CN103091492A - Diagnostic reagent and kit for cancer - Google Patents
Diagnostic reagent and kit for cancer Download PDFInfo
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- CN103091492A CN103091492A CN2011103465825A CN201110346582A CN103091492A CN 103091492 A CN103091492 A CN 103091492A CN 2011103465825 A CN2011103465825 A CN 2011103465825A CN 201110346582 A CN201110346582 A CN 201110346582A CN 103091492 A CN103091492 A CN 103091492A
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Abstract
The present invention relates to a diagnostic reagent and a kit for cancer. The present invention discloses that the existence condition of 5-hydroxymethylcytosine (hmc) in organizations is closely related to the incidence and development of cancer, the significant decrease of the hmc level in the organization indicates incidence or development of cancer, and also the hmc level indicates the severity of cancer. Therefore, the hmc detection reagent can be well applied to cancer diagnosis, prognosis or parting, and has a high clinical application value.
Description
Technical field
The invention belongs to medical domain；More particularly it relates to diagnose the reagent and kit of cancer.
Background technology
DNA methylation is a kind of common epigenetic modification, and the carbon that DNA methylation is primarily referred to as on cytimidine (C) 5 in mammal, which is methylated, is modified into 5-methylcytosine (5mC).CpG dinucleotides is that occur the main site of DNA methylation, and it is in uneven distribution in genome.In some regions, the mainly promoter of gene and First Exon region, CpG is higher than normal probability, and therefore these sections are referred to as CpG islands.In the range of whole gene group, the promoter region that there are about more than 60% gene contains CpG islands.On the whole, 5mC accounts for the 2-7% of C total amounts in mammal, therefore and has the title of " the 5th kind of base ".
DNA methylation plays an important role in normal and abnormal physiological activity.It can be participated in maintaining the structure of chromosome with the expression of suppressor, the important cells function such as regulation x chromosome inactivation, Genomic Imprinting and transposon silencing.
TET1, full name Ten-Eleven Translocation 1.Tet1 is a 2-OG, and 5mC can be changed into 5-hydroxymethyl cytosine (5-HydroxymethylCytosine) (hmc) by the 5mC oxygenases of Fe++ dependences.Meanwhile, hmc presence is have also discovered in brain cell.
TET1 biological function is not known still, because Tet1 is to clone what is come from No. ten derivative chromosomes of Acute Meyloid shape leukaemic, and on transposition to ride on Bus No. 11 chromosome, estimate that it is relevant with cancer generation development, but effects of the Tet1 in cancer is not known still.
Hmc is to be found in bacteriophage nineteen fifty-two.Find within 1972 that hmc is existed in mammal, it is present in the brain DNA of mouse and frog, in the brain and liver that exist in rat, and hmc constitutes about the 20% of total cytimidine in brain, and 6% (40) of total cytimidine are accounted in liver organization.In currently available technology, it has not been found that hmc and the correlation of cancer.
The content of the invention
It is an object of the invention to provide the reagent and kit of diagnosis cancer.
It is used as the purposes of the mark of detection cancer there is provided a kind of 5-hydroxymethyl cytosine in the first aspect of the present invention.
In a preference, described cancer includes but is not limited to：The cancer of the brain (such as glioma), squamous cell carcinoma (such as cancer of the esophagus, cutaneum carcinoma, head and neck cancer), breast cancer, stomach cancer, intestinal cancer (such as colon and rectum carcinoma), kidney, carcinoma of urinary bladder, prostate cancer, lung cancer, liver cancer, leukaemia, lymthoma, oophoroma, the cancer of the uterus, cancer of pancreas, thymic carcinoma, spermatogonium cancer, muscle tumor.
In another aspect of this invention there is provided a kind of 5-hydroxymethyl cytosine (hmc) purposes, the reagent for preparing detection cancer.
In another preference, described detection cancer includes：Determine the order of severity of cancer or carry out the parting of cancer.
In another preference, described 5-hydroxymethyl cytosine is connected with carrier protein, the reagent for preparing detection cancer.
In another preference, described carrier protein is selected from：Bovine serum albumin(BSA) (BSA), oralbumin (OVA), hemocyanin (KLH).
In another aspect of this invention there is provided a kind of antigen, the antigen includes carrier protein (preferably without immunogenicity), and one or more (such as 1-100 be connected on carrier protein；More particularly such as 5,10,12,13,15,20,25,30,35,40,45,50,60,70,80,90) 5-hydroxymethyl cytosine.
In another preference, described carrier protein is selected from：Bovine serum albumin(BSA) (BSA), oralbumin (OVA), hemocyanin (KLH).
In another aspect of this invention there is provided the purposes of described antigen, the reagent for preparing detection cancer.
In another aspect of this invention there is provided a kind of purposes of the reagent of specific recognition 5-hydroxymethyl cytosine (hmc), the kit for preparing detection cancer.
In another preference, described reagent is the antibody (including polyclonal antibody or monoclonal antibody) of specific recognition 5-hydroxymethyl cytosine.
In another aspect of this invention there is provided a kind of polyclonal antibody, it specifically recognizes 5-hydroxymethyl cytosine.
In another preference, the preparation method of described polyclonal antibody is：With described antigen immune rabbit, immune serum is obtained, described polyclonal antibody is obtained after purification.
In another preference, the preparation method of described polyclonal antibody is：Described antigen is mixed with Freund's adjuvant after (preferably 1: 1 mixing), rabbit is expelled to, 0.5 ± 0.2mg of amount is immunized, is immunized 1 time every 1.5-3 weeks (preferably 2 weeks), it is immunized 2-4 times (preferably 3 times), polyclonal antibody is separated from rabbit anteserum.
In another aspect of this invention there is provided a kind of monoclonal antibody, its specific recognition 5-hydroxymethyl cytosine, it is CCTCC No by preserving number：C201191 hybridoma cell strain is produced.
In another aspect of this invention there is provided a kind of hybridoma cell strain, it is CCTCC No in the preserving number of China typical culture collection center：C201191.
In another aspect of this invention there is provided a kind of kit for being used to detect cancer, including the reagent of specific recognition 5-hydroxymethyl cytosine.
In another preference, also contain in described kit：The SABC reagent such as developer.
The other side of the present invention, due to this disclosure, is obvious to those skilled in the art.
Brief description of the drawings
The Mass Spectrometer Method of Fig. 1, MALDI-TOF 4800 is not coupled the molecular weight of product hmc-BSA (above) after control group BSA (figure below) and coupling.
Product (conju) after Fig. 2, SDS-PAGE separation coupling, control group BSA (Control, Ctrl) is not coupled and is compareed without the BSA of reaction.
The Mass Spectrometer Method of Fig. 3, MALDI-TOF 4800 is not coupled the molecular weight of various products after control group OVA (control) and coupling.U-OVA, 5mU-OVA, A-OVA, G-OVA, hmc-OVA are that uracil, methyl uracil, adenine, guanine and 5-hydroxymethyl cytosine are coupled on OVA respectively.
The absworption peak of product after free KLH, hmc, KLH and hmc coupling reaction of Fig. 4, ultraviolet spectro-photometric analysis, to judge whether coupling reaction succeeds.
Fig. 5, immune-blotting method hmc rabbit polyclonal antibodies HMC-PA01 specificity.5mC or hmc (primary quantity is respectively 125 nanograms) is fixed on film with 2 times of decrements, with hmc rabbit polyclonal antibody HMC-PA01 (above) or 5mC antibody (33 D3, Calbiochem, San Diego, CA) after (middle) reaction, shown after secondary antibody and colour developing：Hmc rabbit polyclonal antibody HMC-PA01 only recognizes hmc, basic nonrecognition 5-methylcytosine (5mC) (above).And 5mC antibody only recognizes 5mC, nonrecognition hmc (middle).Ponceau-S shows that each point contains diluent materials (figure below).
Fig. 6, immunofluorescence dyeing detection hmc rabbit polyclonal antibodies HMC-PA01 specificity.Coding Tet1 and GFP plasmid (mTET1-ires-GFP) is transferred into cell, the nucleus (the picture left above), hmc rabbit polyclonal antibodies HMC-PA01 dye hmc (top right plot), 5mC antibody that each cell is contaminated with DAPI contaminate 5mC (lower-left figure), the cell (middle figure below) that green display mTET1-ires-GFP is transferred to, and Merge is that four images are overlapping.
Fig. 7, hmc rabbit polyclonal antibody can be used for co-immunoprecipitation.Mouse 46C ES cells (left figure), 293T (control) (middle) are transferred to Tet1 293T cells (Tet1) and carry out co-immunoprecipitation with 5mC antibody (5mC) or hmc antibody (hmc) respectively, after the DNA of precipitation is separated through Agarose glue, ethidium bromide staining is used.Input is the DNA consumptions for 1/10th of precipitation, and IgG is antibody control.
Fig. 8, immune-blotting method hmc mouse monoclonal antibodies MA7 specificity.5mC or hmc (initial concentration is respectively 5 nanograms) is fixed on film with 10 times of decrements, with hmc mouse monoclonal antibody MA7 (above), contain 5mC antibody (33 D3 simultaneously, Calbiochem, San Diego, CA) and after the reaction of hmc mouse monoclonal antibody MA7 (middle), shown after secondary antibody and colour developing：Hmc mouse monoclonal antibody only recognizes hmc, nonrecognition 5mC (above).And 5mC antibody understanding 5mC (middle).Ponceau-S shows that each point contains diluent materials (figure below).
Fig. 9, immunofluorescence dyeing detection hmc mouse monoclonal antibodies MA7 specificity.Coding Tet1 and GFP plasmid (Tet1-Ires-GFP) is transferred into cell, the cell (middle figure below) that mTET1-ires-GFP is transferred to is shown with the nucleus (the picture left above) of each cell of DAPI dyes, hmc mouse monoclonal antibody MA7 immunostainings (top right plot), hmc rabbit polyclonal antibodies immunostaining (lower-left figure), green, Merge is that four images are overlapping.
Figure 10, hmc mouse monoclonal antibody can be used for co-immunoprecipitation.293T (control) (left figure) and it is transferred to Tet1 293T cells (Tet1) and carries out co-immunoprecipitation with 5mC antibody (5mC) or hmc antibody (hmc) respectively, after the DNA of precipitation is separated through Agarose glue, ethidium bromide staining is used.Input is the DNA consumptions for 1/10th of precipitation, and IgG is antibody control.
Figure 11, hmc mouse monoclonal antibody can be used for co-immunoprecipitation.Mouse 46C ES cells (left figure) and brain tissue (right figure) carry out co-immunoprecipitation with 5mC antibody (5mC) or hmc antibody (hmc) respectively, after the DNA of precipitation is separated through Agarose glue, ethidium bromide staining is used.Input is the DNA consumptions for 1/10th of precipitation, and IgG is antibody control.
Figure 12, the hmc immunohistochemical stainings of the renal carcinoma tissue of pathological number 200401486.The renal carcinoma tissue's section for including cancerous tissue and cancer beside organism carries out immunohistochemical staining with hmc antibody (left figure) or 5mC antibody (right figure), there is high-caliber hmc (the left figure upper right corner) in cancer beside organism, but cancerous tissue is substantially not detectable 5-hmc (the left figure lower right corner).On the contrary, there are similar 5mC levels in cancerous tissue (the right figure upper right corner) and its cancer beside organism (the right figure lower right corner).
Figure 13, the tissue of the kidney patient of pathological number 200309294 are found through immunohistochemical staining：The hmc that there is high intensity in cancer beside organism (a) is dyed, but cancerous tissue is substantially not detectable hmc (b).On the contrary, the hmc that there are similar intensity in cancerous tissue (c) and its cancer beside organism (d) is dyed.
Hmc levels are decreased obviously in Figure 14, renal carcinoma tissue.According to the quantitative integration method of table one, using tinctorial strength and range product as tissue hmc dye levels.Each circle represents a cancerous tissue, and each triangle represents a cancer beside organism, and two horizontal lines are the average value of cancerous tissue and cancer beside organism's hmc dye levels respectively, and there is high-caliber hmc in cancer beside organism, and hmc levels are decreased obviously in cancerous tissue.
The normal urothelium of Figure 15, hmc immunohistochemical staining pathological number 201003780 and 201004888, hmc stained positives.
Figure 16, illustration bladder cancer organize hmc dye levels.The tissue of the (above) of hmc immunohistochemical stainings pathological number 200216104,200300702 (middle) and 200309678 (figure below) bladder cancers, their hmc dye levels are respectively 1 point (feminine gender), 6 points (weakly positives) and 9 points (positive).
Hmc levels are associated with the order of severity of carcinoma of urinary bladder in Figure 17, Bladder Cancer.With reference to foregoing, using tinctorial strength and range product as dye levels, to show the integration of different deterioration degree carcinomas of urinary bladder.Horizontal line is the average value of different deterioration degree carcinoma of urinary bladder cancerous tissue hmc dye levels respectively, and with the intensification of deterioration degree, hmc levels are constantly reduced.LNI：Low level non-infiltration bladder transitional cell carcinoma, Low：Low level bladder transitional cell carcinoma, High：High-level bladder transitional cell carcinoma, HI：High-level wellability bladder transitional cell carcinoma.
Figure 18, hmc immunohistochemical staining normal cerebral tissue, DAPI dyes nucleus, NeuN dye nerve cells, nerve cell hmc stained positives.
Figure 19, hmc immunohistochemical staining normal cerebral tissue, DAPI dyes nucleus, GFAP dye spongiocytes, spongiocyte hmc stained positives.
Figure 20, illustration samples of human glioma hmc dye levels.The tissue of hmc immunohistochemical staining glioma patients, their hmc dye levels are respectively 0 point (A, 0x0), 1 point (B, 1x1), 4 points (C, 2x2) and 9 points (D, 3x3).
Figure 21, samples of human glioma hmc immunohistochemical stainings degree are associated with the severity of cancer of glioma.Glioma is divided into 4 grades according to pathological diagnosis rank：I, II, III and IV grades.With reference to the integration method, quantitative integration analysis is carried out to samples of human glioma hmc coloration results, using tinctorial strength and range product as dye levels, to show the integration of different deterioration degree carcinomas of urinary bladder.Horizontal line is the average value of different deterioration degree glioma cancerous tissue hmc dye levels respectively, and with the intensification of deterioration degree, hmc levels are constantly reduced.A, first group of 55 patient samples, B, second group of 103 patient samples.
Hmc levels may determine that the time-to-live of glioma cancer patient in Figure 22, glioma cancerous tissue.Patient is divided into according to glioma cancerous tissue hmc coloration results by negative (2 points and less), weakly positive (3-4 points), positive (6 points and more than), the Kaplan-Meier time-survivor curves of each class patient are then drawn.A, first group of 55 patient samples, B, second group of 103 patient samples.
Figure 23, pathological diagnosis order of severity identical cancer patient can pass through the further parting of hmc levels.First group of 55 patient and second group of 103 patient samples are combined, and Kaplan-Meier time-survivor curves analyze pathological diagnosis for II grades of cancer specimens (A) or III level cancer specimen (B) respectively.
Figure 24, breast cancer tissue's hmc immunohistochemical stainings.Two include cancer beside organism's (left side figure) and the breast tumor tissue sections of cancerous tissue (the right figure) carry out immunohistochemical staining with hmc antibody, normal breast ductal cells hmc stained positives in cancer beside organism, but its adjacent cancerous tissue hmc dyeing is negative.
Figure 25, breast cancer tissue's hmc immunohistochemical stainings.4 breast cancer tissue's immunohistochemical stainings find that hmc levels have different degrees of decline in breast cancer tissue.
Figure 26, prostate cancer tissue hmc immunohistochemical stainings.The prostate cancer tissue section for including cancer beside organism's (left side figure) and cancerous tissue (the right figure) carries out immunohistochemical staining with hmc antibody (above) or 5mC antibody (figure below), there is high-caliber hmc (the picture left above) in cancer beside organism, but cancerous tissue is substantially not detectable hmc (top right plot).On the contrary, there are similar 5mC levels in cancerous tissue (bottom-right graph) and its cancer beside organism's (lower-left figure).
Figure 27, prostate cancer tissue hmc immunohistochemical stainings.The prostate cancer tissue section for including cancer beside organism's (left figure) and cancerous tissue (right figure) carries out immunohistochemical staining with hmc antibody, there is high-caliber hmc (left figure) in cancer beside organism, although cancerous tissue is substantially not detectable hmc, but still has some cells to contain low-level hmc (right figure).
Figure 28, colon cancer tissue hmc immunohistochemical stainings.The colon cancer tissue section for including cancer beside organism's (the right figure) and cancerous tissue (left side figure) carries out immunohistochemical staining, part Normal Colon gland cell hmc stained positives with hmc antibody, but cancerous tissue is substantially not detectable hmc.
Figure 29, two colon cancer tissues hmc immunohistochemical stainings, cancerous tissue lose hmc.
Figure 30, stomach organization hmc immunohistochemical stainings.The stomach cancer tissue slides for including cancer beside organism's (left side figure) and cancerous tissue (the right figure) carry out immunohistochemical staining, normal gastric gland cell hmc stained positives with hmc antibody, but cancerous tissue is substantially not detectable hmc.
Figure 31, the hmc immunohistochemical stainings of rectum cancer tissue.The rectum cancer tissue's section for including cancer beside organism's (left side figure) and cancerous tissue (the right figure) carries out immunohistochemical staining with hmc antibody, and normal gland cell has certain hmc dyeing, but cancerous tissue is substantially not detectable hmc.
Figure 32, uterus cancerous tissue hmc immunohistochemical stainings.The cancer of the uterus histotomy for including cancer beside organism's (left side figure) and cancerous tissue (the right figure) carries out immunohistochemical staining with hmc antibody, normal smooth muscle cell hmc stained positives in cancer beside organism, but dye the (above) that is negative from the cancerous tissue hmc of same patient.In another example, normal uterus scaly epithelium and interstitial cell have certain hmc dyeing in cancer beside organism, but are negative from the uterus squamous carcinoma tissue hmc dyeing of same patient.
Figure 33, leiomyosarcoma tissue hmc immunohistochemical stainings.The leiomyosarcoma tissue section for including cancer beside organism's (left side figure) and cancerous tissue (the right figure) carries out immunohistochemical staining with hmc antibody, normal smooth muscle cell has certain hmc dyeing in cancer beside organism, but dyes the (above) that is negative from the cancerous tissue hmc of same patient.
Figure 34, cancerous lung tissue hmc immunohistochemical stainings.Cancerous lung tissue section carries out immunohistochemical staining with hmc antibody, and most of cancerous tissue hmc dyeing is negative.
Figure 35, liver cancer tissue hmc immunohistochemical stainings.Liver cancer tissue section carries out immunohistochemical staining discovery with hmc antibody, and cancerous tissue hmc dyeing is negative.
Figure 36, leukaemia tissue hmc immunohistochemical stainings.Leukaemia carries out immunohistochemical staining, the non-lymphatic leukemia hmc stained positives of M2 types or weakly positive with hmc antibody, but the non-lymphatic leukemia hmc dyeing of M4 types is negative.
Figure 37, lymphoma tissue hmc immunohistochemical stainings.Lymphoma tissue section carries out immunohistochemical staining with hmc antibody, and cancerous tissue hmc dyeing is negative.
Embodiment
The present inventor is by extensive research, and the occurrence and development that 5-hydroxymethyl cytosine (hmc) presence situation in the tissue and cancer are disclosed first are closely related, and hmc levels are decreased obviously the generation or development of prompting cancer in tissue；And the height of hmc levels can also indicate the order of severity of cancer.Therefore, detection hmc reagent can be well applied to the diagnosis, prognosis or parting of cancer, with very high clinical value.
As used herein, described " cancer " refers to kinds cancer, as long as hmc level is decreased obviously in its cancerous tissue (as being significantly lower than cancer beside organism).It is preferred that described cancer includes but is not limited to：The cancer of the brain (such as glioma), squamous cell carcinoma (such as cancer of the esophagus, cutaneum carcinoma, head and neck cancer), breast cancer, stomach cancer, intestinal cancer (such as colon and rectum carcinoma), kidney, carcinoma of urinary bladder, prostate cancer, lung cancer, liver cancer, leukaemia, lymthoma, oophoroma, the cancer of the uterus, cancer of pancreas, thymic carcinoma, spermatogonium cancer, muscle tumor.
As used herein, described " diagnosis cancer " includes：The generation that determines cancer, the development for determining cancer, the order of severity for determining cancer, prognosis is carried out to cancer, the susceptibility analysis of cancer is carried out, carrying out parting etc. of cancer.
Hmc and application thereof
5-hydroxymethyl cytosine (5-Hydroxymethyl Cytosine, hmc) is a kind of micromolecular compound.It is connected on the cytimidine of DNA in covalent form, and forefathers' research finds that it is present in the brain and liver of mammal, and is present in embryonic stem cell.
The present inventor discloses hmc presence situations in the tissue for the first time and the occurrence and development of cancer are closely related, and hmc levels are decreased obviously the generation or development of prompting cancer in tissue；And the height of hmc levels can also indicate the order of severity of cancer, hmc levels are lower to imply that cancer is more serious.
New discovery based on the present inventor, it is possible to use hmc：(i) parting, antidiastole, and/or the susceptibility analysis of cancer are carried out；(ii) cancer treatment drugs of assessment correlated crowd, curative effect of medication, prognosis, and select suitable treatment method；(iii) earlier evaluations correlated crowd cancer risk, early monitoring early prevention etc..
After correlations of the hmc with cancer is known, it is convenient to diagnose cancer based on this.Any method for identifying hmc expressions in tissue is included in the present invention.These authentication methods are well-known to those skilled in the art, and more classical is, for example, ImmunohistochemistryMethods Methods or co-immunoprecipitation method.
SABC (Immunohistochemistry) is technology well-known to those skilled in the art, i.e. applied immunology and histochemistry's principle, and qualitative, positioning or quantitative in situ is carried out to some of histotomy or cell specimen chemical composition.ImmunohistochemistryMethods Methods are usually that the distribution with the specific antibody (or antigen) of mark to tissue endoantigen (or antibody) carries out tissue and cell in-situ detection.
Co-immunoprecipitation (Co-Immunoprecipitation) is also technology well-known to those skilled in the art, its be the selectivity effect between antibody and antigen based on be used for the classical way that Way for Studying Protein-Protein Interactions or protein interact with DNA.It is to determine two kinds of protein or protein and the DNA effective ways that physiological interacts in intact cell.
Recognize Hmc reagent and application thereof
Described hmc can be used for the reagent for preparing diagnosis cancer.Because hmc is micromolecular compound, with weaker immunogenicity, it is therefore preferred that connecting (coupling is adhered to) to carrier protein using hmc as haptens, the antigen with immunogenicity is prepared.Described carrier protein is class large biological molecule without or with relatively low immunogenicity itself, and carrier protein is selected from example as mentioned：Bovine serum albumin(BSA) (BSA), oralbumin (OVA), hemocyanin (KLH).Described antigen-immunized animal can obtain specific recognition hmc antibody.
The antibody of the present invention can be prepared by various technologies known to a person skilled in the art.For example, the antigen of purifying can be applied to animal to induce the generation of polyclonal antibody, described animal such as rabbit, mouse, rat etc..A variety of adjuvants can be used for enhancing immune response, including but not limited to Freund's adjuvant etc..
The antibody of the present invention can also be monoclonal antibody.Such monoclonal antibody can be prepared using hybridoma technology (see Kohler et al., Nature 256；495,1975；Kohler et al., Eur.J.Immunol.6：511,1976；Kohler et al., Eur.J.Immunol.6：292,1976；Hammerling et al., InMonoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).As the preferred embodiment of the present invention, described monoclonal antibody is CCTCC No by preserving number：C201191 hybridoma cell strain is produced, and recognizes that the specificity of antigen is good through surveying it.
Described antibody can be used in immunohistochemistry technology, the hmc levels in detection sample, so that for diagnosing cancer.
As the preferred embodiment of the present invention there is provided a kind of polyclonal antibody, the preparation method of the antibody is as follows：By hmc coupling bovine serum albumin(BSA)s (BSA), adaptive immune antigen after the immunizing antigen is mixed with Freund's adjuvant, is expelled to rabbit, and 0.5 ± 0.2mg of amount is immunized, was immunized 1 time every 2 weeks, is immunized 3 times, polyclonal antibody is separated from rabbit anteserum.The antibody specificity is good, and detection sensitivity is high, and accuracy is high, can be well applied to Immunohistochemical detection.
Kit
Present invention also offers the kit for diagnosing cancer, the kit includes：The reagent of specific recognition 5-hydroxymethyl cytosine.Described reagent is, for example,：Monoclonal antibody or polyclonal antibody.
In addition, can also contain in described kit：For the reagent of immunohistochemical analysis, described reagent is for example：Coloring agent, developer etc..
In addition, may also include operation instructions etc. in described kit.
Main advantages of the present invention are：
(1) disclosing hmc first can be as the mark for diagnosing cancer, and it can be used for the diagnosis of kinds cancer wide spectrum, and may indicate that the order of severity of cancer；For certain cancers can also as parting foundation.
(2) specific recognition hmc reagent has been prepared first, the reagent specificity is good, detection sensitivity is high, accuracy is high, with good clinical value.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition such as Sambrook et al., molecular cloning：Lab guide (New York：Cold Spring Harbor Laboratory Press, 2002) described in condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and number are calculated by weight.
Unless otherwise defined, all specialties used in text are identical with meaning known to one skilled in the art with scientific words.In addition, any method similar or impartial to described content and material all can be applied in the present invention.Preferable implementation described in text only presents a demonstration with material to be used.
The preparation of embodiment 1,5-hydroxymethyl cytosine (hmc) antigen
5-hydroxymethyl cytosine (hmc) is micromolecular compound, is injected directly into the internal of rabbit, rat, mouse or other immune animals and is not enough to cause antigen-antibody immune response.Therefore, the present inventor is using hmc as hapten conjugation to carrier proteins such as BSA (bovine serum albumin(BSA)), OVA (oralbumin), KLH (hemocyanin), then it is injected into again and resisting more for identification hmc is prepared in the immune animal body such as rabbit, mouse, and by being merged and screening with clone cell, prepare identification hmc monoclonal antibody.
1. method and product detection that bovine serum albumin(BSA) BSA and 5-hydroxymethyl cytosine hmc is coupled
(1) 10mg hmc (PY7596, Berry ＆ Associates, Inc.USA) are weighed, are put into 1.5ml Ep pipes；
(2) 500 μ l 0.1M NaIO is added4, 20min is reacted in dark place at room temperature with hmc；
(3) 30 μ l 1M ethylene glycol is added, 5min is reacted at room temperature, to react away unnecessary NaIO4；
(4) above-mentioned reaction mixture is added in 1ml 28mg/ml BSA solution to (weighing 28mg BSA powder, (Beijing is prosperous, lot：15310450), with 1ml distilled waters (water of Milli-Q ranks also can) dissolving, then with 5% (w/v) K2CO3Adjust PH to 9.1-9.5, matching while using), adjust PH in agitation, it is ensured that PH is maintained at 9.1-9.5, and 45min is reacted at room temperature；
(5) 1ml 15mg/ml NaBH is added4, 18h is reacted at room temperature；
(6) 500 μ l 1M methanol is added, 1h is reacted at room temperature, to react away unnecessary NaBH4；
(7) 350-450 μ l 1M ammoniacal liquor is added, PH to 8.5 is adjusted；
(8) reaction solution is dialysed 10h in the water of flowing；
(9) mass spectrum or SDS-PAGE detections determine that hmc is successfully coupled on BSA.
The Mass Spectrometer Methods of MALDI-TOF 4800 find that the control group BSA molecular weight not being coupled is 66809, but the molecular weight of product becomes 69022 (Fig. 1) after coupling.Because latter hmc molecular weight of coupling is (274-18), therefore, the hmc numbers being coupled on an average BSA molecule are：(69022-66809)/(274-18)=8.3.
Separated by SDS-PAGE product after control group BSA and coupling is not coupled, dyed and found with Coomassic：BSA ratios are not coupled control group BSA movements slowly after reaction, illustrate that hmc is successfully coupled on BSA (Fig. 2) after reaction.
2. the method that oralbumin OVA and 5-hydroxymethyl cytosine hmc is coupled
(1) 5mg hmc are weighed, are put into 1.5ml Ep pipes；
(2) 500 μ l 0.1M NaIO is added4, 20min is reacted in dark place at room temperature with hmc；
(3) 30 μ l 1M ethylene glycol is added, 5min is reacted at room temperature, to react away unnecessary NaIO4；
(4) above-mentioned reaction mixture is added in 1ml 28mg/ml OVA solution (weigh 28mg OVA powder (Sigma, A5503-1G), with 1ml distilled waters (water of Milli-Q ranks also can) dissolving, then with 5% K2CO3Adjust PH to 9.1-9.5, matching while using), adjust PH in agitation, it is ensured that PH is maintained at 9.1-9.5, and 45min is reacted at room temperature；
(5) 1ml 15mg/ml NaBH is added4, 18h is reacted at room temperature；
(6) 500 μ l 1M methanol is added, 1h is reacted at room temperature, to react away unnecessary NaBH4；
(7) 350-450 μ l 1M ammoniacal liquor is added, PH to 8.5 is adjusted；
(8) reaction solution is dialysed 10h in the water of flowing；
(9) Mass Spectrometer Method determines that hmC is successfully coupled on OVA (Fig. 3).
Same method, by adjusting molar ratio, different bases can be coupled on OVA (table 1).
Table 1, different bases are coupled on OVA used molar ratio
The number such as table 2 for the various nucleosides being coupled on an average OVA molecule
Table 2
3. the method that hemocyanin KLH and 5-hydroxymethyl cytosine hmc is coupled
(1) 10mg hmc are weighed, are put into 1.5ml Ep pipes；
(2) 500 μ l 0.1M NaIO is added4, 20min is reacted in dark place at room temperature with hmc；
(3) 30 μ l 1M ethylene glycol is added, 5min is reacted at room temperature, to react away unnecessary NaIO4；
(4) above-mentioned reaction mixture is added in 1ml 6mg/ml KLH solution (weigh 6mg KLH powder (Sigma, H8283-100MG), with 1ml distilled waters (water of Milli-Q ranks also can) dissolving, then with 5% K2CO3Adjust PH to 9.1-9.5), adjust PH in agitation, it is ensured that PH is maintained at 9.1-9.5, and 45min is reacted at room temperature；
(5) 1ml 15mg/ml NaBH is added4, 18h is reacted at room temperature；
(6) 500 μ l 1M methanol is added, 1h is reacted at room temperature, to react away unnecessary NaBH4；
(7) 350-450 μ l 1M ammoniacal liquor is added, PH to 8.5 is adjusted；
(8) reaction solution is dialysed 10h in the water of flowing；
(9) determine that hmC is successfully coupled on KLH by UV spectrophotometer measuring.
Ultraviolet spectro-photometric analysis finds (Fig. 4)：Free KLH and hmc have different absorbances respectively, the coupled product with other absorbances can be generated after coupling reaction occurs for KLH and hmc, and due to there occurs coupling reaction, free KLH and hmc amount reduction, therefore free KLH and hmc absorbance changes, and illustrates that hmc is successfully coupled on KLH.
The preparation method of embodiment 2,5-hydroxymethyl cytosine (hmc) antibody
The preparation of rabbit polyclonal antibody
The present inventor is injected into the hmc being coupled on BSA carrier proteins in rabbit body, obtains immune serum.Preparation method is as follows：After hmc coupling bovine serum albumin(BSA)s (BSA) are mixed with Freund's adjuvant, rabbit is expelled to, amount 0.5mg is immunized, was immunized 1 time every 2 weeks, is immunized 3 times.The rabbit polyclonal antibody for hmc is obtained after conventional affinity purification.
Immunoblot experiment is shown：Rabbit polyclonal antibody HMC-PA01 only recognizes hmc, basic nonrecognition 5-methylcytosine (5mC) (Fig. 5) and other bases such as adenine, guanine, cytimidine and thymidine etc..
The preparation of mouse monoclonal antibody
The present inventor is injected into the hmc being coupled on BSA carrier proteins in Mice Body, obtain immune serum, the mouse for producing high-titer, the hmc antibody of high specificity is selected by ELISA and immunoblot experiment, after these Mouse spleen cells and clone strain fusion, screening produces high-titer, the hybridoma cell strain (preserving number of high specificity hmc antibody：CCTCC No：C201191), it secretes mouse monoclonal antibody MA7.
In hybridoma cell strain injection Mice Body, it is set to produce ascites, these ascites contain the hmc monoclonal antibodies of high concentration.
Immunoblot experiment is shown：Mouse monoclonal antibody MA7 only recognizes hmc, basic nonrecognition 5-methylcytosine (5mC) (Fig. 8) and other bases such as adenine, guanine, cytimidine and thymidine etc..
The experimental applications of embodiment 3, hmc antibody
1. rabbit polyclonal antibody can specifically detect 5-hydroxymethyl cytosine hmc
In order to detect that can HMC-PA01 polyclonal antibodies be used for immunostaining, the present inventor obtains the cDNA that catalysis produces hmc enzyme Tet1 with RT-PCR method from mouse embryo stem cell, and the amino acid that the cDNA contains C-terminal the 1367 to 2007th (is based on GenBank accession number：NM030625 protein sequence) coded sequence, two ends set Nhe I and Bam HI restriction enzyme sites.PIRES2-EGFP of the insertion containing green fluorescent protein (GFP) coded sequence after Nhe I and BamHI digestions, it is built into a plasmid pmTET1-ires-GFP, and it is transferred to 293T cells (CRL-11268, U.S. ATCC) in, the cell for being transferred to and not being transferred to is distinguished with GFP, the cell GFP being transferred to is positive, on the contrary, the cell GFP not being transferred to is negative.Immunofluorescence dyeing (Fig. 6) is carried out to these cells with hmc rabbit polyclonal antibody HMC-PA01 or 5mC antibody (clone 162 33D3, Calbiochem, CA, the U.S.), as a result shown：The cell hmc for not being transferred to Tet1 is negative staining, and the cell hmc stained positives being transferred to.On the contrary, the cell 5mC for not being transferred to Tet1 is positive, the cell 5mC being transferred to is negative.
Illustrate that hmc rabbit polyclonal antibodies can be used for immunostaining, can specifically detect intracellular hmc.
2.hmc rabbit polyclonal antibodies can be used for co-immunoprecipitation (hmcDIP)
In order to detect that can HMC-PA01 rabbit polyclonal antibodies be used for co-immunoprecipitation, it is intracellular that pmTET1-ires-GFP is transferred to 293T by the present inventor, the cell separation for being transferred to He not being transferred to is come with GFP, these cells are carried out with co-immunoprecipitation experiment (Fig. 7) with hmc rabbit polyclonal antibodies or 5mC antibody.With when not being transferred to 293T cells and carrying out co-immunoprecipitation, 5mC antibody can precipitate DNA, and hmc antibody HMC-PA01 can only precipitate seldom DNA；On the contrary, with when being transferred to Tet1 cells and carrying out co-immunoprecipitation, the cell that the DNA ratios of 5mC antibody precipitation are not transferred to Tet1 is significantly reduced, and demonstrates Tet1 and many 5mC are become hmc；Tested with these cells, hmc antibody HMC-PA01 can just precipitate many DNA, illustrate that hmc rabbit polyclonal antibodies can be used for co-immunoprecipitation.The co-immunoprecipitation carried out with hmc antibody is called hmc co-immunoprecipitations (hmc DNA immunoprecipitation, hmcDIP) by the present inventor.
Similarly, co-immunoprecipitation experiment is found, ES cells 46C (can be obtained from Austin Smith by hmc antibody and 5mC antibody, Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, Britain) in DNA precipitate.
3. mouse monoclonal antibody can specifically detect 5-hydroxymethyl cytosine hmc
In order to detect that can MA7 mouse monoclonal antibodies be used for immunostaining, the present inventor is transferred to pmTET1-ires-GFP plasmids in the cell that 293T is intracellular, is transferred to and is not transferred to GFP differentiations, and the cell GFP being transferred to is positive, on the contrary, the cell GFP not being transferred to is into feminine gender.These cells are carried out with immunofluorescence dyeing (Fig. 9) with hmc mouse monoclonal antibodies or rabbit polyclonal antibody HMC-PA01, is as a result shown：The cell hmc mouse monoclonal antibodies for not being transferred to Tet1 are negative staining, and the cell hmc mouse monoclonal antibody stained positives being transferred to.This is as rabbit polyclonal antibody HMC-PA01 dyeing.
Illustrate that hmc mouse monoclonal antibodies MA7 can be used for immunostaining, can specifically detect intracellular hmc.
4.hmc mouse monoclonal antibodies can be used for co-immunoprecipitation (hmcDIP)
In order to detect that can mouse monoclonal antibody be used for co-immunoprecipitation, it is intracellular that pmTET1-ires-GFP is transferred to 293T by the present inventor, the cell separation for being transferred to He not being transferred to is come with GFP, these cells are carried out with co-immunoprecipitation experiment (Figure 10) with hmc mouse monoclonal antibodies or 5mC antibody.With when not being transferred to 293T cells and carrying out co-immunoprecipitation, 5mC antibody can precipitate DNA, and hmc antibody can hardly precipitate any DNA；On the contrary, with when being transferred to Tet1 cells and carrying out co-immunoprecipitation, the cell that the DNA ratios of 5mC antibody precipitation are not transferred to Tet1 is significantly reduced, and demonstrates Tet1 and many 5mC are become hmc；Tested with these cells, hmc antibody can just precipitate many DNA, illustrate that hmc mouse monoclonal antibodies can be used for co-immunoprecipitation.
Similarly, co-immunoprecipitation experiment is found, hmc antibody can precipitate the DNA in ES cells 46C or in mouse brain (Figure 11).
The medical application of embodiment 4,5-hydroxymethyl cytosine (hmc) antibody
1.hmc dyeing can be used for diagnosing kidney
Can the present inventor be used for the level of hmc in immunohistochemical staining detection renal carcinoma tissue with the inspection of pathological number 200401486 hmC polyclonal antibodies HMC-PA01.The renal carcinoma tissue's section for including cancerous tissue and cancer beside organism carries out immunohistochemical staining with hmc antibody, histotomy is through dewaxing, hydrogen peroxide eliminates the activity of endogenous peroxydase, 2N HCl treatments expose antigen, HMC-PA01 is incubated, and horseradish enzyme mark secondary antibody is incubated, and DAB colour developings are found, most cells hmc stained positives in normal renal tubule in cancer beside organism, but cancerous tissue hmc dyeing in same section is negative (Figure 12 left).In order to illustrate that this difference is not that staining technique is caused, the present inventor dyes adjacent renal carcinoma tissue with 5mC and cut into slices as stain control, as a result shows：Cancer cell and the normal renal tubular cell of its cancer beside organism have similar 5mC staining powers (Figure 12 is right).The immunohistochemical staining result is pointed out：Hmc is lost in renal carcinoma tissue.
The present inventor includes the tissue of the kidney pathological number 200309294 of cancerous tissue and cancer beside organism with hmc and 5mC immunohistochemical stainings, obtains same result (Figure 13).Most cells hmc stained positives in normal renal tubule in cancer beside organism, but cancerous tissue hmc dyeing be negative；Cancer cell and the normal renal tubular cell of its cancer beside organism have similar 5mC staining powers.The immunohistochemical staining result is further pointed out：Hmc is lost in renal carcinoma tissue.
In order to check the generality of hmc forfeitures in renal carcinoma tissue, the present inventor carries out hmc immunohistochemical stainings using HMC-PA01 polyclonal antibodies to 49 renal carcinoma tissue's samples, wherein 40 samples contain cancerous tissue and cancer beside organism's (table 3).In order to analyze hmc coloration results, the present inventor employs a quantitative integration method.Staining power is divided into 4 grades, 0 point=do not colour, 1=weakly positives, the coloring of 2=moderates, the coloring of 3=intensity；It is divided into level Four according to histocyte total amount (range) percentage shared by pigmented cells simultaneously, 0=is less than 5%, 1=5-30%, 2=30-60%, 3=is more than 60%, using tinctorial strength and range product as coloration result, 2 points and following for feminine gender, 3-4 points are weakly positive, and 6 and 9 points are the positive.Cancer beside organism's dyeing display：Most of cancer beside organism hmc is positive (6-9 points) (34/40).And in the renal carcinoma tissue of these patients, in addition to 4 hmc staining powers are identical with cancer beside organism (numbering 24,32,41,7), the hmc staining powers of other 36 have obvious reduction, wherein, 16 hmc feminine genders (0-2 points).These data illustrate that hmc is significantly reduced (Figure 14) in most of (90%) renal carcinoma tissue, and hmc is lost in 40% renal carcinoma tissue.Therefore, hmc dyeing can be used for diagnosing kidney.
Table 3
Table 3, renal carcinoma tissue's hmc immunohistochemical stainings.Quantitative integration analysis is carried out to kidney hmc coloration results.Staining power is divided into 4 grades, 0 point=do not colour, 1=weakly positives, the coloring of 2=moderates, the coloring of 3=intensity；It is divided into level Four according to histocyte total amount (range) percentage shared by pigmented cells simultaneously, 0=is less than 5%, 1=5-30%, 2=30-60%, 3=is more than 60%, using tinctorial strength and range product as coloration result, 2 points and following for feminine gender, 3-4 points are weakly positive, and 6-9 points are the positive.
Hmc dyeing can be used for diagnosing bladder cancer
The present inventor have detected the hmc levels of normal Urothelial cell with polyclonal antibody HMC-PA01 immunohistochemical stainings, as a result show：Normal Urothelial cell has strong hmc dyeing, illustrates that normal Urothelial cell contains high-caliber hmc (Figure 15).
In order to check whether hmc loses and its lose in Bladder Cancer generality, the present inventor carries out hmc immunohistochemical stainings (table 4) (Figure 16) to 54 Bladder Cancer samples, with reference to the integration method of table 3, quantitative integration analysis is carried out to hmc coloration results.Staining power is divided into 4 grades, 0 point=do not colour, 1=weakly positives, the coloring of 2=moderates, the coloring of 3=intensity；It is divided into level Four according to histocyte total amount (range) percentage shared by pigmented cells simultaneously, 0=is less than 5%, 1=5-30%, 2=30-60%, 3=is more than 60%, using tinctorial strength and range product as tissue staining degree, hmc in tissue of patient is horizontally divided into negative (2 points and less), weakly positive (3-4 points), positive (6-9 points).Wherein, 14 hmc positives (6-9 points), 16 hmc are in weakly positive (3-4 points), and 24 are hmc negative (0-2 points), illustrate that 44% Bladder Cancer all loses hmc.
In order to check whether hmc levels are associated with the order of severity of carcinoma of urinary bladder in Bladder Cancer, and carcinoma of urinary bladder is divided into 4 classes by the present inventor according to pathological diagnosis：Low level non-infiltration bladder transitional cell carcinoma, low level bladder transitional cell carcinoma, high-level bladder transitional cell carcinoma and high-level wellability bladder transitional cell carcinoma (table 5), and severity of cancer and hmc levels are associated analysis (Figure 17).As a result show：The average hmc dyeing fractions of low level non-infiltration bladder transitional cell carcinoma are 6 points, the average hmc of low level bladder transitional cell carcinoma dyeing fractions are 3.6 points, the average hmc dyeing fractions of high-level bladder transitional cell carcinoma are 2.8 points, the average hmc dyeing fractions of high-level wellability bladder transitional cell carcinoma are 2.7 points.These data displays：The seriousness of Bladder Cancer hmc levels and cancer is negatively correlated.Therefore, hmc dyeing can be used for the order of severity for judging carcinoma of urinary bladder.
Table 4
| Numbering | Sex | Age | Pathological number | Patient's present circumstances | Pathological diagnosis intensity | Range | As a result |
| 30 | Man | 70 | 200404524 | Recur within 09 year, existing work | Low level bladder transitional cell carcinoma 0 | 0 | 0 |
| 20 | Man | 70 | 200311800 | 2004 dead | High-level bladder transitional cell carcinoma 0 | 0 | 0 |
| 61 | Man | 72 | 200517740 | Recurrence 2 times, now very well | High-level bladder transitional cell carcinoma 0 | 0 | 0 |
| 65 | Female | 55 | 200519995 | 2 recurrences.It is now fine | High-level bladder transitional cell carcinoma 0 | 0 | 0 |
| 3 | Man | 58 | 200215946 | Recur within 2007, existing work | Low level bladder transitional cell carcinoma 1 | 1 | 1 |
| 4 | Man | 76 | 200216104 | Very well | Low level bladder transitional cell carcinoma 1 | 1 | 1 |
| 15 | Man | 64 | 200306201 | Very well | Low level bladder transitional cell carcinoma 1 | 1 | 1 |
| 18 | Female | 54 | 200311399 | Very well | Low level bladder transitional cell carcinoma 1 | 1 | 1 |
| 21 | Man | 82 | 200312533 | Survival | Low level bladder transitional cell carcinoma 1 | 1 | 1 |
| 29 | Man | 73 | 200403832 | Very well | Low level bladder transitional cell carcinoma 1 | 1 | 1 |
| 31 | Man | 69 | 200404531 | Very well | Low level bladder transitional cell carcinoma 1 | 1 | 1 |
| 40 | Man | 62 | 200413881 | Very well | Low level bladder transitional cell carcinoma 1 | 1 | 1 |
| 23 | Man | 65 | 200315649 | 2009 dead | High-level bladder transitional cell carcinoma 1 | 1 | 1 |
| 24 | Female | 78 | 200315824 | Very well | High-level bladder transitional cell carcinoma 1 | 1 | 1 |
| 59 | Man | 77 | 200515958 | Very well | Wellability bladder transitional cell carcinoma 1 | 1 | 1 |
| 56 | Man | 78 | 200512267 | Very well | Low level non-infiltration bladder transitional cell carcinoma 1 | 2 | 2 |
| 2 | Man | 22 | 200214735 | 2007 dead | Low level bladder transitional cell carcinoma 2 | 1 | 2 |
| 47 | Man | 70 | 200503353 | Very well | High-level wellability bladder transitional cell carcinoma 2 | 1 | 2 |
| 9 | Man | 24 | 200304456 | Postoperative half a year recurrence is dead | High-level bladder transitional cell carcinoma 1 | 2 | 2 |
| 11 | Man | 57 | 200305963 | Very well | High-level bladder transitional cell carcinoma 1 | 2 | 2 |
| 42 | Female | 70 | 200414872 | 2004 dead | High-level bladder transitional cell carcinoma 1 | 2 | 2 |
| 48 | Female | 43 | 200503756 | Recur within 09 year, existing work | High-level bladder transitional cell carcinoma 1 | 2 | 2 |
| 49 | Man | 86 | 200505447 | 05 year dead | Wellability bladder transitional cell carcinoma 1 | 2 | 2 |
| 53 | Man | 74 | 200508845 | Very well | Wellability bladder transitional cell carcinoma 1 | 2 | 2 |
| 7 | Man | 74 | 200302398 | Recur within 04 year, existing work | Low level bladder transitional cell carcinoma 1 | 3 | 3 |
| 62 | Man | 68 | 200518515 | Recur within 06 year, now very well | High-level wellability bladder transitional cell carcinoma 1 | 3 | 3 |
| 64 | Man | 74 | 200520330 | 2008 dead | High-level wellability bladder transitional cell carcinoma 1 | 3 | 3 |
| 26 | Man | 51 | 200400744 | 2006 dead because shifting | High-level bladder transitional cell carcinoma 1 | 3 | 3 |
| 45 | Female | 68 | 200415880 | Recur twice, existing work | High-level bladder transitional cell carcinoma 1 | 3 | 3 |
| 50 | Man | 59 | 200506200 | Very well | Wellability bladder transitional cell carcinoma 1 | 3 | 3 |
| 57 | Man | 76 | 200512982 | Very well | Bladder transitional cell carcinoma 1 | 3 | 3 |
| 63 | Man | 79 | 200519964 | Very well | Low level non-infiltration bladder transitional cell carcinoma 2 | 2 | 4 |
| 13 | Female | 55 | 200305858 | Very well | Low level bladder transitional cell carcinoma 2 | 2 | 4 |
| 39 | Female | 71 | 200411739 | Very well | Low level bladder transitional cell carcinoma 2 | 2 | 4 |
| 8 | Man | 33 | 200304135 | Survival | High-level bladder transitional cell carcinoma 2 | 2 | 4 |
| 12 | Female | 64 | 200305648 | Multiple relapse, existing work | High-level bladder transitional cell carcinoma 2 | 2 | 4 |
| 32 | Man | 59 | 200405527 | It is dead | High-level bladder transitional cell carcinoma 2 | 2 | 4 |
| 44 | Female | 50 | 200415906 | Very well | High-level bladder transitional cell carcinoma 2 | 2 | 4 |
| 51 | Man | 56 | 200506818 | Very well | Wellability bladder transitional cell carcinoma 2 | 2 | 4 |
| 54 | Man | 68 | 200508645 | 2008 dead | Wellability bladder transitional cell carcinoma 2 | 2 | 4 |
| 5 | Man | 55 | 200300702 | Very well | Low level bladder transitional cell carcinoma 3 | 2 | 6 |
| 17 | Man | 39 | 200309583 | Very well | Low level bladder transitional cell carcinoma 2 | 3 | 6 |
| 34 | Female | 46 | 200407490 | Recur within 05 year, existing work | Low level bladder transitional cell carcinoma 2 | 3 | 6 |
| 38 | Man | 63 | 200409744 | Very well | Low level bladder transitional cell carcinoma 2 | 3 | 6 |
| 41 | Man | 37 | 200413595 | Shift within 2010, existing work | Low level bladder transitional cell carcinoma 2 | 3 | 6 |
| 46 | Man | 45 | 200416212 | Very well | Low level bladder transitional cell carcinoma 2 | 3 | 6 |
| 25 | Man | 75 | 200400547 | Multiple relapse, existing work | High-level bladder transitional cell carcinoma 2 | 3 | 6 |
| 35 | Man | 44 | 200407782 | Very well | High-level bladder transitional cell carcinoma 2 | 3 | 6 |
| 43 | Female | 79 | 200415729 | It is dead | High-level bladder transitional cell carcinoma 2 | 3 | 6 |
| 58 | Man | 56 | 200514823 | Very well | Wellability bladder transitional cell carcinoma 2 | 3 | 6 |
| 52 | Man | 67 | 200508336 | Very well | Low level non-infiltration bladder transitional cell carcinoma 3 | 3 | 9 |
| 55 | Man | 78 | 200510504 | Very well | Low level non-infiltration bladder transitional cell carcinoma 3 | 3 | 9 |
| 16 | Female | 49 | 200309463 | Recur within 09 year, existing work | Low level bladder transitional cell carcinoma 3 | 3 | 9 |
| 28 | Man | 66 | 200402444 | Recur within 08 year, existing work | Low level bladder transitional cell carcinoma 3 | 3 | 9 |
Table 4, Bladder Cancer hmc immunohistochemical stainings.With reference to aforementioned integral method, quantitative integration analysis is carried out to carcinoma of urinary bladder hmc coloration results.Staining power is divided into 4 grades, 0 point=do not colour, 1=weakly positives, the coloring of 2=moderates, the coloring of 3=intensity；It is divided into level Four according to histocyte total amount (range) percentage shared by pigmented cells simultaneously, 0=is less than 5%, 1=5-30%, 2=30-60%, 3=is more than 60%, using tinctorial strength and range product as dye levels, and less than 3 points are feminine gender, 4-6 points are weakly positive, and more than 6 points are the positive.
Table 5
| Numbering | Sex | Age | Pathological number | Patient's present circumstances | Pathological diagnosis | Intensity | Range | As a result |
| 56 | Man | 78 | 200512267 | Very well | Low level non-infiltration bladder transitional cell carcinoma | 1 | 2 | 2 |
| 63 | Man | 79 | 200519964 | Very well | Low level non-infiltration bladder transitional cell carcinoma | 2 | 2 | 4 |
| 52 | Man | 67 | 200508336 | Very well | Low level non-infiltration bladder transitional cell carcinoma | 3 | 3 | 9 |
| 55 | Man | 78 | 200510504 | Very well | Low level non-infiltration bladder transitional cell carcinoma | 3 | 3 | 9 |
| 30 | Man | 70 | 200404524 | Recur within 09 year, existing work | Low level bladder transitional cell carcinoma | 0 | 0 | 0 |
| 3 | Man | 58 | 200215946 | Recur within 2007, existing work | Low level bladder transitional cell carcinoma | 1 | 1 | 1 |
| 4 | Man | 76 | 200216104 | Very well | Low level bladder transitional cell carcinoma | 1 | 1 | 1 |
| 15 | Man | 64 | 200306201 | Very well | Low level bladder transitional cell carcinoma | 1 | 1 | 1 |
| 18 | Female | 54 | 200311399 | Very well | Low level bladder transitional cell carcinoma | 1 | 1 | 1 |
| 21 | Man | 82 | 200312533 | Survival | Low level bladder transitional cell carcinoma | 1 | 1 | 1 |
| 29 | Man | 73 | 200403832 | Very well | Low level bladder transitional cell carcinoma | 1 | 1 | 1 |
| 31 | Man | 69 | 200404531 | Very well | Low level bladder transitional cell carcinoma | 1 | 1 | 1 |
| 40 | Man | 62 | 200413881 | Very well | Low level bladder transitional cell carcinoma | 1 | 1 | 1 |
| 2 | Man | 22 | 200214735 | 2007 dead | Low level bladder transitional cell carcinoma | 2 | 1 | 2 |
| 7 | Man | 74 | 200302398 | Recur within 04 year, existing work | Low level bladder transitional cell carcinoma | 1 | 3 | 3 |
| 13 | Female | 55 | 200305858 | Very well | Low level bladder transitional cell carcinoma | 2 | 2 | 4 |
| 39 | Female | 71 | 200411739 | Very well | Low level bladder transitional cell carcinoma | 2 | 2 | 4 |
| 5 | Man | 55 | 200300702 | Very well | Low level bladder transitional cell carcinoma | 3 | 2 | 6 |
| 17 | Man | 39 | 200309583 | Very well | Low level bladder transitional cell carcinoma | 2 | 3 | 6 |
| 34 | Female | 46 | 200407490 | Recur within 05 year, existing work | Low level bladder transitional cell carcinoma | 2 | 3 | 6 |
| 38 | Man | 63 | 200409744 | Very well | Low level bladder transitional cell carcinoma | 2 | 3 | 6 |
| 41 | Man | 37 | 200413595 | Shift within 2010, existing work | Low level bladder transitional cell carcinoma | 2 | 3 | 6 |
| 46 | Man | 45 | 200416212 | Very well | Low level bladder transitional cell carcinoma | 2 | 3 | 6 |
| 16 | Female | 49 | 200309463 | Recur within 09 year, existing work | Low level bladder transitional cell carcinoma | 3 | 3 | 9 |
| 28 | Man | 66 | 200402444 | Recur within 08 year, existing work | Low level bladder transitional cell carcinoma | 3 | 3 | 9 |
| 47 | Man | 70 | 200503353 | Very well | High-level wellability bladder transitional cell carcinoma | 2 | 1 | 2 |
| 62 | Man | 68 | 200518515 | Recur within 06 year, now very well | High-level wellability bladder transitional cell carcinoma | 1 | 3 | 3 |
| 64 | Man | 74 | 200520330 | 2008 dead | High-level wellability bladder transitional cell carcinoma | 1 | 3 | 3 |
| 20 | Man | 70 | 200311800 | 2004 dead | High-level bladder transitional cell carcinoma | 0 | 0 | 0 |
| 61 | Man | 72 | 200517740 | Recurrence 2 times, now very well | High-level bladder transitional cell carcinoma | 0 | 0 | 0 |
| 65 | Female | 55 | 200519995 | 2 recurrences.It is now fine | High-level bladder transitional cell carcinoma | 0 | 0 | 0 |
| 23 | Man | 65 | 200315649 | 2009 dead | High-level bladder transitional cell carcinoma | 1 | 1 | 1 |
| 24 | Female | 78 | 200315824 | Very well | High-level bladder transitional cell carcinoma | 1 | 1 | 1 |
| 9 | Man | 24 | 200304456 | Postoperative half a year recurrence is dead | High-level bladder transitional cell carcinoma | 1 | 2 | 2 |
| 11 | Man | 57 | 200305963 | Very well | High-level bladder transitional cell carcinoma | 1 | 2 | 2 |
| 42 | Female | 70 | 200414872 | 2004 dead | High-level bladder transitional cell carcinoma | 1 | 2 | 2 |
| 48 | Female | 43 | 200503756 | Recur within 09 year, existing work | High-level bladder transitional cell carcinoma | 1 | 2 | 2 |
| 26 | Man | 51 | 200400744 | 2006 dead because shifting | High-level bladder transitional cell carcinoma | 1 | 3 | 3 |
| 45 | Female | 68 | 200415880 | Recur twice, existing work | High-level bladder transitional cell carcinoma | 1 | 3 | 3 |
| 8 | Man | 33 | 200304135 | Survival | High-level bladder transitional cell carcinoma | 2 | 2 | 4 |
| 12 | Female | 64 | 200305648 | Multiple relapse, existing work | High-level bladder transitional cell carcinoma | 2 | 2 | 4 |
| 32 | Man | 59 | 200405527 | It is dead | High-level bladder transitional cell carcinoma | 2 | 2 | 4 |
| 44 | Female | 50 | 200415906 | Very well | High-level bladder transitional cell carcinoma | 2 | 2 | 4 |
| 25 | Man | 75 | 200400547 | Multiple relapse, existing work | High-level bladder transitional cell carcinoma | 2 | 3 | 6 |
| 35 | Man | 44 | 200407782 | Very well | High-level bladder transitional cell carcinoma | 2 | 3 | 6 |
| 43 | Female | 79 | 200415729 | It is dead | High-level bladder transitional cell carcinoma | 2 | 3 | 6 |
| 59 | Man | 77 | 200515958 | Very well | Wellability bladder transitional cell carcinoma | 1 | 1 | 1 |
| 49 | Man | 86 | 200505447 | 05 year dead | Wellability bladder transitional cell carcinoma | 1 | 2 | 2 |
| 53 | Man | 74 | 200508845 | Very well | Wellability bladder transitional cell carcinoma | 1 | 2 | 2 |
| 50 | Man | 59 | 200506200 | Very well | Wellability bladder transitional cell carcinoma | 1 | 3 | 3 |
| 51 | Man | 56 | 200506818 | Very well | Wellability bladder transitional cell carcinoma | 2 | 2 | 4 |
| 54 | Man | 68 | 200508645 | 2008 dead | Wellability bladder transitional cell carcinoma | 2 | 2 | 4 |
| 58 | Man | 56 | 200514823 | Very well | Wellability bladder transitional cell carcinoma | 2 | 3 | 6 |
| 57 | Man | 76 | 200512982 | Very well | Bladder transitional cell carcinoma | 1 | 3 | 3 |
Table 5, Bladder Cancer hmc immunohistochemical stainings result is reset by pathological diagnosis.With reference to aforementioned integral method, quantitative integration analysis is carried out to carcinoma of urinary bladder hmc coloration results, dye levels are used as using tinctorial strength and range product.And carcinoma of urinary bladder is divided into according to pathological diagnosis by 4 classes：Low level non-infiltration bladder transitional cell carcinoma, low level bladder transitional cell carcinoma, high-level bladder transitional cell carcinoma and high-level wellability bladder transitional cell carcinoma.
Hmc dyeing can be used for diagnosing glioma
The present inventor have detected the hmc levels of normal cerebral tissue's cell with polyclonal antibody HMC-PA01 immunohistochemical stainings, consistent with the result of report, and nerve cell contains high-caliber hmc (Figure 18).Spongiocyte also has strong dyeing (Figure 19) in normal cerebral tissue, and this is not find in the past, is illustrated in normal cerebral tissue in addition to nerve cell, and spongiocyte also contains high-caliber hmc.
In order to check whether hmc loses and its lose in samples of human glioma generality, the present inventor carries out hmc immunohistochemical stainings (table 6) to 55 samples of human glioma samples, wherein, 24 hmc positives (6-9 points), 18 hmc are in weakly positive (3-4 points), 13 are negative staining (0-2 points), illustrate the hmc (Figure 20) that different samples of human glioma contain varying level.
In order to check whether hmc levels are associated with severity of cancer in samples of human glioma, glioma is divided into 4 grades by the present inventor by tissue of patient according to pathological diagnosis rank：Severity of cancer and hmc levels, are associated analysis by I grades, II grades, III level and IV grades.Pathological diagnosis be I grades of cancer specimen average hmc dyeing fraction be 3 points, the average hmc for the cancer specimen that pathological diagnosis is II grade dyeing fraction be 6.1 points, the average hmc for the cancer specimen that pathological diagnosis is III level dyeing fraction be 4.5 points, the average hmc for the cancer specimen that pathological diagnosis is IV grades dye fraction be 3.9 points (Figure 21 A).Because the cancer specimen that pathological diagnosis is I grades only has 4, do not possess representativeness.Other 3 grades of sample dyeing data display：The seriousness of samples of human glioma hmc levels and cancer is into negative correlation.Therefore, hmc dyeing can be used for the order of severity for judging glioma.
In order to check whether hmc levels in samples of human glioma may determine that the time-to-live of glioma cancer patient, the present inventor carries out quantitative integration analysis with reference to aforementioned integral method to glioma hmc coloration results.Staining power is divided into 4 grades, 0 point=do not colour, 1=weakly positives, the coloring of 2=moderates, the coloring of 3=intensity；It is divided into level Four according to histocyte total amount (range) percentage shared by pigmented cells simultaneously, 0=is less than 5%, 1=5-30%, 2=30-60%, 3=is more than 60%, tissue staining degree is used as using tinctorial strength and range product, hmc in glioma tissue of patient is horizontally divided into negative (2 points and less), weakly positive (3-4 points), positive (6-9 points), the Kaplan-Meier time-survivor curves (Figure 22 A, B) of each class patient are then drawn.As a result show：The time-to-live of hmc patients with negative is significantly shorter than the patient of hmc weakly positives in tissue in tissue, and patient's time-to-live positive hmc in organizing is most long.
These as shown by data：Hmc levels may determine that the time-to-live of glioma cancer patient in glioma cancerous tissue, and level is lower, and the time-to-live is shorter.
Table 6
Table 6, samples of human glioma hmc immunohistochemical stainings.With reference to aforementioned integral method, quantitative integration analysis is carried out to glioma hmc coloration results.Staining power is divided into 4 grades, 0 point=do not colour, 1=weakly positives, the coloring of 2=moderates, the coloring of 3=intensity；It is divided into level Four according to histocyte total amount (range) percentage shared by pigmented cells simultaneously, 0=is less than 5%, 1=5-30%, and 2=30-60%, 3=is more than 60%.The result that tinctorial strength and range product are coloured as tissue.
In order to confirm the above results, the present inventor carries out hmc immunohistochemical stainings (table 7) to another group of 103 samples of human glioma sample.As a result find：Pathological diagnosis be I grades of cancer specimen average hmc dyeing fraction be 5 points, the average hmc for the cancer specimen that pathological diagnosis is II grade dyeing fraction be 5.3 points, the average hmc for the cancer specimen that pathological diagnosis is III level dyeing fraction be 3.1 points, the average hmc for the cancer specimen that pathological diagnosis is IV grades dye fraction be 1.8 points (Figure 21 B).The result confirms the seriousness of samples of human glioma hmc levels and cancer into negative correlation.Kaplan-Meier time-survivor curves result confirms that the time-to-live of hmc patients with negative in tissue is significantly shorter than the patient of hmc weakly positives in tissue, and patient's time-to-live positive hmc in organizing is most long (Figure 22 B).
In order to detect whether hmc levels are unrelated with the severity of cancer of current pathological diagnosis with patient's life span correlation in tissue, the present inventor is combined two groups of patients, and Kaplan-Meier time-survivor curve interpretations of result are carried out for the cancer patient of the same rank order of severity with current pathological diagnosis.Because I grades of cancer patient samples only have 10, it is not enough to carry out Kaplan-Meier time-survivor curve analyses.II grades of a total of 71 of cancer specimen is diagnosed as, Kaplan-Meier time-survivor curve analyses are found：The time-to-live of hmc patients with negative is shorter than the patient of hmc weakly positives in tissue in tissue, and patient's time-to-live positive hmc in organizing is most long (Figure 23 A).Kaplan-Meier time-survivor curve analyses are carried out to the cancer specimen for being diagnosed as III level, also, it was found that hmc negative and weakly positive patient time-to-live is significantly shorter than patient (Figure 23 B) positive hmc in tissue in tissue.These results illustrate the cancer patient that current pathological diagnosis is the same rank order of severity, can also pass through the further parting of hmc levels.
Table 7
Table 7, another group of samples of human glioma hmc immunohistochemical staining.With reference to aforementioned integral method, quantitative integration analysis is carried out to glioma hmc coloration results.Staining power is divided into 4 grades, 0 point=do not colour, 1=weakly positives, the coloring of 2=moderates, the coloring of 3=intensity；It is divided into level Four according to histocyte total amount (range) percentage shared by pigmented cells simultaneously, 0=is less than 5%, 1=5-30%, and 2=30-60%, 3=is more than 60%.The result that tinctorial strength and range product are coloured as tissue.
Hmc dyeing can be used for Diagnosis of Breast cancer
The present inventor detects the level of hmc in breast cancer tissue with hmc polyclonal antibody HMC-PA01 immunohistochemical stainings.Two include cancerous tissue and the breast tumor tissue sections of cancer beside organism carry out immunohistochemical staining discovery with hmc antibody：Normal breast ductal cells hmc stained positives in cancer beside organism, but its adjacent cancerous tissue hmc dyeing is negative (Figure 24).Other 4 breast cancer tissues immunohistochemical staining finds that hmc dyeing has different degrees of decline (Figure 25) in breast cancer tissue.The immunohistochemical staining result is pointed out：Hmc is lost in breast cancer tissue to some extent.
Therefore, hmc dyeing can be used for Diagnosis of Breast cancer, and judge the seriousness of cancer.
Hmc dyeing can be used for diagnosis of prostate cancer
The present inventor detects the level of hmc in prostate cancer tissue with hmc polyclonal antibody HMC-PA01 immunohistochemical stainings.The prostate cancer tissue section for including cancerous tissue and cancer beside organism carries out normal prostate cell hmc stained positives in immunohistochemical staining discovery, cancer beside organism with hmc antibody, but the cancerous tissue hmc dyeing in same patient source is negative (Figure 26).In order to illustrate that this difference is not that staining technique is caused, the present inventor dyes adjacent prostate cancer tissue with 5mC and cut into slices as stain control, as a result shows：Cancer cell and its cancer beside organism's normal prostate cell have similar 5mC staining powers.The immunohistochemical staining result is pointed out：Hmc is lost in prostate cancer tissue.Other prostate cancer tissue dyeing is found：Cancerous tissue does not completely lose hmc, still has some cells to contain low-level hmc (Figure 27).
Therefore, hmc dyeing can be used for diagnosis of prostate cancer, judge the seriousness of its cancer.
Hmc dyeing can be used for diagnosing colon
The present inventor detects the level of hmc in colon cancer tissue with hmC polyclonal antibody HMC-PA01 immunohistochemical stainings.The colon cancer tissue section for including cancerous tissue and cancer beside organism carries out part Normal Colon gland cell hmc stained positives in immunohistochemical staining discovery, cancer beside organism with hmc antibody, but the cancerous tissue hmc dyeing of same section is negative (Figure 28).Other two colon cancer tissues dyeing is found：Cancerous tissue loses hmc (Figure 29).
Therefore, hmc dyeing can be used for diagnosing colon.
Hmc dyeing can be used for diagnosis of gastric cancer
The present inventor detects the level of hmc in stomach organization with hmc polyclonal antibody HMC-PA01 immunohistochemical stainings.The stomach cancer tissue slides for including cancerous tissue and cancer beside organism carry out normal gastric gland cell hmc stained positives in immunohistochemical staining discovery, cancer beside organism with hmc antibody, but are negative (Figure 30) from the cancerous tissue hmc dyeing of same patient.
Therefore, hmc dyeing can be used for diagnosis of gastric cancer.
Hmc dyeing can be used for Diagnose Rectal Cancer
The present inventor detects the level of hmc in rectum cancer tissue with hmC polyclonal antibody HMC-PA01 immunohistochemical stainings.The rectum cancer tissue's section for including cancerous tissue and cancer beside organism carries out normal gland cell in immunohistochemical staining discovery, cancer beside organism with hmc antibody and has certain hmc dyeing, but derives from the cancerous tissue hmc of same patient and dye and be negative (Figure 31).
Therefore, hmc dyeing can be used for Diagnose Rectal Cancer.
Hmc dyeing can be used for diagnosing the cancer of the uterus
The present inventor detects the level of hmc in the cancerous tissue of uterus with hmc polyclonal antibody HMC-PA01 immunohistochemical stainings.The cancer of the uterus histotomy for including cancerous tissue and cancer beside organism carries out immunohistochemical staining discovery with hmc antibody：Normal smooth muscle cell hmc stained positives in cancer beside organism, but be negative (Figure 32) from the cancerous tissue hmc dyeing of same patient.Another cases with uterine cancerous tissue dyeing display：Normal uterus scaly epithelium and interstitial cell have certain hmc dyeing in cancer beside organism, but are negative (Figure 32) from the uterus squamous carcinoma tissue hmc dyeing of same patient.
Therefore, hmc dyeing can be used for diagnosing the cancer of the uterus.
Hmc dyeing can be used for diagnosing leiomyosarcoma
The present inventor detects the level of hmc in leiomyosarcoma tissue with hmc polyclonal antibody HMC-PA01 immunohistochemical stainings.The leiomyosarcoma tissue section for including cancerous tissue and cancer beside organism carries out immunohistochemical staining discovery with hmc antibody：Normal smooth muscle cell has certain hmc dyeing in cancer beside organism, but is negative (Figure 33) from the cancerous tissue hmc dyeing of same patient.
Therefore, hmc dyeing can be used for diagnosing muscle tumor.
Hmc dyeing can be used for diagnosing
The present inventor detects the level of hmc in cancerous lung tissue with hmc polyclonal antibody HMC-PA01 immunohistochemical stainings.Cancerous lung tissue section carries out immunohistochemical staining discovery with hmc antibody：Such as tissue such as squamous cell carcinoma, the small cell carcinoma hmc dyeing of most of lung cancer is negative, but adenocarcinoma cell is positive (Figure 34).
These as shown by data, hmc dyeing can distinguish different lung cancer, can be used for the parting of lung cancer.
Hmc dyeing can be used for diagnosing liver cancer
The present inventor detects the level of hmc in liver cancer tissue with hmc polyclonal antibody HMC-PA01 immunohistochemical stainings.Liver cancer tissue section carries out immunohistochemical staining discovery with hmc antibody：Liver cancer tissue hmc dyeing is negative (Figure 35).
These as shown by data, hmc dyes the diagnosis that can be used for liver cancer.
Hmc dyeing can be used for diagnosing leukaemia
The present inventor detects the level of hmc in leukaemia tissue with hmc polyclonal antibody HMC-PA01 immunohistochemical stainings.The leukaemia of different stage carries out immunohistochemical staining discovery with hmc antibody：The non-lymphatic leukemia hmc stained positives of M2 types or weakly positive, but M4 types non-lymphatic leukemia hmc dyeing is negative (Figure 36).
Therefore, hmc dye levels may determine that the order of severity of leukaemia.
Hmc dyeing can be used for diagnosing lymthoma
The present inventor detects the level of hmc in lymphoma tissue with hmc polyclonal antibody HMC-PA01 immunohistochemical stainings.Lymphoma tissue section carries out immunohistochemical staining discovery with hmc antibody：Lymthoma cancerous tissue hmc dyeing is negative (Figure 37).
Therefore, hmc dyeing can be used for diagnosing lymthoma.
Preservation
Mouse monoclonal antibody MA7 hybridoma cell strain (5hmC mouse monoclonal antibodies hybridoma cell strain) is secreted, China typical culture collection center (CCTCC, Wuhan, China) has been preserved in, preservation registration number is CCTCC No：C201191, preservation date on October 26th, 2011.
All documents referred in the present invention are all incorporated as reference in this application, are individually recited just as each document as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims limited range.
Claims (16)
1. a kind of 5-hydroxymethyl cytosine is used as the purposes of the mark of detection cancer.
2. purposes as claimed in claim 1, it is characterised in that described cancer includes：The cancer of the brain, squamous cell carcinoma, breast cancer, stomach cancer, intestinal cancer, kidney, carcinoma of urinary bladder, prostate cancer, lung cancer, liver cancer, leukaemia, lymthoma, oophoroma, the cancer of the uterus, cancer of pancreas, thymic carcinoma, spermatogonium cancer, muscle tumor.
3. a kind of purposes of 5-hydroxymethyl cytosine, the reagent for preparing detection cancer.
4. purposes as claimed in claim 3, it is characterised in that described detection cancer includes：Determine the order of severity of cancer or carry out the parting of cancer.
5. purposes as claimed in claim 1, it is characterised in that described 5-hydroxymethyl cytosine is connected with carrier protein, the reagent for preparing detection cancer.
6. purposes as claimed in claim 5, it is characterised in that described carrier protein is selected from：Bovine serum albumin(BSA), oralbumin, hemocyanin.
7. a kind of antigen, it is characterised in that the antigen includes carrier protein, and the one or more 5-hydroxymethyl cytosines being connected on carrier protein.
8. ground as claimed in claim 7 antigen, it is characterised in that described carrier protein is selected from：Bovine serum albumin(BSA), oralbumin, hemocyanin.
9. the purposes of the antigen described in claim 7 or 8, the reagent for preparing detection cancer.
10. a kind of purposes of the reagent of specific recognition 5-hydroxymethyl cytosine, the kit for preparing detection cancer.
11. purposes as claimed in claim 10, it is characterised in that described reagent is the antibody of specific recognition 5-hydroxymethyl cytosine.
12. a kind of polyclonal antibody, it specifically recognizes 5-hydroxymethyl cytosine.
13. polyclonal antibody as claimed in claim 12, it is characterised in that its preparation method is：With the antigen immune rabbit described in claim 5, immune serum is obtained, described polyclonal antibody is obtained after purification.
14. a kind of monoclonal antibody, its specific recognition 5-hydroxymethyl cytosine, it is CCTCC No by preserving number：C201191 hybridoma cell strain is produced.
15. a kind of hybridoma cell strain, it is CCTCCNo in the preserving number of China typical culture collection center：C201191.
16. a kind of kit for being used to detect cancer, including the reagent of specific recognition 5-hydroxymethyl cytosine.
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Cited By (6)
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| CN104698060A (en) * | 2013-12-04 | 2015-06-10 | 苏州中赢医疗科技有限公司 | Acute myelogenous leukemia tumor marker and application thereof |
| CN105296520A (en) * | 2015-12-04 | 2016-02-03 | 中国科学院福建物质结构研究所 | Preparation method and enzyme-linked immunosorbent assay kit of human tumor antigen 3H11Ag |
| CN107142320A (en) * | 2017-06-16 | 2017-09-08 | 上海易毕恩基因科技有限公司 | Gene marker for detecting liver cancer and application thereof |
| WO2019024579A1 (en) * | 2017-08-04 | 2019-02-07 | 上海易毕恩生物技术有限公司 | Gene marker for detecting lung cancer, kit and lung cancer detection method |
| WO2019024578A1 (en) * | 2017-08-04 | 2019-02-07 | 上海易毕恩生物技术有限公司 | Gene marker for detecting benign and malignant liver tumors, kit and detection method |
| CN114373511A (en) * | 2022-03-15 | 2022-04-19 | 南方医科大学南方医院 | Intestinal cancer model based on 5hmC molecular marker detection and intestinal cancer model construction method |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104698060A (en) * | 2013-12-04 | 2015-06-10 | 苏州中赢医疗科技有限公司 | Acute myelogenous leukemia tumor marker and application thereof |
| CN104698060B (en) * | 2013-12-04 | 2018-07-06 | 苏州中赢医疗科技有限公司 | A kind of acute myeloid leukemia tumor markers and its application |
| CN105296520A (en) * | 2015-12-04 | 2016-02-03 | 中国科学院福建物质结构研究所 | Preparation method and enzyme-linked immunosorbent assay kit of human tumor antigen 3H11Ag |
| CN105296520B (en) * | 2015-12-04 | 2019-01-15 | 中国科学院福建物质结构研究所 | A kind of preparation method and its enzyme-linked immunologic detecting kit of human tumor antigen 3H11Ag |
| CN107142320A (en) * | 2017-06-16 | 2017-09-08 | 上海易毕恩基因科技有限公司 | Gene marker for detecting liver cancer and application thereof |
| WO2019024579A1 (en) * | 2017-08-04 | 2019-02-07 | 上海易毕恩生物技术有限公司 | Gene marker for detecting lung cancer, kit and lung cancer detection method |
| WO2019024578A1 (en) * | 2017-08-04 | 2019-02-07 | 上海易毕恩生物技术有限公司 | Gene marker for detecting benign and malignant liver tumors, kit and detection method |
| CN114373511A (en) * | 2022-03-15 | 2022-04-19 | 南方医科大学南方医院 | Intestinal cancer model based on 5hmC molecular marker detection and intestinal cancer model construction method |
| CN114373511B (en) * | 2022-03-15 | 2022-08-30 | 南方医科大学南方医院 | Intestinal cancer model based on 5hmC molecular marker detection and intestinal cancer model construction method |
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