WO2019134392A1 - 一种多肽及其用途 - Google Patents

一种多肽及其用途 Download PDF

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WO2019134392A1
WO2019134392A1 PCT/CN2018/105631 CN2018105631W WO2019134392A1 WO 2019134392 A1 WO2019134392 A1 WO 2019134392A1 CN 2018105631 W CN2018105631 W CN 2018105631W WO 2019134392 A1 WO2019134392 A1 WO 2019134392A1
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polypeptide
tumor
human
medicament
cancer cells
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魏敏杰
于丽凤
赵琳
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中国医科大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • the invention belongs to the field of biomedicine, and particularly relates to a polypeptide which has a targeting effect on human colorectal cancer cells, pancreatic cancer cells, breast cancer cells or ovarian cancer cells and uses thereof.
  • Colorectal cancer is one of the most common malignant tumors in the digestive system. According to the 2016 big data research report on the incidence and mortality of malignant tumors in China, the annual incidence of colorectal cancer in China is 331,000, and the incidence rate is ranked among all malignant tumors. Fourth place; 159,000 people die of the disease each year, and the death rate is the fifth leading cause of cancer death. In the United States, colorectal cancer is the third most common malignant tumor, and more than 106,000 people are diagnosed with colorectal cancer each year.
  • screening methods for colorectal cancer such as fecal occult blood test, fecal immunochemistry test and colonoscopy, may not be optimal. These methods may not detect tumor lesions or perform unnecessary endoscopic diagnosis. Moreover, dysplastic lesions may be indistinguishable from epithelial regeneration associated with inflammation. In addition, adverse events such as perforation may also occur in colonoscopy. Therefore, early detection and development of colorectal cancer, sensitive diagnostic techniques, targeted therapy is the focus of research.
  • the existing anticancer drugs mainly include metal anticancer drugs, anticancer active components extracted from natural products, anticancer drug targeted agents, genetic engineering drugs, and nano controlled release anticancer drugs. These drugs usually have toxic side effects. The disadvantages of large dosage, large dosage of drugs, and prone to acquired drug resistance have an impact on the treatment of tumors.
  • Phage display technology is an important technique for screening intermolecular interactions in the field of molecular biology.
  • the main principle of phage display technology is that the gene of interest or the gene encoding the protein and polypeptide is cloned into the appropriate position of the phage surface protein gene by genetic engineering technology, and it is expressed on the surface of the phage with the amplification of phage DNA, due to the foreign gene.
  • the expression product polypeptide or protein of the foreign gene can maintain its original spatial structure and corresponding biological activity.
  • the target cells or target molecules to perform meiotic screening on the phage, and finally select the phage that can specifically bind to the target cell or target molecule from the phage peptide library, and sequence the DNA to obtain the corresponding polypeptide.
  • the coding sequence This technology enables the connection between genotypes and phenotypes of proteins or peptides, and has the characteristics of simple operation and high-throughput detection, thus becoming an efficient means for screening tumor cell-specific binding peptides, for early detection of tumors and Targeted vector research in drug therapy offers new directions.
  • the object of the present invention is to provide a polypeptide which specifically binds to human colorectal cancer cells without affecting human normal intestinal epithelial cells, and also for human pancreatic cancer cells, human breast cancer cells and human ovarian cancer cells. It has a recognition function, which plays an important role in the early diagnosis of colorectal cancer and the development of targeted drugs, and also plays an important role in the early diagnosis of pancreatic cancer, breast cancer, ovarian cancer and targeted drug development.
  • human normal intestinal epithelial cells were used as control
  • human colorectal cancer cell HCT116 was used to screen the phage display peptide library
  • blue-white screening test was used to select positive phage capable of cell-specific binding to colorectal cancer cells.
  • the specificity of phage binding to colorectal cancer cells was verified by ELISA.
  • the purified phage is amplified and the DNA is extracted for sequencing, thereby obtaining a coding sequence of a polypeptide capable of specifically binding to colorectal cancer cells, and synthesizing a fluorescently labeled positive polypeptide for fluorescent labeling-polypeptide Verification of binding to human colorectal cancer cells and tissues provides an experimental basis for early diagnosis and targeted therapy of colorectal cancer.
  • a polypeptide which is (1) the amino acid sequence of the polypeptide is: DYHDPSLPTLRK (SEQ ID No. 1), (2) in the polypeptide molecule of (1)
  • the polypeptide has a targeting effect on tumor cells and specifically binds to tumor cells.
  • the tumor cells are human colorectal cancer cells, human pancreatic cancer cells, human breast cancer cells or human ovarian cancer cells.
  • polypeptide in the preparation of a tumor diagnostic kit comprising the polypeptide or polypeptide conjugate.
  • a polypeptide for the preparation of a medicament for the treatment of a tumor comprising the polypeptide and a pharmaceutically active ingredient, or a polypeptide and a delivery vehicle.
  • the medicament is any pharmaceutically therapeutically acceptable dosage form, and the preferred dosage form of the medicament is an injectable preparation.
  • the drug is any pharmacologically acceptable dose.
  • the invention has the advantages that the phage display technology of the invention has simple operation, high-throughput panning, high efficiency, and can screen mimic epitopes, display polypeptides or proteins and genes contained in the phage.
  • Figure 1 shows the results of positive phage DNA sequencing specifically binding to human colorectal cancer cell HCT16.
  • Figure 2 shows the targeted binding of FITC-DK12 to human normal intestinal epithelial cells HIEC and human colorectal cancer cells HCT116, SW620.
  • Figure 3 shows the targeted binding of FITC-DK12 to human ovarian epithelial cells HOSEpiC and human ovarian cancer gland cells OVCAR-3, SK-OV-3.
  • Figure 4 shows the targeted binding of FITC-DK12 to human embryonic pancreatic tissue-derived cells CCC-HPE-2 and human pancreatic cancer cell line MIA PaCa-2.
  • Figure 5 shows the targeted binding of FITC-DK12 to human mammary epithelial cells MCF-10A and human breast cancer cells MCF-7, MDA-MB-23110A.
  • Figure 6 shows the targeted binding of FITC-DK12 to human colorectal cancer and paracancerous tissues.
  • Figure 7 shows the targeted binding of FITC-DK12 to tumor-bearing mouse colorectal cancer cell line HCT116.
  • Phage 12 peptide library Escherichia coli E. coli ER2738, human normal intestinal epithelial cells HIEC, human colorectal cancer cells HCT116 and SW620, human ovarian epithelial cells HOSEpiC, human ovarian adenocarcinoma cells OVCAR-3 and SK-OV-3, human Embryonic pancreatic tissue-derived cells CCC-HPE-2, human pancreatic cancer cells MIA PaCa-2, human mammary epithelial cells MCF-10A, human breast cancer cells MCF-7 and MDA-MB-231, tumor-bearing mice, human colorectal cancer organization.
  • DEME medium RPMI-1640 medium, trypsin, FITG-labeled rabbit anti-mouse, fetal bovine serum, yeast powder, peptone, agar powder, tetracycline stock solution, Tween-20, bovine serum albumin BSA, M13 phage single-strand DNA extraction kit, IPTG, X-gal, PEG-8000, TMB.
  • Preparing cells The 6-well culture plate was pretreated with poly-lysine, and human colorectal cancer cell HCT116 and human normal intestinal epithelial cells HIEC were taken and treated with trypsin, respectively, and cultured until the cells successfully adhered. After the growth state is good, screening is performed.
  • Serum-free culture The cell culture medium was aspirated, washed once with PBS, and then added to serum-free medium, and placed in a 37 ° C constant temperature cell incubator with 5% CO 2 for 1 h.
  • Closing the cells aspirate the cell culture medium, place the plate on a clean paper towel, remove the remaining medium, and block the human colorectal cancer cell HCT116 and human normal intestinal epithelial cells with a medium containing 1% BSA. HIEC was placed in a 37 ° C thermostatic cell incubator with 5% CO 2 for 1 h.
  • Binding The adsorbed phage supernatant was incubated with human colorectal cancer cell HCT116 for 1 h.
  • Washing Discard the unbound phage, place the microplate on a clean paper towel and pat it vigorously to remove the remaining solution. The plate was washed 5 times with 0.1% TBST as described above.
  • the IPTG/X-gal plate was preheated in an electrothermal incubator at 37 ° C; the appropriate amount of top agar was taken out and heated in a microwave oven, and after it was completely melted, it was taken out, and 3 ml was dispensed in each 10 ml centrifuge tube; After the phage was diluted in a ratio, 10 ⁇ l and 200 ⁇ l of E. coli were mixed and reacted for 5 min, then added to 3 ml of top agar, and then evenly spread on preheated IPTG/X-gal plate, after condensation. The titration results were observed at 37 ° C in an electrothermal incubator overnight.
  • Amplification of phage 20 ml of LB/Tet liquid medium was added to the Erlenmeyer flask, then E. coli bacteria solution and phage to be amplified were added at 1:100, placed at 37 ° C, and shaken vigorously in a constant temperature oscillator 4.5 h, an phage amplification solution is obtained.
  • (1) Cell plating human normal intestinal epithelial cells HIEC, human colon cancer cells HCT116 and SW620, human ovarian epithelial cells HOSEpiC, human ovarian adenocarcinoma cells OVCAR-3 and SK-OV-3, human embryonic pancreatic tissue-derived cells CCC -HPE-2, human pancreatic cancer cell line MIA PaCa-2, human mammary epithelial cell MCF-10A, human breast cancer cell MCF-7 and MDA-MB-231 were plated in a six-well plate for use.
  • DAPI staining washing with PBS 3 times, adding DAPI 100 ⁇ l, room temperature, 15 min
  • Blocking Blocking with 3% BSA/PBS at 37 ° C for 30 min.
  • DAPI staining washing with PBS 3 times, adding DAPI 100 ⁇ l, room temperature, 15 min
  • Tumor-bearing The nude mice were used for four weeks of age, and the human colon cancer cell HCT116 was inoculated into the tumor.
  • Targeted phage DNA sequencing results for human colorectal cancer cell HCT116 as shown in Figure 1, the polypeptide sequence was translated according to the principle of the triplet codon: DYHDPSLPTLRK (SEQ ID No. 1) (DK12).
  • the fluorescently labeled peptide FITC-DK12 was used to detect the targeted binding of the peptide to human colorectal cancer, ovarian cancer, pancreatic cancer and breast cancer cells by immunofluorescence staining, and then further confirmed by immunofluorescence staining.
  • Targeted binding ability of colorectal cancer tissues, and the targeting effect of the polypeptide and human colorectal cancer cell HCT16 was verified using tumor-bearing mice.
  • the polypeptide sequence can bind to human colorectal cancer cells HCT116 and SW620, and the HIEC binding ability to normal intestinal mucosal epithelial cells is weak, and there is a significant difference between the two; at the same time, the polypeptide sequence can also Human ovarian adenocarcinoma cells OVCAR-3 and SK-OV-3 are targeted for binding, while HOSEpiC binding to human normal ovarian epithelial cells is weak, and there is a significant difference between the two (see Figure 3); and the polypeptide sequence can also Human pancreatic cancer cell line MIA PaCa-2 and human breast cancer cells MCF-7 and MDA-MB-231 are targeted for binding, while human embryonic pancreatic tissue-derived cells CCC-HPE-2 and human mammary epithelial cells MCF-10A have better binding ability.
  • the polypeptide sequence can also Human pancreatic cancer cell line MIA PaCa-2 and human breast cancer cells MCF-7 and MDA-MB-231 are targeted for binding, while human embryo

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Abstract

提供了一种多肽，其氨基酸序列为：DYHDPSLPTLRK(SEQ ID No.1)，所述多肽能特异性与人结直肠癌细胞靶向结合，而对人正常肠上皮细胞没有影响，并且对人胰腺癌细胞、乳腺癌细胞及卵巢癌细胞也具有识别作用。还提供了所述多肽在制备肿瘤诊断试剂盒和用于治疗肿瘤的药物中的用途。

Description

一种多肽及其用途 技术领域
本发明属于生物医学领域，具体涉及一种对人结直肠癌细胞、胰腺癌细胞、乳腺癌细胞或卵巢癌细胞有靶向作用的多肽及其用途。
背景技术
结直肠癌是消化系统最常见的恶性肿瘤之一，根据2016年我国恶性肿瘤发病和死亡分析大数据研究报道，我国的结直肠癌每年新发病例33.1万人，发病率在全部恶性肿瘤中排名第四位；每年死于该病的患者有15.9万人，死亡率则位居癌症死亡原因第五位。在美国结直肠癌成为发病率第三高的恶性肿瘤，每年有超过10.6万人被诊断患结直肠癌。与我国不同，尽管美国结直肠癌的发病率高，但死亡率却逐年下降，这主要归功于越来越多的结直肠癌在早期被发现，从而获得被治愈的机会。早期结直肠癌患者的5年生存率达到90％，而转移性或晚期癌仅为10％。在临床实践中，人们普遍认为早期诊断有望实现个性化治疗，有助于改善长期结局。
目前，结直肠癌的筛查方式，如粪便隐血试验，粪便免疫化学试验和结肠镜检查，可能不是最理想的，这些方法可能检测不到肿瘤病变或者进行不必要的内窥镜诊断。而且发育不良的病变可能与炎症相关的上皮再生难以区分。此外，结肠镜检查中还可能会出现不良事件，如穿孔等。因此，早期结直肠癌检测、开发敏感诊断技术，靶向治疗是研究的重点。
现有的抗癌药物主要有金属抗癌药物、天然产物中提取抗癌活性成分、抗癌药物靶向制剂、基因工程药物、纳米控释抗癌药物等五类，这些药物通常具有毒副作用较大、药物用量大、容易产生后天耐药性等缺点，对肿瘤的治疗造成影响。
噬菌体展示技术是分子生物学领域一种重要的筛选分子间相互作用的技术。噬菌体展示技术的主要原理是目的基因或编码蛋白质和多肽的基因通过基因工程技术克隆到噬菌体表面蛋白基因的适当位置上，让其随着噬菌体DNA的扩增而表达在噬菌体表面，由于外源基因和噬菌体基因的兼容性，外源基因的表达产物多肽或蛋白质仍可以保持其原有的空间结构和相应的生物学活性。然后，我们利用靶细胞或靶分子对噬菌体进行减数筛选，最终从噬菌体肽库中筛选出能够与靶细胞或靶分子特异性结合的目的噬菌体，并对其DNA进行测序，即可得到相应多肽的编码序列。这一技术实现了蛋白质或多肽基因型和表现型之间的联系，并且具有操作简便，可以高通量检测的特点，从而成为筛选肿瘤细胞特异性结合肽的高效手段，为肿瘤的早期检测和药物治疗的靶向载体研究提供了新的方向。
发明内容
本发明的目的在于提供一种多肽，能特异性与人结直肠癌细胞靶向结合，而对人正常肠上皮细胞没有影响，并且对人胰腺癌细胞、人乳腺癌细胞及人卵巢癌细胞也具有识别作用，这在结直肠癌的早期诊断和靶向药物的研发等方面具有重要作用，且对胰腺癌、乳腺癌、卵巢癌的早期诊断和靶向药物研发也具有重要作用。
本实验即以人正常肠上皮细胞为对照，采用人结直肠癌细胞HCT116对噬菌体展示肽库进行减数筛选，用蓝白筛选试验挑选出能够与结直肠癌细胞发生细胞特异性结合的阳性噬菌体克隆，并用ELISA实验验证噬菌体与结直肠癌细胞结合的特异性。然后以大肠杆菌为载体，扩增纯化噬菌体后提取其DNA进行测序，即得到能够与结直肠癌细胞发生特异性结合的多肽的编码序列，并人工合成荧光标记的阳性多肽，进行荧光标记-多肽与人结直肠癌细胞和组织的结合 作用的验证，进而为结直肠癌的早期诊断和靶向治疗提供实验基础。
为了实现上述目的，本发明采用如下技术方案：一种多肽，该多肽为(1)多肽的氨基酸序列为：DYHDPSLPTLRK(SEQ ID No.1)，(2)在(1)所述的多肽分子中经过缺失、插入或置换一个或几个氨基酸且与(1)所述的多肽分子具有相同生物学功能的多肽衍生物。
该多肽对肿瘤细胞有靶向作用，与肿瘤细胞特异性结合。
所述的肿瘤细胞为人结直肠癌细胞、人胰腺癌细胞、人乳腺癌细胞或人卵巢癌细胞。
多肽在制备肿瘤诊断试剂盒中的应用，该试剂盒中包含所述多肽或多肽偶联物。
多肽在制备用于治疗肿瘤药物中的应用，该药物包含所述的多肽与药物活性成分，或包含所述的多肽与递药载体。该药物为任何药物治疗学上可接受的剂型，该药物优选的剂型为注射制剂。
该药物为任何药物治疗学上可接受的的剂量。
与现有技术相比，本发明的效果在于：本发明使用噬菌体展示技术具有操作简便、高通量的淘选、高效率，可以筛选模拟表位、展示多肽或蛋白质与其包含在噬菌体内部的基因密码的连接、重组噬菌体易于纯化等优点。我们运用使菌体展示技术所筛选出的肽能够与人结直肠癌细胞特异性结合，而与正常的肠上皮细胞无特异性作用，而且作用效果明显。且实验证明，该肽与人胰腺癌细胞、人乳腺癌细胞及人卵巢癌细胞也有靶向作用。
附图说明
图1为与人结直肠癌细胞HCT16特异性结合的阳性噬菌体DNA测序结果。
图2为FITC-DK12与人正常肠上皮细胞HIEC和人结直肠癌细胞HCT116、 SW620的靶向结合作用。
图3为FITC-DK12与人卵巢上皮细胞HOSEpiC和人卵巢癌腺细胞OVCAR-3、SK-OV-3的靶向结合作用。
图4为FITC-DK12与人胚胎胰腺组织来源细胞CCC-HPE-2和人胰腺癌细胞MIA PaCa-2的靶向结合作用。
图5为FITC-DK12与人乳腺上皮细胞MCF-10A和人乳腺癌细胞MCF-7、MDA-MB-23110A的靶向结合作用。
图6为FITC-DK12与人结直肠癌和癌旁组织的靶向结合作用。
图7为FITC-DK12与荷瘤鼠人结直肠癌细胞HCT116的靶向结合作用。
具体实施方式
以下所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
实施例1
1.实验材料
1.1噬菌体肽库、细胞和宿主菌。
噬菌体12肽库、大肠杆菌E.coli ER2738、人正常肠上皮细胞HIEC、人结直肠癌细胞HCT116和SW620、人卵巢上皮细胞HOSEpiC、人卵巢腺癌细胞OVCAR-3和SK-OV-3、人胚胎胰腺组织来源细胞CCC-HPE-2、人胰腺癌细胞MIA PaCa-2、人乳腺上皮细胞MCF-10A、人乳腺癌细胞MCF-7和MDA-MB-231、荷瘤鼠、人结直肠癌组织。
1.2实验试剂
DEME培养基、RPMI-1640培养基、胰蛋白酶、FITG标记兔抗鼠、胎牛血清、酵母粉、蛋白胨、琼脂粉、四环素贮存液、吐温-20(tween-20)、牛血清蛋白BSA、M13噬菌体单链DNA提取试剂盒、IPTG、X-gal、PEG-8000、TMB。
1.3实验工作液
1×PBS、LB液体培养基、LB-Tet固体平板、顶层琼脂、IPTG/X-gal工作液、IPTG/X-gal平板、PEG-NaCl、TBS缓冲液、0.1％TBST、0.5％TBST、4％多聚甲醛固定液、TBS-NaN3液的配制、3％BSA封闭液、碘化钠缓冲液、TE缓冲液、TMB工作液、四环素贮存液。
2.实验方法
2.1大肠杆菌的培养。
(1)大肠杆菌的复苏：从-80℃冰箱中取出大肠杆菌甘油冻存液，用接种环取少量划线于LB/Tet固体平板上，然后将此LB/Tet固体平板倒置于37℃的电热恒温培养箱中培养过夜，使用时挑取单菌落即可。
(2)大肠杆菌的培养：在15ml离心管中加入10ml LB/Tet液体培养基，挑取大肠杆菌单菌落加入其中。然后将离心管置于恒温振荡器中培养过夜，待菌液的OD600值为0.5时即可进行相关实验。
2.2噬菌体展示肽库的减数筛选。
(1)准备细胞：先将6孔培养板经poly-lysine预处理,取人结直肠癌细胞HCT116和人正常肠上皮细胞HIEC,分别用胰蛋白酶处理后铺于其中,培养至细胞成功贴壁且生长状态良好后进行筛选。
(2)制备菌液：筛选当天,将大肠杆菌ER2738接种于20ml LB/Tet液体培养基中，置于37℃恒温振荡器中震荡培养,待菌液的OD600值为0.5时，用于扩增筛选洗脱的噬菌体。
(3)无血清培养：吸掉细胞培养基，用PBS洗涤1次后加入无血清培养基，置于通有5％CO2的37℃恒温细胞培养箱中1h。
(4)洗涤:吸掉封闭液,用0.1％TBST较轻地洗涤5次,每次均需旋转以使微孔的底部及边缘都被洗涤,甩干。至第2、3轮筛选时分别用0.5％TBST、1.0％TBST。
(5)封闭细胞:吸掉细胞培养基,将平板倒置于干净的纸巾上用力拍甩去除残存的培养基,用含1％BSA的培养基封闭人结直肠癌细胞HCT116和人正常肠上皮细胞HIEC，置于通有5％CO2的37℃恒温细胞培养箱中1h。
(6)吸附:取原始肽库10μl，加入到990μl 0.5％BSA/PBS缓冲液中，将噬菌体稀释为1.5×1011pfu/ml，并将其加入到已封闭的人正常肠上皮细胞HIEC中,37℃ 1h,吸附可以和人正常肠上皮细胞HIEC结合的噬菌体，留取上清。
(7)结合:将吸附后的噬菌体上清液与人结直肠癌细胞HCT116共同孵育1h。
(8)洗涤:弃掉未结合的噬菌体,将微孔板倒置于干净的纸巾上用力拍甩，以去除残存的溶液。按上述方法用0.1％TBST洗板5次。
(9)洗脱:加入0.2M Glycine-HCl(pH2.2)1mg/mlBSA洗脱液1ml,冰上慢摇10min，然后将洗脱液吸出并转移至预先已准备好的150μl中和液(1M Tris-HCl，pH9.1)中。
(10)按照上述步骤重复操作2次。
2.3噬菌体的滴度测定。
将IPTG/X-gal平板预热于37℃的电热恒温培养箱中；取出适量的顶层琼脂在微波炉中加热，待其完全融化后取出，在每个10ml离心管中分装3ml；将待筛选的噬菌体进行等比稀释后，取10μl与200μl的大肠杆菌菌液充分混合反应5min后，加入到3ml的顶层琼脂中，然后均匀地铺在预热的IPTG/X-gal平板上，待冷凝后置于37℃的电热恒温培养箱过夜，观察滴定结果。
2.4噬菌体的扩增和纯化。
(1)噬菌体的扩增：在锥形瓶中加入20mlLB/Tet液体培养基，然后按1:100加入大肠杆菌菌液和待扩增的噬菌体，置于37℃，恒温振荡器中剧烈震荡4.5h，得到噬菌体的扩增液。
(2)噬菌体的纯化：将经上述步骤得到的噬菌体扩增液4℃、12000r/min，离心10min，取上清并加入1/6体积PEG-NaCl沉淀过夜后，12000r/min离心15min，弃去上清液，用TBS缓冲液溶解沉淀，再次给予1/6体积PEG-NaCl，冰上孵育1h。4℃、14000r/min，离心15min，弃去上清，将得到的沉淀用TBS-NaN3溶解后置于4℃冰箱保存。
2.5阳性噬菌体DNA的提取及测序
(1)在上述纯化的噬菌体沉淀中加入100ul碘化物缓冲液,再加入250ul无水乙醇,充分混匀,室温作用20min。
(2)离心：4℃，14,000rpm，10min,弃上清。
(3)清洗：用500ul 70％乙醇洗涤沉淀,短暂离心后真空干燥。
(4)30ulTE(10mM Tris-HCl,pH5.0,1mMEDTA)缓冲液重悬沉淀,制成DNA测序模板液，送与上海生工测序。
2.6细胞免疫荧光实验
(1)细胞铺板：将人正常肠上皮细胞HIEC、人结肠癌细胞HCT116和SW620、人卵巢上皮细胞HOSEpiC、人卵巢腺癌细胞OVCAR-3和SK-OV-3、人胚胎胰腺组织来源细胞CCC-HPE-2、人胰腺癌细胞MIA PaCa-2、人乳腺上皮细胞MCF-10A、人乳腺癌细胞MCF-7和MDA-MB-231铺于六孔板中待用。
(2)固定：用4％多聚甲醛固定15min。
(3)封闭：弃去4％多聚甲醛，PBS洗2次，用3％BSA/PBS于37℃封闭30min。
(4)FITC-DK12孵育：将封闭液擦拭后加入FITC-DK12，37℃1h。
(5)DAPI染色：用PBS洗涤3次后加DAPI 100μl，室温，15min
(6)封片:PBS洗3次后，封片。
2.7组织免疫荧光实验
(1)烤片：60℃，4～6h
(2)切片脱蜡至水：二甲苯Ⅰ15min→二甲苯Ⅱ15min→无水酒精Ⅰ5min→无水酒精Ⅱ5min→95％酒精5min→85％酒精5min→75％酒精5min→蒸馏水5min。
(3)封闭：用3％BSA/PBS于37℃封闭30min。
(4)FITC-DK12孵育：将封闭液擦拭后加入FITC-DK12，37℃1h。
(5)DAPI染色：用PBS洗涤3次后加DAPI 100μl，室温，15min
(6)封片:PBS洗3次后，封片。
2.8荷瘤鼠实验
(1)荷瘤：实验用裸鼠四周龄，腋下接种人结肠癌细胞HCT116使其成瘤。
(2)成像：将多肽用PBS配置成100μM/ml的溶液备用，给与荷瘤鼠尾静脉注射FITC-DK12多肽溶液0.1ml，10分钟后于小动物成像仪上成像。
3.实验结果
针对人结直肠癌细胞HCT116的靶向噬菌体DNA测序结果，如图1所示，按照三联密码子的原则，翻译出多肽序列：DYHDPSLPTLRK(SEQ ID No.1)(DK12)。
荧光标记多肽FITC-DK12，用免疫荧光染色实验检测该多肽分别与人结直肠癌、卵巢癌、胰腺癌和乳腺癌细胞的靶向结合作用，然后进一步用免疫荧光染色实验验证了该多肽与人结直肠癌组织的靶向结合能力，并使用荷瘤鼠验证了该多 肽与人结直肠癌细胞HCT16的靶向作用。如图2所示，该多肽序列能够与人结直肠癌细胞HCT116和SW620靶向结合，而对正常肠粘膜上皮细胞HIEC结合能力较弱，两者有显著性差异；同时该多肽序列也能够与人卵巢腺癌细胞OVCAR-3和SK-OV-3靶向结合，而对人正常卵巢上皮细胞HOSEpiC结合能力较弱，两者有显著性差异(见图3)；并且该多肽序列亦能够与人胰腺癌细胞MIA PaCa-2以及人乳腺癌细胞MCF-7和MDA-MB-231靶向结合，而对人胚胎胰腺组织来源细胞CCC-HPE-2和人乳腺上皮细胞MCF-10A结合能力较弱，两者有显著性差异(见图4、5)；通过进一步实验证实，FITC-DK12能够特异性靶向人结直肠癌组织，与癌旁组织的结合力则比较弱(见图6)，并且与荷瘤鼠人结直肠癌细胞HCT116具有靶向结合能力(见图7)。

Claims (10)

  1. 一种多肽，其特征在于，该多肽为以下任意：
    多肽的氨基酸序列为：DYHDPSLPTLRK(SEQ ID No.1)；
    在(1)所述的多肽分子中经过缺失、插入或置换一个或几个氨基酸且与(1)所述的多肽分子具有相同生物学功能的多肽衍生物。
  2. 根据权利要求1所述的多肽，其特征在于，该多肽对肿瘤细胞有靶向作用，与肿瘤细胞特异性结合。
  3. 根据权利要求1所述的多肽，其特征在于，所述的肿瘤细胞为结肠癌细胞、胰腺癌细胞、乳腺癌细胞或卵巢癌细胞。
  4. 如权利要求1所述的多肽在制备肿瘤诊断试剂盒中的应用。
  5. 根据权利要求1所述的多肽在制备肿瘤诊断试剂盒中的应用，其特征在于，在该试剂盒中包含所述多肽或多肽偶联物。
  6. 如权利要求1所述的多肽在制备用于治疗肿瘤药物中的应用。
  7. 根据权利要求6所述的多肽在制备用于治疗肿瘤药物中的应用，其特征在于，该药物包含所述的多肽与药物活性成分，或包含所述的多肽与递药载体。
  8. 根据权利要求6所述的多肽在制备用于治疗肿瘤药物中的应用，其特征在于，该药物为任何药物治疗学上可接受的的剂型。
  9. 根据权利要求6所述的多肽在制备用于治疗肿瘤药物中的应用，其特征在于，该药物的剂型为注射制剂。
  10. 根据权利要求6所述的多肽在制备用于治疗肿瘤药物中的应用，其特征在于，该药物为任何药物治疗学上可接受的的剂量。
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