CN1296378C - CpG-N ODN gene sequence with high immunological activity CpG-S ODN and antagonism CpG-N ODN and use thereof - Google Patents

CpG-N ODN gene sequence with high immunological activity CpG-S ODN and antagonism CpG-N ODN and use thereof Download PDF

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CN1296378C
CN1296378C CN 200410034787 CN200410034787A CN1296378C CN 1296378 C CN1296378 C CN 1296378C CN 200410034787 CN200410034787 CN 200410034787 CN 200410034787 A CN200410034787 A CN 200410034787A CN 1296378 C CN1296378 C CN 1296378C
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周红
王良喜
郑江
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中国人民解放军第三军医大学
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Abstract

本发明公开了一种高免疫活性的免疫刺激寡核苷酸及其对免疫刺激寡核苷酸拮抗的寡核苷酸的基因序列,同时公开了高免疫活性的免疫刺激寡核苷酸在制备治疗感染性疾病、过敏性疾病、免疫缺陷性疾病和肿瘤药物中的应用;对免疫刺激寡核苷酸拮抗的寡核苷酸在制备治疗SIRS和脓毒症药物中的应用。 The present invention discloses an immunostimulatory oligonucleotide sequence of one gene and the immunological activity of highly immunostimulatory oligonucleotide antagonizing oligonucleotides, also discloses a high immunological activity in the preparation of polynucleotide immunostimulatory oligonucleotides treatment of infectious diseases, allergic diseases, immunodeficiency diseases and tumors use in medicine; in the manufacture of a medicament the treatment of sepsis and SIRS immunostimulatory oligonucleotide of antagonizing oligonucleotides.

Description

高免疫活性的CpG-S ODN和拮抗CpG-S ODN 作用的CpG-N ODN的基因序列及其应用 Highly immunoreactive CpG-S ODN and antagonistic effect of CpG-S ODN CpG-N ODN gene sequence and its application

技术领域 FIELD

本发明涉及一种寡核苷酸及其在医药学上的应用,具体地说,涉及一种免疫刺激寡核苷酸(CpG-S ODN)及对其有拮抗作用的寡核苷酸(CpG-N ODN)。 The present invention relates to oligonucleotides and their application in medicine, in particular, relates to an immunostimulatory oligonucleotides (CpG-S ODN) and its antagonism oligonucleotides (CpG -N ODN).

背景技术 Background technique

含有未甲基化CpG的寡核苷酸片段是细菌DNA的最小作用单位,因此具有免疫刺激作用的含未甲基化CpG的寡核苷酸被称作免疫刺激寡核苷酸(Immnostimulatory oligonucleotides,CpG-S ODN)。 Oligonucleotides containing unmethylated CpG effect is the smallest unit of bacterial DNA, thus containing immunostimulatory effect unmethylated CpG oligonucleotide is referred to as immunostimulatory oligonucleotide (Immnostimulatory oligonucleotides, CpG-S ODN). 其结构特征为含有CpG基元即6个碱基互补回文结构的六聚体,该六聚体中必须至少含有一个CpG二核苷酸,CpG的5'端必须有一个嘌呤,结构类似ApA,GpA或GpT等,3'端必须有二个嘧啶如TpT,目前已知具有免疫刺激作用的六聚体包括AACGTT,AGCGCT等。 Wherein the structure containing a CpG motif i.e., complementary 6 base palindrome hexamers, the hexamers must contain at least one CpG dinucleotides, CpG 5 'ends must be a purine, structurally similar ApA etc., GpA or GpT, 3 'end must have two pyrimidines like TpT, now known to have immunostimulatory hexamer comprising AACGTT, AGCGCT like.

CpG-S ODN具有两方面的作用:小剂量CpG-S ODN对小鼠免疫细胞具有广泛的免疫活性,大剂量则可诱导全身性炎症反应综合征(Systemic inflammatoryresponse syndrome,SIRS)和脓毒症的发生。 CpG-S ODN has two effects: small doses of CpG-S ODN have broad immunocompetent mice immune cells, large doses can induce a systemic inflammatory response syndrome (Systemic inflammatoryresponse syndrome, SIRS) and sepsis occur.

大量研究表明:①小剂量CpG-S ODN能够直接或间接激活树突状细胞、B细胞、单核-巨噬细胞、NK细胞和细胞毒性T细胞(CTL);诱导IFN-γ等Th1型细胞因子释放。 Numerous studies show that: ① low dose of CpG-S ODN capable of directly or indirectly activating dendritic cells, B cells, monocyte - macrophage, NK cells and cytotoxic T cells (the CTL); induction of IFN-γ and other Th1-type cells factor release. 因此,单独使用即可有效抑制小鼠肿瘤生长、促进肿瘤消退,并可形成保护性免疫记忆,从而能够有效防止肿瘤复发。 Thus, it can be used alone effectively suppressed tumor growth in mice, promoting tumor regression, and forming a protective immunological memory, thereby effectively preventing tumor recurrence. 除此之外,由于CpG ODN是一种优良的Th1型疫苗佐剂,因此也可与肿瘤疫苗配伍治疗恶性肿瘤,在肿瘤预防与治疗方面具有良好的应用前景。 In addition, due to the CpG ODN is a good Th1-type vaccine adjuvant, and therefore compatible with the tumor vaccine to treat malignant tumors, have a good prospect in cancer prevention and treatment. 由于目前尚缺乏对人免疫细胞具有高免疫活性的CpG-S ODN,因此需要寻找高免疫活性的CpG-S ODN对肿瘤预防与治疗具有重要意义。 Due to the current lack of a high immunological activity of CpG-S ODN on human immune cells, it needs to find a high immunological activity of CpG-S ODN is important for cancer prevention and treatment. ②CpG-S ODN诱导Th1型细胞因子释放而抑制Th2型类免疫应答,所以可用来治疗Th2型类免疫应答介导的过敏性疾病如哮喘。 ②CpG-S ODN induce Th1 cytokine release based suppressed Th2-type immune response, so the class can be used to treat Th2-type immune response mediated allergic diseases such as asthma. ③CpG-S ODN能活化树突状细胞、B细胞、单核-巨噬细胞等细胞,因此治疗细菌或病毒等病原体引起的感染性疾病、免疫功能低下性疾病。 ③CpG-S ODN can activate dendritic cells, B cells, monocyte - macrophage like cells and therefore treatment of infectious diseases, immune dysfunction and other diseases caused by bacterial or viral pathogens.

由于CpG-S ODN广泛存在于细菌DNA中,当细菌感染发生时,细菌大量繁殖可以导致CpG-S ODN大量复制。 Since the CpG-S ODN widespread in bacterial DNA, bacterial infection occurs when large population of bacteria can result in a large number of CpG-S ODN replication. 过量的CpG-S ODN通过激活单核-吞噬细胞系统大量释放TNF-α、IL-1、IL-6和IL-12等多种细胞因子引起SIRS和脓毒症的发生,其病死率高达50-80%,至今尚无有效的治疗手段。 Excess of CpG-S ODN by activation of mononuclear - phagocytic system large release TNF-α, IL-1, IL-6 and IL-12 and other cytokines lead to the occurrence of SIRS and sepsis, mortality up to 50 -80%, yet no effective treatment. 有文献报告细菌基因组DNA中含有未甲基化的CpG基元不一定具有免疫刺激作用,甚至具有阻断或中和CpG-SODN作用,此类寡核苷酸被称为CpG-N ODN(CpG-neutralizing oligonucleotides,CpG-N ODN),因此研究对CpG-S ODN具有拮抗作用的CpG-N ODN对SIRS和脓毒症的治疗具有重要意义。 There containing unmethylated CpG motifs bacterial genomic DNA reported in the literature does not necessarily have immunostimulatory effect, or even have a blocking effect and CpG-SODN, such oligonucleotides are referred to as CpG-N ODN (CpG -neutralizing oligonucleotides, CpG-N ODN), so the research CpG-N ODN antagonism of CpG-S ODN is important for the treatment of SIRS and sepsis.

发明内容 SUMMARY

本发明的目的在于提供一种免疫刺激寡核苷酸,其具有高免疫活性,可形成保护性免疫记忆,能抑制Th2型类免疫应答。 Object of the present invention is to provide an immunostimulatory oligonucleotide, having a high immunoreactivity, can form a protective immune memory can be suppressed Th2-type immune response Class.

本发明的另一目的在于提供一种高免疫活性的免疫刺激寡核苷酸在预防和治疗感染性疾病、过敏性疾病、免疫缺陷性疾病和肿瘤药物中的应用。 Another object of the present invention is to provide a highly immunoreactive immunostimulatory oligonucleotides infectious diseases, allergic diseases, immunodeficiency diseases and cancer drugs used in the prevention and treatment nucleotides.

本发明的再一目的在于提供一种对免疫刺激寡核苷酸拮抗的寡核苷酸(CpG-NODN),具有阻断或中和CpG-S ODN的作用。 A further object of the present invention is to provide an immunostimulatory oligonucleotide antagonizing oligonucleotides (CpG-NODN), has the effect of blocking or neutralizing the CpG-S ODN.

本发明的还一目的在于提供一种对免疫刺激寡核苷酸拮抗的寡核苷酸在用于制备治疗SIRS和脓毒症药物中的应用。 A further object of the present invention is to provide an oligonucleotide for immunostimulatory oligonucleotides antagonism in the preparation of a medicament for the treatment of sepsis and SIRS nucleotides.

为了实现本发明目的,一种免疫刺激寡核苷酸,具有如下基因序列:GGC GGC GGC GCG;(1)CGC GGC GCC GGG;(2)GGC GGC GGC GGA;(3)CCG CGC GCG GGC;(4)TGG CGC GGG GCG;(5)GCC GGC GCC CGG;(6)GCG CGC GCG GTA;(7)CCA GGC GGC GGG;(8)GGC CGC GGG GGT;(9)CAG CCC GGG GGA;(10)GCT CCC GGG CTT;(11)GCG CTC GGG CTT;(12)CCG CCC GCG CCT;(13)或GCA CCC GCG CGC(14)其中的一种。 To achieve the object of the present invention, an immunostimulatory oligonucleotide, having the following gene sequence: GGC GGC GGC GCG; (1) CGC GGC GCC GGG; (2) GGC GGC GGC GGA; (3) CCG CGC GCG GGC; ( 4) TGG CGC GGG GCG; (5) GCC GGC GCC CGG; (6) GCG CGC GCG GTA; (7) CCA GGC GGC GGG; (8) GGC CGC GGG GGT; (9) CAG CCC GGG GGA; (10) GCT CCC GGG CTT; (11) GCG CTC GGG CTT; (12) CCG CCC GCG CCT; (13) or GCA CCC GCG CGC (14) one of them.

其中,优选的基因序列:GGC GGC GGC GCG;CCG CGC GCG GGC;TGG CGC GGG GCG;GCC GGC GCC CGG;GGC CGC GGG GGT;CAG CCC GGG GGA; Among them, gene sequence: GGC GGC GGC GCG; CCG CGC GCG GGC; TGG CGC GGG GCG; GCC GGC GCC CGG; GGC CGC GGG GGT; CAG CCC GGG GGA;

GCT CCC GGG CTT;GCG CTC GGG CTT;或CCG CCC GCG CCT其中的一种。 GCT CCC GGG CTT; GCG CTC GGG CTT; or one of them CCC GCG CCT CCG.

本发明所述的一种高免疫活性的免疫刺激寡核苷酸具有高免疫活性,可形成保护性免疫记忆,能抑制Th2型类免疫应答,其用于制备治疗感染性疾病、过敏性疾病、免疫缺陷性疾病和肿瘤药物中的应用。 A high-activity immunogens of the present invention is highly immune stimulatory oligonucleotides having activity, can form a protective immune memory can be suppressed Th2-type immune response category, for the preparation of the treatment of infectious diseases, allergic diseases, application of immunodeficiency diseases and cancer drugs.

为了实现另一发明目的,一种对免疫刺激寡核苷酸拮抗的寡核苷酸,具有如下基因序列:GGC CGC GGG GAC;(15)GCC GCC GCC GCC;(16)CGC CTC GGG GAG;(17)TGC CGC GGC AGA;(18)CTC CAC GTG CCG;(19)GCC GCC GCC GTC;(20)或GCT GCC GCC GCC(21)其中的一种。 In order to achieve another object of the invention, a method of antagonizing the immunostimulatory oligonucleotide is an oligonucleotide, having the following gene sequence: GGC CGC GGG GAC; (15) GCC GCC GCC GCC; (16) CGC CTC GGG GAG; ( 17) TGC CGC GGC AGA; (18) CTC CAC GTG CCG; (19) GCC GCC GCC GTC; (20) or GCT GCC GCC GCC (21) one of them.

其优选的基因序列:CGC CTC GGG GAG;TGC CGC GGC AGA;CTC CAC GTG CCG;GCC GCC GCC GTC;或GCT GCC GCC GCC其中的一种。 Preferred gene sequences: CGC CTC GGG GAG; TGC CGC GGC AGA; CTC CAC GTG CCG; GCC GCC GCC GTC; GCT GCC GCC GCC or one of them.

本发明所述的一种对免疫刺激寡核苷酸拮抗的寡核苷酸具有阻断或中和CpG-S ODN诱导hPBMC释放TNF-α的能力,其在用于制备治疗SIRS和脓毒症药物中的应用。 One of the present invention to antagonize the immunostimulatory oligonucleotide or an oligonucleotide having the ability to block TNF-α and the CpG-S ODN induced release hPBMC, which preparation for the treatment of sepsis and SIRS use drugs.

为了得到高活性的CpG-S ODN和CpG-N ODN,本发明的研究人员从病毒的基因组入手进行。 In order to obtain a CpG-S ODN and CpG-N ODN high activity, researchers of the present invention starts from the viral genome.

本领域技术人员了解所有小病毒和逆转录病毒基因组DNA存在众多的未甲基化CpG二核苷酸,其中许多具有CpG-S ODN结构特征。 Those skilled in the art to understand the presence of all the groups of small DNA viruses and retroviruses many genes unmethylated CpG dinucleotides, many of which are structural features CpG-S ODN. 因此理论上推测其应具有很强的免疫刺激作用。 Therefore theorized that it should have a strong immune stimulation. 但事实恰恰相反,病毒DNA不仅可逃脱机体免疫系统对其的免疫识别和清除,而且可在宿主细胞内进行复制,这种能力在逆转录病毒中表现得更为显著。 However, the opposite is true, not only DNA viral escape from immune recognition and clearance of their immune system, and can replicate in a host cell, more in this capacity as a significant retroviruses. 因此病毒DNA中应存在阻断或中和CpG-S ODN作用的寡核苷酸序列,但并不知道其结构特征是什么? Thus blocking oligonucleotide sequence or CpG-S ODN and action should be present in the viral DNA, but do not know what the structure wherein? 有关腺病毒DNA(Adenoviral DNA,Adv DNA)的研究给予了研究人员非常有益的启示。 Study of adenovirus DNA (Adenoviral DNA, Adv DNA) gave very useful insights researchers. 尽管血清型2、5Adv DNA(亚属C)和血清型12Adv DNA(亚属A)中均含有数量相当的未甲基化CpG二核苷酸,因此推测它们均应具有相同的免疫刺激作用。 While serotype 2,5Adv DNA (subgenus C), and serotype 12Adv DNA (subgenus A), contain a considerable amount of unmethylated CpG dinucleotides, it is presumed that they should have the same immune stimulating effect. 但是Krieg等的实验结果显示血清型12Adv DNA具有很强的免疫刺激作用,而血清型2、5Adv DNA则不仅无免疫刺激作用,而且具有阻断CpG-S ODN诱导TNF-α、IL-6分泌的作用。 However, experimental results show Krieg et serotype 12Adv DNA has a strong immunostimulatory effect, while the serotypes 2,5Adv DNA not only the non-immune stimulation, and having a blocking CpG-S ODN-induced TNF-α, IL-6 secretion role. 本发明的研究人员也重复了该实验,结果与Krieg的一致。 The researchers of the present invention is also the experiment was repeated, the results are consistent with Krieg. 该结果说明:(1)血清型12Adv DNA中存在CpG-S ODN,血清型2、5Adv DNA中存在具有阻断或中和EC DNA或CpG-S ODN作用的寡核苷酸序列;(2)血清型12Adv DNA中CpG二核苷酸参与CpG基元的组成并发挥免疫刺激作用;(3)血清型2、5Adv DNA中CpG二核苷酸可能未完全参与CpG-S ODN的组成而发挥免疫刺激作用,因为对血清型2、5Adv DNA序列分析表明,参与CpG基元组成的CpG只占CpG总数量的1/15-30,提示这些CpG基元的作用可能被其它具有中和作用的寡核苷酸所抑制。 This result indicates: (1) serotypes 12Adv DNA present in the CpG-S ODN, an oligonucleotide having a sequence block or neutralize EC DNA or CpG-S ODN effect serotype present 2,5Adv DNA; (2) serotype 12Adv DNA involved in CpG dinucleotides in the CpG motif and composed of immunostimulatory effects play; (3) serotypes 2,5Adv DNA of CpG dinucleotide may not be fully involved in the composition of the CpG-S ODN exert immune stimulation, because the analysis of the sequence showed that serotype 2,5Adv DNA, only the total number of CpG CpG CpG motifs involved in the composition of 1 / 15-30, suggesting a role for these CpG motifs may be another oligonucleotide having a neutralizing effect nucleotide inhibited.

根据以上分析,本发明的研究人员推测血清型12Adv DNA含有具有很强免疫刺激作用的CpG-S ODN,而血清型2、5Adv DNA中存在天然的具有阻断或中和CpG-S ODN的CpG-N ODN,CpG-N ODN的序列特征可能与CpG两端寡核苷酸序列特点改变有关。 According to the above analysis, the present invention is presumed the researchers serotype 12Adv DNA containing CpG-S ODN has a strong immunostimulatory effect of CpG while naturally having block or neutralize the presence of CpG-S ODN 2,5Adv DNA of serotype -N ODN, CpG-N ODN sequence characteristics may be associated with CpG oligonucleotide sequences at both ends to change the characteristics.

因此,本发明的研究人员采用生物信息学技术,以CpG二核苷酸为“电子探针”(CpG二核苷酸为检索“标签”),以CpG-S ODN中的六聚体为比较模板,对血清型2、5和12Adv DNA、EC DNA进行序列分析,寻找血清型2、5Adv DNA中具有共同结构特征的12个碱基的寡核苷酸序列;并通过体外生物活性的筛选后,得到具有阻断或中和CpG-S ODN作用的寡核苷酸(CpG-N ODN);观察CpG-NODN对细菌DNA介导SIRS动物模型小鼠的保护作用,明确CpG-N ODN在SIRS防治中的作用。 Accordingly, the present invention employs the researchers bioinformatics, CpG dinucleotides to as "EPMA" (CpG dinucleotides to retrieve "tag") to CpG-S ODN hexamer in comparison to template, serotypes 2, 5 and 12Adv DNA, EC DNA sequence analysis, to find a nucleotide sequence of an oligonucleotide 12 bases serotypes 2,5Adv DNA have a common structural features; and after in vitro by screening for biological activity to give a block or neutralize the action of CpG-S ODN oligonucleotides (CpG-N ODN); protective effects of bacterial DNA CpG-NODN mediate SIRS mouse model, the CpG-N ODN clearly SIRS the role of prevention and treatment.

本发明的研究人员从PubMed上的Nucleotide数据库中获取了血清型2、5和12Adv DNA、EC DNA序列(ACCESSION:NC_001405、M73260、NC_001460、NC_000913)。 The researchers of the present invention acquired serotypes 2,5 and 12Adv DNA from the Nucleotide database in PubMed, EC DNA sequence (ACCESSION: NC_001405, M73260, NC_001460, NC_000913).

采用生物信息学技术,使用Primer Premier 5、BioEdit version 5.0.6等生物软件,对血清型2、5和12Adv DNA、EC DNA中256种含CpG的六核苷酸序列进行了分析和比较,确定了血清型2、5Adv DNA中特有的19种含CpG的六核苷酸序列;使用BioEdit version 5.0.6,查找出血清型2、5Adv DNA中以这19种CpG六核苷酸为核心的12碱基寡核苷酸(ODN);使用生物软件RNAstructure version 3.71,对12碱基ODN的二级结构进行了预测,找出了4种可能的共同结构特征:(1)cg位于干环顶端;(2)gc位于干环顶端;(3)cg或gc位于干环侧面;(4)无干环结构(见图1),从而完成了对CpG-N ODN分子的初设计。 Bioinformatics techniques using Primer Premier 5, BioEdit version 5.0.6 software, biological, and serotypes 2, 5 12Adv DNA, EC DNA in 256 kinds of hexanucleotide sequences containing CpG were analyzed and compared to determine 19 kinds of hexanucleotide sequences containing a CpG 2,5Adv DNA of serotype-specific; use BioEdit version 5.0.6, serotype 2,5Adv DNA to find out to which 19 kinds of CpG hexanucleotide core 12 base oligonucleotide (ODN); the use of biological software RNAstructure version 3.71, 12-base ODN secondary structure was predicted, identified four possible common structural features: (1) cg top ring located dry; (2) located gc dry top ring; (3) cg gc located or dry side of the ring; (4) uninvolved ring structure (see FIG. 1), thus completing the early design of CpG-N ODN molecule.

根据此设计合成了10条磷酸硫代骨架的ODN,其中cg位于干环顶端的3条(ODN1-3)、gc位于干环顶端的3条(ODN 4-6)、cg或gc位于干环侧面的2条(ODN7-8)、无干环结构形成的2条(ODN9-10)。 According to this Article ODN 10 were designed and synthesized thio phosphate backbone, which is located in the dry cg top ring 3 (ODN1-3), gc is located in the dry top ring 3 (ODN 4-6), cg gc located dry ring or 2 (ODN9-10) 2 (ODN7-8), nothing to the ring structure formed by the side surface. 本发明的研究人员对10条ODN的体外生物学活性进行鉴定,发现2条无明显刺激性的ODN,8条ODN具有诱导hPBMC释放TNF-α的能力,即8条ODN为免疫刺激寡核苷酸(CpG-S ODN)。 The researchers of the present invention on in vitro biological activity of ODN 10 were identified, two found no irritating ODN, ODN 8 hPBMC ability to induce the release of TNF-α, i.e., 8 to ODN immunostimulatory oligonucleotide acid (CpG-S ODN).

使用生物软件RNAstructure version 3.71,对ODN的二级结构自由能进行了分析,在此基础上,对10条ODN的刺激性与结构之间的关系进行了分析并发现,ODN的生物活性可能与预测二级结构的4种共同结构特征无关;而可能与预测二级结构的自由能高低密切相关。 Use of biological software RNAstructure version 3.71, the secondary structure of the free energy of ODN was analyzed on this basis, the relationship between the structure 10 and the stimulatory ODN was analyzed and found, ODN biological activity and possible prediction 4 kinds of common structural features of the secondary structure is independent; and may be closely related to the free energy of the level predicted secondary structure. 因此,使用生物软件RNAstructure version 3.71对CpG-NODN分子进行了再设计。 Therefore, the use of biological software RNAstructure version 3.71 Dui CpG-NODN molecule has been re-designed. 根据此设计合成了11条磷酸硫代骨架的ODN,本发明的研究人员对11条ODN的体外生物学活性进行鉴定,发现5条无明显刺激性的ODN,6条ODN为免疫刺激寡核苷酸(CpG-S ODN)。 According to this design synthesis ODN 11-thio phosphate backbone, the researchers of the present invention on in vitro biological activity of ODN 11 were identified, 5 ODN found no irritating to 6 ODN immunostimulatory oligonucleotide acid (CpG-S ODN).

本发明的研究人员在两次筛选实验中共获得14条免疫刺激寡核苷酸(CpG-SODN),其基因序列为:GGC GGC GGC GCG;CGC GGC GCC GGG;GGC GGC GGCGGA;CCG CGC GCG GGC;TGG CGC GGG GCG;GCC GGC GCC CGG;GCG CGCGCG GTA;CCA GGC GGC GGG;GGC CGC GGG GGT;CAG CCC GGG GGA;GCT CCC GGG CTT;GCG CTC GGG CTT;CCG CCC GCG CCT;或GCA CCC GCGCGC其中的一种;其中优选的为:GGC GGC GGC GCG;CCG CGC GCG GGC;TGG CGC GGG GCG;GCC GGC GCC CGG;GGC CGC GGG GGT;CAG CCC GGGGGA;GCT CCC GGG CTT;GCG CTC GGG CTT;或CCG CCC GCG CCT其中的一种。 The researchers of the present invention in the two experiments were obtained after screening 14 immunostimulatory oligonucleotides (CpG-SODN), which is a gene sequence: GGC GGC GGC GCG; CGC GGC GCC GGG; GGC GGC GGCGGA; CCG CGC GCG GGC; TGG CGC GGG GCG; GCC GGC GCC CGG; GCG CGCGCG GTA; CCA GGC GGC GGG; GGC CGC GGG GGT; CAG CCC GGG GGA; GCT CCC GGG CTT; GCG CTC GGG CTT; CCG CCC GCG CCT; or GCA CCC GCGCGC therein one kind; preferred are: GGC GGC GGC GCG; CCG CGC GCG GGC; TGG CGC GGG GCG; GCC GGC GCC CGG; GGC CGC GGG GGT; CAG CCC GGGGGA; GCT CCC GGG CTT; GCG CTC GGG CTT; or CCG CCC wherein the one kind GCG CCT.

同时本发明的研究人员对7条无明显刺激性的ODN抑制CpG-S ODN的活性进行鉴定,表明该7条ODN均有抑制CpG-S ODN诱导的hPBMC释放TNF-α的能力,即7条ODN为对免疫刺激寡核苷酸拮抗的寡核苷酸(CpG-N ODN),其基因序列为:GGC CGC GGG GAC;GCC GCC GCC GCC;CGC CTC GGG GAG;TGCCGC GGC AGA;CTC CAC GTG CCG;GCC GCC GCC GTC;或GCT GCC GCCGCC其中的一种;优选的为:CGC CTC GGG GAG;TGC CGC GGC AGA;CTC CACGTG CCG;GCC GCC GCC GTC;或GCT GCC GCC GCC其中的一种。 The researchers also ODN of the present invention, no significant inhibitory activity of 7-irritating CpG-S ODN were identified, indicating that the 7 ODN have the ability to inhibit the release of TNF-α hPBMC of CpG-S ODN induced, i.e., 7 ODN immunostimulatory oligonucleotide for the antagonistic oligonucleotides (CpG-N ODN), which is a gene sequence: GGC CGC GGG GAC; GCC GCC GCC GCC; CGC CTC GGG GAG; TGCCGC GGC AGA; CTC CAC GTG CCG ; GCC GCC GCC GTC; or wherein one GCT GCC GCCGCC; preferred are: CGC CTC GGG GAG; TGC CGC GGC AGA; CTC CACGTG CCG; GCC GCC GCC GTC; GCT GCC GCC GCC or one of them.

本发明所述的免疫刺激寡核苷酸(CpG-S ODN)及对免疫刺激寡核苷酸拮抗的寡核苷酸(CpG-N ODN)可采用本领域常用合成方法合成,比如固相亚磷酰胺三酯法。 Immunostimulatory oligonucleotides (CpG-S ODN) according to the present invention and immunostimulatory oligonucleotide antagonizing oligonucleotides (CpG-N ODN) synthetic methods commonly employed in the art synthesis, such as solid phase phosphoramidite triester method.

本发明的研究人员经过对本发明所设计并合成的核苷酸进行药理学分析研究,发现本发明所述的免疫刺激寡核苷酸(CpG-S ODN)具有广泛的免疫活性,直接或间接激活树突状细胞、B细胞、单核-巨噬细胞、NK细胞和细胞毒性T细胞(CTL);诱导IFN-γ等Th1型细胞因子释放,可形成保护性免疫记忆,能抑制Th2型类免疫应答,其用于制备治疗感染性疾病、过敏性疾病如哮喘、免疫缺陷性疾病和肿瘤药物中的应用。 After the design of the present invention is the synthesis of nucleotides present invention researchers pharmacological drug analysis, it found immunostimulatory oligonucleotides (CpG-S ODN) according to the present invention has broad immune activity, directly or indirectly activated dendritic cells, B cells, monocyte - macrophage, NK cells and cytotoxic T cells (the CTL); induction of IFN-γ and other Th1 cytokine release, can form a protective immune memory can be suppressed Th2-type immune class response, which is for treating infectious diseases, allergic diseases such as asthma, immunodeficiency diseases and tumors for use in medicine.

本发明所述的拮抗寡核苷酸(CpG-N ODN)具有阻断或中和CpG-S ODN诱导hPBMC释放TNF-α的能力,其在用于制备治疗SIRS和脓毒症药物中的应用。 The present invention antagonize oligonucleotides (CpG-N ODN) having the ability to block or neutralize TNF-α CpG-S ODN induced hPBMC release, their use in the preparation of a medicament for the treatment of SIRS and sepsis in .

附图说明 BRIEF DESCRIPTION

图1 12碱基ODN预测的二级结构4种可能的共同结构特征:cg位于干环顶端图2 12碱基ODN预测的二级结构4种可能的共同结构特征:gc位于干环顶端图3 12碱基ODN预测的二级结构4种可能的共同结构特征:cg或gc位于干环侧面图4 12碱基ODN预测的二级结构4种可能的共同结构特征:无干环结构具体实施方式下面实施例为进一步描述本发明,但所述实施例仅用于说明本发明而不是限制本发明。 4 possible common structural features of secondary structure predicted nucleotide ODN FIG. 1 12: cg top ring 4 is located in the dry possible common structural features of secondary structure predicted nucleotide ODN 2 12: gc top ring 3 is located in the dry 4 possible common structural features of secondary structure predicted 12-base ODN: gc located CG or dry side view of the ring 4 possible common structural features of secondary structure predicted 412 base ODN: uninvolved ring structure DETAILED DESCRIPTION Example embodiments of the present invention is further described, the embodiments are merely illustrative of the invention and not to limit the present invention.

实验例1本实验例在于通过采用生物信息学技术,获取CpG-S ODN和CpG-N ODN基因序列。 Experimental Example Experimental Example 1 in that by using bioinformatics, obtaining CpG-S ODN and CpG-N ODN gene sequence.

1.从PubMed上的Nucleotide数据库中获取了血清型2、5和12Adv DNA、ECDNA序列(ACCESSION:NC_001405、M73260、NC_001460、NC_000913)。 1. Obtain serotype 5 and 12Adv DNA Nucleotide from the database in PubMed, ECDNA sequence (ACCESSION: NC_001405, M73260, NC_001460, NC_000913).

2.采用生物信息学技术,使用Primer Premier 5、BioEdit version 5.0.6等生物软件,对血清型2、5和12Adv DNA、EC DNA中256种含CpG的六核苷酸序列进行了分析和比较(见表1),根据:①在血清型2、5Adv DNA中出现频率较高;②在Adv 12DNA和EC DNA中出现频率较低;③Adv2:Adv12≥2或Adv2:EC≥5等3条原则,确定了血清型2、5AdvDNA中特有的19种含CpG的六核苷酸序列。 2. bioinformatics techniques using Primer Premier 5, BioEdit version 5.0.6 biological software, the nucleotide sequence of the CpG-containing six serotypes 2,5 and 12Adv DNA, EC DNA in 256 kinds of analyzes and comparison (see Table 1), according to: ① serotype 2,5Adv DNA present in the higher frequency; ② Adv 12DNA in less frequent and the EC DNA; ③Adv2: Adv12≥2 or Adv2: EC≥5 other three principles determining the nucleotide sequence of the CpG-containing six serotypes 2,5AdvDNA peculiar 19 kinds.

表1 Adv 2、5、12DNA和EC DNA中256种含CpG的六核苷酸序列分析和比较 Table 256 kinds hexanucleotide sequence analysis and comparison of CpG containing 1 Adv 2,5,12DNA and the EC DNA

3.使用BioEdit version 5.0.6,查找出血清型2、5Adv DNA中以这19种CpG六核苷酸为核心的12碱基寡核苷酸(ODN);使用生物软件RNAstructure version 3.71(该软件既可预测RNA的二级结构,也可预测DNA的二级结构),对12碱基ODN的二级结构进行了预测,找出了4种可能的共同结构特征:(1)共同特征1:cg位于干环顶端;(2)共同特征2:gc位于干环顶端;(3)共同特征3:cg或gc位于干环侧面;(4)共同特征4:无干环结构(见图1)。 3. BioEdit version 5.0.6, serotype 2,5Adv DNA to find out to which 19 kinds of CpG hexanucleotide core 12 base oligonucleotide (the ODN); the use of biological software RNAstructure version 3.71 (the software RNA secondary structure prediction can also be predicted secondary structure of DNA), the secondary structure of the 12-base ODN is predicted, identified four possible common structural features: (1) a common feature: dry the top ring located cg; (2) common feature 2: gc top ring located dry; (3) common feature 3: cg gc located or dry side of the ring; (4) common feature 4: uninvolved ring structure (see FIG. 1).

4.合成了10条磷酸硫代骨架的12碱基ODN,其中cg位于干环顶端的3条(ODN1-3)、gc位于干环顶端的3条(ODN 4-6)、cg或gc位于干环侧面的2条(ODN7-8)、无干环结构形成的2条(ODN9-10)。 4. Synthesis of the 12-base ODN 10 Article thio-phosphate backbone, which is located in the dry cg top ring 3 (ODN1-3), gc is located in the dry top ring 3 (ODN 4-6), cg gc located, or 2 (ODN9-10) 2 (ODN7-8), nothing to the ring structure formed by the dry side of the ring.

5. 10条ODN的体外生物学活性的鉴定工作:分离并培养人外周血单个核细胞(hPBMC),调整hPBMC细胞悬液浓度为1×106/ml,加入24孔板中,每孔1ml。 Appraisal vitro biological activity of ODN 5.10: isolated and cultured human peripheral blood mononuclear cells (hPBMC), adjust the cell concentration hPBMC suspension of 1 × 106 / ml, added to 24-well plates, each well 1ml. 置37℃ CO2孵箱培养2h后,加入10μg/ml各待筛选的ODN,置37℃ CO2孵箱培养4h后取细胞培养上清,采用双抗体夹心ELISA法测定细胞培养上清中TNF-的浓度,明确10条ODN诱导hPBMC释放TNF-α的能力(见表2),据此筛选出了2条无明显刺激性的ODN:ODN1和ODN 6;而其它8条ODN具有诱导hPBMC释放TNF-α的能力。 Post-culture 37 ℃ CO2 incubator for 2h, 10μg / ml of each of the ODN to be screened, counter 37 ℃ CO2 incubator culture 4h after taking cell culture supernatant, double antibody sandwich ELISA assay of cell culture supernatant TNF- concentration, clear 10 ODN induction ability hPBMC TNF-α (see Table 2) is released, whereby the filter 2 ODN had no irritating: ODN1 and ODN. 6; and the other eight ODN induce release of TNF- hPBMC α ability.

表2.10条寡核苷酸10μg/ml刺激时诱导hPBMC释放TNF-α能力的测定(n=3, x±s) Table 2.10 hPBMC induced oligonucleotide 10μg / ml ability to stimulate the release of TNF-α was determined (n = 3, x ± s)

6.使用生物软件RNAstructure version 3.71,对ODN的二级结构自由能进行了分析,在此基础上,对上述10条ODN的刺激性与结构之间的关系进行了分析并发现,ODN的生物活性可能与预测二级结构的4种共同结构特征无关;而可能与预测二级结构的自由能高低密切相关:不含GGCG结构且自由能大于-1.0的ODN可能为无免疫刺激性的ODN(见表3)。 6. The use of biological software RNAstructure version 3.71, the secondary structure of the free energy of ODN was analyzed on this basis, the relationship between the configuration of the stimulatory ODN 10 were analyzed and found that the biological activity of ODN may be independent of four kinds of common structural features predicted secondary structure; the free energy level may predict the secondary structure is closely related to: free GGCG structure and free energy of greater than -1.0 may be non-ODN immunostimulatory ODN (see table 3). 因此,使用生物软件RNAstructure version 3.71对CpG-N ODN分子进行了再设计,根据此设计合成了11条磷酸硫代骨架的ODN。 Thus, the use of biological RNAstructure version 3.71 software pair of CpG-N ODN molecules redesign, designed and synthesized in accordance with this Article ODN 11-thio phosphate backbone.

表3. 10条ODN的刺激性与结构之间的关系 The relationship between the table and the structure stimulatory ODN of 3.10

7. 11条ODN的体外生物学活性的鉴定工作:方法同步骤5,据此筛选出了5条无明显刺激性的ODN:ODN15、18、19、20、21;而其它6条ODN具有诱导hPBMC释放TNF-α的能力(见表4)。 Appraisal vitro biological activity of ODN 7.11: Step 5 with a method, whereby a screened 5 ODN had no irritating: ODN15,18,19,20,21; while the other six ODN induce TNF-α is the ability (see Table 4) hPBMC release.

表4. 11条寡核苷酸10μg/ml刺激时诱导hPBMC释放TNF-α能力的测定(n=3, x±s) HPBMC induced Table 4.11 oligonucleotide 10μg / ml ability to stimulate the release of TNF-α was determined (n = 3, x ± s)

实验例2本实验例在于研究寡核苷酸的合成方法。 Experimental Example 2 Experimental Example that the method for synthesizing oligonucleotides study.

本发明所述的方法采用固相亚磷酰胺三酯法,简单地说,是溶液中的单体通过缩合反应形成3'→5'磷酸二酯键,从而连接到固相支持物上。 The method of the present invention, the solid phase phosphoramidite triester method, briefly, a solution of the monomers to form 3 '→ 5' phosphodiester bond by a condensation reaction, thereby attached to a solid support.

1.基本材料:(1)支持物:固相合成是将核酸固定在固相载体上完成合成反应的,最常用的固相载体为可控微孔玻璃珠(CPG,controlled pore glass),CPG的孔径根据所合成的寡核苷酸的长度而定,一般合成链长小于60nt时,选择孔径500埃,链长大于60nt时,使用1000埃CPG,使用CPG的缩合效率高达98%-99.9%,可以满足合成长达175nt的寡核苷酸的条件。 1. Materials: (1) support: Solid phase synthesis is complete nucleic acid synthesis reaction on a fixed solid support, the most common solid support is controlled pore glass beads (CPG, controlled pore glass), CPG the pore size of oligonucleotides synthesized on the length, generally less than 60 nt long chain synthetic, 500 Å pore size selected, when the chain length greater than 60 nt, 1000 Å CPG, CPG using condensing efficiency up to 98% -99.9% meet up 175nt synthetic oligonucleotides condition. CPG通过连接化合物与初始核苷酸的羟基共价结合,核苷酸的5'羟基用二甲氧基三苯甲基(DMT)保护。 CPG linker compound with a hydroxyl group by binding covalently initial nucleotides, nucleotide 5 'hydroxyl with dimethoxytrityl (DMT) protected.

(2)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。 (2) Monomer: Synthesis of the monomers are nucleoside phosphoramidite, or a chemically modified nucleotide, containing several functional groups below.

1. 3'位P上二异丙胺基,缩合所用的功能基2. 3'位P上腈乙基,保护基,合成完毕后脱去。 1.3 'functional group at position P diisopropylamine, 2.3 condensed used' off-cyanoethyl the position P, the protecting group, after synthesis is completed.

3. 5'-DMT,保护基,缩合前脱去。 3. 5'-DMT, protecting group removed prior to the condensation.

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.

(3)反应步骤:合成的第一步----去封闭(Deblocking),用三氯乙酸去除CPG所连核苷上的DMT,以暴露5'羟基,供下一步缩合。 (3) reaction step: the first step of the synthesis ---- deblocked (Deblocking), CPG is removed with trichloroacetic acid on DMT nucleosides attached, to expose the 5 'hydroxyl group for the next condensation. 此步需要注意TCA为一较强的酸,可能会有脱嘌呤作用,故TCA与寡核苷酸接触时间不要超过规定时间。 Note that this step TCA is a strong acid, there may be depurination, it is in contact with an oligonucleotide TCA no more than a predetermined time.

第二步----活化(Activation),在缩合之前,单体与四唑混合并进入合成柱,此时四唑提供一个质子给3'磷酸上二异丙胺基的N原子,质子化的二异丙胺是一个良好的游离基团,与四唑形成亚磷酰胺四唑这种活性中间体。 ---- The second step activation (Activation), prior to the condensation, and the monomer is mixed with tetrazole to the synthesis column, this time to provide a proton-tetrazol-N atom 3 'phosphorylated diisopropylamine groups, protonated diisopropylamine is a good free radicals, tetrazole forming phosphoramidite and tetrazole intermediates such activity. 此步四唑过量保证了单体活化充分。 This step ensures that the excess monomer tetrazole full activation.

第三步----连接(Coupling),亚磷酰胺四唑与CPG所连的核苷酸碰撞时,与其5'羟基发生亲核反应,发生缩合并脱掉四唑,合成的寡核苷酸链延长一个。 ---- connecting the third step (Coupling), phosphoramidite and tetrazole attached CPG nucleotide collision, its 5 'hydroxyl nucleophilic reactions, the occurrence of condensation and off-tetrazole, synthetic oligonucleotide a chain extension. 此步单体相对于CPG所连核苷酸上5'-羟基过量保证了连接的高效率。 This step CPG monomer to the 5'-hydroxyl group attached polynucleotide excess ensures efficient connection.

第四步----盖帽(Capping),为了防止未反应的与CPG相连的5'羟基在随后的循环中被延长,需要在连接反应充分进行之后使之封闭,常用乙酰化来封闭此羟基。 The fourth step ---- cap (Capping), in order to prevent the 5 'hydroxyl group attached to CPG unreacted subsequent cycle is extended, it is necessary to make the closure, conventional acetylation reaction sufficiently proceeds after the connection is closed this hydroxy . 临用前混合乙酸酐和N-甲基咪唑等形成一活性很强的乙酰化试剂,与少量为参与连接反应的5羟基缩合成酯键。 Mixed immediately prior to use acetic anhydride and N- methylimidazole formed a highly active acetylating agent, and a small amount of 5-hydroxy-coupling reaction involved condensation of an ester bond. 由于须封闭的羟基少且乙酰化试剂活性高和充分过量,此步反应速度很快,几秒钟即足够。 To be closed due to the small hydroxyl groups and high acetylating agent activity and sufficient excess, this reaction step is fast, a few seconds is sufficient. 太长的盖帽时间有在非预期位置发生乙酰化反应的可能,且增加乙酸酐与痕量水形成酸攻击新生成的亚磷酯键的危险性。 Cap long time acetylation reaction may occur in a non-intended position, and increases with acetic anhydride to form an acid attack traces of water newly generated hazardous phosphorous ester bond.

第五步----氧化(Oxidation),连接反应后新加上的核苷酸通过亚磷酯键(磷为三价)与CPG上相连的寡核苷酸链连接,此亚磷酯键不稳定,易被酸、碱水解,因此需将此处三价磷氧化为五价的磷。 ---- fifth step oxidation (Oxidation), the reaction after the newly added nucleotides are connected by an ester bond phosphoramidite (trivalent phosphorus) is connected to the oligonucleotide strands connected on CPG, this phosphorous ester bond unstable and susceptible to acid, base hydrolysis, and therefore need to herein trivalent phosphorus is oxidized to pentavalent phosphorus. 常用的氧化剂为碘的四氢呋喃溶液。 Commonly used oxidizing agent is a solution of iodine in tetrahydrofuran. 此步反应速度亦很快。 This step reaction speed is also fast. 应注意最后一个循环时,氧化步骤不可省略。 It is noted that when the last cycle, the oxidation step can not be omitted. 此外亦可用其他氧化剂来完成氧化,从而得到各种符合实验需要的寡核苷酸。 Also other oxidants can also be used to complete the oxidation, to obtain a variety of oligonucleotide with the experimental needs.

循环上述一至五步,寡核苷酸链即可延伸至所需长度时即可完成。 To a five-step cycle described above, the oligonucleotide chain can be extended to the desired length to complete.

2.合成后处理: 2. Synthesis of post-treatment:

(1.)切割:将合成好的寡核苷酸链从支持物上化学切割下来。 (1) Cutting: good synthetic oligonucleotide strands from the support chemically cleaved. 常用新鲜的浓氨水来裂解CPG上连接化合物与初始核苷间的酯键。 Fresh concentrated aqueous ammonia used to cleave the ester linkage between the linker compound with the initial CPG nucleoside. 断裂下来的寡核苷酸带有自由的3'羟基。 3 'hydroxyl oligonucleotide cleaved with freedom.

(2.)脱保护基:须在此步前选好后续的纯化方法。 (2) Deprotection: the subsequent purification method to be selected before this step. 一般用新鲜的浓氨水处理较长时间以脱掉腈乙基、苯甲酰基、异丙基,用三氟乙酸脱5'-DMT。 Usually longer treated with fresh concentrated ammonia at off-cyanoethyl, benzoyl group, an isopropyl group, a 5'-DMT off with trifluoroacetic acid.

(3.)纯化:根据所合成寡核苷酸的组成和应用来选定纯化的方法。 (3) Purification: Purified be selected according to the composition and method of application of the synthetic oligonucleotide. 常用的纯化方法有:C18柱、OPC柱、PAGE和HPLC。 Conventional purification methods: C18 column, OPC column, PAGE, and HPLC.

(4.)定量。 (4) quantitatively. 根据寡核苷酸在260nm处的紫外吸收来定量。 Oligonucleotides ultraviolet absorption at 260nm according quantified.

(5.)储存。 (5) storage. 通常一次合成提供的寡核苷酸的量会比所需要的量大得多,所以会涉及到寡核甘酸的储存问题。 Typically the amount of time provided by the synthesis of oligonucleotides is much greater than the amount required, so the problem will involve storage of Oligonucleotides. 一般来说,这不成为问题,因为寡核甘酸的稳定性很好。 In general, this is not a problem, because a good stability of Oligonucleotides. 需要注意的是避免紫外线等剧烈的物理环境,避免反复冻融,-20℃可稳定保存一年以上。 Note that to avoid strenuous physical environment such as ultraviolet rays, to avoid repeated freezing and thawing, -20 ℃ stable for more than one year.

实验例3本实施例是对7条无明显刺激性的ODN抑制CpG-S ODN的活性进行鉴定。 Experimental Example 3 This example is in no stimulatory ODN inhibitory activity of CpG-S ODN were identified to 7.

分离并培养人外周血单个核细胞(hPBMC),调整hPBMC细胞悬液浓度为1×106/ml,加入24孔板中,每孔1ml。 Isolated and cultured human peripheral blood mononuclear cells (hPBMC), adjust the cell concentration hPBMC suspension of 1 × 106 / ml, added to 24-well plates, each well 1ml. 置37℃ CO2孵箱培养2h后,加入10μg/ml各待筛选的ODN 30分钟后,再加入5μg/ml的CpG-S ODN(TGG CGC GGGGCG),置37℃ CO2孵箱培养4h后取细胞培养上清,采用双抗体夹心ELISA法测定细胞培养上清中TNF-α的浓度,明确上述7条ODN抑制CpG-S ODN诱导hPBMC释放TNF-α的能力,此次实验结果表明上述7条ODN均有抑制CpG-S ODN诱导hPBMC释放TNF-α释放的能力,因此我们正式称之为CpG-N ODN(见表5)。 After 2h facing CO2 incubator cultured 37 ℃, addition of 10μg / ml of each to be screened ODN 30 minutes, then added 5μg / ml of CpG-S ODN (TGG CGC GGGGCG), opposing 37 [deg.] C CO2 incubator culture 4h after taking cells culture supernatant, double antibody sandwich ELISA assay of cell culture supernatant TNF-α concentrations of ODN clearly above 7 the ability to inhibit TNF-α induced by CpG-S ODN hPBMC released, the above results show that ODN 7 inhibition of CpG-S ODN were induced TNF-α release from hPBMC release capability, so we formally called CpG-N ODN (Table 5).

表5.CpG-N ODN对CpG-S ODN诱导人PBMC释放TNF-α的影响(n=6, x±s) Table 5.CpG-N ODN affect the release of TNF-α induced by CpG-S ODN on human PBMC (n = 6, x ± s)

★:与CpG-S ODN组比较,p<0.01 ★: Comparison and CpG-S ODN group, p <0.01

实验例4本实施例在于研究CpG-N ODN对CpG-S ODN攻击小鼠的保护作用。 Experimental Example 4 This example is to study the protective effect of CpG-N ODN CpG-S ODN challenged mice.

清洁级昆明种小白鼠90只(第三军医大学实验动物中心提供),体重19.9±O.5g/只,雌雄不拘,随机分为对照组、CpG-S ODN、ODN1、ODN6、ODN15、ODN18、ODN19、ODN20、ODN21组。 Kunming species mice cleaning stage 90 (Experimental Animal Center, Third Military Medical University), weighing 19.9 ± O.5g / only, either male or female, were randomly divided into the control group, CpG-S ODN, ODN1, ODN6, ODN15, ODN18, ODN19, ODN20, ODN21 group. 对照组给予注射用水,注射体积为200μl/20g体重。 Control group was given water for injection, an injection volume of 200μl / 20g body weight. CpG-S ODN组,给予80μg/20g体重的CpG-S ODN;其余各组在给予相应的CpG-NODN 400μg/20g体重后,立即给予80μg/20g CpG-S ODN;给药方式为尾静脉注射,观察7天内小鼠一般情况及死亡率(表6)。 CpG-S ODN group received 80μg / 20g body weight of CpG-S ODN; in the other groups to give the corresponding CpG-NODN 400μg / 20g body weight, administered immediately 80μg / 20g CpG-S ODN; administration is intravenous injection , the mice were observed for 7 days, and general mortality (table 6). 结果显示CpG-S ODN可导致小鼠3天内全部死亡,7条CpG-N ODN可降低CpG-S ODN攻击小鼠的死亡率。 The results show that CpG-S ODN may result in death of all mice 3 days, 7 CpG-N ODN can reduce mortality CpG-S ODN challenged mice.

表6. 7条CpG-N ODN对CpG-S ODN攻击小鼠的保护作用 Table 6.7 Protective effects of CpG-N ODN of CpG-S ODN challenged mice

★:与CpG-S ODN组比较,p<0.05 ★: Comparison and CpG-S ODN group, p <0.05

SEQUENCE LISTING&lt;110&gt;中国人民解放军第三军医大学&lt;120&gt;高免疫活性CpG-S ODN和拮抗CpG-S ODN作用的CpG-N ODN的基因序列及其应用&lt;130&gt;2672-107&lt;140&gt;200410034787.X&lt;141&gt;2004-05-17&lt;160&gt;21&lt;170&gt;PatentIn version 3.1&lt;210&gt;1&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;1ggcggcggcg cg 12&lt;210&gt;2&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;2cgcggcgccg gg 12&lt;210&gt;3&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;3ggcggcggcg ga 12&lt;210&gt;4&lt;211&gt;12&lt;212&gt;DNA SEQUENCE LISTING & lt; 110 & gt; Third Military Medical University & lt; 120 & gt; highly immunoreactive CpG-S ODN and antagonistic CpG-S ODN effect of CpG-N ODN gene sequence and its application & lt; 130 & gt; 2672-107 & lt; 140 & gt ; 200410034787.X & lt; 141 & gt; 2004-05-17 & lt; 160 & gt; 21 & lt; 170 & gt; PatentIn version 3.1 & lt; 210 & gt; 1 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt; 1ggcggcggcg cg 12 & lt; 210 & gt; 2 & lt ; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt; 2cgcggcgccg gg 12 & lt; 210 & gt; 3 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt; 3ggcggcggcg ga 12 & lt; 210 & gt; 4 & lt; 211 & gt; 12 & lt ; 212 & gt; DNA

&lt;213&gt;未知&lt;400&gt;4ccgcgcgcgg gc 12&lt;210&gt;5&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;5tggcgcgggg cg 12&lt;210&gt;6&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;6gccggcgccc gg 12&lt;210&gt;7&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;7gcgcgcgcgg ta 12&lt;210&gt;8&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;8ccaggcggcg gg 12&lt;210&gt;9&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;9ggccgcgggg gt 12 & Lt; 213 & gt; unknown & lt; 400 & gt; 4ccgcgcgcgg gc 12 & lt; 210 & gt; 5 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt; 5tggcgcgggg cg 12 & lt; 210 & gt; 6 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt; 6gccggcgccc gg 12 & lt; 210 & gt; 7 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt; 7gcgcgcgcgg ta 12 & lt; 210 & gt; 8 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt ; 8ccaggcggcg gg 12 & lt; 210 & gt; 9 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt; 9ggccgcgggg gt 12

&lt;210&gt;10&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;10cagcccgggg ga 12&lt;210&gt;11&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;11gctcccgggc tt 12&lt;210&gt;12&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;12gcgctcgggc tt 12&lt;210&gt;13&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;13ccgcccgcgc ct 12&lt;210&gt;14&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;14gcacccgcgc gc 12&lt;210&gt;15&lt;211&gt;12&lt;212&gt;DNA & Lt; 210 & gt; 10 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt; 10cagcccgggg ga 12 & lt; 210 & gt; 11 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt; 11gctcccgggc tt 12 & lt; 210 & gt; 12 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt; 12gcgctcgggc tt 12 & lt; 210 & gt; 13 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt; 13ccgcccgcgc ct 12 & lt; 210 & gt; 14 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt; 14gcacccgcgc gc 12 & lt; 210 & gt; 15 & lt; 211 & gt; 12 & lt; 212 & gt; DNA

&lt;213&gt;未知&lt;400&gt;15ggccgcgggg ac 12&lt;210&gt;16&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;16gccgccgccg cc 12&lt;210&gt;17&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;17cgcctcgggg ag 12&lt;210&gt;18&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;18tgccgcggca ga 12&lt;210&gt;19&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;19ctccacgtgc cg 12&lt;210&gt;20&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;20gccgccgccg tc 12 & Lt; 213 & gt; unknown & lt; 400 & gt; 15ggccgcgggg ac 12 & lt; 210 & gt; 16 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt; 16gccgccgccg cc 12 & lt; 210 & gt; 17 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt; 17cgcctcgggg ag 12 & lt; 210 & gt; 18 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt; 18tgccgcggca ga 12 & lt; 210 & gt; 19 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt ; 19ctccacgtgc cg 12 & lt; 210 & gt; 20 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt; 20gccgccgccg tc 12

&lt;210&gt;21&lt;211&gt;12&lt;212&gt;DNA&lt;213&gt;未知&lt;400&gt;21gctgccgccg cc 12 & Lt; 210 & gt; 21 & lt; 211 & gt; 12 & lt; 212 & gt; DNA & lt; 213 & gt; unknown & lt; 400 & gt; 21gctgccgccg cc 12

Claims (6)

1.一种高免疫活性的免疫刺激寡核苷酸,其特征在于,具有如下基因序列:GGC GGC GGC GCG;CGC GGC GCC GGG;GGC GGC GGC GGA;CCG CGC GCG GGC;TGG CGC GGG GCG;GCC GGC GCC CGG;GCG CGC GCG GTA;CCA GGC GGC GGG;GGC CGC GGG GGT;CAG CCC GGG GGA;GCT CCC GGG CTT;GCG CTC GGG CTT;CCG CCC GCG CCT;或GCACCC GCG CGC其中的一种。 A highly immunoreactive immunostimulatory oligonucleotide, wherein the gene has the following sequence: GGC GGC GGC GCG; CGC GGC GCC GGG; GGC GGC GGC GGA; CCG CGC GCG GGC; TGG CGC GGG GCG; GCC GGC GCC CGG; GCG CGC GCG GTA; CCA GGC GGC GGG; GGC CGC GGG GGT; CAG CCC GGG GGA; GCT CCC GGG CTT; GCG CTC GGG CTT; CCG CCC GCG CCT; GCACCC GCG CGC or one of them.
2.根据权利要求1所述的一种高免疫活性的免疫刺激寡核苷酸,其特征在于,具有如下基因序列:GGC GGC GGC GCG;CCG CGC GCG GGC;TGG CGC GGG GCG;GCC GGC GCC CGG;GGC CGC GGG GGT;CAG CCC GGG GGA;GCT CCC GGG CTT;GCG CTC GGG CTT;或CCG CCC GCG CCT其中的一种。 The immunological activity of a highly immunostimulatory oligonucleotide according to claim 1, wherein the gene has the sequence: GGC GGC GGC GCG; CCG CGC GCG GGC; TGG CGC GGG GCG; GCC GGC GCC CGG ; GGC CGC GGG GGT; CAG CCC GGG GGA; GCT CCC GGG CTT; GCG CTC GGG CTT; CCG CCC GCG CCT or one of them.
3.一种对免疫刺激寡核苷酸拮抗的寡核苷酸,其特征在于,具有如下基因序列:GGC CGC GGG GAC;GCC GCC GCC GCC;CGC CTC GGG GAG;TGC CGC GGC AGA;CTC CAC GTG CCG;GCC GCC GCC GTC;或GCT GCC GCC GCC其中的一种。 An immunostimulatory oligonucleotide of antagonizing oligonucleotides, wherein the gene has the sequence: GGC CGC GGG GAC; GCC GCC GCC GCC; CGC CTC GGG GAG; TGC CGC GGC AGA; CTC CAC GTG CCG; GCC GCC GCC GTC; GCT GCC GCC GCC or one of them.
4.根据权利要求3所述的对免疫刺激寡核苷酸拮抗的寡核苷酸,其特征在于,具有如下基因序列:CGC CTC GGG GAG;TGC CGC GGC AGA;CTC CAC GTG CCG;GCC GCC GCC GTC;或GCTGCC GCC GCC其中的一种。 According to claim immunostimulatory oligonucleotide for antagonizing an oligonucleotide of claim 3, wherein the gene has the following sequence: CGC CTC GGG GAG; TGC CGC GGC AGA; CTC CAC GTG CCG; GCC GCC GCC GTC; GCTGCC GCC GCC or one of them.
5.权利要求1或2所述的高免疫活性的免疫刺激寡核苷酸在用于制备治疗感染性疾病、过敏性疾病、免疫缺陷性疾病和肿瘤药物中的应用。 Treatment of infectious diseases, allergic diseases, immunodeficiency diseases and tumors in the preparation of medicaments for the high immunocompetent immunostimulatory oligonucleotide according to claim 1 or 2 nucleotides.
6.权利要求3或4所述的对免疫刺激寡核苷酸拮抗的寡核苷酸在用于制备治疗SIRS和脓毒症药物中的应用。 For antagonistic immunostimulatory oligonucleotide or oligonucleotide 3 according to claim 4 in the manufacture of a medicament for the treatment of sepsis and SIRS nucleotides.
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