甲型H1N1流感病毒HA基因的原核表达及免疫反应性分析

目的构建甲型H1N1流感病毒HA基因的原核表达质粒,获得融合表达蛋白,并对表达蛋白的免疫反应性进行分析。方法人工合成A/California/05/2009 H1N1流感病毒的HA基因,以合成基因为模板,通过PCR方法扩增出去除信号肽的HA部分基因片段,然后将去除信号肽的HA基因克隆至pET30a(+)原核表达载体中,构建出原核表达质粒,再将重组质粒转化E.coliBL21(DE3)表达菌株;重组菌经IPTG诱导后,收集菌体进行SDS-PAGE电泳分析,Western blot分析表达产物的免疫反应性。结果获得了HA基因的原核表达重组菌,细菌经IPTG诱导表达后,SDS-PAGE分析可见约67.4 ku大小的目的蛋白表达条带,Western blot结果显示,表达产物与人甲型H1N1流感患者阳性血清具有反应性。结论成功表达出甲型H1N1流感病毒的HA蛋白,该蛋白具有良好的免疫反应性,为甲型H1N1流感的快速诊断方法的建立提供了生物材料。     

重组沙门菌表达结核分枝杆菌ESAT-6及其特异性免疫应答分析

Objective To determine the immune responses induced by recombinant Salmonella ty-phimurium expressing the secreting antigen ESAT-6 of Mycobacterium tuberculosis. Methods ESAT-6 cod-ing gene was cloned and identified by PCR and sequencing. Prokaryotic expression plasmid pYA33-esat car-rying the ESAT-6 coding sequence was constructed firstly and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria was named as X4550(33-esat). C57BL/6 mice were immunized intranasally (I. N) with 108 CFU recombinant bacteria at day 0 and 18. Cells from spleen, lung, mesenteric lymph node (MLN) and Peyer's patch (PP) were collected from mice after second immu-nization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected by ELISPOT assay u-sing ESAT-6 peptide as stimulus. Furthermore, CTL effects were in vivo evaluated by CFSE assay. Results The results showed that cellular immune responses specific for ESAT-6 could be detected by ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 were biased toward Th1 type, the frequency of IFN-γ-secreting cells was much higher than that of IL-4-secreting cells. In splenocytes and MLN cells, the anti-gen specific immune responses acted as Thl and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. CFSE assay indicated that recombinant bacteria could induce the high level of CTL effects specific for ESAT-6 peptide. Conclusion These results suggested that recombinant Sal-monella typhimurium X4550(33-esat) not only can induce cellular immune responses, but also can elicit specific CTL responses after I. N immunization. It also provided the useful information for the control of infec-tious disease of tuberculosis.     

鸡白痢沙门菌SpiC蛋白的原核表达及其应用

目的进行鸡白痢沙门菌Spi C蛋白原核表达并建立以Spi C蛋白为检测抗原的间接ELISA。方法以鸡白痢沙门菌S06004基因组DNA为模板,PCR扩增384 bp的spi C基因。将spi C基因克隆至原核表达载体p ET30a中,构建重组原核表达质粒p ET30a-spi C,再转化到表达宿主菌E.coli BL21(DE3)中,异丙基β-D-硫代半乳糖苷(IPTG)诱导表达,SDS-PAGE和Western blot分析鉴定,纯化His-Spi C蛋白,建立Spi C蛋白为检测抗原的间接ELISA并检测临床血清样品。结果通过对诱导后重组菌裂解产物进行SDS-PAGE和Western blot分析,重组菌可以表达相对分子质量(Mr)约19 200的可溶性重组蛋白His-Spi C。重组蛋白GST-Spi C免疫无特定病原体鸡,所获得的高免血清能识别重组蛋白His-Spi C,表明体外表达的重组蛋白His-Spi C有良好的免疫反应原性。所建立的以重组蛋白His-Spi C介导的间接ELISA有较好的特异性,能够区分spi C基因缺失型疫苗候选株免疫与野生菌自然感染。结论重组蛋白His-Spi C呈可溶性表达,具有良好的免疫原性,以重组蛋白His-Spi C建立的间接ELISA,可以作为区分感染和免疫动物(DIVA)鸡白痢疫苗免疫的鉴定方法。     

重组沙门菌表达结核分枝杆菌ESAT-6及其特异性免疫应答分析

Objective To determine the immune responses induced by recombinant Salmonella ty-phimurium expressing the secreting antigen ESAT-6 of Mycobacterium tuberculosis. Methods ESAT-6 cod-ing gene was cloned and identified by PCR and sequencing. Prokaryotic expression plasmid pYA33-esat car-rying the ESAT-6 coding sequence was constructed firstly and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria was named as X4550(33-esat). C57BL/6 mice were immunized intranasally (I. N) with 108 CFU recombinant bacteria at day 0 and 18. Cells from spleen, lung, mesenteric lymph node (MLN) and Peyer's patch (PP) were collected from mice after second immu-nization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected by ELISPOT assay u-sing ESAT-6 peptide as stimulus. Furthermore, CTL effects were in vivo evaluated by CFSE assay. Results The results showed that cellular immune responses specific for ESAT-6 could be detected by ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 were biased toward Th1 type, the frequency of IFN-γ-secreting cells was much higher than that of IL-4-secreting cells. In splenocytes and MLN cells, the anti-gen specific immune responses acted as Thl and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. CFSE assay indicated that recombinant bacteria could induce the high level of CTL effects specific for ESAT-6 peptide. Conclusion These results suggested that recombinant Sal-monella typhimurium X4550(33-esat) not only can induce cellular immune responses, but also can elicit specific CTL responses after I. N immunization. It also provided the useful information for the control of infec-tious disease of tuberculosis.     

肠炎沙门菌ΔspiCΔcrp基因工程疫苗候选株的构建

目的 构建肠炎沙门菌ΔspiCΔcrp双基因缺失株,以探索其作为新型基因工程疫苗的可能性。方法 以等位基因同源重组方法,在构建单基因缺失肠炎沙门菌ΔspiC基础上,运用λRed重组酶系统构建双基因缺失株肠炎沙门菌ΔspiCΔcrp。结果 PCR和抗生素抗性结果表明肠炎沙门菌ΔspiCΔcrp成功构建;生物学鉴定显示,与野生菌相比,其生长速度与生化特性发生了变化,LD₅₀提高约1 000倍,毒力显著降低。结论 双基因缺失株肠炎沙门菌ΔspiCΔcrp被成功构建,为其作为疫苗的免疫学评价奠定基础。     

我国地方品种鸡IL-17cDNA的克隆、表达及鉴定

目的:克隆我国地方品种鸡IL-17 cDNA,构建该基因的原核表达质粒,获得融合表达蛋白并鉴定其免疫特性。方法:利用特异性引物,通过RT-PCR方法扩增得到隐性白羽鸡IL-17(ChIL-17)的基因片段,PCR产物克隆至原核表达载体pGEX-6P-1中,构建重组表达质粒pGEX-6P-1-ChIL17。将重组质粒pGEX-6P-1-ChIL17转化E.coliBL21,IPTG诱导表达目的蛋白,应用SDS-PAGE和Western blot分析鉴定表达产物。结果:成功扩增出ChIL-17基因片段,大小约510 bp,序列与GenBank登录的序列(NM 204460)相比,核苷酸同源性为99.8%,第477位碱基由G变为A,氨基酸同源性为100%。酶切鉴定结果表明,ChIL-17基因正确克隆入原核表达载体pGEX-6P-1中。重组质粒pGEX-6P-1-ChIL17在大肠杆菌中获得表达,SDS-PAGE结果显示出Mr约46 000大小的目的蛋白表达条带;Western blot结果表明,表达产物与小鼠IL-17抗体具有良好的反应性。结论:成功克隆并表达出我国地方品种鸡ChIL-17基因,为抗ChIL-17单...     

红细胞富集与多重RT-PCR联合检测禽流感病毒

目的对禽流感病毒H5、H7亚型进行更快速、更灵敏检测。方法根据禽流感病毒(AIV)核蛋白基因和血凝素基因设计3对特异性引物,建立在结合红细胞富集AIV病毒的基础上进行多重RT-PCR检测方法。结果该方法在1个反应体系中不仅能够鉴定A型流感病毒,而且可以同时确定是否为H5和H7亚型AIV;该方法的检测下限可达2.5pg的病毒RNA;对于参考毒株都可以扩增出预期大小的基因片段,而对新城疫病毒和传染性支气管炎病毒的平行对照结果均呈阴性。结合AIV能够吸附鸡红细胞并在一定条件下解离的特点,利用鸡红细胞对48份已确诊样品进行病毒富集,然后用建立的多重RT-PCR方法检测富集产物结果显示48个样品中,30个为H5亚型AIV阳性,与病毒分离结果完全一致。结论由以上结果初步表明本研究建立的检测方法快速,特异,在禽流感临床筛检中显示出良好的应用前景。     

重组沙门菌表达结核分枝杆菌ESAT-6及其特异性免疫应答分析

Objective To determine the immune responses induced by recombinant Salmonella ty-phimurium expressing the secreting antigen ESAT-6 of Mycobacterium tuberculosis. Methods ESAT-6 cod-ing gene was cloned and identified by PCR and sequencing. Prokaryotic expression plasmid pYA33-esat car-rying the ESAT-6 coding sequence was constructed firstly and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria was named as X4550(33-esat). C57BL/6 mice were immunized intranasally (I. N) with 108 CFU recombinant bacteria at day 0 and 18. Cells from spleen, lung, mesenteric lymph node (MLN) and Peyer's patch (PP) were collected from mice after second immu-nization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected by ELISPOT assay u-sing ESAT-6 peptide as stimulus. Furthermore, CTL effects were in vivo evaluated by CFSE assay. Results The results showed that cellular immune responses specific for ESAT-6 could be detected by ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 were biased toward Th1 type, the frequency of IFN-γ-secreting cells was much higher than that of IL-4-secreting cells. In splenocytes and MLN cells, the anti-gen specific immune responses acted as Thl and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. CFSE assay indicated that recombinant bacteria could induce the high level of CTL effects specific for ESAT-6 peptide. Conclusion These results suggested that recombinant Sal-monella typhimurium X4550(33-esat) not only can induce cellular immune responses, but also can elicit specific CTL responses after I. N immunization. It also provided the useful information for the control of infec-tious disease of tuberculosis.     

沙门菌果蝇感染致死模型构建及其评价

目的 为了比较不同沙门菌对果蝇致死毒力,建立果蝇沙门菌肠道感染模型,并进行模型评价。方法 将鼠伤寒沙门菌SL1344、14028S和肠炎沙门菌C50041及其突变株C50041ΔspiC,以OD₆₀₀为100的高浓度菌液通过饲喂法和针刺法,分别感染30只CS型果蝇和yw型果蝇,每隔3 h观察活果蝇数,计算存活率,分析沙门菌毒力。结果 肠炎沙门菌C50041和C50041ΔspiC饲喂果蝇,120小时后CS型果蝇存活率0%,yw型果蝇的存活率低于20%,而针刺法感染CS型果蝇和yw型果蝇存活率则高于80%;鼠伤寒沙门菌饲喂法感染CS型果蝇和yw型果蝇,其存活率均高于90%。针刺法存活率均高于60%。比较分析显示,鼠伤寒沙门菌肠道感染耐受和肠炎沙门菌肠道感染敏感,存在明显的差异;肠炎沙门菌对果蝇肠道感染模式的毒力明显高于非肠道感染模式,不同果蝇品系之间则无明显的差异。结论 本研究中,果蝇沙门菌肠道感染模型被成功建立,并能够评价不同沙门菌的毒力,其中肠炎沙门菌C50041果蝇肠道感染呈高度敏感,为后续肠炎沙门菌功能基因鉴定研究奠定基础,便于深入研究沙门菌致病机理。     

Real-Time PCR探针法定量检测沙门菌方法的建立及应用

目的建立快速、特异性好、灵敏度高的Real-Time PCR方法定量检测沙门菌。方法根据编码沙门菌肠毒素基因stn的核苷酸序列,设计荧光探针和一对引物,通过对荧光定量PCR反应体系和反应条件的摸索,建立定量检测沙门菌的方法。结果建立的Real-Ti me PCR方法有很好的特异性与敏感性,所检测沙门菌结果均为阳性,而非沙门菌均为阴性;标准曲线相关系数为R2=0.993,其敏感性为5CFU。运用该方法对108份鸡粪便、50份鸡肉以及58份水样进行检测,阳性率分别为3.7%(6/108)、4%(2/50)和3.4%(2/58),与传统细菌分离检测结果相符。结论结果表明该方法具有简便、快速、特异性强、敏感性高等特点,此研究为环境及疾病诊断中沙门菌快速检测提供了新方法。