CN111138541B

CN111138541B - Tumor stem cell detection kit

Info

Publication number: CN111138541B
Application number: CN202010265776.1A
Authority: CN
Inventors: 王阳; 金鑫
Original assignee: Hebei Yihe Medical Laboratory Co Ltd
Current assignee: Hebei Yihe medical laboratory Co., Ltd
Priority date: 2020-04-07
Filing date: 2020-04-07
Publication date: 2020-08-07
Anticipated expiration: 2040-04-07
Also published as: CN111138541A

Abstract

The invention relates to a tumor stem cell detection kit, which comprises an antibody capable of specifically binding short isoform DC L K1 protein, can indicate the existence and distribution of colon cancer stem cells, and has a wide application prospect.

Description

Tumor stem cell detection kit

Technical Field

The invention relates to the field of biological detection, in particular to a tumor stem cell detection kit.

Background

In china, colorectal cancer is the fourth most common cancer and the fifth most common cause of death, with an estimated 33.13 million new patients and 15.93 million dead patients each year [1], and the incidence of which is increasing with the age of the patients. With the progress of research, it is increasingly recognized that most colorectal cancers (CRC) originate from colorectal adenomas (CRA), which are abnormal growths and processes on the surface of the colorectal and are a common disease in the digestive system. Adenomas develop slowly, sometimes undergoing malignant transformation through a series of genetic and epigenetic changes, and may take at least 10 years to progress from a detectable adenoma to cancer [2 ]. However, the disease is hidden, and the clinical manifestations lack specificity and are not easy to be found. Thus, through appropriate screening and monitoring, these polyps can be detected and resected early, thereby reducing the incidence of cancer. But the early cancer screening rate is still not ideal. Early diagnosis of colorectal cancer is still one of the great challenges facing home and abroad at present. Because the examination at the cellular level has limitations, such as early detection and early warning signals of Fecal Occult Blood Test (FOBT) and invasive colonoscope combined pathological biopsy are low, the missed diagnosis rate is high, the complications are many, and the result is easily influenced by multiple factors; researchers are therefore forced to search for effective non-invasive methods from the molecular level [3 ]. The emergence of the tumor stem cell theory provides a new target for the diagnosis and treatment of tumors, and the selection of surface markers of the tumor stem cells becomes a prerequisite for early diagnosis and tumor fighting [4 ].

Tumor Stem cells (CSC) are present in tumor tissues, are few in number, have unlimited proliferation potential, can differentiate into tumor cells, and play a decisive role in the development, development and metastasis of tumors, and are also root-origin of tumor recurrence, metastasis and treatment failure [5 ]. known tumor Stem Cell surface markers are CD133[6], CD44[7], CD117[8] and Doublecin-like kinase-1 [9], (Doublecin.1ike kinase kin 1, DC 3874 Kl), etc.. DC 585K 1 is a member of the protein kinase superfamily and the doublecin family, whose coding genes are located in human chromosome 13q13[10 ]. recent studies found that DC L Kl is poorly expressed in cytoplasm and stroma of most malignant tumor tissues such as breast Cancer [11], pancreatic Cancer [12] and prostate Cancer [13] epithelial cells, and also found in connection with tumor metastasis and prognosis, DC L expression has two more favorable translational markers for early stage tumor metastasis and colorectal Cancer initiation, as a marker for colorectal Cancer initiation marker, wherein the promoter is expressed in the promoter of Kl T.7. DC L, T. As a diagnostic marker for early stage Cancer, T. 2, T. T.

Disclosure of Invention

Based on the above findings, the primary object of the present invention is to provide a method for improving the accuracy of early diagnosis of colorectal cancer and sample classification by detecting the presence and content of short isoform DC L K1 protein in a sample.

The invention provides the following technical scheme:

an anti-short isoform DC L K1(DC L K1-S) antibody consisting of a heavy chain and a light chain comprising variable and constant regions, respectively, wherein the heavy chain variable region comprises 3 complementarity determining regions designated VH-CDR1, VH-CDR2, VH-CDR3, and likewise the light chain variable region comprises 3 complementarity determining regions designated V L-CDR 1, V L-VDR 2, V L-CDR 3. the heavy chain of said DC L K1-S antibody comprises the amino acid sequence VH-CDR1 shown in SEQ ID NO:6, and the amino acid sequence VH-CDR2 shown in SEQ ID NO:7, and the amino acid sequence VH-CDR3 shown in SEQ ID NO:8, and the light chain of said DC L K7-S antibody comprises the amino acid sequence V L-CDR 1 shown in SEQ ID NO:9, and the amino acid sequences V1, V1 and V1 shown in SEQ ID NO: 87411.

The DC L K1-S antibody of the invention comprises a heavy chain variable region shown in SEQ ID NO. 4 and a light chain variable region shown in SEQ ID NO. 5.

The heavy chain variable region and the light chain variable region of the DC L K1-S antibody comprise framework regions, the antibody light chain variable region further comprises light chain framework regions of human kappa, lambda chains or variants thereof, and the antibody heavy chain variable region further comprises heavy chain framework regions of murine IgG1, IgG2a, IgG2b or IgG3 or IgM or variants thereof.

The DC L K1-S antibody of the invention comprises a constant region, and the constant region comprises a heavy chain constant region of mouse IgG1 and a light chain constant region comprising a kappa subtype.

In some embodiments, the DC L K1-S antibody binds only to the short isoform DC L K1.

The invention aims to provide an antibody for detecting the existence and the content of a short isoform DC L K1 protein in a sample.

Another object of the present invention is to provide an antibody for detecting colon tumor stem cells in a sample.

Another objective of the invention is to provide a method for improving the early diagnosis of colorectal cancer and the accuracy of sample classification by detecting the presence and the content of the short isoform DC L K1 protein in a sample.

Another objective of the present invention is to provide a method for improving the accuracy of early diagnosis of colorectal cancer and classification of samples by detecting the presence and amount of colon tumor stem cells in the samples.

The invention also aims to provide a kit for detecting the existence and the content of the short isoform DC L K1 protein in a sample, which comprises an antibody capable of specifically binding to the short isoform DC L K1 protein.

It is still another object of the present invention to provide a kit for detecting the presence and amount of colon tumor stem cells in a sample, which comprises an antibody that specifically binds to the short isoform of DC L K1 protein.

The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, and encompasses full-length antibodies (e.g., IgG1 or IgG4 antibodies), various functional fragments thereof (e.g., may comprise only antigen binding portions, such as Fab, Fab ', F (ab') 2). Examples of antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, Fab ', F (ab') 2 fragments, and the like.

The term "monoclonal antibody" refers to an antibody derived from a single clonal cell line, not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombinant technology, phage display technology, synthetic techniques (e.g., CDR-grafting), or other known techniques. A Fab fragment "consists of one light and one heavy chain of CH1 and the variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab ' fragment" contains the VH and CH1 domains of one light and one heavy chain and the constant region portion between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of the two Fab ' fragments to form the F (ab ') 2 molecule. An "F (ab') 2 fragment" contains the VH and CH1 domains of two light and two heavy chains and a constant region portion between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, a F (ab ') 2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

The term "hybridoma" or "hybridoma cell" refers to a cancer cell or cell line (typically a myeloma or lymphoma cell) that is fused to an antibody-producing lymphocyte and is not producing an antibody and is capable of proliferating indefinitely. As known to those of ordinary skill in the art, hybridomas proliferate and continuously supply production of a particular monoclonal antibody. Methods for producing hybridomas are known in the art. When referring to the term "hybridoma" or "hybridoma cell", it also includes subclones and progeny cells of the hybridoma.

The term "hypervariable region" or "CDR region" or "complementarity determining region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. CDR region sequences can be defined by Kabat, Chothia method definition or the field of any known CDR region sequence determination method and identification of the variable region within amino acid residues. The methods used in the present invention may utilize or be defined according to CDRs defined by any of these methods, including but not limited to any of the Kabat definitions, Chothia definitions. In particular, the CDR sequences provided herein are according to the Kabat definition.

The target antigen DC L K1 of the monoclonal antibody 8F7-2 is expressed on the surfaces of living cells of various tumor stem cells, the expression form of the target antigen in the colon tumor stem cells is different from that in normal cells, the expression form of the target antigen in the colon tumor stem cells is short isoform DC L K1., the antibody 8F7-2 can recognize short isoform DC L K1 but not long isoform DC L K1, and the monoclonal antibody 8F7-2 is a monoclonal antibody specifically directed at the colon tumor stem cells.

The antibody 8F7-2 of the present invention specifically recognizes colon tumor stem cells. Accordingly, the present invention also provides a method of detecting the presence of colon tumor stem cells in a biological sample, comprising: a) Contacting the biological sample with a monoclonal antibody or antigen-binding fragment thereof of the invention; b) Detecting binding of the monoclonal antibody or antigen binding fragment thereof of the invention to a target antigen in the biological sample, wherein detection is representative of colon tumor stem cells in the biological sample.

In some embodiments of the above-described detection methods of the invention, the monoclonal antibody or antigen-binding fragment thereof of the invention is further conjugated to a fluorescent dye, chemical, polypeptide, enzyme, isotope, tag, or the like for detection or detectable by other reagents. Wherein the amount of colon tumor stem cells in the biological sample is represented by the intensity of fluorescence.

Methods for detecting antibody-antigen binding are known in the art, e.g., E L ISA, immunohistochemistry, and the like.

The kit of the invention comprises the monoclonal antibody or antigen-binding fragment thereof of the invention, and instructions for use. The kit may further comprise at least one additional detection reagent for detecting the presence of a monoclonal antibody of the invention. The kit generally includes a label indicating the intended use and/or method of use of the kit contents. The term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.

Advantageous effects

The invention has the beneficial effects that the first 9 amino acids of the short isoform DC L K1 protein are used as antigens, and a new monoclonal antibody which specifically binds to the short isoform DC L K1 and does not bind to the long isoform DC L K1 is obtained by immunizing a mouse, so that a new tool is provided for early diagnosis and experimental research of colorectal cancer.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

FIG. 1 shows the subtype identification of murine antibody 8F 7-2.

FIG. 2 shows the binding of Western-blotting detection antibody 8F7-2 to two different DC L K1 isomers.

FIGS. 3a and 3b show Western-blotting detection of the binding of anti-DC L K1 antibody to two different DC L K1 isomers in colon cancer cell lines.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

Example 1 preparation of DC L K1 protein antigen

DC L K1 short isomer (SEQ ID NO: 1) differs from the long isomer (SEQ ID NO: 2) only in the first 6 amino acids of the short isomer from the long isomer so we prepared antibodies specifically binding only the DC L K1 short isomer using the first 9 amino acids of the short isomer (SEQ ID NO: 3) as antigen the synthetic technique known in the art was to synthesize a plasmid containing 6 repeated SEQ ID NO:3 sequences (purchased from Beijing optisco Biotech Co., Ltd.) denoted pET-43.1B (+) -sDC L K1-6, transform the plasmid into E.coli competent B L (DE 3), add the competent to 2ml L B culture containing ampicillin resistance overnight, next day, inoculate to 20 m 3B culture medium containing ampicillin resistance in an amount of 1%, shake culture for 3H at 37 ℃, add G in a final concentration of 0.5/mmol L to 30 ml of B culture for overnight, collect the protein after centrifugation of the supernatant by adding DNA chip-DNA chip DNA, obtain a protein, and obtain a protein cross-link with antigen by centrifugation using DNA chip DNA.

Example 2 anti-DC L K1 antibody preparation and purification

The antigen prepared in example 1 was injected into the female Balb/c mouse intraperitoneally together with Freund's complete adjuvant which caused the immune reaction, and the primary immunization was performed, and then the same amount of antigen was injected into the female Balb/c mouse intraperitoneally every 2 weeks, and the immunization was continued, and finally the final immunization was performed 3 days before the fusion with myeloma cells, and after 100. mu.g of the DC L K1 antigen in example 1 was mixed with the immune adjuvant, the mice were subjected to the intraperitoneal final immunization, and spleen cells of the immunized Balb/c mice were taken, fused with the myeloma Sp2/0 cell line, and the fused cells were diluted with Iscove medium (0.1 mM hypoxanthine, 0.4. mu.M aminopterin, and 16. mu.M thymidine) containing 10% serum, added to 96-well plates for culture, after 10 days, cell supernatants were taken, and ISA detection was performed in high-throughput E L ISA assay, and primary isoform protein of DC L K1 was again performed, and the culture of the cells were again subjected to the final hybridoma cloning, and screening was performed in E L wells, andobtaining the positive hybridoma cell strain 8F 7-2. The hybridoma cell line 8F7-2 was expanded and cultured, and the hybridoma cells were harvested, resuspended in PBS and prepared to 10%⁷Hybridoma cell suspension in ml. Freund's incomplete adjuvant was used as an inducer. Injecting Freund's incomplete adjuvant into Balb/c abdominal cavity for about 8 weeks, and injecting 500ul of 10-concentration adjuvant into Balb/c mouse abdominal cavity after 3 days⁷And (2) carrying out precipitation by saturated ammonium sulfate (PH = 7.8) to obtain a crude DC L K1 monoclonal antibody, and carrying out protein G column chromatography to obtain a DC L K1 antibody with higher purity.

Example 3 anti-DC L K1 antibody subtype identification

The subtype of the anti-DC L K1 monoclonal antibody obtained in example 2 was identified using a mouse monoclonal antibody subtype identification kit (Proteitech), the microplate provided in the kit was pre-coated with specific antibodies against mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgM, Kappa light chain, and lambda light chain, the anti-DC L K1 antibody sample purified in example 2 was added to the sample well 50. mu.l per well without incubation, 1X goat anti-mouse IgM + IgG-HRP was added to the sample well 50. mu.l per well, after mixing gently, incubation for 1h, tapping off the liquid in the well, washing the well with 1 BST 3 times, blotting the excess water with a blotting paper, 100. mu.l per well, developing color at room temperature in the dark place for 15min, 100. mu.l of a stop solution was added, the detection was performed with a microplate reader at 450nm, and the result is shown in FIG. 1, and the developing color of the anti-DC 5634K 4645 monoclonal antibody obtained in our sample was a monoclonal antibody subtype 48335, which was a heavy chain and the Kappa light chain was 1.

Example 4.8 binding Capacity of F7-2 antibody to two different DC L K1 isoforms

The binding affinity of the anti-DC L K1 antibody 8F7-2 obtained above to two different DC 5730K 1 isoform proteins at 50% effective concentration (EC50) was evaluated by E L ISA experiments, two different recombinant DC L K1 isoform proteins were diluted with PBS 1000ng/m L, added to 96 plates at 100 μ L per well, coated overnight at 4 ℃, the solid phase was removed by reverse centrifugation, a blocking agent was added at 200 μ L per 1 well, left to stand at room temperature for 30 minutes, the above DC L K-1 solid phase plates were washed with TBS, and anti-DC L K1 antibody 8F7-2 was added at 100 μ l per well, anti-DC 7K 1 antibody 8F7-2 was diluted with medium to 1000ng/ml, 250/ml, 62.5ng/ml, 15.63ng/ml, 3.91ng/ml, 0.98ng/ml, 4 ng/ml, PBS 8742 was diluted with medium to 1000ng/ml, 250ng/ml, 62.5ng/ml, 15.63ng/ml, 15 ng/ml, 3ng/ml, 7 nm, the absorbance of the absorbance was measured after incubation with PBS, the absorbance data was obtained by a final incubation with 100 nm, the absorbance value was determined by adding a light absorbance test using a light absorbance gradient after incubation gradient, the anti-absorbance test, the anti-DC absorbance test results were performed at room temperature, and after incubation with PBS 3 nm.

TABLE-determination of the binding Capacity of the 8F7-2 antibody to DC L K1

Name (R)	EC50（ng/ml）
		Short isoform DC L K1	50.3±3.5
Long isomeric form DC L K1	>1000

Example 5 Western-blotting detection of antibody binding to two different DC L K1 isoforms

The monoclonal antibody 8F7-2 specific to the DC L K1 short isomer prepared in example 2 and the antibody Ab106635 (purchased from Abcam) specific to the DC L K1 long isomer were assayed by Western-blotting (WB) method HEK293 cells were transfected respectively by DC L K1 short isomer plasmid (Flag-DC L K1-S) and DC L K1 long isomer plasmid (Flag-DC L K1-L) (purchased from Beijing Okagaku Biotech Co., Ltd.), cells were harvested after 24h, XS4 DS lysate was added and boiled in water for 10min, WB samples were obtained after centrifugation at 12000rpm after 10min, proteins in the PVDF samples were transferred by SDS-PAGE and semidry membrane method, after blocking for 1h with 5% degreasing, the monoclonal antibody 8F 3753 specific to the DC L K1 short isomer and the antibody Ab 10672-K863 were incubated in a shaker for staining with the antibody, and the antibody was washed off by a specific antibody Ab 10672, as a secondary antibody, and the antibody was added to the goat yellow antibody Ab 10672, after staining for the antibody was added for staining for 5 h, the antibody was added to the mouse antibody, and the antibody was washed once again, and the antibody was detected by the antibody was added to obtain a secondary antibody No. 7, the detection method of HRP 10672, the antibody was added to obtain a specific antibody of the antibody of IgG 10672, and the antibody of IgG 1068F 8F 3714K 7.

Example 6 Western-blotting detection of antibody binding to two different DC L K1 isoforms in colon cancer cell lines

The method comprises the steps of culturing a colon cancer cell line HCT116 and a normal colon cell line CCD841 respectively, collecting cells after the cells grow out of a culture dish, adding 4XSDS lysate to boil for 10min, centrifuging at 12000rpm, and obtaining a WB sample after 10min, transferring proteins in the WB sample to a PVDF membrane by SDS-PAGE electrophoresis and a semi-dry transfer membrane method, after blocking for 1h by 5% skim milk, respectively, incubating a monoclonal antibody 8F7-2 specific to a DC L K1 short isomer and an antibody Ab106635 specific to a DC L K1 long isomer as a primary antibody overnight, adding TBST to wash for 3 times, placing the cells in a shaker at 40rpm each time, washing for 5min, adding an HRP-conjugated goat anti-mouse secondary antibody to incubate for incubation for 1h, washing for 3 times in the same manner, washing for unspecifically bound antibodies, adding a developing solution, placing the cells in a developing instrument to image, wherein after incubating the antibody with an 8F7-2, only the short colon cancer cell line HCT116 can be detected, only after incubating the antibody CD841 is capable of detecting the presence of the short isoform CD 89116 in the colon cancer cell line HCT 638K, and the antibody of the normal colon cancer cell line is further capable of detecting the presence of the normal colon cancer cell line as an early-CD 968 DC dry isoform 1 in the colon cancer cell line as an early diagnosis index of a colon cancer cell line 369K-CD 638 CD-CD 638 CD.

Example 7 sequence determination of monoclonal antibodies

The cultured hybridoma cell line 8F7-2 was lysed with TRNzol-A +, and placed in a centrifuge tube, 200. mu.l of chloroform was added to each ml of TRNzol-A +, vortex shaking was carried out for 15 seconds, the cell solution was left for 3 minutes, centrifuged at 13000 rpm for 10 minutes at 4 ℃ and the Trizol-A + cell solution was divided into three layers, i.e., an upper colorless aqueous phase, a middle layer and a lower yellow organic phase, the aqueous phase in which RNA was dissolved was transferred to the centrifuge tube, an equal volume of isopropanol was added to the aqueous phase, mixed, left to stand at room temperature for 25 minutes, centrifuged at 13000 rpm for 10 minutes at 4 ℃, waste liquid was discarded to obtain RNA precipitate which was deposited at the bottom, after washing the RNA precipitate twice with 75% ethanol, PEDC RNA was dissolved with water, and stored at 80 ℃ C.C.A first-chain cDNA was synthesized with a reverse transcription kit SMERARCE, and the first-chain cDNA was used as a subsequent template to amplify the antibody variable region DNA sequence corresponding to the hybridoma cell, whereby the antibody variable region DNA sequences of the first-CDR 3, CDRs of the IgG1 subtype, Kappa light chain region, CDR3, CDRs were designed, CDRs for which were amplified by PCR using primers designed, CDRs for the constant PCR amplification of the heavy chain region of the heavy chain CDR3 and CDR3 CDR 5, CDRs from the heavy chain region of the antibody sequences of the heavy chain CDR3, CDRs equivalent to the heavy chain region of the heavy chain CDR 5, CDRs equivalent to the heavy chain region of the heavy chain CDR3 CDR 5, CDRs equivalent to the heavy chain CDR 3.

Reference to the literature

[1]CHEN W, ZHENG R, ZUO T, et al. National cancer incidence andmortality in China, 2012 [J]. Chin J Cancer Res, 2016, 28(1): 1-11.

[2]LIAO Y, LI S, CHEN C, et al. Screening for colorectal cancer inTianhe, Guangzhou: results of combining fecal immunochemical tests and riskfactors for selecting patients requiring colonoscopy [J]. Gastroenterol Rep(Oxf), 2018, 6(2): 132-6.

[3] Li Xin, Yanghua, Jiangyonggen, research progress of early molecular diagnosis of colorectal cancer [ J ]. Shanghai preventive medicine 2015, 27(02): 97-9.

[4] The application of the Lizee, Nie-Cai-Hui, Zuijun, et al. tumor markers and the research progress of screening technology thereof [ J ]. pharmaceutical progress, 2014, 38(01): 1-13.

[5]KOBAYASHI N, NAVARRO-ALVAREZ N, SOTO-GUTIERREZ A, et al. Cancerstem cell research: current situation and problems [J]. Cell Transplant,2008, 17(1-2): 19-25.

[6]SONG W, LI H, TAO K, et al. Expression and clinical significanceof the stem cell marker CD133 in hepatocellular carcinoma [J]. Int J ClinPract, 2008, 62(8): 1212-8.

[7]BAUMANN M, KRAUSE M. CD44: a cancer stem cell-related biomarkerwith predictive potential for radiotherapy [J]. Clin Cancer Res, 2010, 16(21): 5091-3.

[8]JING H, LIU X Y, CHEN Y L, et al. [Expression Level of Membrane-associated Proteins Numb in Epithelial Ovarian Carcinoma and Its Relationshipwith Ovarian Cancer Stem Cell Markers CD117, CD133, ALDH1.][J]. Sichuan DaXue Xue Bao Yi Xue Ban, 2016, 47(6): 878-82.

[9]IKEZONO Y, KOGA H, AKIBA J, et al. Pancreatic NeuroendocrineTumors and EMT Behavior Are Driven by the CSC Marker DCLK1 [J]. Mol CancerRes, 2017, 15(6): 744-52.

[10]BANG Y J, KIM Y W, YANG H K, et al. Adjuvant capecitabine andoxaliplatin for gastric cancer after D2 gastrectomy (CLASSIC): a phase 3open-label, randomised controlled trial [J]. Lancet, 2012, 379(9813): 315-21.

[11]HAAKENSEN V D, BJ RO T, L DERS T, et al. Serum estradiol levelsassociated with specific gene expression patterns in normal breast tissue andin breast carcinomas [J]. BMC Cancer, 2011, 11(332): 332.

[12]SUREBAN S M, MAY R, LIGHTFOOT S A, et al. DCAMKL-1 regulatesepithelial-mesenchymal transition in human pancreatic cells through a miR-200a-dependent mechanism [J]. Cancer Res, 2011, 71(6): 2328-38.

[13]SUREBAN S M, MAY R, MONDALEK F G, et al. Nanoparticle-baseddelivery of siDCAMKL-1 increases microRNA-144 and inhibits colorectal cancertumor growth via a Notch-1 dependent mechanism [J]. J Nanobiotechnology,2011, 9(40): 40.

[14] Acacia, horse courts, liu bin, et al, DC L K1 +/Ki67 in colorectal cancer tissues-morphology and distribution of tumor stem cell-like cells [ J ]. chinese tissue engineering studies 2015, 19(10): 1575-9.

[15]GAGLIARDI G, GOSWAMI M, PASSERA R, et al. DCLK1 immunoreactivityin colorectal neoplasia [J]. Clin Exp Gastroenterol, 2012, 5(35-42).

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

465 470 475 480

Asn Lys Tyr Thr Glu Arg Asp Ala Ser Gly Met Leu Tyr Asn Leu Ala

485 490 495

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

500 505 510

Lys Pro Glu Asn Leu Leu Val Tyr Glu His Gln Asp Gly Ser Lys Ser

515 520 525

Leu Lys Leu Gly Asp Phe Gly Leu Ala Thr Ile Val Asp Gly Pro Leu

530 535 540

Tyr Thr Val Cys Gly Thr Pro Thr Tyr Val Ala Pro Glu Ile Ile Ala

545 550 555 560

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

565 570 575

Thr Tyr Ile Leu Leu Cys Gly Phe Pro Pro Phe Arg Gly Ser Gly Asp

580 585 590

Asp Gln Glu Val Leu Phe Asp Gln Ile Leu Met Gly Gln Val Asp Phe

595 600 605

Pro Ser Pro Tyr Trp Asp Asn Val Ser Asp Ser Ala Lys Glu Leu Ile

610 615 620

Thr Met Met Leu Leu Val Asp Val Asp Gln Arg Phe Ser Ala Val Gln

625 630 635 640

Val Leu Glu His Pro Trp Val Asn Asp Asp Gly Leu Pro Glu Asn Glu

645 650 655

His Gln Leu Ser Val Ala Gly Lys Ile Lys Lys His Phe Asn Thr Gly

660 665 670

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

675 680 685

His Gly Phe Thr Ile Lys Arg Ser Gly Ser Leu Asp Tyr Tyr Gln Gln

690 695 700

Pro Gly Met Tyr Trp Ile Arg Pro Pro Leu Leu Ile Arg Arg Gly Arg

705 710 715 720

Phe Ser Asp Glu Asp Ala Thr Arg Met

725

<210>3

<211>9

<212>PRT

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>3

Met Leu Glu Leu Ile Glu Val Asn Gly

1 5

<210>4

<211>120

<212>PRT

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>4

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Ile Leu Tyr Ser Gln Met

20 25 30

Ala Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Met Ala Thr Ser His Gly Gln Asp Asp Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 7580

Lys Val Asn Asn Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Arg

85 90 95

Ala Met Gly Val Lys Tyr Ser Phe Glu Gln Thr Ile Lys Trp Gly Ala

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210>5

<211>107

<212>PRT

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>5

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gly Tyr Ser Lys Asp Glu

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Glu Gly Thr Leu Lys Leu Leu Ile

35 40 45

Tyr Ser Glu Tyr Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Lys Asn Arg Ser Trp Thr Pro Asn

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>6

<211>5

<212>PRT

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>6

Gln Met Ala Val Asn

1 5

<210>7

<211>16

<212>PRT

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>7

Met Ala Thr Ser His Gly Gln Asp Asp Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210>8

<211>12

<212>PRT

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>8

Met Gly Val Lys Tyr Ser Phe Glu Gln Thr Ile Lys

1 5 10

<210>9

<211>11

<212>PRT

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>9

Arg Ala Ser Gly Tyr Ser Lys Asp Glu Leu Asn

1 5 10

<210>10

<211>7

<212>PRT

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>10

Ser Glu Tyr Arg Leu His Ser

1 5

<210>11

<211>9

<212>PRT

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>11

Lys Asn Arg Ser Trp Thr Pro Asn Thr

1 5

Claims

1. An antibody specifically binding to short isoform DC L K1 is characterized in that the CDR1 sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 6, the CDR2 sequence is shown in SEQ ID NO. 7 and the CDR3 sequence is shown in SEQ ID NO. 8, the CDR1 sequence of the light chain variable region is shown in SEQ ID NO. 9, the CDR2 sequence is shown in SEQ ID NO. 10 and the CDR3 sequence is shown in SEQ ID NO. 11.

2. The antibody of claim 1, wherein: the heavy chain variable region sequence is SEQ ID No. 4; the light chain variable region sequence is SEQ ID No. 5.

3. The antibody of any one of claims 1-2, wherein: the antibody is a full-length antibody.

4. A kit for detecting colon tumor stem cells in a sample, which is characterized in that: comprising the antibody of claim 1 or 2.

5. The kit of claim 4, the antibody is further conjugated to a fluorescent dye, chemical, polypeptide, enzyme, isotope, or label for detection or detectable by other reagents.

6. Use of the antibody of any one of claims 1-2 in the manufacture of a kit for detecting the short isoform DC L K1.

7. Use of an antibody according to any one of claims 1-2 in the manufacture of a kit for the detection of colon cancer tumors.