共查询到18条相似文献，搜索用时 343 毫秒
1.
为获得对猪具有免疫刺激活性的含CpG序列的重组质粒,设计合成了11条CpG序列,应用MTS比色法测定合成CpG ODNs刺激猪PBMC增殖的能力,结果有10条CpG ODN对猪PBMC具有刺激活性(SI>2);以对猪PBMC具有较高刺激活性的CpG06和CpG08为核心,分别合成含6次重复序列的重组质粒pCpG06和pCpG08,并检测其对猪PBMC的刺激活性,结果重组质粒pCpG06和pCpG08对猪PBMC的刺激指数分别可达4.97和5.32,并可刺激猪PBMC产生较高水平的IL-4。研究获得的重组质粒pCpG06和pCpG08对猪PBMC具有较好的刺激活性,可为猪用CpG佐剂的研究提供参考。 相似文献
2.
CpG ODN对传染性腔上囊病病毒活疫苗免疫效果的影响 总被引：2，自引：0，他引：2
将120只来航系SPF鸡随机分成对照组、注射CpG ODN组、IBDV活疫苗点眼免疫组及IBDV活疫苗 CpG ODN免疫组，每组30只，于7日龄时对试验鸡进行初次免疫，21日龄时进行加强免疫，28日龄时用IBDV野毒株攻毒。检测不同时间的IBDV特异性抗体效价、血液和脾淋巴细胞的Con A刺激指数(SI)。结果表明，CpG ODN处理后脾细胞的增殖转化能力强于血液淋巴细胞的增殖转化能力；CpG ODN的使用使血清中IBDV特异性抗体产生时间提前7d，抗体效价升高；与对照组相比，仅用CpG ODN处理就可使1／3的鸡攻毒后迅速产生高效价(约1：3000)的特异性抗体，降低了发病率和死亡率，证明CpG ODN能增强IBDV活疫苗的免疫效果。 相似文献
3.
为了探讨CpG寡核苷酸（CpG oligonucleotide,CpG ODN）对鸡新城疫疫苗免疫效力的影响,将CpG2007与鸡淋巴细胞共孵育,测定淋巴细胞增殖率,结果发现CpG2007对鸡淋巴细胞具有显著的刺激活性。将CpG2007与不同浓度的新城疫抗原混合,制备灭活疫苗,免疫健康雏鸡。分别于免疫后不同时间采血,测定抗体效价和细胞因子表达量,并进行攻毒保护试验。结果发现,添加CpG ODN佐剂的试验组均比对应相同抗原剂量的免疫对照组的抗体水平高,产生抗体速度快;抗原剂量降低10倍的佐剂试验组与高抗原剂量免疫对照组抗体水平和攻毒保护率均相当,表明CpG ODN能显著增强新城疫疫苗的免疫效力,能促进机体产生更强烈的免疫应答,是有效的疫苗佐剂候选物质。 相似文献
4.
囊素是存在于禽腔上囊中能诱导B细胞分化的活性肽类物质,其化学组成为三肽Lys-His-Gly-NH2。本试验探讨了猪外周血单个核细胞的分离培养条件,并研究了人工合成的囊素对体外培养的猪外周血单个核细胞（PBMC）的免疫刺激活性。采用猪PBMC体外增殖实验,3H-脱氧胸苷（3H-TdR）掺入法测定刺激指数（SI）。结果表明,用猪淋巴细胞分离液（1.110g/cm3）在1500r/min、30min条件下细胞得率及细胞活力最高,分别为7.26×107、91.5%。体外培养的PBMC经不同浓度的囊素刺激24h后,细胞体积明显增大、数目增多,在同一视野中细胞聚集成团的数目明显增多。4μg囊素/孔与8μg囊素/孔组每分钟脉冲数（cpm值）分别达821.60±6.90和916.70±9.25,刺激指数（SI值）分别达4.32±0.12、4.83±0.15,表明囊素对体外培养的猪PBMC具有明显的免疫刺激活性,且在一定浓度范围内囊素对猪PBMC的免疫刺激活性有剂量依赖关系。 相似文献
5.
CpG ODN对仔猪淋巴细胞增殖和细胞因子诱生的影响:在体和离体试验 总被引：1，自引：0，他引：1
将CpGODN、non CpG ODN分别接种初生仔猪，同时分离仔猪的外周血单核细胞，以CpGODN、non CpG ODN对外周血单核细胞体外培养，分别检测仔猪Th1型细胞因子分泌情况。结果表明，与non CpG ODN和对照组相比，CpG ODN能在体内显著提高仔猪的淋巴细胞白介素-2诱生活性、淋巴细胞增殖、血清中IFN-γ和IL-12水平（P〈0．05），亦能在体外刺激仔猪的外周血单核细胞分泌IFN-γ和IL-12（P〈0．01）。这显示CpG—ODN能显著增强动物的免疫应答能力。 相似文献
6.
CpG-DNA免疫刺激活性研究进展 总被引：2，自引：0，他引：2
CpG—DNA是一些具有免疫激活功能的以未甲基化的CpG基序(CpG motif)为核心的DNA序列，它包括含CpG基序的人工合成的寡聚脱氧核苷酸(oligodeoxvnucleotides，ODN)和自然界中某些低等生物的基因组DNA。近几年研究证实，CpG基序能在体内诱导强烈的免疫反应，主要包括激活B细胞增殖、分化、分泌白细胞介素-12(IL-12)及其他细胞因子，激活自然杀伤细胞(NK)的裂解活性，分泌 相似文献
7.
CpG ODN对鸡新城疫LaSota活疫苗的免疫增强效应 总被引：4，自引：0，他引：4
将3种不同的未甲基化CpGODN分别与新城疫LaSota活疫苗混合后，经滴鼻和点眼免疫鸡，通过检测鸡血清中HI抗体、外周血T淋巴细胞增殖活性、诱导巨噬细胞分泌NO含量，以及刺激外周血淋巴细胞表达IFN-γ、IL-6与IL-1β mRNA量，分析各CpGODN对新城疫LaSota活疫苗免疫效果的影响。结果表明，经2次免疫后，含GTCGTT核心基序的CpG ODN1组，鸡血清平均HI抗体效价最高达8．2log2、淋巴细胞刺激指数达9．836、NO分泌量达35．833μmol／L，分别比疫苗单独免疫组高出2个滴度（P〈0．05）、4．4（P〈0．01）、27．6μmol／L（P〈0．01）；含GACGTT核心基序的CpG ODN2组增强作用不明显．与疫苗单独免疫组无差异；而CpGODN3的免疫刺激活性由于受其侧翼序列的影响，作用明显减弱甚至丧失。对细胞因子的影响，CpGODN3组IFN-γ mRNA表达量稍高于CpG ODN1组，而其余细胞因子均以CpG ODN1组表达水平最高（P〈0．05）。由此证明CpG ODN1能显著增强鸡对新城疫LaSota活疫苗的体液和细胞免疫反应，可以作为高效的免疫增强剂。 相似文献
8.
为构建长链CpG ODN DNA,将合成的含有CpG的GTCGTT核心序列以TT间隔开后重复3次,并在3′端添加polyG结构,而后在其两翼人为添加Xba Ⅰ的黏性末端(简称为A链)后合成与其互补的互补链(简称为B链),通过退火形成CpG ODN DNA单体,以DNA连接酶将CpG单体连接为多聚体后再以A链和B链作为引物,利用SOE-PCR技术对poly CpG ODN DNA进行长链扩增,分别将600～700 bp片段克隆到pMD-18T载体中,并获得相应的重组子pUC44、pUC26及pUC30.序列分析结果表明:3个重组子与理论上产生的含有10,11,13个CTAGATCGT(CG)TTTTGT(CG)TTTTGT(CG)TTGAGGGGGGAT拷贝长链CpG ODN DNA标准序列的同源性分别为97.4%、97.9%和97.1%. 相似文献
9.
10.
旨在克隆获得水貂DCT基因5′UTR序列并分析其结构特征,预测转录调控元件并检测启动子活性,为探究DCT基因在调控水貂毛皮颜色形成中的作用提供理论依据。本研究利用PCR扩增黑貂、白貂和咖啡貂DCT基因5′UTR,构建咖啡貂DCT基因5′UTR的pGL3-1～pGL3-7和黑貂pGL3-4～pGL3-6缺失片段的荧光素酶报告基因重组质粒,检测各片段的启动子活性;利用亚硫酸氢盐法检测3种毛色水貂DCT基因启动子区CpG岛甲基化水平。结果,克隆获得水貂DCT基因长8 203 bp的5′UTR序列,发现g.7133-7336为长204 bp的转座元件,与其高相似度的100条序列中,一条为蜕皮动物总门线虫纲的索巴利吸虫,其他均来自犬形亚目。P3和P4片段具有显著的启动子活性(P<0.05);咖啡貂的CpG岛甲基化水平显著高于黑貂和白貂(P<0.05);咖啡貂CC单倍型启动子活性显著低于黑貂的TT单倍型片段(P<0.05)。结果表明,水貂DCT基因5′UTR长204 bp的犬形亚目特异短散在元件Can-SINEs由蜕皮动物门的索巴利吸虫侵入动物基因组形成;基因上游32 bp元件和近端域共同作用发挥启动子活性,而GC-box和CpG岛结构沉默水貂DCT基因启动;g.-684和g.-621位点的T> C突变形成的CC单倍型导致咖啡貂DCT基因的高甲基化与低启动子活性,从而抑制真黑素合成,产生咖啡色被毛特征。 相似文献
11.
Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides 总被引：10，自引：0，他引：10
Exposure to bacterial DNA generates a "danger signal" that stimulates cellular elements of the mammalian immune system to proliferate and/or secrete cytokines. Stimulation is critically dependent on hexameric motifs that contain an unmethylated CpG dinucleotide: these are commonly found in bacterial but not vertebrate DNA. Different motifs are optimally stimulatory in different species. This work examines whether oligodeoxynucleotides (ODNs) containing CpG motifs stimulate peripheral blood mononuclear cells from pigs. Results show that pigs respond to CpG ODN by proliferating and secreting IL-6, IL-12 and TNF-alpha. By screening a large panel (>100) of ODNs, the palindromic hexamer 'ATCGAT' was identified as being optimally active in all animals examined (N=10). These findings are the first to establish the immunostimulatory activity of CpG ODN in pigs, and suggest that the therapeutic uses envisioned for these ODNs (as vaccine adjuvants and immunoprotective agents) may be applicable to husbandry animals. 相似文献
12.
Dar A Nichani AK Benjamin P Lai K Soita H Krieg AM Potter A Babiuk LA Mutwiri GK 《Veterinary immunology and immunopathology》2008,123(3-4):324-336
The immune stimulatory effects of synthetic CpG DNA, on porcine peripheral blood mononuclear cells (PBMC) have been reported, but little is known about CpG-induced responses in other lymphoid tissues of pigs. We investigated innate immune responses induced by CpG DNA in cells from blood, lymph nodes (LN) and spleens of pigs. Porcine PBMC and lymph node cells (LNC) were stimulated in vitro with three classes (A-, B- and C-class) of CpG oligodeoxynucleotides (ODNs), and a non-CpG control ODN. All three classes of CpG ODNs induced significant production of IFNalpha, TNFalpha, IL-1, IL-6 and IL-12 in PBMC. In contrast, in LNC, only IL-12 was stimulated by all three classes of CpG ODNs, while IFNalpha, and IL-6 were induced by A- and C-class ODNs. No TNFalpha was induced in LNC by any of the ODNs. Significant lymphocyte proliferation was induced in PBMC by all three classes of CpG ODNs and non-CpG control. However, in LNC, B- and C-class ODNs induced significant proliferation, while no proliferation was seen with A-class and non-CpG control ODN. All three classes of ODNs induced NK-like cytotoxicity in PBMC and spleen cells, but were less effective in inducing NK cytotoxicity in LNC. We then investigated the reasons for the relatively poor CpG-induced responses in LNC. Our investigations revealed that LNC had a lower frequency of IFNalpha-secreting cells and expressed low levels of TLR9 mRNA compared to PBMC. We conclude that the lower number of IFNalpha-secreting cells and receptor expression may contribute to the attenuated responses in LNC following stimulation with CpG ODN. 相似文献
13.
Monocytes are required for optimum in vitro stimulation of bovine peripheral blood mononuclear cells by non-methylated CpG motifs 总被引：7，自引：0，他引：7
Pontarollo RA Rankin R Babiuk LA Godson DL Griebel PJ Hecker R Krieg AM van Drunen Littel-van den Hurk S 《Veterinary immunology and immunopathology》2002,84(1-2):43-59
Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs within certain flanking base pairs are recognized as a danger signal by the innate immune system of vertebrates. Using lymphocyte proliferative response (LPR) and IFN-gamma secretion assays, a panel of 38 ODN was screened for immunostimulatory activity on bovine peripheral blood mononuclear cells. ODN composed of a nuclease resistant phosphorothioate backbone and a leading 5'-TCGTCGTT-3' motif with two 5'-GTCGTT-3' motifs were highly stimulatory in both assays. Flow cytometric analysis and cell-specific surface marker labeling determined that B-cells (surface IgM(+)) were the primary cell population responding in the LPR assay. Depletion of T cells (CD3(+)) from the PBMC population did not affect IFN-gamma secretion or B-cell proliferation when cultured with CpG-ODN. However, depletion of monocytes (DH59B(+)) completely abrogated the ability of CpG-ODN to stimulate IFN-gamma secretion, and significantly reduced the B-cell proliferative response. These data establish the identity of an optimal immunostimulatory CpG motif for cattle and demonstrate that monocytes play a pivotal role in the ability of cell populations to respond to CpG-ODN. These data provide insight for future studies investigating the mechanism of CpG-ODN bioactivity and its application in novel vaccine formulations and immunotherapy. 相似文献
14.
Domeika K Magnusson M Eloranta ML Fuxler L Alm GV Fossum C 《Veterinary immunology and immunopathology》2004,101(1-2):87-102
The immunostimulatory effects of oligodeoxyribonucleotides (ODN) containing unmethylated CpG dinucleotides (CpG-ODN) in certain base contexts have been extensively studied in man and mice. One major action is their ability to trigger production of massive amounts of interferon-alpha (IFN-alpha) by plasmacytoid dendritic cells (PDC), also referred to as natural IFN-alpha/beta producing cells (NIPC). The present study using porcine PBMC activated by CpG-ODN or plasmid DNA revealed a considerable variation in the IFN-alpha production in response to various CpG-ODN constructs. Several phosphodiester ODNs, such as 5' TTTTCAATTCGAAGATGAAT 3' (ODN H), and the plasmid pcDNA3 all required pre-incubation with lipofectin in order to induce IFN-alpha. Intact unmethylated CpGs were also important, because methylation or substitution of the cytosines and CpG-inversion strongly reduced the IFN-alpha induction by single- or double-stranded forms of ODN H. Certain CpG-ODNs that contained flanking phosphorothioate or phosphodiester poly-G sequences were potent inducers of IFN-alpha without pre-incubation with lipofectin, for instance the ODN 2216 (5' GGGGGACGATCGTCGGGGGG 3'). While poly-G sequences have been suggested to increase uptake of ODNs by cells, they did not obviate the need for lipofectin when added to the ODN H. However, they resulted in up to five-fold increases of the IFN-alpha levels caused by ODN H upon lipofection, indicating other enhancing effects of poly-G sequences on the induction of IFN-alpha. The identity of the IFN-alpha producing cells (IPC) stimulated by CpG-ODN or plasmid DNA was studied by means of flow cytometry using combined staining for intracellular IFN-alpha and surface markers. Approximately 1-3 IPC/10(3) PBMC were detected, compared to only 3 IPC/10(4) PBMC stimulated by Aujeszky's disease virus. The IPC frequencies were confirmed by detection of IFN-alpha mRNA positive cells by in situ hybridisation. The IPC induced by CpG-ODN or plasmid DNA had a similar phenotype, expressing CD2 and CD4 and intermediate levels of MHC class II and the myeloid marker SWC3, but not the markers of T and B cells or monocytes (CD3, CD21 and CD14). Consequently, porcine IPC that respond to CpG-DNA seem to correspond to the PDC/NIPC. 相似文献
15.
Poultry coccidiosis is the major parasitic disease of poultry and, until now, no recombinant vaccine has been developed. Short oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs (CpG ODNs) have been shown to be effective immunoprotective agents and vaccine adjuvants in mammalian systems. Their use in poultry to protect against intracellular parasites has not been reported to date. The present work investigated the effects of CpG ODN treatment on host susceptibility to Eimeria infection in two chicken strains with different genetic background, SC and TK. The data show that CpG ODN enhanced the birds' resistance to coccidiosis in a normally susceptible chicken strain (TK), as shown by reduced oocyst shedding and improved weight gain. CpG treatment had a differential effect on body weight gains and serum antibody responses, depending on the chicken strain and ODN dose, delivery route, and backbone. This study shows for the first time that CpG ODNs could be used as immunoprotective agents in Eimeria-infected chickens to enhance resistance to the pathogen and improve performance. Future research is needed to optimize their use alone and as vaccine adjuvants that may lead to better and more efficient vaccine applications. 相似文献
16.
Catherine M Wernette Bruce F Smith Zachary L Barksdale Rolf Hecker Henry J Baker 《Veterinary immunology and immunopathology》2002,84(3-4):223-236
Oligodeoxynucleotides (ODNs) with unmethylated CpG dinucleotide motifs may be useful as non-specific immune system stimulants and adjuvants for protein or nucleic acid vaccines in humans and other primates. They may also be useful in cancer immunotherapy and in the modulation of allergic responses or mucosal immunity. To begin to determine the potential utility of CpG ODN technology in small animal veterinary medicine, we developed procedures to analyze the effects of CpG ODN on canine and feline blood, spleen and lymph node (LN) cells. We find that certain CpG ODN cause good lymphocyte proliferation (as monitored by [(3)H]-thymidine incorporation) in both canine and feline spleen and LN cells, but not in blood. This overall stimulatory effect of CpG ODN on spleen and LN cells is CpG dependent. The reverse sequences, GpC ODNs, do not cause significant lymphocyte proliferation in the cat; however, dogs are more sensitive to stimulation by the non-specific immune effects of the phosphorothioate backbone. We conclude that unmethylated CpG ODNs may also have potential uses as immune stimulants for vaccines and other antimicrobial agents in veterinary medicine for companion animals. 相似文献
17.
Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) prevent development of T-helper type 2 (Th2) immune response and reverse established allergic responses in mouse models. However, little work on immune responses in piglets has been conducted in vivo. In this report, the ability of a porcine-specific CpG ODN to act as an immunostimulant and enhance immune responses of piglets to swine Pasteurella multocida living vaccine (SPML vaccine) was determined. The titre of IgG and IgG1/IgG2 isotype to SPML vaccine in serum, the proliferation of lymphocytes, SPML-specific interferon-gamma (IFN-gamma) and IL-6, TNF-alpha, IL-4 production of PBMCs in vitro and IFN-gamma, IL-6, TNF-alpha, IL-4, IL-10 in piglets serum were examined to identify the immune responses of the piglets. Immune responses of the piglets vaccinated with SPML and CpG ODN were significantly stronger than responses of piglets vaccinated with SPML alone. All these data summarized that immunostimulatory CpG ODN could modulate the immune response towards a Th1-like response when co-administered to piglets during SPML vaccination, which suggested that the therapeutic uses envisioned for these ODNs (as vaccine adjuvants and immunoprotective agents) may be applicable to husbandry animals. 相似文献
18.
Mutwiri G Pontarollo R Babiuk S Griebel P van Drunen Littel-van den Hurk S Mena A Tsang C Alcon V Nichani A Ioannou X Gomis S Townsend H Hecker R Potter A Babiuk LA 《Veterinary immunology and immunopathology》2003,91(2):89-103
Bacterial DNA contains a much higher frequency of CpG dinucleotides than are present in mammalian DNA. Furthermore, bacterial CpG dinucleotides are often not methylated. It is thought that these two features in combination with specific flanking bases constitute a CpG motif that is recognized as a "danger" signal by the innate immune system of mammals and therefore an immune response is induced when these motifs are encountered. These immunostimulatory activities of bacterial CpG DNA can also be achieved with synthetic CpG oligodeoxynucleotides (ODN). Recognition of CpG motifs by the innate immune system requires engagement of Toll-like receptor 9 (TLR-9), which induces cell signaling and subsequently triggers a pro-inflammatory cytokine response and a predominantly Th1-type immune response. CpG ODN-induced innate and adaptive immune responses can result in protection in various mouse models of disease. Based on these observations, clinical trials are currently underway in humans to evaluate CpG ODN therapies for cancer, allergy and infectious disease. However, potential applications for immunostimulatory CpG ODN in species of veterinary importance are just being explored. In this review, we will highlight what is presently known about the immunostimulatory effects of CpG ODN in domestic animals. 相似文献