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1.
细粒棘球蚴Eg95s基因的串联表达及表达系统优化   总被引：2，自引：2，他引：0  
为研制新型棘球蚴病基因工程疫苗,根据GenBank公布的细粒棘球蚴Eg95基因序列,针对其中一段348 bp高度亲水的优势表位序列设计引物,扩增Eg95s基因,利用同尾酶法基因同向串联方案,获得3拷贝Eg95s串联体。分别用两种表达载体pGEX-6p-1和pET-32a构建重组质粒,转化入3种大肠杆菌表达宿主BL21(DE3)、Rosseta(DE3)和Origami (DE3),对其进行IPTG诱导表达,经免疫印迹分析结果表明,均能正确表达具有免疫原性的Eg95s蛋白。通过重组蛋白的表达量、可溶性、纯化方法等方面的比较,对Eg95s重组表达系统的载体及宿主菌进行了优化,结果表明,3拷贝的串联Eg95s在以pET-32a为表达载体,Rosseta(DE3)为宿主菌的系统中表达最佳。最终获得了表达量高、可溶性好、纯化方便的Eg95s重组蛋白表达系统,为大规模生产棘球蚴病基因工程疫苗提供了科学依据。  相似文献   

2.
本试验初次建立了CpG ODN联合重组乳酸乳球菌滴鼻免疫BALB/c小鼠模型。结果得出CpG ODN佐剂协同重组菌抗原组血清中抗PRRSV特异性抗体IgG和呼吸道黏膜抗体s-Ig A均高于单独重组菌组(P>0.05)和单独佐剂组(P<0.01),佐剂CpG ODN协同重组菌抗原组的Th1型细胞因子IL-2、IFN-γ及黏膜淋巴因子IL-5的活性高于单独抗原组(P>0.05)及单独佐剂组(P<0.01)。试验结果说明CpG ODN作为佐剂与重组菌鼻腔免疫小鼠后,可以提高重组菌诱导的黏膜免疫反应和Th1细胞参与的系统免疫反应,为研究安全有效的重组菌黏膜疫苗奠定了基础。  相似文献   

3.
将构建的含鸡传染性法氏囊病病毒（IBDV）JS超强毒株VP2基因的重组真核表达质粒与人参皂苷Rg1和CpG DNA联合佐剂免疫SPF鸡,检测免疫后的抗体水平,并观察超强毒株攻毒后的免疫保护效果。试验结果显示：Rg1-CpG佐剂与重组真核表达质粒DNA疫苗联合免疫后的抗体水平比单一使用DNA疫苗高,二者间有显著性差异;Rg1-CpG佐剂组的发病率、死亡率均低于非佐剂组,保护率则高于非佐剂组,且有显著性差异;而与法氏囊病中等毒力（NF8株）活疫苗免疫组的抗体水平及保护率相一致。表明Rg1-CpG佐剂显著增强了IBDV DNA疫苗的免疫效果。  相似文献   

4.
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   

5.
探讨CpG寡聚脱氧核苷酸(CpG oligodeoxynucleotides, CpG ODN)和ICTYOLANETM31微乳佐剂对重组表达的ASFV-CD2v蛋白在小鼠体内免疫效果的影响。以杆状病毒表达的CD2v蛋白为抗原，分别配伍CpG寡脱氧核苷酸(CpG oligonucleotide, CPG ODN)和ICTYOLANETM31微乳佐剂，皮下免疫BALB/c小鼠。首次免疫后14 d进行二免，采集首免后0、14 d以及二免后7、14及28 d血清，通过间接ELISA检测血清中CD2v抗体水平；采集二免后35 d小鼠分离脾脏淋巴细胞，通过流式细胞术分析IFN-γ水平。结果显示，间接ELISA结果显示在二免后7 d可检测到较高水平的CD2v抗体，ICTYOLANETM31佐剂和CPG ODN佐剂组抗体水平明显高于蛋白组。T细胞亚群分析结果显示，在二免后35 d, CpG ODN佐剂组的CD4+T淋巴细胞的比例最高，达到81.34%,ICTYOLANETM31佐剂组CD8<...  相似文献   

6.
为分析CpG基序寡聚脱氧核苷酸（CpG DNA）对H9亚型禽流感病毒（AIV-H9）灭活疫苗的免疫增强作用，利用商品化的含有CpG DNA的重组质粒作为AIV-H9灭活抗原的免疫佐剂，经肌内注射1、7、21日龄的SPF鸡，通过检测鸡血清中HI抗体和外周血淋巴细胞CD4、CD8基因相对表达量，分析CpG DNA重组质粒对不同日龄SPF雏鸡首次免疫AIV-H9灭活疫苗免疫效果的影响。结果显示：1、7和21日龄SPF鸡首免后，CpG DNA免疫增强剂添加组的HI抗体滴度明显高于未添加组（P≤0.05），HI抗体峰值滴度升高了近2log2滴度，差异极显著（P≤0.01），且上升速度更快；CpG DNA免疫增强剂添加组，在21日龄免疫时的免疫效果明显优于1日龄和7日龄免疫组，表现为HI抗体上升速度更快，峰值更高，离散度更小；首免3周后，7日龄和21日龄SPF鸡CpG DNA免疫增强剂添加组的CD4、CD8基因相对表达量均高于同日龄未添加组，且明显高于1日龄SPF鸡组（P≤0.01）。结果表明，CpG DNA 能显著增强鸡对AIV-H9灭活疫苗的特异性体液免疫反应，可以作为高效的免疫增强剂，尤其在21日龄使用时免疫效果更好。本研究为CpG DNA重组质粒在禽流感疫苗上的联合应用以及研制新型复合佐剂疫苗提供了依据。  相似文献   

7.
为研制新型棘球蚴病基因工程疫苗,根据GenBank公布的细粒棘球蚴Eg95基因序列,针对其中一段348 bp高度亲水的优势表位序列设计引物,扩增Eg95s基因,利用同尾酶法基因同向串联方案,获得3拷贝Eg95s串联体.分别用两种表达载体pGEX-6p-1和pET-32a构建重组质粒,转化入3种大肠杆菌表达宿主BL21(DE3)、Rosseta(DE3)和Orjgami(DE3),对其进行IPTG诱导表达,经免疫印迹分析结果表明,均能正确表达具有免疫原性的Eg95s蛋白.通过重组蛋白的表达量、可溶性、纯化方法等方面的比较,对Eg95s重组表达系统的载体及宿主菌进行了优化,结果表明,3拷贝的串联Eg95s在以pET-32a为表达载体,Rosseta(DE3)为宿主菌的系统中表达最佳.最终获得了表达量高、可溶性好、纯化方便的Eg95s重组蛋白表达系统,为大规模生产棘球蚴病基因工程疫苗提供了科学依据.  相似文献   

8.
细粒棘球蚴Eg95重组蛋白疫苗临床免疫效果研究   总被引：1，自引：0，他引：1  
为评价细粒棘球蚴Eg95重组蛋白的临床免疫效果,本研究利用ELISA试剂盒对3个地区12个养殖场的1 637头3月龄~4月龄绵羊与山羊进行了18个月的免疫后抗体滴度水平监测。结果显示,Eg95重组蛋白疫苗在14 d时可以诱导免疫动物产生特异性抗体,抗体滴度水平在28 d达到峰值,免疫保护大于3个月。而且在首次免疫后第28 d进行第二次免疫,则抗体滴度水平呈显著增加(p0.05),并可持续12个月以上。在第二次免疫后12个月再次进行免疫,动物的Eg95特异性抗体水平显著性提高,并且差异极显著(p0.01),抗体滴度长期保持在3.5以上。因此,细粒棘球蚴Eg95重组蛋白免疫效果良好,可以有效预防棘球蚴(包虫)病。  相似文献   

9.
试验旨在通过大肠杆菌表达系统表达鼠源重组CD40L蛋白,探讨其对河豚毒素（tetrodotoxin,TTX）人工完全抗原在免疫BALB/c小鼠过程中的免疫增强作用。用Trizol试剂提取BALB/c小鼠脾脏总RNA并反转录成cDNA,根据CD40L CDS区设计引物,PCR扩增目的基因,构建pGEX4T-1重组载体,进行原核表达,并纯化重组CD40L蛋白;根据曼尼希反应原理,用甲醛法制备TTX免疫原TTX-BSA和检测原TTX-OVA;以人工重组蛋白CD40L佐剂组为试验组,弗氏佐剂组作为对照组,免疫BALB/c小鼠,用ELISA方法结合SPSS 19.0软件分析各组免疫效果,探讨重组CD40L蛋白在TTX-BSA免疫过程中对免疫效果的影响。结果显示,本试验成功扩增了783 bp的CD40L目的基因,原核表达并纯化了融合GST标签的55 ku鼠源CD40L重组蛋白;与人工制备的TTX-BSA协同免疫小鼠试验结果显示,在免疫初期与弗氏佐剂相比,CD40L具有极显著的免疫增强效果（P<0.01）。综上所述,重组蛋白CD40L与TTX-BSA完全抗原协同免疫小鼠,在免疫初期CD40L具有增强机体对半抗原的应答强度的作用,为进一步开发适于小分子半抗原抗体高效制备的免疫增强佐剂奠定基础。  相似文献   

10.
为研究细粒棘球蚴14-3-3(r Eg14-3-3)重组蛋白免疫小鼠的细胞免疫机制,本研究利用r Eg14-3-3、弗氏佐剂和PBS分别免疫3组ICR小鼠,每隔两周免疫一次,在第三次免疫后4周,以原头蚴攻击感染,采用流式细胞术分别检测免疫后0、2、4、6、10、18、30周小鼠外周血中CD4~+、CD8~+T淋巴细胞的百分率。攻击感染24周后迫杀鼠,检获棘球蚴包囊数并计算减囊率。结果表明:与对照组相比,r Eg14-3-3免疫组CD4~+T细胞和CD8~+T细胞在原头蚴感染后均显著增高,rEg14-3-3蛋白免疫组小鼠的免疫保护力为84.47%。本研究表明r Eg14-3-3免疫小鼠在原头蚴攻击感染后T淋巴细胞数量显著增加,该重组蛋白能够诱导有效的抗感染细胞免疫应答。  相似文献   

11.
To study the immune enhancement activity of CpG motifs on the recombinant Eg95 antigen of Echinococcus granulosus,Escherichia coli BL21(DE3) containing pET-32a-3Eg95 was grown in LB medium,and IPTG was then added for induction.The recombinant Eg95 protein was expressed and later harvested by affinity chromatography.The obtained antigen was mixed either with the conventional adjuvant Quli-A or with different types of CpG motifs (pUC18-CpG or CpG ODN),to prepare vaccine candidates for mice immunization.The average levels of serum IgG antibody were tested by indirect ELISA and the quantification of cytokine expression were determined by Real-time quantitative PCR after in vitro stimulation.The results identified that the size of the recombinant protein was 56 ku as predicted,and the recombinant protein could be recognized by serum from Echinococcus granulosus-infected sheep.The average levels of serum IgG antibody (D450 nm) of the pUC18-CpG and CpG ODN groups on 14,28 and 42 d were 3.10,3.03,3.22 and 2.98,3.12, 3.27,respectively.Antibody levels from both CpG groups were significantly higher compared to the conventional adjuvant Quli-A group (P<0.05) The average level of serum IgG antibody in CpG ODN group was a little higher than that in pUC18-CpG group(P>0.05).After stimulation by recombinant protein 3Eg95,the relative expression levels of IFN-β,IFN-γ and TNF-α of pUC18-CpG and CpG ODN groups were 6.88,2.35,6.28 and 5.03,2.85,7.07,respectively,and both were higher than that of the Quli-A group (P<0.05).The results indicated that pUC18-CpG and CpG ODN had significant immune enhancement property on Eg95 antigen in both humoral and cellular immune response,and the immune enhancement activity of CpG ODN was slightly better than that of the pUC18-CpG.Hence,pUC18-CpG and CpG ODN could be used as efficient immune adjuvants enhancer in future vaccines against Echinococcus granulosus infection.  相似文献   

12.
In order to develop novel immunoadjuvants to boost immune response of conventional vaccines, experiments were conducted to investigate the regulating effects of porcine interleukin-6 gene and CpG motifs as the molecular adjuvants on immune responses of mice that were co-inoculated with trivalent vaccines against Swine fever, the Pasteurellosis and Erysipelas suis. Synthetic oligodeoxynuleotides containing CpG motifs were ligated into pUC18, forming recombinant pUC18-CpG plasmid. Eukaryotic plasmid expressing porcine interleukin-6 (VPIL-6) were also constructed as molecular adjuvants in an attempt to enhance levels of immune responses of mice co-administered with the trivalent vaccines in this paper. The cellular and humoral immune responses of mice were systematically analysed, and the experimental results were observed that the number of white blood cells, monocytes, granuloytes and lymphocytes significantly increased, respectively, in the mice immunized with VPIL-6, compared with those of the control; the IgG content and titre of specific antibodies to the trivalent vaccine mounted remarkably in the sera from the VPIL-6 vaccinated mice; the proliferation of lymphocytes and induced IL-2 activities were significantly increased in the vaccinated groups. The above-mentioned immune responses of mice co-inoculated with pUC18-CpG plasmid were significantly stronger than those of co-inoculated with pUC18 plasmid, suggesting that the immunostimulatory effect of oligodeoxynuleotides CpG is closely connected with the number of CpG motifs. These results suggest that the porcine IL-6 gene and CpG motifs could be employed as effective immunoadjuvants to elevate immunity to conventional vaccines.  相似文献   

13.
为建立特异性和敏感性高的检验犬细粒棘球绦虫感染的方法。用细粒棘球绦虫(简称,Eg)成虫抗原分别免疫兔和绵羊,收集高免血清,纯化的高免抗体。依据抗体夹心ELISA工作原理,以兔抗体包被,检测感染Eg、不同犬带科绦虫的实验犬和空白犬粪样,绵羊抗体扑捉抗原,HRP标记兔抗绵羊IgG(1∶8 000)催化显色,用酶标仪测定OD 405nm吸光度,用以确定其特异性和敏感性。试验结果表明,敏感性为82.69%(43/52),特异性为85.88%(140/163);粪抗原在感染细粒棘球绦虫16d后可检出,最低抗原浓度为9.7ng/mL即犬感染5条成虫时可检测出阳性。该检测方法具有较好的特异性和灵敏性,为进一步研制检测细粒棘球绦虫虫体抗原ELISA检测试剂盒奠定了基础。  相似文献   

14.
Porcine reproductive and respiratory syndrome (PRRS) is now among the most important swine diseases that affect the Chinese swine industry. Both killed and live attenuated vaccines are currently used against the disease, but neither of them could provide full protection after vaccination. In the present study, the adjuvanticity of a plasmid containing CpG motifs (pUC18-CpG) was introduced to enhance the efficacy of a commercial PRRS live attenuated vaccine. After vaccination, PRRSV-specific antibodies, PRRSV-specific cytokines, and clinical parameters were studied and compared between different vaccinated groups. During a following challenge study, co-administration of pUC18-CpG with the vaccine could confer higher protection rate. Our results have shown that co-administration of pUC18-CpG with the vaccine could elicit more potent adaptive immune response and provide better protection.  相似文献   

15.
Immunostimulatory CpG oligodeoxynucleotides (ODN) have been tested as immunoadjuvants for various vaccines in mice and human. Findings from previous reports suggest that CpG ODN can be used to enhance magnitude and balance of an immune response while reducing undesirable side effects of commercial vaccine, when delivered by parenteral route. Recently, it has been showed that CpG ODN is a promising mucosal adjuvant in mice, but data on mucosal immune responses induced by CpG ODN in other animals, especially in chickens, are scarce. Herein, we evaluated intranasal (IN) delivery of CpG ODN with newcastle disease (ND) vaccine (NDV) to determine its potential as a mucosal adjuvant to a commercial vaccine. CpG ODN augmented systemic (IgG in serum, T cell proliferation) and mucosal (IgA in intestinal washings and feces) immune responses against antigen. CpG ODN stimulated effectively both systemic and mucosal immune responses when delivered intranasally. Results from this study indicate that stimulatory CpG ODN is a potential effective mucosal adjuvant for the NDV in SPF chickens and may be applicable to husbandry animals.  相似文献   

16.
Immunostimulatory CpG oligodeoxynucleotides (ODN) have been tested as immunoadjuvants for various vaccines in mice and human. Findings from previous reports suggest that CpG ODN can be used to enhance magnitude and balance of an immune response while reducing undesirable side effects of commercial vaccine, when delivered by parenteral route. Recently, it has been showed that CpG ODN is a promising mucosal adjuvant in mice, but data on mucosal immune responses induced by CpG ODN in other animals, especially in chickens, are scarce. Herein, we evaluated intranasal (IN) delivery of CpG ODN with newcastle disease (ND) vaccine (NDV) to determine its potential as a mucosal adjuvant to a commercial vaccine. CpG ODN augmented systemic (IgG in serum, T cell proliferation) and mucosal (IgA in intestinal washings and feces) immune responses against antigen. CpG ODN stimulated effectively both systemic and mucosal immune responses when delivered intranasally. Results from this study indicate that stimulatory CpG ODN is a potential effective mucosal adjuvant for the NDV in SPF chickens and may be applicable to husbandry animals.  相似文献   

17.
A recombinant replication-defective adenovirus expressing the major epitopes of porcine circovirus-2 (PCV-2) capsid protein (rAd/Cap/518) was previously constructed and shown to induce mucosal immunity in mice following intranasal delivery. In the present study, immune responses induced by intranasal immunization with a combination of rAd/Cap/518 and cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) were evaluated in mice. The levels of PCV-2-specific IgG in serum and IgA in saliva, lung, and intestinal fluids were significantly higher in the group immunized with rAd/Cap/518 and CpG ODN than animals immunized with rAd/Cap/518 alone. The frequencies of IL-2-secreting CD4+ T cells and IFN-γ-producing CD8+ T cells were significantly higher in the combined immunization group than mice immunized with rAd/Cap/518 alone. The frequencies of CD3+, CD3+CD4+CD8-, and CD3+CD4-CD8+ T cells in the combined immunization group were similar to that treated with CpG ODN alone, but significantly higher than mice that did not receive CpG ODN. PCV-2 load after challenge in the combined immunization group was significantly lower than that in the phosphate-buffered saline placebo group and approximately 7-fold lower in the group treated with CpG ODN alone. These results indicate that rAd/Cap/518 combined with CpG ODN can enhance systemic and local mucosal immunity in mice, and represent a promising synergetic mucosal vaccine against PCV-2.  相似文献   

18.
Serum antibody responses in sheep naturally or experimentally infected with Echinococcus granulosus and/or other larval cestodes were examined using an enzyme-linked immunosorbent assay (ELISA) with various antigens prepared from sheep hydatid cyst fluid ( SHCF ). Serum donors included: sheep experimentally infected with E. granulosus and their age-matched non-infected controls; sheep experimentally infected with other helminth parasites; sheep naturally infected with E. granulosus both from Tasmania and the Australian mainland; sheep from Tasmania naturally infected with larval cestodes other than E. granulosus; and naturally reared sheep completely free from infection with larval cestodes. Attempts were made to eliminate serological reactions which were not specific for E. granulosus by using a series of antibody affinity chromatography steps to deplete crude SHCF antigen; these included adsorption with a monoclonal antibody, 3EgH 29-2, removal of host IgG using rabbit anti-sheep IgG antibody, and removal of antigens which bound non-specifically to normal sheep immunoglobulin. The final affinity-depleted antigen product was designated AD SHCF . Specific serological reactivity in infected sheep was very low. Affinity depletion of SHCF using 3EgH 29-2 did not appear to increase the specificity of serological diagnosis of E. granulosus infection when experimentally infected sheep were compared with their non-infected controls provided the latter were age-matched with experimental animals. The other affinity adsorption steps significantly reduced non-specific background binding to antigen by normal sheep serum. Despite this reduction in background in the ELISA, only low levels of antibody could be detected in naturally-infected sheep.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

19.
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   

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