300
Ala Arg Ile Glu Ile Glu Ser Phe Tyr Glu Gly Glu Asp Phe Ser Glu
305 310 315 320
Thr Leu Thr Arg Ala Lys Phe Glu Glu Leu Asn Met Asp Leu Phe Arg
325 330 335
Ser Thr Met Lys Pro Val Gln Lys Val Leu Glu Asp Ser Asp Leu Lys
340 345 350
Lys Ser Asp Ile Asp Glu Ile Val Leu Val Gly Gly Ser Thr Arg Ile
355 360 365
Pro Lys Ile Gln Gln Leu Val Lys Glu Phe Phe Asn Gly Lys Glu Pro
370 375 380
Ser Arg Gly Ile Asn Pro Asp Glu Ala Val Ala Tyr Gly Ala Ala Val
385 390 395 400
Gln Ala Gly Val Leu Ser Gly Asp Gln Asp Thr Gly Asp Leu Val Leu
405 410 415
Leu Asp Val Cys Pro Leu Thr Leu Gly Ile Glu Thr Val Gly Gly Val
420 425 430
Met Thr Lys Leu Ile Pro Arg Asn Thr Val Val Pro Thr Lys Lys Ser
435 440 445
Gln Ile Phe Ser Thr Ala Ser Asp Asn Gln Pro Thr Val Thr Ile Lys
450 455 460
Val Tyr Glu Gly Glu Arg Pro Leu Thr Lys Asp Asn His Leu Leu Gly
465 470 475 480
Thr Phe Asp Leu Thr Gly Ile Pro Pro Ala Pro Arg Gly Val Pro Gln
485 490 495
Ile Glu Val Thr Phe Glu Ile Asp Val Asn Gly Ile Leu Arg Val Thr
500 505 510
Ala Glu Asp Lys Gly Thr Gly Asn Lys Asn Lys Ile Thr Ile Thr Asn
515 520 525
Asp Gln Asn Arg Leu Thr Pro Glu Glu Ile Glu Arg Met Val Asn Asp
530 535 540
Ala Glu Lys Phe Ala Glu Glu Asp Lys Lys Leu Lys Glu Arg Ile Asp
545 550 555 560
Thr Arg Asn Glu Leu Glu Ser Tyr Ala Tyr Ser Leu Lys Asn Gln Ile
565 570 575
Gly Asp Lys Glu Lys Leu Gly Gly Lys Leu Ser Ser Glu Asp Lys Glu
580 585 590
Thr Met Glu Lys Ala Val Glu Glu Lys Ile Glu Trp Leu Glu Ser His
595 600 605
Gln Asp Ala Asp Ile Glu Asp Phe Lys Ala Lys Lys Lys Glu Leu Glu
610 615 620
Glu Ile Val Gln Pro Ile Ile Ser Lys Leu Tyr Gly Ser Ala Gly Pro
625 630 635 640
Pro Pro Thr Gly Glu Glu Asp Thr Ala Glu Lys Asp Glu Leu
645 650

Claims (5)

1. A tumor diagnostic reagent capable of reacting with a substrate of horseradish peroxidase and developing a color, wherein the diagnostic reagent is:
a nanoparticle loaded with a substance with peroxidase catalytic activity and fused with the targeting polypeptide SP94 on the surface, wherein the nanoparticle is a ferritin particle, and the substance with peroxidase catalytic activity is cobaltosic oxide;
the tumor is liver cancer;
the ferritin particles are 24-polymer particles of furiosus ferritin, and the amino acid sequence of the furiosus ferritin is shown as SEQ ID NO. 2;
the amino acid sequence of the targeting polypeptide SP94 is shown in SEQ ID NO. 1.
2. The reagent of claim 1, wherein the targeting polypeptide SP94 is linked to the nanoparticle protein by a linker sequence to form a fusion protein.
3. The reagent for diagnosing tumor according to claim 2, wherein the linker sequence is represented by SEQ ID No. 3.
4. Use of the tumor diagnostic reagent of any one of claims 1 to 3 for the preparation of a diagnostic kit for diagnosing liver cancer or judging the degree of differentiation of liver cancer and the degree of invasion of liver cancer.
5. The use according to claim 4, wherein the test sample of the diagnostic kit is a biopsy specimen, a post-operative pathological section, a necropsy tissue, or exfoliated cells, lysed cells, or cultured cells in vitro in the circulatory system of the body.
CN201910044299.3A 2019-01-17 2019-01-17 Specific target spot and diagnostic reagent for liver cancer diagnosis Active CN109852666B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910044299.3A CN109852666B (en) 2019-01-17 2019-01-17 Specific target spot and diagnostic reagent for liver cancer diagnosis

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910044299.3A CN109852666B (en) 2019-01-17 2019-01-17 Specific target spot and diagnostic reagent for liver cancer diagnosis

Publications (2)

Publication Number Publication Date
CN109852666A CN109852666A (en) 2019-06-07
CN109852666B true CN109852666B (en) 2021-01-01

Family

ID=66895019

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910044299.3A Active CN109852666B (en) 2019-01-17 2019-01-17 Specific target spot and diagnostic reagent for liver cancer diagnosis

Country Status (1)

Country Link
CN (1) CN109852666B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113995829B (en) * 2021-10-25 2023-05-05 重庆医科大学 Inhibitors of GRP78 modulation of anti-hepatitis B virus particle secretion

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102778567A (en) * 2011-05-12 2012-11-14 中国科学院生物物理研究所 Difunctional tumor diagnosis reagent and method thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102778567A (en) * 2011-05-12 2012-11-14 中国科学院生物物理研究所 Difunctional tumor diagnosis reagent and method thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A Nanocarrier Based on a Genetically Engineered Protein Cage to Deliver Doxorubicin to Human Hepatocellular Carcinoma Cells;Riki Toita等;《Chem Commun》;20130627;第49卷(第67期);参见第7442-7444页，尤其是第7442页右栏第1段-第7443页左栏第1段、图1 *
GRP78在人肝细胞癌组织中的表达及临床意义，E072-185;曹雪霞;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20131115(第11期);参见全文，尤其是正文第24页第4段-第25页第2段 *
铁蛋白的生物工程应用;王玲等;《中国生物工程杂志》;20180630;第38卷(第6期);参见第77-85页，尤其是第78页左栏第3段、第79页右栏第1段、第80页左栏第2段 *

Also Published As

Publication number Publication date
CN109852666A (en) 2019-06-07

Similar Documents

Publication Publication Date Title
Fathi et al. Ultra-sensitive detection by metal nanoparticles-mediated enhanced SPR biosensors
JP6117185B2 (en) Bifunctional tumor diagnostic reagent and method
US11965881B2 (en) Nanosensors and methods for detection of biological markers
KR101153748B1 (en) NOVEL Au/Ag CORE SHELL COMPOSITE USEFUL FOR BIOSENNOVEL Au/Ag CORE SHELL COMPOSITE USEFUL FOR BIOSENSOR SOR
Han et al. Glypican-3-targeted precision diagnosis of hepatocellular carcinoma on clinical sections with a supramolecular 2D imaging probe
WO2018054391A1 (en) Intracellular atp circular dichroism spectrum real-time detection method
WO2023274252A1 (en) Polymer-modified magnetic nanomaterial, and preparation method therefor and use thereof
Li et al. Rapid and specific detection nanoplatform of serum exosomes for prostate cancer diagnosis
Cao et al. Application of functional peptides in the electrochemical and optical biosensing of cancer biomarkers
CN109852666B (en) Specific target spot and diagnostic reagent for liver cancer diagnosis
da Fonseca Alves et al. A novel peptide-based electrochemical biosensor for breast cancer characterization over a poly 3-(3-aminophenyl) propionic acid matrix
Shu et al. A novel chemiluminescence biosensor based on dual aptamers bound nanoparticles with multi-site signal amplification for sensitive detection of carcinoembryonic antigen
Zhang et al. Janus nanozyme based satellite structure immunosandwich colorimetric strategy for glycoproteins visual detection
Kong et al. Ultrasensitive detection of tumor-derived small extracellular vesicles based on nonlinear hybridization chain reaction fluorescence signal amplification and immunomagnetic separation
CN110907513B (en) Electrochemical detection method of stem cells
Xia et al. Ex vivo identification of circulating tumor cells in peripheral blood by fluorometric “turn on” aptamer nanoparticles
Yang et al. Multiplex analysis of tumor multidrug-resistance genes expression with photonic suspension array
CN104020287B (en) A kind of blood serum special active protease containing radioactive nuclide detects nanometer kit
CN112048554A (en) Liposome nanoparticle chip, application of liposome nanoparticle chip in preparation of pancreatic cancer diagnosis product and corresponding marker
Katafigioti et al. In the search of novel urine biomarkers for the early diagnosis of prostate cancer. Intracellular or secreted proteins as the target group? Where and how to search for possible biomarkers useful in the everyday clinical practice
CN112521455B (en) Polypeptide for detecting bladder cancer antigen protein specificity and application thereof
Ren et al. Identifying a membrane-type 2 matrix metalloproteinase-targeting peptide for human lung cancer detection and targeting chemotherapy with functionalized mesoporous silica
Ma et al. Recent progress in quantitative technologies for analysis of cancer-related exosome proteins
Chen et al. Mg-based micromotors for electrochemical detection of Parkinson's disease blood biomarkers
CN111018991B (en) anti-CA50 antibody and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant