CN100486987C - Antivira and antitumor deoxyoligonucleotide containing CpG single strand - Google Patents

Antivira and antitumor deoxyoligonucleotide containing CpG single strand Download PDF

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CN100486987C
CN100486987C CN 03119840 CN03119840A CN100486987C CN 100486987 C CN100486987 C CN 100486987C CN 03119840 CN03119840 CN 03119840 CN 03119840 A CN03119840 A CN 03119840A CN 100486987 C CN100486987 C CN 100486987C
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CN1526718A (en
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于永利
包木胜
王丽颖
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长春华普生物技术有限公司
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    • HELECTRICITY
    • H01BASIC ELECTRIC ELEMENTS
    • H01LSEMICONDUCTOR DEVICES; ELECTRIC SOLID STATE DEVICES NOT OTHERWISE PROVIDED FOR
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    • H01L2924/10Details of semiconductor or other solid state devices to be connected
    • H01L2924/11Device type
    • H01L2924/14Integrated circuits
    • HELECTRICITY
    • H01BASIC ELECTRIC ELEMENTS
    • H01LSEMICONDUCTOR DEVICES; ELECTRIC SOLID STATE DEVICES NOT OTHERWISE PROVIDED FOR
    • H01L2924/00Indexing scheme for arrangements or methods for connecting or disconnecting semiconductor or solid-state bodies as covered by H01L24/00
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Abstract

本发明提供了系列含CpG单链脱氧寡核苷酸,它们由含一个或多个CpG的脱氧寡核苷酸单链DNA分子构成,其能够刺激人外周血单个核细胞(PBMC)杀伤人的白血病细胞和刺激人外周血单个核细胞产生α干扰素;以及这些含CpG单链脱氧寡核苷酸用于制备治疗肿瘤性疾病和病毒感染性疾病的药物的用途。 The present invention provides a series of CpG-containing single-stranded oligodeoxynucleotides, they are composed of oligodeoxynucleotide single stranded DNA molecule containing one or more CpG, which can stimulate human peripheral blood mononuclear cells (PBMC) of anti-human leukemia cells and stimulation of human peripheral blood mononuclear cells to produce interferon α; and the use of the CpG-containing single-stranded oligodeoxynucleotides used for treating neoplastic diseases and viral infectious diseases medicament.

Description

抗病毒和抗肿瘤的含CpG单链脱氧寡核苷酸 Antiviral and antitumor CpG-containing single-stranded oligodeoxynucleotides

发明领域 Field of the Invention

本发明涉及数种含CpG单链脱氧寡核苷酸,特别是涉及对肿瘤性疾病和病毒感染性病疾具有治疗作用的含CpG单链脱氧寡核苷酸,以及这些含CpG单链脱氧寡核苷酸用于制备治疗肿瘤性疾病和病毒感染性疾病的药物的用途。 The present invention relates to several CpG-containing single-stranded oligodeoxynucleotides, particularly to CpG-containing single-stranded oligodeoxynucleotide having a therapeutic effect on the disease neoplastic diseases and viral sexually transmitted diseases, as well as the CpG-containing single-stranded oligodeoxynucleotide core nucleotide manufacture of a medicament for the treatment of neoplastic diseases and viral infectious diseases.

发明背景 BACKGROUND OF THE INVENTION

100多年前,Coley在世界上第一个开展了应用细菌提取物治疗细菌感染性疾病的实验,他得出结论:细菌的提取物是一种可以用于治疗传染性疾病的制剂。 100 years ago, Coley first in the world to carry out experiments using bacterial extracts treatment of bacterial infectious diseases, he concluded: bacteria can be used to extract is a preparation of infectious diseases.

1984年,Tokunaga T等发现从卡介苗(BCG)提取的DNA能抑制多种动物肿瘤的生长,具有激活人外周血单个核细胞和小鼠脾脏天然杀伤(NK)细胞的活性,诱导干扰素(IFN)的产生。 1984, Tokunaga T et al found that extracted from BCG (BCG) DNA can inhibit the growth of various tumors in animals, and mononuclear cells having the activity of mouse spleen natural killer (NK) cells activated human peripheral blood, induction of interferon (IFN ) generation. 非脊椎动物的DNA 有上述功能,脊椎动物和植物的DNA则不然,这些功能依赖于DNA 分子中CpG结构(Tokunaga T, Antitumor activity of deoxyribonucleic acid fraction from Mycobacterium bovis BCG.工. Isolation, physicochemical characterization, and antitumor activity, JNC工,72:955. 1984)。 Non-vertebrate DNA has the above functions, vertebrate and plant DNA is not the case, these functions rely on CpG DNA molecule structure (Tokunaga T, Antitumor activity of deoxyribonucleic acid fraction from Mycobacterium bovis BCG. Workers. Isolation, physicochemical characterization, and antitumor activity, JNC Engineering, 72: 955 1984).

CpG是由胞嘧啶和鸟嘌呤通过磷酸连接成的二核苷酸。 CpG is connected by cytosine and guanine through phosphoric acid into dinucleotide. C代表胞嘧啶,G代表鸟嘌呤,p代表磷酸,胞嘧啶位于5'端。 C represents Cytosine, G indicates Guanine, P representative of phosphoric acid, cytosine at the 5 'end. 多种具有CpG结构的细菌和病毒DNA对脊椎动物的免疫系统均为危险的信号,可激活多种免疫细胞启动对细菌和病毒的抵抗机制。 Having a variety of viral and bacterial DNA CpG configuration of a vertebrate immune system are the danger signals, cellular promoters can activate various immune resistance mechanism of bacteria and viruses. 近年来的研究表明,人工合成的含一个或多个CpG的寡核苷酸单链DNA (CpG ODN)也可表现强效的免疫增强和免疫调节作用。 Recent studies show, synthetic CpG containing one or more single-stranded oligonucleotide DNA (CpG ODN) and may also exhibit immune enhancement potent immunomodulatory effects. CpG ODN可强力增强B细胞、T 细胞、NK细胞、抗原提呈细胞(单核细胞、巨噬细胞和树突状细胞) CpG ODN can be strongly enhanced B cells, T cells, NK cells, antigen-presenting cells (monocytes, macrophages and dendritic cells)

和中性粒细胞的功能,表现出明显的临床应用前景(Weiner GJ, Thei画unobiology and clinical potential of i咖unostimulatory CpG oligodeoxy而cleotides. J Leukoc Biol 2000 Oct; 68(4) :455-63)。 And neutrophil function, showed significant clinical application (Weiner GJ, Thei Videos unobiology and clinical potential of i and coffee unostimulatory CpG oligodeoxy cleotides J Leukoc Biol 2000 Oct; 68 (4):. 455-63).

由于序列,尤其是CpG两侧的序列的不同,CpGODN可有多种多样的形式,表现不同性质、不同强度的免疫增强和免疫调节作用。 Since the sequence, particularly the sequence of different sides of the CpG, CpGODN can have a wide variety of forms, the performance of different nature, enhance immune and immunomodulatory effects of different intensities. 有些CpGODN可表现出种属依赖性,即对一种动物表现免疫增强功能的CpG ODN,在另一种动物或人则未必表现出同样的或等效的免疫增强功能 Some CpGODN may exhibit species-dependent, i.e., CpG ODN showed enhanced immune function of an animal, another animal or the human is not necessarily exhibit the same immune enhancement or equivalent

(Kamstrup S, et al. Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides, Vet Microbiol 2001 Feb 26; 78(4)352-62; Gunther Hartmann, et al. Delineation of a CpG Phosphorothioate Oligodeoxynucleotide for Activating Primate Immune Responses In Vitro and In Vivol The Journal of Immunology, 2000' 164: 1617-1624)。 (Kamstrup S, et al Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides, Vet Microbiol 2001 Feb 26;. 78 (4) 352-62;. Gunther Hartmann, et al Delineation of a CpG Phosphorothioate Oligodeoxynucleotide for Activating Primate Immune Responses In Vitro and In Vivol The Journal of Immunology, 2000 '164: 1617-1624).

CpG-DNA具有广泛的免疫调节作用。 CpG-DNA has wide immunomodulatory effects. CpG-DNA作用于TLR-9,对巨噬细胞、树突状细胞和B细胞有很强的剌激作用。 CpG-DNA acting on TLR-9, macrophages, dendritic cells and B cells have a strong stimulating effect. CpG是细菌感染引起炎症的重要因子。 CpG is an important factor of a bacterial infection caused by inflammation. 原发小胶质细胞表达TLR-9 mRNA.在体外CpG-DNA 激活小胶质细胞产生TNF-。 Primary microglia expression of TLR-9 mRNA. Microglial activation in vitro CpG-DNA produced TNF-. , IL-12p40, IL-12p70和N0.此外,还可上调MHC II类分子,使B7-l, B7-2和CD40分子上调,吞噬活性增强。 , IL-12p40, IL-12p70 and N0. In addition, MHC II molecules may be regulated, so that B7-l, B7-2 and CD40 molecules upregulated enhanced phagocytic activity. 脑室内注射CpG-DNA,刺激小胶质细胞表达TOF-cv和IL-12p40 tmRNA Intraventricular injection of CpG-DNA, stimulated microglia expression TOF-cv and IL-12p40 tmRNA

(Alexander H. Dalpke et al. Immunostimulato:ry CpG-DNA Activates Murine Microglia1 r力e Jo"777s7 of J卿moJo^7, 2002, 168: 4854-4863)。 (Alexander H. Dalpke et al Immunostimulato:. Ry CpG-DNA Activates Murine Microglia1 r force e Jo "777s7 of J State moJo ^ 7, 2002, 168: 4854-4863).

PDC和B细胞(而非单核细胞(monocytes) ), NK细胞或T细胞是外周血CpG ODN的耙细胞。 PDC and B-cells (but not monocytes (monocytes)), NK cells or T cells are peripheral blood cells rake CpG ODN. TLR9是PDC和B细胞识别CpG ODN的受 TLR9 is identified PDC and B cells by CpG ODN

体(Veit Hornung , et al. Quantitative Expression of Toll-Like Rec印tor 1— 10 mRNA in Cellular Subsets of Human Peripheral Blood Mononuclear Cells and Sensitivity to CpG Oligodeoxynucleotides1 77?e /。"zy737 of /腳w?o2ogy, 2002, 168: 4531-4537)。 Body (Veit Hornung, et al. Quantitative Expression of 1- 10 mRNA Toll-Like Rec printed tor in Cellular Subsets of Human Peripheral Blood Mononuclear Cells and Sensitivity to CpG Oligodeoxynucleotides1 77? E /."zy737 of / foot w? O2ogy, 2002 , 168: 4531-4537).

在人的外周血单个核细胞中有天然杀伤细胞(NK)。 There are natural killer (NK) cells in human peripheral blood mononuclear cells. NK是一类无吞噬作用的非粘附性细胞,胞质内有许多嗜苯胺颗粒,是一种起源于骨髓的大颗粒淋巴细胞。 NK is a class of non-adherent cells without phagocytosis, there are many intracytoplasmic addicted aniline particles is originated in the bone marrow of large granular lymphocytes. 具有广谱的抗肿瘤作用,能杀伤同系、同种及异种的肿瘤细胞,尤其对淋巴瘤和白血病最为有效。 Having broad-spectrum anti-tumor effect, kill or homologs, isoforms and xenogeneic tumor cells, in particular the most effective for lymphoma and leukemia. 干扰素(interferon, IFN)是最先发现的细胞因子。 Interferon (interferon, IFN) cytokines was first discovered. I型干扰素是一种重要的抗病毒细胞因子。 Type I Interferon is an important antiviral cytokines. 受到病毒感染的细胞可合成和分泌IFN , 这些IFN可刺激邻近的细胞合成抑制RNA及DNA病毒复制的酶类使其进入抗病毒状态,从而使这些细胞受到保护。 By virus-infected cells and secretion of IFN synthesis, IFN these cells can stimulate neighboring RNA and DNA synthesis inhibition virus replication enzymes it into an antiviral state, so that these cells are protected.

发明内容发明概述 SUMMARY SUMMARY OF THE INVENTION

本发明的目的之一是提供系列含CpG单链脱氧核苷酸,特别是刺激人天然杀伤细胞活性和诱生a干扰素的含CpG单链脱氧核苷酸。 One object of the present invention is to provide a series of CpG-containing single-stranded oligodeoxynucleotides, particularly stimulating CpG-containing single-stranded oligodeoxynucleotides human natural killer cell activity and interferon induced a. 它们由含一个或多个CpG的寡核苷酸单链DNA分子构成,其磷酸二酯键可以是部分硫化的,全部硫化的, 也可以是未硫化的。 They consist of a single-stranded oligonucleotide DNA molecule containing one or more CpG, the phosphodiester linkage may be partially cured, the entire vulcanized, may be uncured.

优选地,本发明的含CpG单链脱氧寡核苷酸具有SEQ ID NO: 1-95 所示的序列。 Preferably, the CpG-containing single-stranded oligodeoxynucleotides according to the present invention having SEQ ID NO: 1-95 of the sequence shown.

本发明的目的之二是提供本发明的含CpG单链脱氧寡核苷酸用于制备治疗肿瘤性疾病和病毒感染性疾病的药物的用途。 Second object of the present invention is to provide a use of the inventive CpG-containing single-stranded oligodeoxynucleotides used medicament for the treatment of tumor diseases and viral infectious diseases.

另外,需要指出的是,在本申请的上下文的公开内容的基础上,本发明的其它具有实质性特点的方面和创造性的有益效果对本领域的普通技术人员来说是可以直接推知的。 Further, to be noted that, on the basis of the disclosure of the present application on the context, other substantive features and inventive aspects of the present invention, the beneficial effects of ordinary skill in the art that can be directly inferred.

具体实施方式 Detailed ways

在本发明的上下文中,所使用的术语除非另外说明, 一般具有本领域的普通技术人员通常理解的含义。 In the present context, the term is used unless stated otherwise, meaning having ordinary skill in the art generally understood. 特别地,下列术语具有如下的含义: 优选地,本发明的含CpG单链脱氧寡核苷酸具有如下所示的序列: In particular, the following terms have the following meanings: Preferably, the CpG-containing single-stranded oligodeoxynucleotides according to the present invention has the sequence shown below:

101 5, -TcgTCgAgggCgCCggTgAC-3' (SEQ ID N0:1) 101 5, -TcgTCgAgggCgCCggTgAC-3 '(SEQ ID N0: 1)

102 5, -TCgTCgCCggTgggggTgTg-3 , (SEQ ID N0:2) 102 5, -TCgTCgCCggTgggggTgTg-3, (SEQ ID N0: 2)

103 5' -TCgTCgTACgCAATTgTCTT-3' (SEQ ID N0:3) 103 5 '-TCgTCgTACgCAATTgTCTT-3' (SEQ ID N0: 3)

104 5, -TCgCCTCgTCgCCTTCgAgC-3 , (SEQ ID N0:4) 104 5, -TCgCCTCgTCgCCTTCgAgC-3, (SEQ ID N0: 4)

201 5, -TCgCCCACCggTgggggggg-3 , (SEQ ID N0:5) 201 5, -TCgCCCACCggTgggggggg-3, (SEQ ID N0: 5)

202 5, -TCgTCgCAgACCggTCTgggg-3' (SEQ ID N0:6) 202 5, -TCgTCgCAgACCggTCTgggg-3 '(SEQ ID N0: 6)

203 5, -gggggACgTCgCCggggggg-3' (SEQ ID N0:7)<formula>formula see original document page 6</formula>615 5' -TCgTTgCCgTCggCCCCCC-3, (SEQ ID N0:37) 203 5, -gggggACgTCgCCggggggg-3 '(SEQ ID N0: 7) <formula> formula see original document page 6 </ formula> 615 5' -TCgTTgCCgTCggCCCCCC-3, (SEQ ID N0: 37)

616 5, -TCgTTgCCgTCggCCCCC-3, (SEQ ID NO:38) 616 5, -TCgTTgCCgTCggCCCCC-3, (SEQ ID NO: 38)

617 5, -TCgTTgCCgTCggCCCC-3, (SEQ ID NO:39) 617 5, -TCgTTgCCgTCggCCCC-3, (SEQ ID NO: 39)

618 5, -TCgTTgCCgTCggCCCCCCC-3, (SEQ ID N0:40) 618 5, -TCgTTgCCgTCggCCCCCCC-3, (SEQ ID N0: 40)

619 5, -TCgTTgCCgTCgg-3, (SEQ ID NO:41) 619 5, -TCgTTgCCgTCgg-3, (SEQ ID NO: 41)

620 5, -TCgTTgCCgTCggg-3, (SEQ ID NO:42) 620 5, -TCgTTgCCgTCggg-3, (SEQ ID NO: 42)

621 5, -TCgTTgCCgTCgggg-3' (SEQ ID NO:43) 621 5, -TCgTTgCCgTCgggg-3 '(SEQ ID NO: 43)

622 5, -TCgTTgCCgTCggggg-3' (SEQ ID NO:44) 622 5, -TCgTTgCCgTCggggg-3 '(SEQ ID NO: 44)

623 5, -TCgTTgCCgTCgggggg-3' (SEQ ID NO:45) 623 5, -TCgTTgCCgTCgggggg-3 '(SEQ ID NO: 45)

624 5, -TCgTTgCCgTCggggggg-3' (SEQ ID NO:46) 624 5, -TCgTTgCCgTCggggggg-3 '(SEQ ID NO: 46)

625 5, -TCgTTgCCgTCgggggggg-3, (SEQ ID NO:47) 625 5, -TCgTTgCCgTCgggggggg-3, (SEQ ID NO: 47)

626 5, -TCgTTgCCgTCggggggggg-3, (SEQ ID NO:48) 626 5, -TCgTTgCCgTCggggggggg-3, (SEQ ID NO: 48)

627 5' -TCgAggACAAgATTCTCgT-3, (SEQ ID NO:49) 627 5 '-TCgAggACAAgATTCTCgT-3, (SEQ ID NO: 49)

628 5' -TCCCgCTggACgTT-3, (SEQ ID NO:50) 628 5 '-TCCCgCTggACgTT-3, (SEQ ID NO: 50)

629 5, -TCggCACgCgACgTgCTggCCgTCgTT-3, (SEQ ID NO:51) 629 5, -TCggCACgCgACgTgCTggCCgTCgTT-3, (SEQ ID NO: 51)

631 5, -TCgTCgCgCCgTCACgggggg-3, (SEQ ID NO:52) 631 5, -TCgTCgCgCCgTCACgggggg-3, (SEQ ID NO: 52)

632 5, -TCgTgTgCgTgCCgTTggg-3, (SEQ ID NO:53) 632 5, -TCgTgTgCgTgCCgTTggg-3, (SEQ ID NO: 53)

633 5, -TCgTCgCCgTTgggCggg-3, (SEQ ID NO:54) 633 5, -TCgTCgCCgTTgggCggg-3, (SEQ ID NO: 54)

634 5, -TCgTCgACgTCgTTgggCggg-3, (SEQ ID NO:55) 634 5, -TCgTCgACgTCgTTgggCggg-3, (SEQ ID NO: 55)

635 5, -TCgCAgTTgTCgTAACgTTgggCggg-3, (SEQ ID NO:56) 635 5, -TCgCAgTTgTCgTAACgTTgggCggg-3, (SEQ ID NO: 56)

636 5, -TTACCggTTAACgTTggCCggCC-3, (SEQ ID NO:57) 636 5, -TTACCggTTAACgTTggCCggCC-3, (SEQ ID NO: 57)

637 5, -ACCggTTAACgTTgTCCCCgggg-3, (SEQ ID NO:58) 637 5, -ACCggTTAACgTTgTCCCCgggg-3, (SEQ ID NO: 58)

638 5, -TCgTCgTTggTATgTT-3, (SEQ ID NO:59) 638 5, -TCgTCgTTggTATgTT-3, (SEQ ID NO: 59)

639 5, -TCgTCgTCgTCgTTgTCgTT -3, (SEQ ID NO:60) 639 5, -TCgTCgTCgTCgTTgTCgTT -3, (SEQ ID NO: 60)

640 5' -TCgTCgTCgTCgTTgTCgTTgggg-3, (SEQ ID NO:61) 640 5 '-TCgTCgTCgTCgTTgTCgTTgggg-3, (SEQ ID NO: 61)

641 5, -TCgTTCggggTgCCg-3, (SEQ ID NO:62) 641 5, -TCgTTCggggTgCCg-3, (SEQ ID NO: 62)

642 5, -TCgTTCggggTAACgATT-3, (SEQ ID NO:63) 642 5, -TCgTTCggggTAACgATT-3, (SEQ ID NO: 63)

643 5' -TCgTTCggggTAACgTT-3' (SEQ ID NO:64) 643 5 '-TCgTTCggggTAACgTT-3' (SEQ ID NO: 64)

644 5, -TCgTTCggggTACCgAT -3, (SEQ ID NO:65) 644 5, -TCgTTCggggTACCgAT -3, (SEQ ID NO: 65)

645 5' -TCgTTCggggTACCgATgggg-3' (SEQ ID NO:66)646 5' -TCgTTgCgCTCCCATgCCgggggg-3' (SEQ ID NO:67) 645 5 '-TCgTTCggggTACCgATgggg-3' (SEQ ID NO: 66) 646 5 '-TCgTTgCgCTCCCATgCCgggggg-3' (SEQ ID NO: 67)

647 5' -TCgTCgTTTCgTCgTTgggg-3, (SEQ ID NO:68) 647 5 '-TCgTCgTTTCgTCgTTgggg-3, (SEQ ID NO: 68)

648 5' -TCgTTgTCgTTTCgCTgCCggCggggg-3' (SEQ ID NO:69) 648 5 '-TCgTTgTCgTTTCgCTgCCggCggggg-3' (SEQ ID NO: 69)

649 5, -CgTTgACgATCgTCCCATggCggg-3, (SEQ ID NO:70) 649 5, -CgTTgACgATCgTCCCATggCggg-3, (SEQ ID NO: 70)

650 5' -TCTgCggCCTTCgTCg-3, (SEQ ID NO:71) 650 5 '-TCTgCggCCTTCgTCg-3, (SEQ ID NO: 71)

651 5, -TAgTAACCggTCCggCgCCCCC-3, (SEQ ID NO:72) 651 5, -TAgTAACCggTCCggCgCCCCC-3, (SEQ ID NO: 72)

652 5, -TTgCAgCgCTgCCggTggg-3, (SEQ ID NO:73) 652 5, -TTgCAgCgCTgCCggTggg-3, (SEQ ID NO: 73)

653 5, -TCgTACggCCgCCgTACggCggg-3, (SEQ ID NO:74) 653 5, -TCgTACggCCgCCgTACggCggg-3, (SEQ ID NO: 74)

654 5' -CggCCCATCgAgggCgACggC-3, (SEQ ID NO:75) 654 5 '-CggCCCATCgAgggCgACggC-3, (SEQ ID NO: 75)

655 5, -TCgCgTCgACTCCCCTCgAgggg-3, (SEQ ID NO:76) 655 5, -TCgCgTCgACTCCCCTCgAgggg-3, (SEQ ID NO: 76)

656 5, -TCgTCgTCgACTCgTggTCggggg-3, (SEQ ID NO:77) 656 5, -TCgTCgTCgACTCgTggTCggggg-3, (SEQ ID NO: 77)

657 5, -TCgggCgCCCgATCgggggg-3' (SEQ ID NO:78) 657 5, -TCgggCgCCCgATCgggggg-3 '(SEQ ID NO: 78)

658 5, -TCgTCggTCTTTCgAAATT-3, (SEQ ID NO:79) 658 5, -TCgTCggTCTTTCgAAATT-3, (SEQ ID NO: 79)

659 5, -TCgTgACgTCCTCgAgTT-3' (SEQ ID NO:80) 659 5, -TCgTgACgTCCTCgAgTT-3 '(SEQ ID NO: 80)

660 5' -ggACgATCgATCgTgggggg-3' (SEQ ID N0:82) 660 5 '-ggACgATCgATCgTgggggg-3' (SEQ ID N0: 82)

661 5' -gggATgCATCgATgCATCgggggg-3' (SEQ ID N0:83) 661 5 '-gggATgCATCgATgCATCgggggg-3' (SEQ ID N0: 83)

662 5' -gggggAATCgATTCgggggg-3' (SEQ ID N0:84) 662 5 '-gggggAATCgATTCgggggg-3' (SEQ ID N0: 84)

663 5' -ggTgCgACgTCgCAgggggg-3' (SEQ ID N0:85) 663 5 '-ggTgCgACgTCgCAgggggg-3' (SEQ ID N0: 85)

664 5' -TCgTCgggTgCATCgATgCAgggggg-3' (SEQ ID N0:86 664 5 '-TCgTCgggTgCATCgATgCAgggggg-3' (SEQ ID N0: 86

665 5' -ggTgCATCgTACgATgCAgggggg-3' (SEQ ID N0:87) 665 5 '-ggTgCATCgTACgATgCAgggggg-3' (SEQ ID N0: 87)

666 5' -gggACgTACgTCgggggg-3, (SEQ ID N0:88) 666 5 '-gggACgTACgTCgggggg-3, (SEQ ID N0: 88)

667 5' -TCggggACgATCgTCgggggg-3' (SEQ ID N0:89) 667 5 '-TCggggACgATCgTCgggggg-3' (SEQ ID N0: 89)

668 5' -gggggATCgATATCgATCgggggg-3' (SEQ ID N0:90) 668 5 '-gggggATCgATATCgATCgggggg-3' (SEQ ID N0: 90)

669 5' -gggggATCgACgTCgATCgggggg-3' (SEQ ID N0:91) 669 5 '-gggggATCgACgTCgATCgggggg-3' (SEQ ID N0: 91)

670 5' -ggTgCATCgATCgATgCAgggggg-3' (SEQ ID N0:92) 670 5 '-ggTgCATCgATCgATgCAgggggg-3' (SEQ ID N0: 92)

671 5' -ggATCgATCgATCgATgggggg-3' (SEQ ID N0:93) 671 5 '-ggATCgATCgATCgATgggggg-3' (SEQ ID N0: 93)

672 5, -ggCgATCgATCgATCggggggg-3, (SEQ ID N0:94) 672 5, -ggCgATCgATCgATCggggggg-3, (SEQ ID N0: 94)

673 5, -ggggTCgATCgATCgAgggggg—3' (SEQ ID N0:95) 673 5, -ggggTCgATCgATCgAgggggg-3 '(SEQ ID N0: 95)

674 5, -ggTgCgATCgATCgCAgggggg-3' (SEQ ID N0:96) 674 5, -ggTgCgATCgATCgCAgggggg-3 '(SEQ ID N0: 96)

675 5, -ggTCgCgATCgCgAgggggg-3, (SEQ ID N0:97)676 5'- -TCgTCTTTCgACTCgTTCTC-3' (SEQ ID N0:98) 675 5, -ggTCgCgATCgCgAgggggg-3, (SEQ ID N0: 97) 676 5'- -TCgTCTTTCgACTCgTTCTC-3 '(SEQ ID N0: 98)

677 5,- -TCgTCgTTTTgCgTTCTC-3, (SEQ ID N0:99) 678 5,- -TCATCgACTCTCgAgCgTTC-3' (SEQ ID NO:100) 677 5, - -TCgTCgTTTTgCgTTCTC-3, (SEQ ID N0: 99) 678 5, - -TCATCgACTCTCgAgCgTTC-3 '(SEQ ID NO: 100)

679 5,- -ATCgTCgACTCTCgTgTTCTC-3, (SEQ ID NO:101) 679 5, - -ATCgTCgACTCTCgTgTTCTC-3, (SEQ ID NO: 101)

680 5,- -TCgACTTTCgTCgTTCTgTT-3' (SEQ ID NO:102) 680 5, - -TCgACTTTCgTCgTTCTgTT-3 '(SEQ ID NO: 102)

681 5,- -TCgTCgTTTCgTCgTTCTC-3" (SEQ ID NO:103) 681 5, - -TCgTCgTTTCgTCgTTCTC-3 "(SEQ ID NO: 103)

682 5'- -TCgTCgTCgTCgTTgTCgTT-3' (SEQ ID NO:104) 682 5'- -TCgTCgTCgTCgTTgTCgTT-3 '(SEQ ID NO: 104)

683 5'- -TCgTTCTCgACTCgTTCTC-3' (SEQ ID NO:105) 683 5'- -TCgTTCTCgACTCgTTCTC-3 '(SEQ ID NO: 105)

684 5,- -TCgACgTTCgTCgTTCgTCgTTC- -3, (SEQ ID NO:106) 684 5, - -TCgACgTTCgTCgTTCgTCgTTC- -3, (SEQ ID NO: 106)

685 5'- -TCgTCgACgTCgTTCgTTCTC-3' (SEQ ID NO:107) 685 5'- -TCgTCgACgTCgTTCgTTCTC-3 '(SEQ ID NO: 107)

其中的磷酸二酯键可以是部分硫化的,全部硫化的,也可以是未硫化的。 Wherein the phosphodiester bond may be partially cured, the entire vulcanized, may be uncured.

本发明的CpG单链脱氧核苷酸可通过己知的方法生产,例如采用固相亚磷酰胺三酯法进行生产。 CpG single-stranded oligodeoxynucleotides according to the present invention can be produced by known methods, for example, produced using the solid phase phosphoramidite triester method. 以下的实施例详细地例举了一种生产本发明的CpG单链脱氧核苷酸的方法。 The following examples illustrate the present invention, a method of producing a single-stranded CpG oligodeoxynucleotides in detail.

在这些含CpG单链脱氧寡核苷酸用于制备用于制备治疗肿瘤性疾病和病毒感染性疾病的药物的用途中,与治疗肿瘤性疾病和病毒感染性疾病的药物的使用量的比例为l: 1-100:1 (摩尔比)。 In these applications, CpG-containing single-stranded oligodeoxynucleotides preparation of a medicament for the treatment of neoplastic diseases and viral infectious diseases, the drug treatment of neoplastic diseases and viral infectious disease is used in an amount ratio l: 1-100: 1 (molar ratio).

本发明的含CpG单链脱氧寡核苷酸的使用方式包括单独应用、两种或两种以上的CpG单链脱氧寡核苷酸联合应用、与治疗肿瘤性疾病和病毒感染性疾病的药物混合使用,与治疗肿瘤性疾病和病毒感染性疾病的药物通过交联剂共价偶联使用; Use CpG-containing single-stranded oligodeoxynucleotides according to the present invention comprises a separate application, of two or more single-stranded CpG oligodeoxynucleotides in combination, mixed with a pharmaceutical treatment of neoplastic diseases and viral infectious diseases use, drug use and treatment of neoplastic diseases, viral infectious diseases by covalent coupling a crosslinking agent;

应用方式包括皮下应用、鼻腔粘膜应用、肌肉应用、胃肠应用、腹腔应用、静脉注射等常用的方式。 Application mode including conventional manner subcutaneous application, the application nasal mucosa, muscle application, parenteral application, intraperitoneal application, intravenous injection and the like.

CpG单链脱氧寡核苷酸使用时间可在使用治疗肿瘤性疾病和病毒感染性疾病的药物之前应用、与治疗肿瘤性疾病和病毒感染性疾病的药物同时应用、在治疗肿瘤性疾病和病毒感染性疾病的药物后应用。 CpG single-stranded oligodeoxynucleotides time can be used prior to drug treatment of neoplastic diseases and viral infectious diseases applications, drug treatment of neoplastic diseases and viral infectious diseases applied simultaneously, in the treatment of neoplastic diseases and viral infections drug-induced diseases after application.

在小鼠试验中CpG单链脱氧寡核苷酸的使用量为0-500微克/小鼠, 如进行人体试验则按标准的换算方法进行换算。 The amount of the test in the mouse CpG single-stranded oligodeoxynucleotides of 0-500 [mu] g / mouse, such as human trials are shown at standard conversion methods for conversion.

下面结合具体的制备实施例和生物学效果实施例,并参照附图进一步详细地描述本发明。 The following specific embodiments in conjunction with the preparation of Examples and biological effects, and the present invention is described in further detail with reference to the drawings. 应理解,这些实施例只是为了举例说明本发明,而非以任何方式限制本发明的范围。 It should be understood that these examples are merely to illustrate the invention without limiting the scope of the invention in any manner. 实施例 Example

在如下实施例中,未详细描述的各种过程和方法是本领域中公知的常规方法,例如合成采用固相亚磷酰胺三酯法。 In the following examples, the various processes and methods not described in detail are conventional methods well known in the art, for example, synthesized by the solid phase phosphoramidite triester method.

在如下实施例中,所用试剂的来源、商品名和/或有必要列出其组成成分者,均只标明一次。 In the following examples, reagents used are derived, trade names and / or which are necessary components listed only once are marked. 在其后所用相同试剂如无特殊说明,不在赘述上述内容。 Thereafter the same reagents Unless otherwise specified, the above is not repeated.

实施例l CpG单链脱氧核苷酸的制备(上海生工合成) Preparation Example l CpG single-stranded deoxyribonucleotides (Shanghai Sangon synthesis) embodiment

合成采用固相亚磷酰胺三酯法,合成方法(上海生工提供)如下: 材料和方法: Synthesized by the solid phase phosphoramidite triester method, synthetic methods (Shanghai Sangon provided) as follows: Materials and Methods:

三氯乙酸(TrichloroaceticAcid, TCA)、可控多孔玻璃(Controlled PoreGlass)、 DMT(二甲氧基三苯甲基)、四氮唑活化剂、乙酸酐、N-甲基咪唑、ABIDNA合成仪、高效液相色谱层析仪等。 Trichloroacetic acid (TrichloroaceticAcid, TCA), controlled pore glass (Controlled PoreGlass), DMT (dimethoxytrityl), tetrazolium activator, acetic anhydride, N- methylimidazole, ABIDNA synthesizer efficient liquid chromatography instrument.

A、 T、 C、 G四种核苷酸单体:合成所用单体为核苷亚磷酰胺, 是经过化学修饰的核苷酸,含下面几个功能基团: A, T, C, G the four nucleotide monomers: synthesis with phosphoramidite nucleoside monomers are chemically modified nucleotides, containing the following a few functional groups:

1. 3'位P上二异丙胺基,縮合所用的功能基 1.3 apos diisopropylamine position P, the condensation with a functional group

2. 3'位P上腈乙基,保护基,合成完毕后脱去。 2.3 'cyanoethyl the position P, the protecting group, after synthesis is completed removed.

3. 5' -Dmt,保护基,縮合前脱去。 3. 5 '-Dmt, a protecting group, prior to the condensation off.

4. A和C的杂环氨基上的苯甲酸保护基,合成完毕后脱去。 Acid protecting group on the heterocyclic amino 4. A and C, removed after the synthesis is completed.

5. G上嘌呤环氨基上的异丙酰保护基,合成完毕后脱去。 5. G isopropyl upper acyl protecting group on the purine ring group, removed after the synthesis is completed. 具体的反应步骤如下: Specific reaction steps are as follows:

、脱保护基 , Deprotection

用三氯乙酸(Trichloroacetic Acid, TCA)脱去连结在可控多孔玻璃(Controlled Pore Glass)上的核苷酸的保护基团二甲氧基三苯甲基(匿T),获得游离的5'-羟基端,以供下一步縮合反应。 Off nucleotides coupled to the controlled pore glass (Controlled Pore Glass) of trichloroacetic acid (Trichloroacetic Acid, TCA) protective dimethoxytrityl group (hide T), to give the free 5 ' - hydroxyl end for the next condensation reaction. ......-:、活化 ......-:,activation

将亚磷酰胺保护的核苷酸单体与四氮唑活化剂混合并进入合成柱, 形成亚磷酰胺四唑活性中间体(其3'-端己被活化,但5'-端仍受DMT保护),此中间体将与可控多孔玻璃上的已脱保护基的核苷酸发生缩合反应。 Mixing the protected phosphoramidite nucleotide monomer tetrazolium activator and into synthesis column to form a tetrazolyl phosphoramidite reactive intermediate (3'-end of which had been activated, but still subject to the 5'-terminal DMT protection), the condensation reaction of this intermediate is deprotected with a nucleotide on the controlled pore glass.

三、 连接 Third, the connection

亚磷酰胺四唑活性中间体遇到可控多孔玻璃上己脱保护基的核苷酸 Tetrazolyl phosphoramidite reactive intermediate encountered on controlled pore glass hexyl deprotected nucleotide

时,将与其5'-羟基发生亲合反应,縮合并脱去四唑,此时合成的寡核 When the 5'-hydroxyl its affinity reaction occurs, tetrazole condense off, then synthetic oligonucleotide

苷酸链向前延长一个碱基。 A nucleotide chain extending forwardly base.

四、 封闭 Fourth, closed

縮合反应后为了防止连在可控多孔玻璃上的未参与反应的5'-羟基在随后的循环反应中被延伸,常通过乙酰化来封闭此端羟基, 一般乙酰化试剂是用乙酸酐和N-甲基咪唑等混合形成的。 After the condensation reaction in order to prevent the unreacted 5'-hydroxyl group attached to the controlled pore glass is extended in the subsequent cycle reaction, this terminal hydroxyl group is often closed by acetylation, generally acetylating agent is acetic anhydride and N - mixed methylimidazole formed.

五、 氧化 Fifth, oxidation

縮合反应时核苷酸单体是通过亚磷酯键与连在可控多孔玻璃上的寡核苷酸连接,而亚磷酯键不稳定,易被酸、碱水解,此时常用碘的四氢呋喃溶液将亚磷酰转化为磷酸三酯,得到稳定的寡核苷酸。 Nucleotide phosphoramidite monomer by ester linkage to the oligonucleotide attached to the controlled pore glass when the condensation reaction is connected, and the phosphorous ester bond labile and susceptible to acid, alkaline solution, typically at a iodine in tetrahydrofuran the solution was converted to the phosphoramidite triester phosphate, a stable oligonucleotide. 经过以上五个步骤后, 一个脱氧核苷酸就被连到可控多孔玻璃的核苷酸上,同样再用三氯乙酸脱去新连上的脱氧核苷酸5'-羟基上的保护基团DMT后,重复以上的活化、连接、封闭、氧化过程即可得到一DNA片段粗品。 After above five steps, a deoxynucleotide was attached to controlled pore glass nucleotide, also trichloroacetic acid and then removing the protecting group on the 5'-hydroxyl group of deoxynucleotide new connection after the DMT group, repeat the above activation, connection closed during oxidation to obtain a DNA fragment crude product. 最后对其进行切割、脱保护基(一般对A、 C碱基采用苯甲酰基保护;G碱基用异丁酰基保护;T碱基不必保护;亚磷酸用腈乙基保护)、 纯化(常用的有HAP, PAGE, HPLC, C18, 0PC等方法)、定量等合成后处理即可得到符合实验要求的寡核苷酸片段。 Finally, its cutting, deprotection (typically for A, C using benzoyl protecting bases; G nucleotide protection isobutyryl; T bases do not have protection; nitrile ethyl phosphite protection), purified (common there HAP, PAGE, HPLC, C18, 0PC the like), quantification can be obtained by post-synthesis treatment with the experimental oligonucleotides requirements.

未硫化的CpG单链脱氧寡核苷酸在ABI 3900 DNA合成仪上合成(上海生工生物技术服务有限公司),全硫化及部分硫化CpG单链脱氧寡核苷酸的合成采用置换法,在ABI 394 DNA合成仪上合成(上海生工生物技术服务有限公司)。 Unvulcanized CpG single-stranded oligodeoxynucleotides synthesized (Shanghai Sangon Biological Engineering Technology Services Ltd.), and partially cured fully cured CpG single-stranded oligodeoxynucleotides synthesized by the displacement method on ABI 3900 DNA synthesizer, in synthesis of the ABI 394 DNA synthesizer (Shanghai biological technology service Co., Ltd.).

实施例2人外周血单个核细胞杀伤人白血病细胞(K562细胞) Example 2 Peripheral blood mononuclear cells kill human leukemia cells (K562 cells)

一、K562细胞的培养试剂和材料: A, K562 cell culture reagents and materials:

KM2细胞:为人的白血病细胞株(美国ATCCCCL-243)。 KM2 cells: human leukemia cell line (American ATCCCCL-243). 细胞培养常用的仪器设备:低温冰箱、二氧化碳孵箱、超净工作台、倒置显微镜、液氮罐、蒸馏水器、真空泵、细胞培养瓶、滤菌器、滤过瓶、 各种规格的吸管、加样器、滴管等。 Commonly used cell culture equipment: cryogenic refrigerator, carbon dioxide incubator, clean bench, inverted microscope, a liquid nitrogen tank, distilled water, a vacuum pump, cell culture flask, a bacteria filter, filtration bottles, a variety of straw, plus sampler, dropper.

RPMI1640培养液: RPMI1640 medium:

含L一谷氨酰胺的RPMI1640(GIBC0BRL) 10. 4克碳酸氢钠2. 0克 10.4 g of sodium bicarbonate containing L-glutamine RPMI1640 (GIBC0BRL) 2. 0 g of

庆大霉素10万单位 Gentamicin 100,000 units

加三蒸水至体积1000毫升0. 22微米的滤膜真空泵抽滤除菌、分装。 Was added to a volume of triple-distilled water 1000 ml vacuum filtration membrane filtering 0.22 microns dispensing. 小牛血清灭活:小牛血清(Invitrogen)—2(TC取出,4°C冰箱融化后, 56""C水浴30min。 Inactivated fetal bovine serum: calf serum (Invitrogen) -2 (TC removed after the 4 ° C refrigerator thawing, 56 "" C water bath for 30min.

10%小牛血清的RPMI1640: 10% calf serum RPMI1640:

灭活的小牛血清10毫升 Inactivated fetal bovine serum 10 ml

RPMI 1640培养液90毫升 RPMI 1640 culture medium 90 ml

冻存液的配制:200微升小牛血清,100微升二甲基亚砜(DMSO), 700微升无血清RPMI1640培养基。 Frozen liquid formulation: 200 microliters calf serum, 100 [mu] l dimethylsulfoxide (DMSO), 700 [mu] l serum-free RPMI1640 medium.

1. K562细胞的复苏:从液氮罐中取出冻存的K562细胞,37"C水浴迅速融化,加5毫升RPMI1640培养液稀释,水平离心机以l,OOOXg 离心3min,弃上清,加入10%小牛血清的RPMI1640 2毫升,滴管吹打均匀,转移至50毫升培养瓶中,补加RPMI1640培养液至终体积5毫升。 K562 Cells 1. Recovery: frozen in liquid nitrogen tank was removed from K562 cells, 37 "C water bath and quickly thawed, diluted with 5 ml RPMI1640 culture medium, centrifuged at level l, OOOXg centrifugation 3min, the supernatant discarded, and 10 2% calf serum RPMI1640 ml dropper uniformly by pipetting, transferred to a 50 ml flask, supplemented RPMI1640 medium to a final volume of 5 ml.

2. K562细胞的培养:带10X小牛血清的RPMI1640作为培养基,50 毫升细胞培养瓶,37°C 5%二氧化碳孵箱培养。 2. K562 cells in culture: 10X with fetal calf serum as RPMI1640 medium, 50 ml cell culture flasks, 37 ° C 5% carbon dioxide culture incubator.

3. K562细胞的传代:生长至一定密度的K562细胞,滴管轻轻吹打均匀,转移至10毫升离心管中,水平离心机以1,000 X g离心力离心3min,弃上清,加入10%小牛血清的RPMI1640 2毫升,滴管吹打均匀,分别转移到两个50毫升培养瓶中,分别补加培养液至终体积5毫升,37。 3. K562 passaged cell: K562 grown to a certain cell density, a uniform dropper gently pipetting, transferred to a 10 ml centrifuge tube, centrifuged at 1,000 X g level centrifuged at 3min, the supernatant discarded, and 10% calf serum RPMI1640 2 mL dropper evenly pipetting were transferred to two 50 ml flasks were supplemented culture medium to a final volume of 5 ml, 37. C5^二氧化碳孵箱培养。 C5 ^ carbon dioxide culture incubator.

4. K562细胞的冻存:生长良好的细胞,滴管轻轻吹打均匀,转移至IO毫升离心管中,水平离心机以1,000 Xg离心力离心3min, 弃上清,冻存液1毫升,滴管轻轻吹打均匀,转移至冻存管中, 梯度降温(降温rC/l一2h),降至一70。 4. K562 frozen cells: The cells grew well, gently pipetting uniform dropper, IO ml was transferred to a centrifuge tube, centrifuged at 1,000 Xg horizontal centrifuged at 3min, the supernatant was discarded, 1 ml was frozen, dropper homogeneous by gently pipetting, transferred to cryovials, gradient cooling (cooling rC / l a 2H), down to a 70. C左右,然后,转移到液氮罐中。 C or so, then transferred to a liquid nitrogen tank.

二、人外周血单个核细胞的分离 Second, human peripheral blood mononuclear cells

1、试剂和材料: 1, Reagents and Materials:

肝素抗凝的人全血:长春市中心血站。 Heparinized human whole blood: Changchun city blood bank.

聚蔗糖-泛影葡胺:比重1.077士0.001,北京鼎国生物技术有限公司。 Ficoll - hypaque: specific gravity 1.077 0.001 disabilities, Beijing Ding States Biological Technology Co., Ltd.

细胞培养常用的仪器设备:低温冰箱、二氧化碳孵箱、超净工作台、倒置显微镜、液氮罐、蒸馏水器、真空泵、细胞培养瓶、滤菌器、 滤过瓶、各种规格的吸管、加样器、滴管、血球计数板、水平式离心机等。 Commonly used cell culture equipment: cryogenic refrigerator, carbon dioxide incubator, clean bench, inverted microscope, a liquid nitrogen tank, distilled water, a vacuum pump, cell culture flask, a bacteria filter, filtration bottles, a variety of straw, plus sampler, a dropper, hemacytometer, centrifuge horizontal.

RPMI1640培养液: RPMI1640 medium:

含L一谷氨酰胺的RPMI1640(GIBCOBRL) 10. 4克碳酸氢钠2. 0克 10.4 g of sodium bicarbonate containing L-glutamine RPMI1640 (GIBCOBRL) 2. 0 g of

庆大霉素10万单位 Gentamicin 100,000 units

加三蒸水至体积1000毫升0.22微米的滤膜真空泵抽滤除菌、分装。 To a volume of triple-distilled water was added 1,000 ml of a 0.22 micron vacuum filter filtration sterilization, packaging.

小牛血清灭活:小牛血清(Invitrogen)—20。 Inactivated fetal bovine serum: calf serum (Invitrogen) -20. C取出,4。 Remove C, 4. C冰箱融化后,56 C refrigerator after thawing, 56

. C水浴30min。 C water bath for 30min.

10%小牛血清的RPM工1640: 10% calf serum work 1640 RPM:

灭活的小牛血清10毫升 Inactivated fetal bovine serum 10 ml

RPMI 1640培养液90毫升 RPMI 1640 culture medium 90 ml

Hank's液(无钙离子、镁离子)的配制: Hank's solution (without calcium ions, magnesium ions) formulation:

氯化钠8. 0克 Sodium chloride 8.0 g

氯化钾0. 4克磷酸氢二钠(带一个结晶水) 0. 06克 Potassium chloride 0.4 g disodium hydrogen phosphate (with one crystal water) 0.06 g

磷酸二氢钾0. 06克 0.06 g of potassium dihydrogen phosphate

碳酸氢钠0. 35克 0.35 g of sodium bicarbonate

葡萄糖lO克酚红0. 02克 LO grams of glucose phenol red 0.02 g

加入双蒸水至1000毫升。 Was added to 1000 ml double distilled water. 将上列成分混合后溶化,8磅15min灭菌,4。 After mixing above ingredients melted, 8 lbs of sterilization 15min, 4. C冰箱保存。 Save C freezer. 临用时 Extemporaneous

用7. 4% NaHC03调pH至7. 3〜7. 6。 With 7. 4% NaHC03 adjusted to pH 7. 3~7. 6.

2%苔酚兰染色液: Moss 2% phenol blue staining solution:

苔酚兰2克生理盐水100毫升 Moss 2 g of phenol blue physiological saline 100 ml

2、方法: 2. Method:

肝素抗凝的人外周血缓慢沿管壁缓慢加于比重为1.077±0.001的聚蔗糖-泛影葡胺淋巴细胞分层液面上,分离液与外周血的比例约为2:1。 Human peripheral blood heparinized was slowly along the wall slowly added to a specific gravity of 1.077 ± 0.001 Ficoll - Hypaque on Ficoll level, and Peripheral separated liquid ratio of about 2: 1.

水平离心机离心1,000xg 15 — 20min,离心后管内分为3层,用吸管吸取白色云雾层狭窄带,置入另一管中。 Horizontal centrifuge 1,000xg 15 - 20min, after the inner tube is divided into three layers, with a narrow pipette with white clouds layer, into another tube.

加入等倍或以上体积的Hank's液(无血清),800—1,000 xg 10-15min,弃上清,加入Hank's液,洗涤细胞两次。 An equal volume of times or more Hank's solution (serum-free), 800-1,000 xg 10-15min, the supernatant discarded, Hank's solution was added, cells were washed twice.

末次离心后,弃上清,加入培养基2ml,重悬细胞。 After the last centrifugation, the supernatant was discarded, the medium was added 2ml, resuspend the cells.

取一滴细胞悬液与一滴0. 2%苔酚兰染液混合,于血球计数板计数四个大方格内的细胞总数,单个核细胞浓度(细胞数/ 1毫升细胞悬液) =4个大方格内细胞总数/4xl0、2(稀释倍数)。 Cells taken mixed liquor 0.2% phenol moss and blue suspension drop by drop, in a hemocytometer counting total number of cells in the four large squares, mononuclear cell concentration (cells / 1 ml cell suspension) = 4 the total number of cells in the large squares / 4xl0,2 (dilution factor). 三、K562细胞的标记试剂和材料: Three, K562 cells labeled reagents and materials:

细胞培养常用的仪器设备:低温冰箱、二氧化碳孵箱、超净工作台、 倒置显微镜、液氮罐、蒸馏水器、真空泵、细胞培养瓶、各种规格的吸管、加样器、滴管等。 Commonly used cell culture equipment: cryogenic refrigerator, carbon dioxide incubator, clean bench, inverted microscope, a liquid nitrogen tank, distilled water, a vacuum pump, cell culture flask, a variety pipette, pipette, dropper.

放射性同位素:镉51 (5lCr)(美国PerkinElmer公司) Y射线计数器(美国Beckman公司)、同位素标记管RPMI1640培养液: Radioisotope: Cd 51 (5lCr) (PerkinElmer, Inc. USA) Y-ray counter (Beckman, USA), RPMI1640 medium isotopically labeled tube:

含L一谷氨酰胺的RPMI1640(GIBC0BRL) 10. 4克碳酸氢钠2. 0克 10.4 g of sodium bicarbonate containing L-glutamine RPMI1640 (GIBC0BRL) 2. 0 g of

庆大霉素10万单位加三蒸水至体积iooo毫升 100,000 units of gentamicin plus three milliliters distilled water to a volume iooo

0. 22微米的滤膜真空泵抽滤除菌、分装。 0.22 micron filter sterilized vacuum filtration, packaging.

小牛血清灭活:小牛血清(Invitrogen)—2(TC取出,4匸冰箱融化后,56 Inactivated fetal bovine serum: calf serum (Invitrogen) -2 (TC removed Xi refrigerator melted after 4, 56

. C水浴30min。 C water bath for 30min. 10X小牛血清的RPMI1640: 10X calf serum RPMI1640:

灭活的小牛血清10毫升 Inactivated fetal bovine serum 10 ml

RPMI 1640培养液90毫升 RPMI 1640 culture medium 90 ml

方法: method:

1. 取生长良好的K562细胞,吸管吹打均匀后吸到IO毫升离心管中, 水平离心机以800 X g离心力离心5min,弃上清,加入1毫升培养基,吸管吹打均匀,取少量计数,调节细胞浓度为&107个/ 1. Take K562 cells grow well, even after pipetting sucked IO mL centrifuge tubes, centrifuged at 800 X g level centrifuged 5min, discarded the supernatant, added 1 ml of culture medium, evenly pipetting, counted small amount, cell concentration was adjusted to 107 & /

2. 吸取100微升(1><106个细胞)放入塑料管中,加入100微升放射性同位素镉51。 2. 100 microliters of suction (1> <106 cells) were placed in a plastic tube was added 100 microliters of 51 Cd radioisotope.

3. 放入37。 3. Add 37. C 5%二氧化碳孵箱中培养lh,每隔5 — 8min混匀一次。 C 5% carbon dioxide incubator lh, every 5 - 8min once mixed.

4. 用10%小牛血清的RPMI1640洗涤三次,然后10%小牛血清的RPMI1640重悬细胞。 4. washed three times with RPMI1640 with 10% fetal calf serum, 10% fetal calf serum and resuspended in RPMI1640 cells.

四、杀伤K562细胞的实验试剂和材料: Fourth, killing K562 cells of laboratory reagents and materials:

细胞培养常用的仪器设备:低温冰箱、二氧化碳孵箱、超净工作台、 倒置显微镜、液氮罐、蒸馏水器、真空泵、细胞培养瓶、各种规格的吸管、加样器、滴管等。 Commonly used cell culture equipment: cryogenic refrigerator, carbon dioxide incubator, clean bench, inverted microscope, a liquid nitrogen tank, distilled water, a vacuum pump, cell culture flask, a variety pipette, pipette, dropper. RPMI1640培养液: RPMI1640 medium:

含L一谷氨酰胺的RPMI1640(G工BC0BRL) 10. 4克 L-glutamine-containing RPMI1640 (G ENGINEERING BC0BRL) 10. 4 g of

庆大霉素10万单位 Gentamicin 100,000 units

加三蒸水至体积1000毫升0.22微米的滤膜真空泵抽滤除菌、分装。 To a volume of triple-distilled water was added 1,000 ml of a 0.22 micron vacuum filter filtration sterilization, packaging. 小牛血清灭活:小牛血清(Invitrogen)—2(TC取出,4。C冰箱融化后,56 。C水浴30min。10%小牛血清的RPMI1640: Inactivated fetal bovine serum: calf serum (Invitrogen) -2 (TC removed 4.C refrigerator after thawing, 56 .C 30min.10% calf serum in a water bath of RPMI1640:

灭活的小牛血清io毫升 Io ml of inactivated fetal bovine serum

RPMI 1640培养液90毫升 RPMI 1640 culture medium 90 ml

TE缓冲液:10毫摩尔Tris, 1毫摩尔EDTA,盐酸调节pH值7.0。 TE buffer: 10 mM Tris, 1 mmole EDTA, 7.0 hydrochloric acid to adjust the pH. l摩尔/升的盐酸:盐酸10毫升,加三蒸水至终体积IOO毫升 l mol / l hydrochloric acid: 10 ml of hydrochloric acid, distilled water was added to a final volume of three ml IOO

方法: method:

1. 用10X小牛血清的RPMI1640培养基调节人外周血单个核细胞至终浓度为2><106个/毫升,96孔板每孔加入IOO微升,即每孔细胞数为2xl()5个。 RPMI1640 medium 1. 10X calf serum regulation of human peripheral blood mononuclear cells to a final concentration of 2> <106 / ml, 96 [mu] l added to each well IOO, i.e. the number of cells per well 2xl () 5 a.

2. 加入用TE缓冲液稀释的CpG单链脱氧核苷酸至每孔终浓度120 微克/毫升,每种CpG单链脱氧核苷酸三个复孔,设一不加CpG 单链脱氧核苷酸的对照孔。 2. diluted with TE buffer CpG single-stranded oligodeoxynucleotides each well to a final concentration of 120 g / ml, CpG single-stranded oligodeoxynucleotides each triplicate wells, disposed without a single-stranded CpG deoxynucleoside control wells acid.

3. 设最大释放孔6复孔,最大释放组每孔加100微升1摩尔/升的盐酸,自发释放孔6复孔,每孔加100微升10%小牛血清的RPMI1640培养基,混匀。 3. Set the highest release hole 6 wells, maximal release group 1 mol l L HCl / 100 added to each well, spontaneous release hole 6 wells, 100 microliters per well of RPMI1640 medium containing 10% calf serum, mixed uniform.

4, 37°C 5%二氧化碳孵箱中培养至18h。 4, 37 ° C 5% carbon dioxide incubator to 18h. 取标记好的K562细胞, 用10%'小牛血清的RPMI1640培养基调节细胞浓度至lxlOS个/ The labeled K562 cells, cell concentration was adjusted to lxlOS with a 10% 'RPMI1640 medium calf serum /

5. 在96孔板中每孔加100微升,设最大释放组和自发释放组,37 。 5. 96-well plate was added 100 microliters per well, provided the maximum release spontaneous release group and the group 37. C 5%二氧化碳孵箱中培养他。 C 5% carbon dioxide incubator him.

6. 水平离心机1,000 Xg离心5min每孔取上清150微升,加入塑闪瓶中,再加3—5毫升液闪液,测量CPM值。 6. centrifuge for 5min centrifugation 1,000 Xg 150 microliters per well of the supernatant, added flash plastic bottle, together with 3-5 ml of liquid scintillation fluid, measured CPM value.

7. 计算杀伤率:杀伤率=(实验组CPM值一自发释放组CPM值) / (最大释放组CPM值一自发释放组CPM值)X100% 7. The killing rate is calculated: killing rate = (experimental release spontaneous CPM value set a CPM value) / (maximum release spontaneous release a set value set CPM CPM value) X100%

实验结果(见下表)表明,CpG ODN对人外周血单个核细胞杀伤K562 细胞用明显的剌激作用。 The results (see table below) shows that, CpG ODN on human peripheral blood mononuclear cells against K562 cells with significant stimulation effect.

第一组实验的结果:j1^一」一CPG俩瑤宗iL窈雄隳^甚^统樹(。/0) The results of the first set of experiments: j1 ^ a "a CPG both cases iL Yao-yao Hung destroy even ^ ^ system tree (./0)

301 5",T门gT门gTTA门门gATgA门g,r门g门门gT-3" 払2.1 301 5 ", T gT door door door door gATgA door gTTA g, r g door door door gT-3" partial payment 2.1

302 5 >-TCgTCgggTgCgACgTCgCAgggggg-3> 41.2 302 5> -TCgTCgggTgCgACgTCgCAgggggg-3> 41.2

303 5广TcgTcgggTgcgAcgATCgTcgggggg-4 46.8 3035 Canton TcgTcgggTgcgAcgATCgTcgggggg-4 46.8

304 5,TcgTcgTTTgcATCgATgcAgTcgTcgTT-y 35.7 304 5, TcgTcgTTTgcATCgATgcAgTcgTcgTT-y 35.7

305 5rTcgTcgTTTgcATCgATgcAggggggg-y 43.1 305 5rTcgTcgTTTgcATCgATgcAggggggg-y 43.1

306 5广AccggTATCgATgccggTgggggg-3。 3065 broad AccggTATCgATgccggTgggggg-3. 55.6 55.6

307 5广ggggTCCATgAcgTTCCTgAAgggggg】 27.8 3075] 27.8 wide ggggTCCATgAcgTTCCTgAAgggggg

308 5"-TcgTcgTTTTgAcgATCgTcgggggg一35.8 308 5 "-TcgTcgTTTTgAcgATCgTcgggggg a 35.8

309 ^-TTCgTcgTTTgATCgATgTTCgTTgggggg一33.4 309 ^ -TTCgTcgTTTgATCgATgTTCgTTgggggg a 33.4

310 5广TTCgTcgTTgTgATCgATgggggg-r 48.1 3105 broad TTCgTcgTTgTgATCgATgggggg-r 48.1

311 5ZTATCgATgTTTTCgTcgTcgTTgggggg-y 46.0 311 5ZTATCgATgTTTTCgTcgTcgTTgggggg-y 46.0

312 5》-TTCgTTgcATCgATgcATCgTTgggggg-3> 2S.2 312 5 "-TTCgTTgcATCgATgcATCgTTgggggg-3> 2S.2

313 5〕-TT门g门TT门g门TTTT门g门TT门g门TT-3y 27.8 Gate 313 5 TT TT] -TT door door door g g g door TTTT door door door TT-3y 27.8 g

^当, CPG4潔宗掷荡难隳都^ ,密栅(%) When ^, CPG4 were clean swing throw are difficult to destroy ^, dense grid (%)

601 ^-T门gAggACAAgATT门TcgTgc-3" 60.1 601 ^ -T door gAggACAAgATT door TcgTgc-3 "60.1

602 5广TcgAggACAAgATTCTCgTgcAggcc-3" 21.7 6025 Canton TcgAggACAAgATTCTCgTgcAggcc-3 "21.7

603 5:TcgTgcAggccAACgAgg。 603 5: TcgTgcAggccAACgAgg. cg-r 77 cg-r 77

604 「-AccgccAAggAgAAgccgcAggAggg-3" 81 604 "-AccgccAAggAgAAgccgcAggAggg-3" 81

605 5广TcgTTgccgTcggccc-:T 103 6055 Canton TcgTTgccgTcggccc-: T 103

606 5"-TACAACggcgAggAATACC-3" 78 606 5 "-TACAACggcgAggAATACC-3" 78

607 5,T门gg门A门g门gA门gTg门Tgg门门gT门gTTT门门-3^ 91 607 5, T gate door A door g gg door door Tgg gTg gA Gate Gate Gate Gate Gate gTTT gT door 91 ^ -3

608 5u-gTACAACggcgAggAATACCT-3" 99 608 5u-gTACAACggcgAggAATACCT-3 "99

609 5:A门门gT门gTTg门门gT门gg门门门-:r 88<formula>formula see original document page 18</formula><table>table see original document page 19</column></row> <table><table>table see original document page 20</column></row> <table> 609 5: A door door gT door gTTg Gate Gate gT door gg Gate Gate Gate -: r 88 <formula> formula see original document page 18 </ formula> <table> table see original document page 19 </ column> </ row > <table> <table> table see original document page 20 </ column> </ row> <table>

实施例3 CpG ODN刺激产生人干扰素(HIFNa) Example 3 CpG ODN stimulate the production of interferon (HIFNa)

本实验采用PBL Biomedical Laboratories检测人IFNcd式剂盒(批号: 1755)进行。 This experiment detecting human PBL Biomedical Laboratories IFNcd formula cartridge (lot: 1755) performed.

一.人外周血单个核细胞的分离1、试剂和材料: A human peripheral blood separation 1, the reagents and materials mononuclear cells:

肝素抗凝的人全血:长春市中心血站。 Heparinized human whole blood: Changchun city blood bank.

聚蔗糖-泛影葡胺:比重1.077士0.001,北京鼎国生物技术有限公司。 Ficoll - hypaque: specific gravity 1.077 0.001 disabilities, Beijing Ding States Biological Technology Co., Ltd. 细胞培养常用的仪器设备:低温冰箱、二氧化碳孵箱、超净工作台、倒置显微镜、液氮罐、蒸馏水器、真空泵、细胞培养瓶、滤菌器、 滤过瓶、各种规格的吸管、加样器、滴管、血球计数板、水平式离心机等。 Commonly used cell culture equipment: cryogenic refrigerator, carbon dioxide incubator, clean bench, inverted microscope, a liquid nitrogen tank, distilled water, a vacuum pump, cell culture flask, a bacteria filter, filtration bottles, a variety of straw, plus sampler, a dropper, hemacytometer, centrifuge horizontal.

RPMI1640培养液: RPMI1640 medium:

含L一谷氨酰胺的RPMI1640(GIBC0BRL) 10. 4克碳酸氢钠2. 0克 10.4 g of sodium bicarbonate containing L-glutamine RPMI1640 (GIBC0BRL) 2. 0 g of

庆大霉素10万单位加三蒸水至体积1000毫升0.22微米的滤膜真空泵抽滤除菌、分装。 Gentamicin plus three units of 100,000 to a volume of 1,000 ml distilled water, 0.22 micron filter sterilized vacuum filtration, packaging.

小牛血清灭活:小牛血清(Irwitrogen)—2(TC取出,4。C冰箱融化后,56 Inactivated fetal bovine serum: calf serum (Irwitrogen) -2 (TC removed, 4.C refrigerator after thawing, 56

. C水浴30min。 C water bath for 30min.

10%小牛血清的RPMI1640: 10% calf serum RPMI1640:

灭活的小牛血清10毫升 Inactivated fetal bovine serum 10 ml

RPMI 1640培养液90毫升 RPMI 1640 culture medium 90 ml

Hank's液(无钙离子、镁离子)的配制: Hank's solution (without calcium ions, magnesium ions) formulation:

氯化钠8. 0克 Sodium chloride 8.0 g

氯化钾0.4克磷酸氢二钠(带一个结晶水) 0.06克 Potassium chloride 0.4 g disodium hydrogen phosphate (with a crystal water) 0.06 g

磷酸二氢钾0. 06克 0.06 g of potassium dihydrogen phosphate

碳酸氢钠0. 35克 0.35 g of sodium bicarbonate

葡萄糖lO克 LO grams of glucose

酚红0. 02克加入双蒸水至1000毫升。 0.02 g phenol red were added to 1000 ml double distilled water.

将上列成分混合后溶化,8磅15min灭菌,4。 After mixing above ingredients melted, 8 lbs of sterilization 15min, 4. C冰箱保存。 Save C freezer. 临用吋用7. 4% NaHC03调pH至7. 3〜7. 6。 Inch clinical use with 7. 4% NaHC03 adjusted to pH 7. 3~7. 6. 2%苔酚兰染色液: Moss 2% phenol blue staining solution:

苔酚兰2克生理盐水100毫升 Moss 2 g of phenol blue physiological saline 100 ml

2、方法: 2. Method:

肝素抗凝的人外周血缓慢沿管壁缓慢加于比重为1.077士0.001的聚蔗糖-泛影葡胺淋巴细胞分层液面上,分离液与外周血的比例约为2:1。 Human peripheral blood heparinized was slowly along the wall slowly added to a specific gravity of 0.001 to 1.077 persons Ficoll - Hypaque on Ficoll level, and Peripheral separated liquid ratio of about 2: 1.

水平离心机离心1,000 xg 15 —20min,离心后管内分为3层,用吸管吸取白色云雾层狭窄带,置入另一管中。 Horizontal centrifuge 1,000 xg 15 -20min, the inner tube is divided into three layers after the centrifuge, a white cloud Pipet layer narrow band, placed in another tube.

加入等倍或以上体积的Hank's液(无血清),800—1,000 xg 10-15min,弃上清,加入Hank's液,洗涤细胞两次。 An equal volume of times or more Hank's solution (serum-free), 800-1,000 xg 10-15min, the supernatant discarded, Hank's solution was added, cells were washed twice.

末次离心后,弃上清,加入培养基2ml,重悬细胞。 After the last centrifugation, the supernatant was discarded, the medium was added 2ml, resuspend the cells.

取一滴细胞悬液与一滴0. 2%苔酚兰染液混合,于血球计数板计数四个大方格内的细胞总数,单个核细胞浓度(细胞数/ 1毫升细胞悬液) A drop of cell suspension was mixed dye with one drop of 0.2% of phenol blue moss, counted in a hemocytometer large total number of cells in four squares, mononuclear cell concentration (cells / 1 ml cell suspension)

二4个大方格内细胞总数/4xl0、2(稀释倍数)。 The total number of cells in 4 large squares two / 4xl0,2 (dilution factor). 人外周血单个核细胞的 Human peripheral blood mononuclear cells

分离 Separate

二人干扰素(HIFNot)的检测 Two interferon (HIFNot) detecting

试剂和材料: Reagents and materials:

细胞培养常用的仪器设备:低温冰箱、二氧化碳孵箱、超净工作台、 倒置显微镜、液氮罐、蒸馏水器、真空泵、细胞培养瓶、各种规格的吸管、加样器、滴管等。 Commonly used cell culture equipment: cryogenic refrigerator, carbon dioxide incubator, clean bench, inverted microscope, a liquid nitrogen tank, distilled water, a vacuum pump, cell culture flask, a variety pipette, pipette, dropper.

RPMI1640培养液: RPMI1640 medium:

含L一谷氨酰胺的RPMI1640(GIBCOBRL) 10. 4克 L-glutamine-containing RPMI1640 (GIBCOBRL) 10. 4 g of

碳酸氢钠2. 0克 2.0 g of sodium bicarbonate

庆大霉素10万单位 Gentamicin 100,000 units

加三蒸水至体积1000毫升 Was added to a volume of triple-distilled water 1000 ml

0.22微米的滤膜真空泵抽滤除菌、分装。 0.22 micron filter sterilized vacuum filtration, packaging. 小牛血清灭活:小牛血清(Invitrogen)—20。 Inactivated fetal bovine serum: calf serum (Invitrogen) -20. C取出,4。 Remove C, 4. C冰箱融化后,56 C refrigerator after thawing, 56

. C水浴30min。 C water bath for 30min. 10%小牛血清的RPMI1640: 10% calf serum RPMI1640:

灭活的小牛血清10毫升 Inactivated fetal bovine serum 10 ml

RPMI 1640培养液90毫升 RPMI 1640 culture medium 90 ml

24孔板(平底)(Costar)。 24-well plate (flat bottom) (Costar). 方法: method:

1. 用10%小牛血清的RPMI1640培养基调节人外周血单个核细胞至终浓度为2xlos个/毫升。 1. RPMI1640 medium with 10% calf serum regulation of human peripheral blood mononuclear cells to a final concentration 2xlos / ml.

2. 24孔板每孔加入1000微升,即每孔细胞数为2xlos个。 2.24 1000 microliters added to each well plates, i.e. the number of cells per well of a 2xlos. 加入用TE 缓冲液稀释的CpG单链脱氧核苷酸至每孔终浓度6微克/毫升。 Nucleotide with TE buffer was added diluent single-stranded CpG-deoxy-6 to each well to a final concentration g / ml.

3. 37°C 5%二氧化碳孵箱中培养至24小时,收上清200微升。 3. 37 ° C 5% carbon dioxide incubator to 24 hours, received 200 microliters of supernatant.

4. 继续培养24小时,收上清200微升。 4. continued for 24 hours, 200 microliters of supernatant was closed.

5. 采用PBL Biomedical Laboratories检测人IFNcd式剂盒(批号:1755) 进行IFNa的检测。 The use of human PBL Biomedical Laboratories detection kit IFNcd formula (lot: 1755) for the detection of IFNa.

HIFNa的检测试剂准备:A瓶:浓縮洗液(50ml) HIFNa detection reagent preparation: A bottle: Wash Concentrate (50ml)

B (小)瓶:HIFNa标准品(1.5ml) B (small) bottles: HIFNa standard (1.5ml)

C瓶:稀释缓冲液(50 ml) Bottle C: dilution buffer (50 ml)

D (小)瓶:抗体浓缩液(0.25 ml) D (small) bottles: antibody concentrate (0.25 ml)

E (小)瓶:辣根过氧化物酶试剂(HRP)浓縮液(20ul) F瓶:辣根过氧化物酶试剂(HRP)稀释液(25 ml) G瓶:辣根过氧化物酶底物反应液(TMB) (25 ml) H瓶:终止液(25 ml) E (small) bottles: horseradish peroxidase reagent (HRP) concentrate (20ul) F Bottle: horseradish peroxidase reagent (HRP) diluted solution (25 ml) G Bottle: horseradish peroxidase The reaction substrate solution (TMB) (25 ml) H bottle: stop solution (25 ml)

步骤: step:

(1) 配制稀释洗液(工作液) (1) Preparation of diluted wash solution (working solution)

A (瓶)液50 ml A (bottle) 50 ml solution

双蒸水加到1000 ml即成工作洗液 Was added to 1000 ml distilled water washes work Serve

此工作洗液配制后应存放与4"C冰箱中,用前须充分混匀。冲洗步骤均应在室温(24°C)下用冷洗液(2-6°C)进行冲洗。 After this job lotion formulation should be stored and 4 "C in a refrigerator, to be mixed thoroughly before use. Rinsing step should be rinsed with cold wash solution (2-6 ° C) at room temperature (24 ° C).

(2) 绘制标准曲线 (2) standard curve

系列稀释HIFNa标准品:HIFNa标准品(小瓶B, 1.5ml)加稀释缓冲液(C瓶),使之配成0-500 pg/ ml (高敏度)或0-5000 pg/ ml (广范围)系列稀释液。 Standard serial dilutions HIFNa: HIFNa Standard (vial B, 1.5ml) and diluted with buffer (C bottle), so dubbed 0-500 pg / ml (high sensitivity) or 0-5000 pg / ml (wide range) serial dilutions. 所提供的样品曲线谨供参考。 Samples curve is provided for reference.

方法:标记7个聚丙烯小管(分别为S6-Si以及BK),所有稀释步骤均在其中进行,枪头须每次更换。 Method: 7 labeled polypropylene vials (S6-Si, respectively, and BK), all the steps were diluted therein, each pipette tip to be replaced. 干扰素浓度待测的样品亦须用同样方法稀释。 Interferon concentration of the sample shall be tested is diluted in the same way.

(3) 设计并确定96孔微量板上测试样品所需孔数:建议选16个孔用于HIFNa标准品。 (3) 96-well microplate design is performed and the test sample plates required number of wells: 16 holes for the proposed election HIFNa standards. 其中空白对照(BK,只加缓冲液BK) 4个孔,HIFNa 标准品(S6-S。 12个孔(各二复孔)。另选16个孔用于干扰素浓度待测的样品。去掉多余的微量板条块,封于锡箔袋内置冰箱(2-6°C)待用。 Wherein the blank (BK, only buffer was added BK) 4 holes, HIFNa standard (S6-S. 12 wells (two wells each). Alternatively, 16-well sample to be tested for the concentration of interferon in removing the excess microplate strips, sealing the foil pouch built in refrigerator (2-6 ° C) until use.

(4) 精确汲取100ul步骤(2)中配制的HIFNct标准品稀释液及待测的 (4) accurate 100ul drawn in step (2) prepared in HIFNct standard dilution and tested

样品稀释液,加入到选定的微量板各复孔中(包括标准品和待测的样 Each sample dilution in duplicate, is added to the selected microplates (including standards and test samples

口n入 The port n

(5) 加盖,室温(24°C)下孵育lh.避免干燥及其他温度的影响。 (5) capped and incubated lh at room temperature (24 ° C). Avoid drying temperatures and other influence. (6) 此间,可配制抗体溶液,以便在步骤(8)中使用。 (6) During this period, the antibody solution can be formulated for use in step (8). 为了避免丢失, 可先将抗体浓縮液(小瓶D)离心几秒钟,使液体集中于瓶底。 To avoid losing, the antibody may be first concentrated solution (vial D) centrifugation for several seconds, the liquid concentrated in the bottom of the bottle.

方法: method:

(高敏感度)抗体浓縮液(小瓶D) 6.7 iil 或(广范围)抗体浓縮液(小瓶D) 2.2 ul 加稀释缓冲液(C瓶) 1 ml 此量为单排孔用量。 (High sensitivity) antibody concentrate (vial D) 6.7 iil or (wide range) antibody concentrate (vial D) 2.2 ul diluted with buffer (C bottle) 1 ml This amount is the amount of a single row of holes. 复排用量倍增。 Complex multiplying the amount of discharge.

(7) 孵育后,去掉孔内液体,用步骤(1)中配制的工作洗液冲洗每个孔一次。 (7) After incubation, removing the liquid hole, the step (1) prepared in Working Rinse once each well. 冲洗时每孔应充满。 Each hole should be filled with flushing. 最好使用自动冲洗器(如Nunc Immunowasher),尽量不用手工操纵的移液器冲洗。 Preferably using an automatic washer (e.g., Nunc Immunowasher), do not try to manually operated pipette rinse. 冲洗后将微量板翻转,置于脱纤维吸水纸上,轻轻叩打,令其干燥。 After rinsing inverted microplate, placed on absorbent paper defibering, tapping gently, and allowed to dry. 样品中如含有害物质, 须采取防护措施。 Samples such as those containing hazardous substances, must take protective measures.

(8) 冲洗后,每孔加100p1步骤(6)中所配制的抗体溶液,加盖, 室温下孵育lh.。 (8) rinse, add 100p1 bore step (6) in each of the formulated antibody solution, capped, LH incubated at room temperature ..

(9)在此孵育期间,配制浓縮的服P溶液,以便下一步步骤(10)使用。 Period (9) In this incubation, a concentrated formulation P was served to the next step (10) used. 为了避免丢失,可先将小瓶E离心几秒钟,使液体集中于瓶底。 To avoid losing, centrifugation vial E can first few seconds, the liquid concentrated in the bottom of the bottle. 具体配制方法如下: Specific preparation methods are as follows:

服P原液(小瓶E) 20^1 加入服P稀释液(F瓶)140 y 1 轻轻混匀,必要时可再次离心。 P serving stock solution (vial E) 20 ^ 1 P diluent added service (F bottle) 140 y 1 mix gently, if necessary, can be centrifuged again. 此即浓縮的HRP溶液。 Namely HRP solution was concentrated.

(10)配制HRP稀释液: 取步骤(9 )中所配制的浓縮的HRP溶液1 li 1 (10) Preparation of HRP dilution: Take the step (9) in the formulated solution was concentrated HRP 1 li 1

(如为高敏)加入(F瓶)HRP稀释液1 ml即成(高敏)稀释液 (Such as high sensitivity) was added (F bottle) 1 ml dilution of the HRP Serve (high sensitivity) dilution

(如为广范围)则需将HRP浓縮液进一步稀释。 (Such as a wide range) need further diluted HRP concentrate. 将经步骤(9 )稀释的HRP溶液存放于一7 0 r待用,未经稀释的HRP 浓縮液则放在4"C冰箱中。 By the step (9) was diluted HRP stored in a stand 7 0 r, HRP undiluted concentrate on the 4 "C in a refrigerator.

(11) 孵育后,去掉微量板孔内液体,用步骤(1)中配制的工作洗液冲洗, 每个孔洗3次,同步骤(7)。 (11) After incubation, plate wells remove all traces of liquid, the step (1) prepared in working Rinse, each well was washed three times with step (7). 洗后,将微量板翻转,置于脱纤维吸水纸上,轻轻叩打,令其干燥。 After washing the microplate inverted and placed on absorbent paper defibering, tapping gently, and allowed to dry. 样品中如含有害物质,须采取防护措施。 Samples such as those containing hazardous substances, must take protective measures.

(12) 每孔加入稀释的HRP溶液100y 1,封盖,室温孵育lh。 (12) added to each well diluted HRP solution 100y 1, the closure and incubated lh room temperature. (13)室温下(24°C)预温TMB底物溶液(G瓶)。 (13) at room temperature (24 ° C) of pre-warmed TMB substrate solution (G bottle).

(14) 孵育后去掉微量板孔内液体,用步骤(1)中配制的工作洗液冲洗,每个孔洗4次,同步骤(7),去掉微量板孔内液体,用步骤(1)中配制的工作洗液冲洗每个孔三次,同步骤(7)。 (14) is removed after incubation hole microplate liquid, the step (1) prepared in working Rinse, each well was washed 4 times with step (7), remove the liquid hole microplate, the step (1) formulated to work rinse each hole three times, the same step (7). 洗后,将微量板翻转, 置于脱纤维吸水纸上,轻轻叩打,令其干燥。 After washing the microplate inverted and placed on absorbent paper defibering, tapping gently, and allowed to dry. 样品中如含有害物质,须采取防护措施。 Samples such as those containing hazardous substances, must take protective measures.

(15) 每孔加TMB底物溶液(G瓶)100 u 1,封盖,室温暗处孵育15min.。 (15) was added per well TMB substrate solution (G bottle) 100 u 1, cover, incubated for 15min at room temperature in the dark .. (16)孵育后,每孔加终止液(H瓶)lOOul,涡旋或叩打轻轻, (16) After the incubation, each well was added Stop Solution (H bottle) lOOul, gently vortexing or percussion,

令其混匀。 Make it mix.

(17) 酶标仪于450nm处5min内测出吸收值。 (17) in a microplate reader at 450nm 5min absorbance was measured.

(18) 绘出标准曲线。 (18) draw the standard curve. 待测样品中HIFNa的滴度可从标准曲线上求出。 HIFNa titer test sample can be determined from the standard curve. 所提供的标准曲线可供参考。 The standard curve provided for reference.

由于样品中干扰素的滴度是根据标准测得的,因此其单位可以是units/ml或pg/ml,转换系数为3 — 5pg/unit。 Since the sample interferon titer is measured according to the standard, therefore the unit can be units / ml or pg / ml, the conversion factor of 3 - 5pg / unit. 这个转换系数只是一 This is just a conversion factor

个近似值。 An approximation.

三、结果见下表: Third, the results are as follows:

CpG 24h 48h CpG 24h 48h

OD450 a干扰素(pg) OD450 a干扰素(pg) OD450 a interferon (pg) OD450 a interferon (PG)

660 0. 419 214. 82 0. 4335 223. 3 660 0.419 214.82 223.3 0.4335

661 0. 207 90.795 0. 195 83.775 661 0.207 90.795 0.195 83.775

662 0. 212 93. 72 0. 276 131.16 662 93.72 0.212 0.276 131.16

663 0. 159 62.715 0. 117 38.145 663 0.159 0.117 38.145 62.715

664 0. 883 486. 26 0. 899 495. 62 664 0.883 486.26 0.899 495.62

665 0. 2465 113. 9 0. 1675 67.688 113.9 665 0.2465 67.688 0.1675

666 0. 0705 10.943 0, 176 72. 66 0.0705 10.943 666 0 176 72.66

667 0. 831 455. 84 0. 7505 408.74 667 0.831 455.84 408.74 0.7505

668 0. 4375 225.64 0. 3865 195.8 668 0.4375 225.64 0.3865 195.8

669 0. 19 80. 85 0. 18 75670 0. 227 102. 5 0. 2155 95.768 669 0.19 80.85 0.18 75670 0.227 0.2155 95.768 102.5

671 0. 802 438.87 0. 789 431.27 671 0.802 438.87 0.789 431.27

672 0. 5065 266 0. 475 247. 58 672 0.5065 266 0.475 247.58

673 0. 3655 183.52 0. 3595 180.01 673 0.3655 183.52 0.3595 180.01

674 0. 233 106.01 0. 23 104.25 674 0.233 106.01 0.23 104.25

675 0. 471 245.24 0. 5335 281,8 675 0.471 245.24 0.5335 281,8

302 1. 3785 776. 12 1. 227 687.5 302 1.3785 776.12 1.227 687.5

647 0. 074 12. 99 0. 08 16. 5 647 0.074 0.08 12.99 16.5

TE 0. 072 11.82 0. 078 15. 33 TE 0. 072 11.82 0. 078 15. 33

注: Note:

CpG纵列:CpGODN的编号,其序列如前文表所示。 CpG column: CpGODN number, as previously shown the sequence table.

24h OD450纵列:代表用CpG纵列所示的CpG ODN刺激人外周血单 24h OD450 columns: columns representative of CpG ODN with CpG stimulate human peripheral blood mononuclear shown

个核细胞24h收获上清中ct干扰素测定实验中测得的光密度值(波长为 Ct interferon measured optical density measured in experiments in nuclear cell supernatants were harvested 24h (wavelength

450nm)。 450nm).

24h oc干扰素(pg)纵列:代表用CpG纵列所示的CpG ODN刺激人外周血单个核细胞24h收获上清中ot干扰素的含量。 24h OC interferon (PG) column: column representative of CpG ODN with CpG stimulated human peripheral blood content shown supernatants were harvested 24h mononuclear cells ot interferon.

48h OD450纵列:代表用CpG纵列所示的CpG ODN刺激人外周血单个核细胞48h收获上清中a干扰素测定实验中测得的光密度值(波长为450nm)。 Column of OD450 48h: Representative CpG ODN with CpG column shown stimulate peripheral blood mononuclear cells supernatants were harvested 48h interferon a measured optical density values ​​measured in the experiment (wavelength of 450nm).

48h a干扰素(pg)纵列:代表用CpG纵列所示的CpG ODN刺激人外周血单个核细胞48h小时收获上清中a干扰素的含量。 48h a interferon (PG) column: column shown represent the CpG ODN with CpG stimulate human peripheral blood mononuclear cells were harvested 48h h interferon content of a supernatant. SEQUENCE LISTING <110>长春华普生物技术有限公司<120>抗病毒和抗肿瘤的含CpG单链脱氧寡核苷酸 SEQUENCE LISTING <110> Changchun Maple Biotechnology Ltd. <120> anti-viral and anti-tumor CpG-containing single-stranded oligodeoxynucleotides

<130> 1030021 <130> 1030021

<160〉 107 <160> 107

<170〉 Patentln version 3.1 <170> Patentln version 3.1

<210〉 1 〈211〉 20 <212〉 DNA<213〉 Artificial <210> 1 <211> 20 <212> DNA <213> Artificial

<400> 1 <400> 1

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tcgtcgtacg caattgtctt 20 tcgtcgtacg caattgtctt 20

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tcgcctcgtc gccttcgagc 20 tcgcctcgtc gccttcgagc 20

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〈400〉 11 <400> 11

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〈210〉 17 <210> 17

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ggggtccatg 3Cgttcctga agggggg ggggtccatg 3Cgttcctga agggggg

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〈211〉 26 <211> 26

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<211> 30 <211> 30

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〈210〉 26 <210> 26

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accgccaagg agaagccgca ggaggg accgccaagg agaagccgca ggaggg

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accgtcgttg ccgtcggccc accgtcgttg ccgtcggccc

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<210> 35 <210> 35

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<210> 36 <210> 36

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<400〉 36 <400> 36

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<210> 37 <210> 37

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说明书第42/75页 Instructions Page 42/75

20 20

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18<210> 40 18 <210> 40

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<2i0> 41 <2i0> 41

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〈400〉 41 tcgttgccgt egg <400> 41 tcgttgccgt egg

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<400> 42 tcgttgccgt cggg <400> 42 tcgttgccgt cggg

<210> 43 <210> 43

<211> 15 <211> 15

〈212〉 DNA <212> DNA

<213> Artificial <213> Artificial

<400〉 43 <400> 43

tcgttgccgt cgggg 15 tcgttgccgt cgggg 15

<210> 44 〈211〉 16<212〉 DNA <210> 44 <211> 16 <212> DNA

<213> Artificial <213> Artificial

<400> 44 tcgttgccgt cggggg <400> 44 tcgttgccgt cggggg

〈210〉 45 <210> 45

<211> 17 <211> 17

<212> DNA <212> DNA

<213> Artificial <213> Artificial

<400> 45 <400> 45

tcgUgccgt cgggggg tcgUgccgt cgggggg

<210> 46 <211> 18 <212> DNA<formula>formula see original document page 49</formula><400> 48 <210> 46 <211> 18 <212> DNA <formula> formula see original document page 49 </ formula> <400> 48

tcgttgccgt cggggggggg tcgttgccgt cggggggggg

〈210〉 49 <210> 49

<211〉 19 <211> 19

<212> DNA <212> DNA

〈213〉 Artificial <213> Artificial

<400> 49 <400> 49

tcgaggacaa gattctcgt tcgaggacaa gattctcgt

<210〉 50 <210> 50

<211〉 14 <211> 14

〈212〉 DNA <212> DNA

<213〉 Artificial <213> Artificial

20 20

19〈400〉 50 19 <400> 50

tcccgctgga cgtt 14 tcccgctgga cgtt 14

<210> 51 <210> 51

<211> 27 <211> 27

<212> 腿 <212> leg

<213〉 Artificial <213> Artificial

<400> 51 <400> 51

tcggcacgcg acgtgctggc cgtcgtt 27 tcggcacgcg acgtgctggc cgtcgtt 27

<210> 52 <210> 52

<211> 21 <211> 21

<212> DM <212> DM

<213〉 Artificial <213> Artificial

■> 52 ■> 52

tcgtcgcgcc gtcacggggg g 21〈210> 53 tcgtcgcgcc gtcacggggg g 21 <210> 53

<211> 19 <211> 19

<212> 腿 <212> leg

<213> Artificial <213> Artificial

<400〉 53 <400> 53

tcgtgtgcgt gccgttggg tcgtgtgcgt gccgttggg

<2i0〉 54 <2i0> 54

<211〉 18 <211> 18

〈212〉 DNA <212> DNA

<213〉 Artificial <213> Artificial

<400〉 54 <400> 54

tcgtcgccgt tgggcggg<formula>formula see original document page 53</formula><211〉 23 tcgtcgccgt tgggcggg <formula> formula see original document page 53 </ formula> <211> 23

<212> DNA <212> DNA

〈213> Artificial <213> Artificial

〈400〉 57 <400> 57

ttaccggtta acgttggccg gcc 23 ttaccggtta acgttggccg gcc 23

<210〉 58 <21】〉23 <210> 58 <21]> 23

<212> DNA <212> DNA

<213> Artificial <213> Artificial

〈400> 58 <400> 58

accggttaac gttgtccccg ggg 23 accggttaac gttgtccccg ggg 23

<210> 59 <211> 16<212> DNA <210> 59 <211> 16 <212> DNA

〈213〉 Artificial <213> Artificial

<400> 59 tcgtcgttgg tatgtt <400> 59 tcgtcgttgg tatgtt

<210> 60 <210> 60

<211〉 20 <211> 20

<212> DNA <212> DNA

<213〉 Artificial <213> Artificial

〈400〉 60 <400> 60

tcgtcgtcgt cgttgtcgU tcgtcgtcgt cgttgtcgU

<210> 6] <210> 6]

<211> 24 <211> 24

<212〉 腿 <212> leg

16 16

20<213> Artificial 20 <213> Artificial

<400> 61 <400> 61

tcgtcgtcgt cgUgtcgtt gggg tcgtcgtcgt cgUgtcgtt gggg

〈210〉 62 <210> 62

<2H〉 15 <2H> 15

<212> DNA <212> DNA

<213> Artificial <213> Artificial

62 62

tcgttcgggg tgccg tcgttcgggg tgccg

〈210> 63 <210> 63

<211> 18 <211> 18

<212> 腿 <212> leg

<213〉 Artificial <213> Artificial

24 twenty four

15<formula>formula see original document page 57</formula><400> 65 15 <formula> formula see original document page 57 </ formula> <400> 65

tcgttcgggg taccgat tcgttcgggg taccgat

<210〉 66 <210> 66

<211> 21 <211> 21

<212〉 脆 <212> brittle

<213> Artificial <213> Artificial

<400> 66 <400> 66

tcgttcgggg taccgatggg g tcgttcgggg taccgatggg g

<210> 67 <210> 67

<211> 24 <211> 24

DNA DNA

<213> Artificial <213> Artificial

<400> 67 <400> 67

tcgttgcgct cccatgccgg gggg tcgttgcgct cccatgccgg gggg

17 17

21 twenty one

24〈210〉 68 24 <210> 68

<211> 20 <211> 20

〈212> 舰 <212> Ship

〈213〉 Artificial <213> Artificial

<400> 68 <400> 68

tcgtcgtUc gtcgttgggg tcgtcgtUc gtcgttgggg

<210> 69 <210> 69

<2il> 27 <2il> 27

<212> DNA <212> DNA

<213> Artificial <213> Artificial

<400> 69 <400> 69

tcgttgtcgt Ucgctgccg gcgg腦 tcgttgtcgt Ucgctgccg gcgg brain

20 20

27〈210> 70 27 <210> 70

<211> 24 <211> 24

<212> DNA <212> DNA

<213〉 Artificial <213> Artificial

<400> 70 <400> 70

cgttgacgat cgtxccatgg cggg cgttgacgat cgtxccatgg cggg

<210〉 71 <210> 71

<211> 16 <211> 16

<212〉 DNA <212> DNA

<213〉 Artificial <213> Artificial

〈400〉 71 tctgcggcct tcgtcg <400> 71 tctgcggcct tcgtcg

<210> 72<211> 22 <210> 72 <211> 22

〈212〉腿 <212> leg

<2]3> Artificial <2] 3> Artificial

〈400〉 72 . tagtaaccgg tccggcgccc cc 22 <400> 72. Tagtaaccgg tccggcgccc cc 22

〈210> 73 <211>〗9 <212>腿 <210> 73 <211> 9〗 <212> leg

<213〉 Artificial <213> Artificial

<400〉 73 <400> 73

ttgcagcgct gccggtggg 19 ttgcagcgct gccggtggg 19

<210〉 74 <211〉 23<212>腿 <210> 74 <211> 23 <212> leg

〈213〉 Artificial <213> Artificial

<400> 74 <400> 74

tcgtacggcc gccgtacggc ggg tcgtacggcc gccgtacggc ggg

<210> 75 <210> 75

<211> 21 <211> 21

<212> DNA <212> DNA

<213〉 Artificial <213> Artificial

<400> 75 <400> 75

cggcccatcg agggcgacgg c cggcccatcg agggcgacgg c

<210> 76 <210> 76

<211> 23 <211> 23

<212> 脆 <212> brittle

23 twenty three

2i<213> Artificial 2i <213> Artificial

<400> 76 <400> 76

tcgcgtcgac tcccctcgag ggg 23 tcgcgtcgac tcccctcgag ggg 23

〈210〉 77 <210> 77

<211> 24 <211> 24

<212> DNA <212> DNA

〈213〉 Artificial <213> Artificial

〈400> 77 <400> 77

tcgtcgtcga ctcgtggtcg gggg 24 tcgtcgtcga ctcgtggtcg gggg 24

<210〉 78 <210> 78

<211〉 20 <211> 20

<212〉 DNA <212> DNA

<2〗3> Artificial〈400〉 78 <2〗 3> Artificial <400> 78

tcgggcgccc gatcgggggg tcgggcgccc gatcgggggg

<210> 79 <210> 79

〈211〉 ]9 <211>] 9

<212> 廳 <212> Hall

<213> Artificial <213> Artificial

〈400〉 79 <400> 79

tcgtcggtct ttcgaaatt tcgtcggtct ttcgaaatt

〈210> 80 <210> 80

<211> 18 <211> 18

〈212〉 DNA <212> DNA

<213〉 Artificial <213> Artificial

20 20

19<400> 80 19 <400> 80

tcgtgacgtc ctcgagtt tcgtgacgtc ctcgagtt

<210> 81 <210> 81

<211> 16 <211> 16

<212> DNA <212> DNA

<213> Artificial <213> Artificial

<400> 81 tcgttgccgt cggccc <400> 81 tcgttgccgt cggccc

<210> 82 <210> 82

<211> 20 <211> 20

<212> DNA <212> DNA

<213> Artificial <213> Artificial

<• 82 <• 82

ggacgatcga tcgtgggggg ggacgatcga tcgtgggggg

18 18

16 16

20〈210〉 83 20 <210> 83

<211> 24 <211> 24

<212> DNA <212> DNA

〈213〉 Artificial <213> Artificial

<400> 83 <400> 83

gggatgcatc gatgcat.cgg gggg gggatgcatc gatgcat.cgg gggg

<210> 84 <210> 84

〈211〉 20 <211> 20

<212〉 DNA <212> DNA

<213〉 Artificial <213> Artificial

<400> 84 <400> 84

gggggaatcg attcgggggg gggggaatcg attcgggggg

24 twenty four

20〈210〉 85 20 <210> 85

〈211> 20 <211> 20

<212> DNA <212> DNA

<213> Artificial <213> Artificial

<400> 85 <400> 85

ggtgcgacgt cgcagggggg ggtgcgacgt cgcagggggg

<210> 86 <210> 86

<211〉 26 <211> 26

<212〉 腿 <212> leg

〈213〉 Artificial <213> Artificial

<400> 86 <400> 86

tcgtxgggtg catcgatgca gggggg tcgtxgggtg catcgatgca gggggg

20 20

26 26

<210〉 87<211〉 24 <210> 87 <211> 24

<212> DNA <212> DNA

<213> Artificial <213> Artificial

〈400〉 87 <400> 87

ggtgcatcgt acgatgcagg gggg ggtgcatcgt acgatgcagg gggg

<210〉 88 <210> 88

<211〉 18 <211> 18

<212> DNA <212> DNA

<213〉 Artificial <213> Artificial

<400〉 88 <400> 88

gggacgtacg tcgggggg gggacgtacg tcgggggg

<210> 89 <210> 89

<211〉 21 <211> 21

24 twenty four

18<formula>formula see original document page 69</formula><213〉 Artificial <400〉 91 18 <formula> formula see original document page 69 </ formula> <213> Artificial <400> 91

gggggatcga cgtcgatcgg gggg gggggatcga cgtcgatcgg gggg

<210〉 92 <210> 92

<211> 24 <211> 24

<212〉 腿 <212> leg

〈213〉 Artificial <213> Artificial

<400> 92 <400> 92

ggtg.catcga tcgatgc鄉gggg ggtg.catcga tcgatgc Township gggg

<210> 93 <210> 93

<211〉 22 <211> 22

<212〉 DNA <212> DNA

<213> Artificial〈400〉 93 <213> Artificial <400> 93

ggatcgatcg atcgatgggg gg 22 ggatcgatcg atcgatgggg gg 22

<210> 94 <210> 94

〈211〉 22 <211> 22

<212> DNA <212> DNA

<213> Artificial <213> Artificial

<400〉 94 <400> 94

ggcgatcgat cgatcggggg gg 22 ggcgatcgat cgatcggggg gg 22

<210〉 95 <210> 95

<211> 22 <211> 22

〈212> 腿 <212> leg

<213> Artificial<formula>formula see original document page 72</formula><210〉 98 <213> Artificial <formula> formula see original document page 72 </ formula> <210> 98

〈211〉 20 <211> 20

<212〉 DNA <212> DNA

〈213〉 Artificial <213> Artificial

〈400〉 98 <400> 98

tcgtctttcg actcgttctc tcgtctttcg actcgttctc

<210〉 99 <210> 99

<211> 18 <211> 18

<212〉 DNA <212> DNA

<213> Artificial <213> Artificial

<400〉 99 <400> 99

tcgtcgtttt gcgttctx tcgtcgtttt gcgttctx

20 20

18〈210〉 ]00 18 <210>] 00

〈211〉 20 <211> 20

<212〉 DNA <212> DNA

<213> Artificial <213> Artificial

<400> 100 <400> 100

lxatcgactc tcgagcgttc lxatcgactc tcgagcgttc

<210〉 101 <210> 101

<211> 21 <211> 21

<212> 腿 <212> leg

<213〉 Artificial <213> Artificial

<400〉 101 <400> 101

atcgtcgact ctcgtgttct c atcgtcgact ctcgtgttct c

20 20

21 twenty one

〈210〉 102《211> 20 <212> DNA <213> Artificial <210> 102 "211> 20 <212> DNA <213> Artificial

<400> 102 <400> 102

tcgactttcg tcgttctgtt 20 tcgactttcg tcgttctgtt 20

<210> 103 <210> 103

<211〉 19 <211> 19

<212> DNA <212> DNA

<213> Artificial <213> Artificial

<400> 103 <400> 103

tcgtcgtttc gtcgttctc 19 tcgtcgtttc gtcgttctc 19

<210> 104 <211〉 20<formula>formula see original document page 76</formula><213> Artificial <210> 104 <211> 20 <formula> formula see original document page 76 </ formula> <213> Artificial

<400〉 106 <400> 106

tcgacgttcg tcgttcgtcg ttc tcgacgttcg tcgttcgtcg ttc

〈210〉 107 <210> 107

<211〉 21 <211> 21

<212> DNA <212> DNA

<213〉 Artificial <213> Artificial

<400> 107 <400> 107

tcgtcgacgt cgttcgttct c tcgtcgacgt cgttcgttct c

Claims (6)

1. 一种人工合成的含CpG的单链脱氧寡核苷酸,其核苷酸序列为SEQ ID NO:107所示的序列。 An artificial CpG-containing single-stranded oligodeoxynucleotide, the nucleotide sequence of SEQ ID NO: 107 in the sequence shown in FIG.
2. 根据权利要求1所述的单链脱氧寡核苷酸,其中磷酸二酯键被非硫代修饰、硫代修饰、或部分硫代修饰。 The single-stranded oligodeoxynucleotide according to claim 1 nucleotide, wherein the phosphodiester bond is non-thio modified, phosphorothioate-modified, or partially modified thio.
3. 权利要求1 — 2中任一项的单链脱氧寡核苷酸在制备用于治疗肿瘤性疾病或病毒感染性疾病的药物中的应用。 2 single-stranded oligodeoxynucleotide according to any manufacture of a medicament for the treatment of neoplastic diseases or viral infectious disease nucleotide --1 claim.
4. 权利要求3所述的应用,其中所述药物为适于皮下应用、鼻腔粘膜应用、肌肉应用、胃肠应用、腹腔应用、或静脉注射的形式。 Use according to 3 wherein the medicament is adapted for subcutaneous application, application of the nasal mucosa, muscle application, parenteral application, intraperitoneal application, or intravenous form of claim.
5. 权利要求1-2中任一项所述的单链脱氧寡核苷酸在制备用于激活人外周单个核细胞杀伤人白血病细胞的药物中的应用。 1-2 in a single-stranded oligodeoxynucleotide according to any one of the claim in the manufacture of a medicament nucleotide activating human peripheral mononuclear cell killing in human leukemia cells.
6. 权利要求1-2中任一项所述的单链脱氧寡核苷酸在制备用于诱导人外周单个核细胞产生cc干扰素的药物中的应用。 1-2 in a single-stranded oligodeoxynucleotide according to any one of the claim in the manufacture of a medicament polynucleotide induced human peripheral mononuclear cells to produce interferon in cc.
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CN1304412C (en) * 2003-09-05 2007-03-14 长春华普生物技术有限公司 Artificial synthesized deoxyoligonucleotide containing CpG single chain and anti single strand linear chain RNA virus
JP2008521385A (en) * 2004-11-29 2008-06-26 長春華普生物技術有限公司 CpG-containing single strand deoxynucleotide as adjuvants
CN1847256B (en) * 2005-04-13 2011-06-15 长春华普生物技术有限公司 Antiviral prepn containing both CpG single-strand deoxyoligonucleotide and ribavirin
CN1865275B (en) * 2005-05-17 2011-06-15 长春华普生物技术有限公司 Artificial single-chain deoxynucleotide having therapeutic effect to human B cell tumour
WO2007012285A1 (en) * 2005-07-28 2007-02-01 Changchun Huapu Biotechnology Co., Ltd. Viral infection resistent single strand deoxynucleosides
CN100402090C (en) 2006-06-15 2008-07-16 南京农业大学 Mucosa-immune composite adjuvant
CN101148467B (en) 2006-09-20 2012-03-14 长春华普生物技术有限公司 Oligonucleotide and use thereof for treating lung cancer
CN101161288B (en) * 2006-10-13 2013-07-03 长春华普生物技术有限公司 Enhanced tumour cell cracking article of oligonucleotide and application thereof in preparing medicine for treating tumour
MX2009005849A (en) 2006-12-04 2009-08-12 Univ Illinois Compositions and methods to treat cancer with cupredoxins and cpg rich dna.
CN106893724A (en) * 2015-12-17 2017-06-27 苏州派动生物技术有限公司 Oligonucleotide with antigen synergetic effect and tumor-treating function

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CN1271733A (en) 2000-04-04 2000-11-01 中国预防医学科学院病毒学研究所 CpG oligonucleotide with specific immunostimulation activity to human immune cell

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CN1271733A (en) 2000-04-04 2000-11-01 中国预防医学科学院病毒学研究所 CpG oligonucleotide with specific immunostimulation activity to human immune cell

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