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1.
中国农业科学院哈尔滨兽医研究所侯艳霞等为验证禽流感病毒(AIV)核蛋白(NP)中的3条T细胞表位候选肽NP89-97(PKKTGGPIY)、NP198-206(KRGINDRNF)和NP64-71(ERMVLSAF)的免疫原性,构建了含有NP基因的重组质粒pCAGGS-NP,  相似文献   

2.
为鉴定禽流感病毒核蛋白(NP)中的T细胞表位,本研究根据蛋白结构预测技术预测了4个潜在细胞毒性T淋巴细胞(CTL)表位(NP120-128、NP163-172、NP85-92、NP67-74),并合成相应的候选短肽。构建了含有NP基因的重组质粒p CAGGS-NP,将其转染293T细胞。间接免疫荧光和western blot结果表明NP蛋白能够在293T细胞中获得表达。重组质粒免疫SPF鸡后能够诱导鸡体产生特异的NP抗体。在合成的4条短肽中,NP67-74体外刺激免疫过p CAGGS-NP的鸡脾淋巴细胞可以显著提高脾淋巴细胞IFN-γ的分泌量,提示短肽NP67-74能够在体外诱导活化鸡淋巴细胞产生CTL,可能是AIV NP的CTL表位。该研究结果对流感病毒免疫机制和通用疫苗研究具有借鉴意义。  相似文献   

3.
利用生物结构技术预测传染性支气管炎S1蛋白的1条细胞毒性T淋巴细胞(CTL)表位,并合成相应的候选多肽(Sp6)。为了确定S1蛋白中确实存在该T细胞表位,并鉴定这条T细胞表位的正确性,构建了含有S1基因的重组质粒pCAGGS-S1,通过间接免疫荧光和Western blot确定该重组质粒可以在真核细胞表达后,将该重组质粒免疫SPF鸡,间接ELISA检测血清中的抗体。采集免疫鸡的脾淋巴细胞并用合成的候选多肽刺激,通过实时荧光定量PCR检测脾淋巴细胞中IFN-γ的分泌量以及流式细胞术检测CD8+T淋巴细胞的增殖情况,初步确定该候选肽的正确性。间接免疫荧光、Western blot和间接ELISA试验结果表明,S1蛋白能够在293T细胞中获得表达并能够引起机体的体液免疫,说明S1蛋白具有反应原性,为进一步验证T细胞表位打下了基础;荧光定量PCR结果显示Sp6刺激后的鸡的脾淋巴细胞中IFN-γ的分泌量明显增加;流式细胞检测发现经Sp6和不相关多肽NP89-97刺激后及空白对照,CD8+T淋巴细胞增殖分别为34.8%、2.6%、0。以上结果表明,多肽Sp6能在体外诱导活化的鸡淋巴细胞产生细胞毒性T淋巴细胞反应,是IBV S1的CTL表位。该研究结果对传染性支气管炎病毒免疫机制和通用疫苗研究具有借鉴意义。此次研究结果表明多肽Sp6能在体外诱导活化的鸡淋巴细胞产生CTL,是IBV S1的CTL表位。该研究结果对传染性支气管炎病毒免疫机制和通用疫苗研究具有借鉴意义。  相似文献   

4.
H9亚型禽流感病毒血凝素特异性单因子血清制备   总被引：1，自引：0，他引：1  
为制备特异性的H9亚型禽流感病毒(AIV)单因子血清,本研究分别将6株不同亚群的H9亚型AIV的血凝素(HA)基因以鸡偏嗜的密码子进行优化,经全基因合成插入高效真核表达载体pCAGGS中,构建的真核重组质粒转染293T细胞进行瞬时表达,间接免疫荧光试验结果表明,重组质粒中的HA目的基因获得表达.将重组质粒以200μg/只的剂量免疫1月龄SPF鸡,6周后采血分离血清.交叉微量血凝抑制试验结果表明,血凝抑制效价可达8 long2～12 log2,灵敏度高,与其他AIV亚型抗原无交叉反应,型特异性强.  相似文献   

5.
鸡白细胞介素15真核表达质粒构建及其体外表达   总被引：5，自引：0，他引：5  
应用反转录一聚合酶链式反应(RT-PCR)技术AkConA刺激20 h的白菜航鸡脾脏T淋巴细胞中扩增出鸡白细胞介素15(ChIL-15)全基因片段.将其连接到克隆载体pMD18-T中得到重组克隆质粒pMD18-T-ChIL-15.阳性克隆质粒经鉴定正确后将ChIL-15亚克隆入真核表达载体pcDNA3.1(+)中,得到重组阳性表达质粒pcDNA3.1(+)-ChIL-15.阳性表达质粒经鉴定正确后应用磷酸钙法体外转柒293T细胞,通过间接免疫荧光试验(IFA)检测到了ChIL-15蛋白在293T细胞中的表达.  相似文献   

6.
为研制和更新针对H5亚型禽流感病毒(AIV)流行株的DNA疫苗,本研究将新近分离的H5亚型AIV Clade2.3.2e分支代表株A/Duck/Anhui/S1246/2015 (DK/AH/S1246/15)密码子优化的HA基因定向克隆至载体pCAGGS中构建重组质粒pCA-S1246,将该质粒转染293T细胞。利用间接免疫荧光和western blot检测,结果显示, HA蛋白可以在293T细胞中正确表达。将15μg、 30μg和60μg的p CA-S1246质粒分别免疫3周龄的SPF鸡, 3周以后以相同的剂量加强免疫后1周,检测其HI抗体平均效价分别可达1∶56、 1∶16和1∶37;加强免疫1周后用105EID50的DK/AH/S1246/15进行攻击时,免疫组的保护率为100%。本研究为DNA质粒pCA-S1246作为防控Clade.2.3.2e AIV的候选DNA疫苗株提供了实验依据。  相似文献   

7.
分别将靶向禽流感病毒(AIV)多靶点siRNA和鸡Mx基因克隆到p201慢病毒表达载体,采用鸡β-actin启动子替换p201慢病毒载体CMV启动子构建重组慢病毒载体p201-β-Mx-siRNA。并将其与辅助包装质粒共转染293T细胞,72h后收集细胞上清,实时荧光定量PCR测定重组慢病毒(rLV-MX-siR-NA)与对照组重组慢病毒(rLV-GFP)CT值,两者比较确定rLV-MX-siRNA滴度。rLV-Mx-siRNA以MOI=4感染猪胎儿成纤维细胞(PEF)并传代培养,提取后代细胞RNA检测外源基因转录以及应用间接免疫荧光试验检测鸡Mx蛋白的表达。结果表明,rLV-Mx-siRNA浓缩后滴度与已知滴度的rLV-GFP相等,为2×108 TU/mL,感染PEF效率>95%。感染rLV-Mx-siRNA的细胞传至第5代和第10代检测到外源基因转录以及鸡Mx蛋白的表达。表达鸡Mx基因和靶向AIV多靶点siRNA重组慢病毒滴度的测定与感染效率的分析,其结果为研究抗AIV转基因猪及其抗AIV的体外评价奠定了基础。  相似文献   

8.
选择H9N2亚型禽流感病毒(AIV)血凝素(HA)的两个保守T细胞表位基因和M2基因胞外保守序列(M2e),经人工合成并通过PCR扩增获得HA-M2融合表位基因,与载体p Fast Bac HTA连接,构建重组真核表达质粒p Fast HTA-HA-M2。阳性重组质粒转化DH10Bac感受态细胞,转染Sf9昆虫细胞获得含HA-M2基因的重组杆状病毒,重组杆状病毒感染Sf9细胞72 h后,进行SDS-PAGE、间接免疫荧光试验和Western blot鉴定。利用目的蛋白免疫3周龄SPF鸡,采用ELISA测定抗体滴度,MTT试验检测T淋巴细胞增殖。结果显示,目的蛋白HA-M2在昆虫细胞中有特异性表达,能被H9亚型AIV免疫阳性血清所识别,可诱导高滴度的AIV特异性抗体。试验结果为新型禽流感通用疫苗的研制奠定了基础。  相似文献   

9.
为检测H7N9禽流感病毒(AIV) DNA疫苗的免疫保护效力,本研究将H7N9禽流感疫苗株A/Chicken/Guangxi/SD098/2017 (H7N9)[CK/GX/SD098/17 (H7N9)]的HA基因密码子优化后,定向克隆至载体pCAGGS中构建重组质粒pCA-SD098。将pCA-SD098转染至293T细胞后,采用间接免疫荧光(IFA)和western blot检测,结果显示,HA蛋白可以在293T细胞中正确表达。分别利用15μg和20μg重组质粒pCA-SD098免疫3周龄SPF鸡,3周后以相同的剂量和方式加强免疫,加强免疫一周后通过鼻腔分别感染105EID50的同源高致病性AIV (HPAIV)CK/GX/SD098/17 (H7N9)和异源低致病性AIV (LPAIV) A/Chicken/Chongqing/SD057/2017 (H7N9)[CK/CQ/SD057/17 (H7N9)],攻毒10 d内记录免疫鸡发病和死亡情况,检测免疫鸡HI抗体水平,并于攻毒后第3 d、第5 d和第7 d无菌采集所有鸡的喉头和泄殖腔拭子,采用红细胞凝集试验(HA)检测病毒的滴度。结果显示,重组质粒pCA-SD098免疫后可以诱导SPF鸡产生较高水平的HI抗体,15μg pCA-SD098剂量免疫鸡后能够完全抵御H7N9 HPAIV的致死性攻击,20μg pCA-SD098剂量可以有效阻断H7N9 LPAIV的感染。攻毒后,所有免疫鸡无发病、无死亡、无排毒,本研究为质粒pCA-SD098作为防控H7N9禽流感的候选DNA疫苗提供了实验依据。  相似文献   

10.
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11.
The influenza A nucleoprotein (NP) is an attractive target for avian flu vaccine development because of its high conversancy in the evolutionary chain of the virus. Here we identified two novel HLA-A*0201 restricted NP epitopes, named H5N1 NP373-381 AMDSNTLEL (NP373) and NP458-466 FQGRGVFEL (NP458), using computational bioinformatic analysis. The NP peptides showed a high binding affinity to HLA-A*0201 on T2 cells, and were able to induce the activation of the cytotoxic T cells in the human peripheral blood mononuclear cells. We examined the potential of using NP373 and NP458 peptide sequences supplemented with a single-chain trimer as potential DNA vaccine candidates in an HHD transgenic mouse model. A gene gun delivery system was used for administrating the vaccine candidates into the animals. The results from cytotoxicity and ELISPOT assays indicated that a significant amount of IFN-γ was secreted by the T cells of the vaccinated mice, and the T cells were able to eliminate the corresponding peptide-loaded T2 cells. The discovery of these novel immunogenic NP peptides provides valuable information for avian flu vaccine design and construction.  相似文献   

12.
Chickens were inoculated with serotype 3 Pasteurella multocida cells or purified lipopolysaccharide (LPS), and their serologic responses to LPS and heat-stable antigens of 16 serotypes were compared. Chickens inoculated with cells or LPS had antibodies against LPS as determined by indirect hemagglutination tests; titers were highest 2-4 weeks after the initial inoculation. Sera from chickens inoculated with cells reacted with unheated and heated cell antigen in a tube-agglutination test. Sera from chickens inoculated with LPS reacted only with heated cell antigen in the tube-agglutination test. Nonspecific reactions with heat-stable antigens of other serotypes occurred in the gel-diffusion-precipitin test with sera from chickens inoculated with cells but not with sera from chickens inoculated with LPS. Antisera prepared against LPS could be used for serotyping field isolates of P. multocida.  相似文献   

13.
A Marek's disease (MD) lymphoblastoid cell line, MDCC-MSB1-41C, was highly transplantable and lethal for chickens. Autopsies showed extensive metastasis in various organs. The transplantabilities of the parent cell line, MDCC-MSB1, and another derivative line, MDCC-MSB1-33C, were transient. MD virus (MDV) could be isolated from the kidneys but not from the peripheral blood leukocytes of chickens inoculated with the MSB1-41C cell line. In addition, anti-MDV antibodies were produced both in chickens inoculated with this cell line and in controls raised with inoculated chickens, but several attempts to isolate MDV from this cell line in vitro failed.  相似文献   

14.
We had examined the immunogenicity of a series of plasmid DNAs which include neuraminidase (NA) and nucleoprotein (NP) genes from avian influenza virus (AIV). The interleukin-15 (IL-15) and interleukin-18 (IL-18) as genetic adjuvants were used for immunization in combination with the N1 and NP AIV genes. In the first trial, 8 groups of chickens were established with 10 specific-pathogen-free (SPF) chickens per group while, in the second trial 7 SPF chickens per group were used. The overall N1 enzyme-linked immunosorbent assay (ELISA) titer in chickens immunized with the pDis/N1 + pDis/IL-15 was higher compared to the chickens immunized with the pDis/N1 and this suggesting that chicken IL-15 could play a role in enhancing the humoral immune response. Besides that, the chickens that were immunized at 14-day-old (Trial 2) showed a higher N1 antibody titer compared to the chickens that were immunized at 1-day-old (Trial 1). Despite the delayed in NP antibody responses, the chickens co-administrated with IL-15 were able to induce earlier and higher antibody response compared to the pDis/NP and pDis/NP + pDis/IL-18 inoculated groups. The pDis/N1 + pDis/IL-15 inoculated chickens also induced higher CD8+ T cells increase than the pDis/N1 group in both trials (P < 0.05). The flow cytometry results from both trials demonstrated that the pDis/N1 + pDis/IL-18 groups were able to induce CD4+ T cells higher than the pDis/N1 group (P < 0.05). Meanwhile, pDis/N1 + pDis/IL-18 group was able to induce CD8+ T cells higher than the pDis/N1 group (P < 0.05) in Trial 2 only. In the present study, pDis/NP was not significant (P > 0.05) in inducing CD4+ and CD8+ T cells when co-administered with the pDis/IL-18 in both trials in comparison to the pDis/NP. Our data suggest that the pDis/N1 + pDis/IL-15 combination has the potential to be used as a DNA vaccine against AIV in chickens.  相似文献   

15.
The effects of chemically or virus-induced immunodepression on the infection profile (development of viremia and antibody) and shedding of avian leukosis virus (ALV) were studied in progeny chickens of experimental or commercial breeder flocks. Chickens were infected with ALV subgroup A by contact at hatching and by oral inoculation at 4-5 weeks of age. In the first experiment, chickens were inoculated with a virulent strain of infectious bursal disease virus (IBDV) at 1 day or 6 weeks of age. In the second experiment, chickens were neonatally treated with cyclophosphamide (CY), or were inoculated with strain T of reticuloendotheliosis virus (REV) at hatching, or were inoculated with strain JM of Marek's disease virus (MDV) at 2 weeks of age. The infection profile and cloacal shedding of ALV in chickens exposed to ALV and inoculated with immunodepressive viruses or CY were compared with those in hatchmates exposed only to ALV. In two of four chicken lines tested in the first experiment, shedding of ALV, as determined by virological assays of cloacal swabs at 22 weeks of age, was significantly higher in chickens infected with IBDV at 1 day of age than in uninfected hatchmates. The rate of shedding of ALV in one of these two lines was also significantly higher in chickens infected with IBDV at 6 weeks of age than in uninfected chickens. Further, the frequency of ALV-antibody detection at 22 weeks of age was significantly lower in chickens of these two lines infected with IBDV at 1 day of age than in uninfected chickens. In the second experiment, neonatal treatment with CY significantly increased the frequency of viremic chickens of both experimental and commercial flocks. The frequency of ALV-viremic chickens at 22 weeks of age was considerably higher in the REV- and MDV-inoculated groups (54% and 44%, respectively) than in control hatchmates (29%), but only in chickens of the commercial line. These findings suggest that chemically or virus-induced immunodepression may lead to an increase in rates of viremia and shedding of ALV in chickens infected with virus after hatching, especially in certain genetic lines.  相似文献   

16.
Transmissible viral proventriculitis (TVP) was experimentally reproduced in 2-wk-old specific-pathogen-free chickens and commercial broiler chickens by eyedrop inoculation of adenovirus-like virus (AdLV), isolate R1 1/3. No clinical signs and no weight gain depression were observed in chickens inoculated with AdLV (R11/3); however, gross and microscopic lesions characteristic of TVP were present in proventriculi of inoculated chickens. Proventriculi of AdLV (R11/3)-inoculated chickens were markedly enlarged, compared with sham-inoculated controls, by day 7 postinoculation (PI). Microscopic lesions in proventriculi of inoculated chickens were detected beginning on day 3 PI and consisted of degeneration and necrosis of glandular epithelium, ductal epithelial hyperplasia, replacement of glandular epithelium with ductal epithelium, and diffuse interstitial lymphoid infiltration; no microscopic lesions were observed in other tissues. AdLV (R11/3) antigens were detected in proventriculi by immunohistochemistry on days 3-10 PI in inoculated SPF chickens and days 3-21 PI in inoculated commercial broiler chickens; no viral antigens were detected in other tissues. AdLV (R11/3) was reisolated from proventriculi of inoculated SPF and commercial broiler chickens on days 5 and 7 PI. No virus, viral antigens, or lesions were detected in proventriculi collected from sham-inoculated chickens. These findings indicate an etiologic role for AdLV (R11/3) in TVP.  相似文献   

17.
To better understand the transmission route of H9N2 avian influenza virus (AIV), two duplicate trials were conducted to observe the process of aerosol infection and direct contact in specific pathogen free chickens. Fifteen chickens (G1) were inoculated with H9N2 AIV and housed together with another 15 chickens (G2) in the same positive-negative-pressure isolator (A). Fifteen chickens (G3) were bred in another isolator (B) which was connected with A so that air could flow unidirectionally from A to B. Air, oropharyngeal and cloacal swabs, and blood samples were collected for the detection of aerosolized virus, virus shedding, and seroconversion. AIV aerosols were initially detected at day 2-3 post inoculation (dpi), reaching peak concentrations at 7 dpi. Virus shedding was detected in all chickens of G2, but only in a part in G3 (T1: 87%, T2: 80%). Antibodies were initially detected at 4-5 dpi, peaking at 14-21 dpi. The results showed that H9N2 AIV could be transmitted by both aerosol exposure and direct contact.  相似文献   

18.
Jin M  Wang G  Zhang R  Zhao S  Li H  Tan Y  Chen H 《Avian diseases》2004,48(4):870-878
During the avian influenza outbreak of 2003-04 in Southeast Asia, two avian influenza viruses (AIV), one of H5N1 subtype and the other H9N2 subtype, were isolated and identified from local farms. The nudeoprotein (NP) gene of the H5N1 AI isolate was cloned, and the segment encoding amino acid 47-384, which covers its major antigenic domains, was subcloned and expressed in E. coli. Subsequently, the NP (47-384) expression product was purified and used as the diagnostic antigen to develop a NP-based type-specific indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to AI from chicken sera. The ELISA is shown to be specific for AIV and does not cross-react with chicken sera that has antibodies to other avian viruses. The NP(47-384)-ELISA was compared with a hemagglutination inhibition test and a commercial AIV ELISA kit in evaluating 150 sera samples from experimentally AIV-infected or vaccinated specific-pathogen-free (SPF) chickens. Our NP(47-384)-ELISA was more sensitive than the two tests and showed an 82% agreement ratio with the HI test and an 80.67% agreement ratio with the commercial kit. The NP(47-384)-ELISA and the commercial AIV ELISA were used to evaluate 448 field sera samples from diseased chickens or vaccinated chickens during the 2003-04 AI outbreak in China. The two ELISA tests had a 95% agreement ratio. We conclude that the NP(47-384)-ELISA developed in our laboratory was specific and sensitive and it has great application potential in China's long-term prevention and control of AI.  相似文献   

19.
The pathogenesis of six Newcastle disease virus (NDV) isolates recovered from chickens (Ckn-LBM and Ckn-Australia) and wild (Anhinga) and exotic (YN parrot, pheasant, and dove) birds was examined after the isolates had been passaged four times in domestic chickens. Groups of 10 4-wk-old specific-pathogen-free white leghorn chickens were inoculated intraconjunctivally with each one of the isolates. The infected birds were observed for clinical disease and were euthanatized and sampled at selected times from 12 hr to 14 days postinoculation or at death. Tissues were examined by histopathology, by immunohistochemistry (IHC) to detect viral nucleoprotein (IHC/NP), and by in situ hybridization to detect viral mRNA and were double labeled for apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling ([TUNEL] or IHC/caspase-3) and viral nucleoprorein (IHC/NP). Birds infected with the three low virulence viruses (Ckn-LBM, YN parrot, and Ckn-Australia) did not develop clinical disease. Microscopic lesions were observed only at the inoculation site and in organs of the respiratory system. The detection of viral nucleoprotein (N) was restricted to the inoculation site. The pheasant and dove isolates were highly virulent for chickens with marked tropism for lymphoid tissues, confirmed by the presence of large numbers of cells positive for viral N protein and viral mRNA. Viral N protein was detected early in the cytoplasm of cells in the center of the splenic ellipsoids. The apoptosis assays (TUNEL and IHC/caspase-3) showed increased apoptosis in the splenic ellipsoids as well. Apparently, apoptosis is an important mechanism in lymphoid depletion during NDV infection.  相似文献   

20.
Marek's disease virus (MDV) infection in the brain was studied chronologically after inoculating 3-week-old chickens of two genetic lines with two strains of serotype I MDV representing two pathotypes (v and vv+). Viral replication in the brain was strongly associated with the development of lesions. Three viral antigens (pp38, gB, and meq) were detected in the brain of infected chickens. Marked differences between v and vv+ pathotypes of MDV were identified for level of virus replication, time course of brain lesions, and expression of major histocompatibility complex (MHC) antigens. Two pathologic phenomena (inflammatory and proliferative) were detected in the brain of chickens inoculated with vv+MDV, but only inflammatory lesions were observed in those inoculated with vMDV. Inflammatory lesions, mainly composed of macrophages, CD4+ T cells, and CD8+ T cells, started at 6-10 days postinoculation (dpi) and were transient. Proliferative lesions, characterized by severe infiltrates of CD4+CD8- T cells (blasts), started at 19-26 dpi and persisted. Expression of MHC antigens in endothelial cells and infiltrating cells within the brain was influenced by MDV infection. Upregulation of MHC class II antigen occurred in all treatment groups, although it was more severe in those inoculated with vv+MDV. MHC class I antigen was downregulated only in those groups inoculated with vv+MDV. These results enhance our understanding of the nature and pattern of MDV infection in the brain and help to explain the neurovirulence associated with highly virulent MDV.  相似文献   

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