共查询到20条相似文献，搜索用时 218 毫秒
1.
分子佐剂C3d增强猪流感病毒HA-DNA疫苗免疫原性的研究 总被引：1，自引：0，他引：1
为了探讨分子佐剂C3d对猪流感病毒HA-DNA疫苗免疫原性的影响,本研究构建了3种表达不同形式HA的重组质粒,分别为:表达分泌型HA的质粒p-sHA、表达全长HA的质粒P-tmHA和融合表达3拷贝鼠C3d与HA的质粒P-sHA/mC3d3.3种质粒与空载体pCAGGS分别免疫BALB/c小鼠后,采用ELISA和HI试验检测抗体水平.结果显示,免疫后第8周,P-sHA组与P-tmHA组的抗体水平没有明显差别,而P-sHA/mC3d3免疫组的抗体水平显著高于这两组,说明分子佐剂C3d增强了抗体应答水平.淋巴细胞增殖试验及细胞因子检测实验结果显示,免疫后第8周,P-tmHA诱导了更强的淋巴细胞增殖反应和更高水平的IFN-,而p-sHA/mC3d3诱导了更高水平的IL-4,说明HA表达形式的变化影响了免疫反应的类型,C3d与HA融合后通过诱导IL-4的产生而使免疫反应倾向于Th2型.总之,本研究证实3拷贝分子佐剂C3d与分泌型HA融合后显著提高了DNA疫苗诱导的抗体水平.这提示质粒P-sHA/mC3d3免疫可能对接种动物提供高水平的保护,有希望成为抗猪流感病毒的候选疫苗. 相似文献
2.
CpG ODN的最新研究进展 总被引：1，自引：0，他引：1
CpG C9DN是指含有CpG基序(以非甲基化胞嘧啶鸟嘌呤为基元构成的特定核苷酸序列结构)的C3DN。近期研究显示,CpG ODN作为佐剂,可明显促进Th1型免疫应答的产生。其与不完全弗氏佐剂(IFA)联合应用时,其效应甚至强于完全弗氏佐剂(CFA)。即使与Th2型佐剂如氢氧化铝合用亦可明显促进免疫应答的产生,且Th1型应答明显强于Th2型应答。CpG ODN能直接激活B细胞、树突状细胞、 相似文献
3.
为表达禽流感病毒M2蛋白与单拷贝及双拷贝鸡补体C3d的融合蛋白,本实验分别将单拷贝和双拷贝的鸡补体因子C3d基因同禽流感病毒M2基因5'端连接,再将串联基因定向克隆到pET-32a表达载体中进行诱导表达,表达产物用SDS-PAGE和western blot分析。结果表明,表达的2个融合蛋白大小分别约为64ku和97ku,以包涵体形式存在。Western blot分析结果显示,2种重组蛋白可被M2多克隆抗体识别,表明2种重组蛋白具有反应抗原性,为研制以鸡补体C3d为分子佐剂的禽流感新型疫苗奠定了基础。 相似文献
4.
《中国兽医学报》2015,(8):1254-1259
为研制鸡体内补体因子C3d抗体的检测方法,采用RT-PCR方法从鸡肝脏组织细胞的总RNA中扩增出1 002bp的鸡补体因子C3d基因全长,然后将其克隆到杆状病毒载体pFB-LIC-Bse中获得重组质粒pFB-C3d。将pFB-C3d转化到带有杆状病毒基因组的DH10Bac感受态细胞中,制备重组杆粒rBacmid-C3d。再将该重组杆粒转染到昆虫细胞sf9中,通过病毒拯救技术构建携带C3d基因的重组杆状病毒。Western blot试验结果表明,重组杆状病毒能在昆虫细胞sf9中表达出大小约为35 000的C3d-his融合蛋白。以该融合蛋白作为抗原建立的Western blot和间接免疫荧光试验(IFA)显示,经C3d-M2-GST融合蛋白免疫的鸡血清能够与杆状病毒表达的C3d结合,说明本研究构建的重组杆状病毒可以用来检测C3d作为分子佐剂及其融合蛋白免疫鸡后产生针对C3d自身的抗体,为今后建立体内C3d水平监测的指示系统奠定基础。 相似文献
5.
为了构建融合表达分子佐剂猪补体C3d与猪流感病毒血凝素(HA)的真核表达载体,论文克隆了猪C3d全长基因与H3N2亚型猪流感病毒的HA基因,并构建了包含3拷贝的C3d和经过改造的HA(替换信号肽并去除跨膜区)的质粒pHA-C3d3.序列分析表明,获得的猪C3d与参考序列相比核苷酸同源性达到99.7%,氨基酸同源性达99... 相似文献
6.
《动物医学进展》2015,(7)
为探讨补体因子C3d在家禽健康诊断中的意义,将鸡补体因子C3d全长基因克隆到原核表达载体pET28a中构建重组表达质粒pET28a-C3d,然后将pET28a-C3d转化到大肠埃希菌BL21(DE3)并以IPTG诱导表达。表达产物采用氯化钾染色切胶法进行纯化,获得分子质量约35ku的C3d目的蛋白。将所得蛋白与弗氏佐剂制备疫苗免疫小鼠,制备抗鸡补体因子C3d血清。用制备的血清与重组杆状病毒表达的C3d蛋白进行Western blot和间接免疫荧光(IFA)鉴定血清的特异性。进一步应用该血清对不同健康状态的鸡血清进行C3d相关补体成份相对含量的检测。结果鸡补体因子C3d在大肠埃希菌中成功表达,纯化的C3d蛋白免疫小鼠后制备的抗血清经ELISA检测抗体效价达1∶256 000。Western blot和IFA结果显示,该血清能与重组杆状病毒表达的C3d蛋白发生特异性反应。临床应用试验表明,该血清不仅能用于检测不同健康状态鸡血浆中C3d的表达状况,还能用于ELISA检测不同鸡血清中C3d相关补体成分的相对含量差异。研究还证实鸡血浆中C3d相关补体成分的表达因机体免疫状态不同而存在差异,结果为了解C3d与鸡体健康状况之间的关系提供了依据。 相似文献
7.
《中国兽医学报》2015,(11)
为评价佐剂对金黄色葡萄球菌(S.aureus)CP8-FnBPB-ClfA免疫效果的影响,用纯化的S.aureus血清8型荚膜多糖与FnBPB-ClfA蛋白偶联的偶联物配合不同佐剂对健康成年新西兰大耳白母兔进行免疫接种,第1、2、3、4组的佐剂分别为弗氏佐剂、铝盐佐剂、脂质体佐剂及大肠杆菌不耐热肠毒素B(LTB),第5组为对照组用PBS免疫。用间接ELISA法和Western blot对各组兔血清中的抗体进行监测,评价体液免疫效果;通过检测Th1/Th2类细胞因子含量变化评价机体免疫被激活的程度;T淋巴细胞增殖试验评价细胞免疫,并对三免后2周的白兔进行攻毒保护试验。结果显示,以脂质体为佐剂的组7d时开始产生抗体,为产生抗体最早组,14d后抗体水平就开始下降;以LTB为佐剂的组抗体水平最高,持续时间最长。细胞免疫水平检测结果显示,经ConA刺激后,各组T淋巴细胞普遍增殖,无显著差异,刺激指数都在1.0左右;用抗原刺激的组中,以LTB为佐剂的组T淋巴细胞增殖能力最强,刺激指数为2.23,对白兔的攻毒保护率为80%。 相似文献
8.
为比较不同动物的补体C3d基因序列,本研究参考已报道的人、鼠、牛的C3d基因序列设计引物,分别从小鼠、猪、羊的肝脏组织中扩增获得鼠、猪、羊的补体C3d基因.以鼠、猪、羊的肝脏中提取总RNA为模板,通过RT-PCR扩增出C3d分子的cDNA克隆到pMD18-T载体中,转化大肠杆菌,经酶切鉴定后进行序列测定,并利用生物信息学工具软件对3种C3d蛋白的理化和生物学性质进行分析.鼠C3d基因与人、猪、羊C3d基因的同源性分别为83%、82%、81%,牛和羊C3d基因的同源性为94%.鼠、猪、羊的补体C3d分子与受体CR2结合的功能区同源性很高,只有个别位置氨基酸发生了突变,可能具有相同的结构和功能. 相似文献
9.
目的:研究复合多糖佐剂对猪O型口蹄疫(FMDV)灭活抗原的佐剂效应。方法:以对猪O型FMDV灭活抗原具有佐剂效应的黄芪多糖(APS)、人参多糖(GPS)、木蟞子多糖(HPS)、茯苓多糖(LBPS)和云芝多糖(YPS)等五种多糖为佐剂成分,以各多糖的最佳佐剂剂量复合成多糖佐剂配方(CPS),以市售油佐剂为阳性对照,PBS为阴性对照,与FMDV灭活抗原配制成疫苗并免疫猪后,测定比较血清中FMDV灭活抗原诱导特异性抗体水平和不同佐剂诱导分泌IL-2、TNF-α、IFN-γ的变化。结果:CPS佐剂组在一免疫后抗体效价高于阴性对照组,与其他阳性对照组无显著差异,在二免疫后抗体效价高于ISA206油佐剂组,与其他阳性对照无显著差异,且抗体水平显著高于阴性对照组。二免后血清中三种细胞因子含量均高于其他试验组。结论:CPS作为佐剂,能显著提高抗体水平(GMT值),同时促进Th1和Th2型免疫应答,CPS作为新型佐剂具有应用价值。 相似文献
10.
为了比较猪、鼠、鸭C3d基因的不同,以便为下一步研究不同动物C3d作为分子佐剂核酸疫苗的效果,试验对3种基因进行克隆和序列分析。首先提取猪、鼠、鸭的肝脏总RNA,以RNA为模板,通过RT-PCR方法,分别扩增C3d分子的cDNA,再将3种动物的cDNA分别克隆到pMDl8-T载体中,然后转化BL21大肠杆菌,经酶切鉴定后进行序列测定,根据测序结果利用生物信息学工具软件对3种C3d进行生物学性质分析。结果发现,猪与鼠和鸭的C3d基因的同源性分别为81.9%和65.6%,鼠与鸭C3d基因的同源性为65.8%;鼠、猪的补体C3d分子与受体CR2结合的功能区有28个氨基酸,鸭有29个氨基酸。 相似文献
11.
Fusion of C3d molecule with bovine rotavirus VP7 or bovine herpesvirus type 1 glycoprotein D inhibits immune responses following DNA immunization 总被引：14，自引：0，他引：14
Suradhat S Braun RP Lewis PJ Babiuk LA van Drunen Littel-van den Hurk S Griebel PJ Baca-Estrada ME 《Veterinary immunology and immunopathology》2001,83(1-2):79-92
The binding of the complement C3d molecule with receptors on B cells and/or follicular dendritic cells (FDCs) influences the induction of humoral immune responses. For example, C3d fused to an antigen has been shown to have a strong adjuvant effect on antibody production. We investigated the possibility that co-expression of antigen and C3d as a fusion protein could enhance antigen-specific immune responses, following plasmid immunization. One or two copies of murine C3d-cDNA, C3d or (C3d)(2), respectively, were cloned together with bovine rotavirus (BRV) VP7 or bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) genes. All constructs contained a signal peptide that resulted in the secretion of the expressed proteins. In vitro, the characterization of the chimeric proteins indicated that both VP7 and gD retained their antigenicity and the C3d remained biologically active. However, immunization with plasmids encoding VP7-C3d chimeras did not enhance rotavirus-specific antibody responses and the frequency of BRV-specific IFN-gamma secreting cells in the spleens were significantly lower in mice immunized with pVP7-(C3d)(2) when compared with mice immunized with plasmid encoding VP7. The same pattern of immune responses was observed for plasmids encoding gD-C3d. Both gD-specific antibody responses and the frequency of gD-specific IFN-gamma secreting cells were significantly lower in mice immunized with plasmid expressing gD-C3d chimeras when compared with mice immunized with plasmid encoding gD alone. These results indicate that co-expression of C3d with an antigen actually inhibit both humoral and cell-mediated antigen-specific immune responses. 相似文献
12.
Cloning of a gene fragment encoding bovine complement component C3d with expression and characterization of derived fusion proteins 总被引：2，自引：0，他引：2
Firth MA Prando Moore D Pei Y Shewen PE Lo RY Yoo D Hodgins DC 《Veterinary immunology and immunopathology》2006,114(1-2):61-71
The gene fragment coding for bovine C3d gene (boC3d) was cloned and expressed as a component of fusion proteins destined for use in vaccine studies in cattle, and for in vitro experiments. This fragment of complement protein C3 (C3d) has been shown to enhance B cell responses when complexed with antigen. Three potential vaccine constructs were engineered to contain one, two or three boC3d units linked to a fragment of the leukotoxin of Mannheimia haemolytica A1, an economically important pathogen of cattle that causes a fibrinous pneumonia in calves. A recombinant biotinylated boC3d protein (for use in in vitro studies) was generated by endogenous biotinylation in Escherichia coli by means of the BirA holoenzyme synthetase. All recombinant proteins incorporated polyhistidine tags and were purified by nickel-agarose chromatography, then analyzed by SDS-PAGE and Western immunoblot. The identity of boC3d was confirmed by mass spectrometry, since monoclonal antibodies to boC3d were not available. To date, published research into the adjuvant activities of C3d has been limited to experiments in mice and rabbits, using antigens unrelated to diseases occurring naturally in these species. The boC3d fusion proteins expressed in this study will provide the basis for immunization trials in cattle and studies of receptor binding and cell activation of bovine lymphocytes. 相似文献
13.
Pasteurella haemolytica: purification of saline-extractable proteins by isoelectrofocusing 总被引：1，自引：0，他引：1
K L McKinney A W Confer J A Rummage M J Gentry J A Durham 《Veterinary microbiology》1985,10(5):465-480
A procedure was developed for separating antigens associated with a saline extract of Pasteurella haemolytica serotype 1. Seven antigens were identified by immunoelectrophoresis to be associated with the extract. The extract was subjected to preparative isoelectrofocusing in a pH range of 3-10. The majority of extracted proteins were found to have pI's of 4-6, whereas the carbohydrate antigen(s) were distributed over a pI range of 3.0-8.0. The fractions that were of interest were pooled and refocused in a narrower pH range to improve resolution of the protein antigens. Specific antigens from defined pH ranges were pooled to form 6 antigen groups. These antigen groups were examined further by immunoelectrophoresis, analytical isoelectrofocusing, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The molecular weights of the proteins found in the capsular extracts ranged from 33 k to greater than 80 k. Injection of mice with capsular extract or antigen Groups 1-6 in Freund's incomplete adjuvant resulted in a serum antibody response to the various antigens as detected by an enzyme-linked immunosorbent assay. Significant protection (P less than 0.05) against challenge with virulent P. haemolytica was seen in mice injected with antigen Groups 2 and 4. Six calves were immunized with saline extract. These calves had greater resistance to experimental pneumonic pasteurellosis than did 6 non-vaccinated calves. A serum antibody response to the crude extract and to each antigen group was detected in vaccinated calves by an enzyme-linked immunosorbant assay. 相似文献
14.
The influence of a water-in-oil emulsion on humoral immunity 总被引：1，自引：0，他引：1
B A Bokhout A T Bianchi P J van der Heijden J W Scholten W Stok 《Comparative immunology, microbiology and infectious diseases》1986,9(2-3):161-168
A strategy for research of immunostimulants is best served choosing a way by which the immune response can be studied without being complicated by a combination of effects originating both in adjuvant and antigen. This means that adjuvant and antigen preferably are applied with an interval and by different routes. In our model an adjuvant (a W/O emulsion) was applied intraperitoneally, whereas the antigen was injected intravenously. The stimulatory effect on the splenic plaque forming B-cell response depended on the dose of antigen, on the interval between adjuvant and antigen application, on the mouse strain used, and on the quality of the antigen with respect to the intrinsic adjuvanticity of the antigens. 相似文献
15.
Separation of the capsular antigen and endotoxin from saline extracts of Pasteurella multocida type B was achieved by fractional precipitation from aqueous solution by addition of polar organic solvents. Biological tests for the presence of endotoxin showed that it was absent from capsular antigen preparations so obtained. Properties of the capsular antigen suggested that it was a high molecular weight acidic polysaccharide. The solvent fractionation method was found to be equally applicable to separation of capsular antigen and endotoxin of P multocida type E. The type B capsular antigen in the presence of aluminium hydroxide gel adjuvant, was poorly immunogenic in rabbits. In cattle, however, a dose-dependent serological response was obtained as demonstrated by the mouse passive protection test. 相似文献
16.
利用PGEX-6P-1融合表达系统将SO7基因在大肠杆菌中进行融合表达,对表达产物进行初步纯化和复性,制备免疫原。分别使用不同剂量的人参总皂甙和重组γ-干扰素以及弗氏完全佐剂作为免疫佐剂,于5日龄、12日龄和19日龄对雏鸡进行3次免疫,同时设立蛋白口服免疫组、蛋白皮下注射免疫组、卵囊口服免疫组、未免疫未攻毒组和未免疫攻毒组作对照,于26日龄用105个柔嫩艾美耳球虫卵囊进行攻毒,8d后扑杀,对各组的存活率、相对增重率、病变减少率、相对卵囊产量和抗球虫指数ACI等指标进行统计分析,比较免疫保护效果。与非免疫攻毒组以及不加佐剂的免疫攻毒组相比,佐剂使用后各组的增重、病变记分、相对卵囊产量、ACI等指标均有一定的改善,对柔嫩艾美耳球虫的人工感染可以提供部分保护。3种佐剂中,γ-干扰素和人参总皂甙能增强重组蛋白的免疫保护效果,且佐剂的剂量对免疫保护的效果有一定的影响,以5000Uγ-干扰素效果最佳,效果与E.tenella活卵囊免疫相当,弗氏完全佐剂的免疫增强效果不明显。单独使用重组蛋白皮下注射或口服免疫,虽有一定的保护作用,但不明显。一定剂量的γ-干扰素对SO7抗原的免疫保护效果起明显的增强作用,是一个具有很好临床应用前景的新型佐剂。 相似文献
17.
LONG Chong-chong DENG Wei-xi MAO Yi-zhi ZENG Gui-ying WU Bo-tao XIONG Chao-li XU Mao-wen XIA Peng WEN Ming 《中国畜牧兽医》2016,43(11):2886-2891
To explore the feasibility of entering clinical trials of the goat Chlamydophila abortus eukaryotic plasmids,Chlamydophila abortus OmpA gene was amplified by PCR and cloned into the eukaryotic expressing plasmid pcDNA3.1(+）to construction the recombinant vetor.After identification by PCR and restriction enzyme digestion,this vector was transfected into PK-15 cells and its expression were observed by fluorescent antibody test,the distribution of serum antibodies and plasmid were detected in mice after the immunization of the recombinant vector and molecular adjuvant which was single-stranded DNA of E.coli bacterial genome.The results showed that the recombinant plasmid pcDNA3.1-OmpA was successfully constructed after detecting by PCR,enzyme digestion and sequencing.The OmpA gene was effectively expressed in PK-15 cells.The anti-OmpA antibody levels of the pcDNA3.1-OmpA with molecular adjuvant group was significantly higher than that in pcDNA3.1-OmpA group and the inactivated vaccine group at 14 d after immunization（P<0.05).This levels showed an upward trend following numbers of immunization and times.The highest levels was at 35 d after immunization.Then the antibody titers were gradually decreased which still maintain a higher antibody levels than before.The pcDNA3.1-OmpA could be detected in the heart,liver,spleen,kidney,lung,brain,jejunum and leg muscle of mice on 21 d after immunization,and couldn't be detected in any organs at 49 d after immunization.The results above indicated that the the recombinant vetor based on OmpA gene of Chlamydophila abortus was successfully constructed in this experiment,after joining the molecular adjuvant could induce to a higher level of serum antibodies in mice. 相似文献
18.
为探讨山羊流产嗜衣原体重组真核质粒进入临床试验的可行性,本试验用PCR方法扩增出山羊流产嗜衣原体OmpA基因,克隆至真核表达载体pcDNA3.1（+）中,经PCR和双酶切鉴定后,将重组质粒pcDNA3.1-OmpA转染至PK-15细胞,观察目的基因表达情况,并将制备的大肠杆菌基因组单链DNA作为分子佐剂与重组质粒共同免疫小鼠,检测重组质粒在小鼠体内分布情况及血清抗体水平。结果表明,经PCR、双酶切和测序鉴定表明成功构建重组质粒pcDNA3.1-OmpA;转染PK-15细胞后,荧光抗体试验结果证实重组质粒pcDNA3.1-OmpA得到有效表达。首免后14 d,重组质粒加分子佐剂组的抗体效价显著高于重组质粒组和灭活疫苗组（P<0.05）;随着免疫次数增加和时间推移,免疫小鼠抗体水平均呈现上升趋势,至35 d时达到最高峰,此后抗体滴度逐渐下降,但仍维持较高水平。首免后21 d,在小鼠心脏、肝脏、脾脏、肾脏、肺脏、脑、空肠和腿肌中均可检测到质粒的分布,此后逐渐消失,49 d在所检组织中均未检测到重组质粒的存在。表明试验成功构建了基于OmpA基因的山羊流产嗜衣原体重组真核质粒,且加入分子佐剂后可诱导小鼠产生较高水平的血清抗体。 相似文献
19.
选用中华硬蜱叮咬中肠抗原免疫接种组及初次叮咬组和佐剂对照组宿主并分别在不同时间(叮咬24、48、72h以及5、8d)对蜱中肠蛋白质、糖原及多糖进行了动态观察,并对消化细胞核DNA含量进行测定。结果显示:蜱叮咬中肠抗原免疫接种宿主后可使中肠上皮发生破坏,消化细胞破损,基膜可出现松散和断裂,消化细胞蛋白质含量较初次叮咬组和佐剂对照组宿主明显降低,而糖原及多糖含量基本不变。消化细胞DNA在叮咬无免疫力宿主(初次叮咬组和佐剂对照组)早期24h主要为2倍体,随着吸血量增加、细胞核倍体数加大,第5天达7.06倍体,而叮咬中肠抗原免疫接种组宿主后,消化细胞细胞核倍体数增加不明显,与叮咬无免疫力宿主有显著性差异(P>0.01),提示中华硬蜱叮咬中肠抗原免疫接种宿主后,可干扰中肠消化细胞蛋白质和核酸(DNA)的合成代谢,为进一步揭示抗蜱免疫机理提供了理论依据。 相似文献