问号钩端螺旋体

摘要： 目的 探讨问号钩端螺旋体溶血素Sph2经高迁移率组蛋白B1(high mobility group box-1,HMGBl)放大炎症反应的可能机制.方法 一定浓度的rSph2与小鼠J774A.1巨噬细胞共孵育一定时间,乳酸脱氢酶(LDH)量的变化检测细胞的膜结构损伤情况;冷冻电镜观察细胞结构的改变;ELISA方法检测细胞上清液中HMGB1的含量变化.商品化的HMGB1与小鼠J774A.1巨噬细胞共孵育一定时间,Western blot检测NF-κB、p38-MAPK及JNK炎症信号通路关键分子的磷酸化水平;ELISA检测IL-1β、IL-6、KC(I L-8)等促炎细胞因子的表达情况.结果 重组问号钩端螺旋体溶血素rSph2可导致J774A.1细胞明显的细胞核消失、细胞膜结构损伤、细胞裂解、膜泡胀;LDH释放量及HMGB1的含量明显增加;HMGB1增加NF-κB、p38-MAPK及JNK信号通路关键蛋白质的磷酸化水平;HMGB1上调J774A.1细胞IL-1β、IL-6、KC的表达且该上调作用可被3条通路抑制剂所抑制.结论 重组问号钩端螺旋体溶血素rSph2诱导小鼠J774A.1巨噬细胞产生损伤并释放大量的HMGB1,HMGB1经NF-κB、p38-MAPK及JNK信号通路调控下游促炎细胞因子的表达,从而放大rSph2造成的炎症效应.

摘要： 目的 探讨问号钩端螺旋体(简称钩体)跨血管内皮细胞转运分子机制.方法 采用Transwell法了解问号钩体赖株穿越人脐静脉血管内皮细胞(HUVEC)单层能力.采用透射电镜和激光共聚焦显微镜法检测HUVEC内吞问号钩体后形成内吞泡情况.采用细胞内吞抑制试验确定HUVEC内吞问号钩体方式.采用激光共聚焦显微镜法检测HUVEC胞内问号钩体与溶酶体标志分子LAMP1共定位情况.采用暗视野显微镜Petroff-Hausser计数法检测HUVEC胞内问号钩体出胞情况.结果 问号钩体赖株能迅速穿越HUVEC细胞单层.问号钩体通过PI3K-微丝依赖的细胞内吞方式进入HUVEC并形成内吞泡.HUVEC胞内问号钩体不与LAMP1共定位,提示问号钩体内吞泡不与溶酶体融合.HUVEC胞内问号钩体通过FAK-微丝/微管途径出胞.结论 问号钩体通过跨细胞转运方式穿越人血管内皮细胞导致体内播散并加重病情.

摘要： 目的 确定问号钩端螺旋体vWF-A基因产物结合人胶原蛋白的作用与机制.方法 采用生物信息学软件分析问号钩端螺旋体黄疸出血群赖型赖株vWF-A基因(LA_ 0012、LA _0697和LA_4207)产物结构与功能.构建上述基因vWF-A功能结构域片段原核表达系统,采用SDS-PAGE和Ni-NTA亲和层析法检查目的重组蛋白rLep0012、rLep0697和rLep4207表达情况和提纯效果.采用ELISA和表面等离子共振法(SPR)检测目的重组蛋白与人I、Ⅲ、Ⅳ和Ⅵ型胶原蛋白(hCOL1/3/4/6)的结合能力.采用实时荧光定量RT-PCR和Western blot检测问号钩端螺旋体赖株感染人和小鼠血管内皮细胞(HUVEC和EOMA)时vWF-A基因转录和表达水平变化.结果 vWF-A基因产物均为含vWF-A超家族结构域表面或跨膜蛋白,但LA_0697和LA_4207基因还含有金属离子依赖性黏附位点(MIDAS).所构建的原核表达系统能有效表达目的重组蛋白,Ni-NTA亲和层析法提纯后SDS-PAGE检测均显示为单一蛋白条带.ELISA结果显示rLep0697与hCOL3/6、rLep4207与hCOL1/4呈强结合.SPR结果显示rLep0697快速结合hCOL3/6但快速解离(Kp值=5.71×10-8和5.89×10-8 mol/L),rLep4207快速并稳定结合hCOL1/4(Kp值=6.4×10-9和3.2×10-9 mol/L).感染HUVEC和EOMA细胞时,上述vWF-A基因转录和表达水平均显著升高(P＜0.05).结论 LA_0697和LA._4207基因产物可作为问号钩端螺旋体感染过程中的黏附因子.

摘要： 目的 了解铁对问号钩端螺旋体生长繁殖和能量代谢的影响,确定LA_2690与LA_3598基因产物为问号钩端螺旋体储铁蛋白及其亚铁氧化酶功能.方法 采用Petroff-Hausser计数法了解缺铁对问号钩端螺旋体黄疸出血群赖型56601株在EMJH培养基中生长繁殖的影响.分光光度法和化学发光法检测缺铁抑制问号钩端螺旋体DNA和ATP合成的作用.采用生物信息学软件分析问号钩端螺旋体LA_ 2690和LA_3598基因结构与功能.构建LA_2690和LA_3598基因原核表达系统,Ni-NTA亲和层析法提纯目的重组蛋白rLep2690和rLep3598.分光光度法检测rLep2690和rLep3598亚铁氧化酶活性.实时荧光定量RT-PCR检测问号钩端螺旋体56601株感染人脐静脉内皮细胞(HUVEC)和单核细胞(THP-1)时LA_2690和LA_3598基因转录水平变化.结果 在缺铁EMJH培养基中,问号钩端螺旋体生长繁殖能力、DNA和ATP合成水平均显著下降(P＜0.05).LA_2690和LA_3598基因产物分别为细菌铁蛋白(Bfr)和细菌DNA结合铁蛋白(Dps),均含有双亚铁氧化酶中心,但后者缺乏亚铁血红素结合位点和亚铁氧化酶核心.LA_ 2690和LA_3598基因原核表达系统能高效表达目的重组蛋白rLep2690和rLep3598,提纯后经SDS-PAGE检测均显示为单一的蛋白质条带.rLep2690和rLep3598亚铁氧化酶活性分别为1 238.619和60.052 U/L.感染细胞时,LA_2690和LA_3598基因mRNA水平均显著升高(P＜0.05).结论 铁离子参与问号钩端螺旋体生长繁殖、DNA和ATP合成.LA 2690和LA 3598基因(Bfr和Dps)均为问号钩端螺旋体感染细胞时所需,其中LA_2690基因产物具有较强亚铁氧化酶活性.

摘要： Objective To analyze the enzymatic activity of Leptospira interrogans ( L. interrogans) LA_2144 gene product to hydrolyze platelet activating factor acetylhydrolase ( PAF-AH) and phosphatidase A2(PLA2). Methods Bioinformatic softwares were used to predict transmembrane regions, signal peptides and domains of the LA_2144 gene of L. interrogans strain Lai. A prokaryotic expression system for signal peptide-free LA_2144 gene was established. The expressed target recombinant protein rLep2144 was extrac-ted by Ni-NTA affinity chromatography and then renatured. Spectrometry was used to detect the activity of rLep2144 to hydrolyze PAF-AH substrate 2-thio PAF and the Km and Kcat values as well as the activity to hy-drolyze PLA2 substrate arachidonoyl 2-thio PC. Real-time fluorescence quantitative RT-PCR and Western blot were performed to detect the transcription, protein expression and secretion of LA_2144 gene during infection of human and mouse vascular endothelial cells ( HUVEC and EOMA) with L. interrogans. Results L. interrogans LA_2144 gene contained a signal peptide and a domain belonging to SGNH hydrolase super-family, but no transmembrane regions. The established prokaryotic expression system for signal peptide-free LA_2144 gene could efficiently express rLep2144. The extracted rLep2144 was shown as a single protein fragment in separation gel and then successfully renatured. rLep2144 had a stronger PAF-AH activity with the Km and Kcat values of 688. 235 μmol/L and 0. 976/s, but its PLA2 activity was relatively weak. Expres-sion of the LA_2144 gene at mRNA and protein levels in HUVEC and EOMA was rapidly increased after the cells were infected with L. interrogans (P<0. 05) and the secretion of LA_2144 gene product could be detec-ted. Conclusions L. interrogans LA_2144 gene product had a stronger PAF-AH and a certain PLA2 activi-ty, which might involve in the hemorrhage and inflammatory response in leptospirosis.%目的 了解问号钩端螺旋体LA_2144基因产物血小板活化因子乙酰水解酶(PAF-AH)和磷脂酶A2(PLA2)的酶活性.方法 生物信息学软件预测问号钩端螺旋体赖株LA_2144基因跨膜区、信号肽和结构域.构建无信号肽LA_2144基因原核表达系统,Ni-NTA亲和层析法提纯目的重组蛋白rLep2144后复性.采用分光光度法检测rLep2144水解PAF-AH底物2-thio PAF的活性及其Km和Kcat值以及水解PLA2底物花生四烯酸硫代卵磷脂的活性.采用实时荧光定量RT-PCR和West-ern blot法检测问号钩端螺旋体感染人和小鼠血管内皮细胞(HUVEC和EOMA)后LA_2144基因转录、蛋白质表达和外分泌情况.结果 LA_2144基因无跨膜区但有信号肽和SGNH水解酶超家族结构域.所构建的无信号肽LA_2144基因原核表达系统能高效表达rLep2144,提纯后的rLep2144在分离胶上为单一蛋白质条带并成功复性.rLep2144有较强PAF-AH活性,其Km和Kcat值分别为688.235μmol/L和0.976/s,但PLA2活性较弱.问号钩端螺旋体感染HUVEC和EOMA后LA_2144基因mRNA转录和蛋白质表达水平迅速升高(P<0.05)且外分泌.结论 问号钩端螺旋体LA_2144基因产物具有较强PAF-AH活性和一定的PLA2活性,可在钩端螺旋体病出血性病变和炎症反应中发挥作用.

摘要： Objective To understand the differences in engulfing ability and phagolysosome for-mation between mononuclear-macrophages and neutrophils during Leptospira interrogans infection. Methods Human THP-1 monocytes and HL-60 cells were pretreated with PMA ( phorbol-12-myristate-13-acetate) and ATRA ( all-trans retinoic acid) to differentiate them into mononuclear-macrophages and neutrophils, respec-tively. The phagocytosis of Leptospira interrogans in THP-1-PMA mononuclear-macrophages and HL-60-AT-RA neutrophils was detected by confocal microscopy. The morphology of intracellular Leptospira was deter-mined by transmission electron microscopy. The viability of phagocytized Leptospira and the percentages of dead Leptospira were analyzed by confocal microscopy and spectrofluorimetry, respectively. Confocal micros-copy was used to measure the formation of phagolysosomes in different phagocytes. Results Both THP-1-PMA mononuclear-macrophages and HL-60-ATRA neutrophils could phagocytize Leptospira interrogans, but the phagocytic ability of the former was notably stronger than that of the latter (P<0. 05). Intracellular Lep-tospira were surrounded by phagocytic vesicles in both types of phagocytes. THP-1-PMA mononuclear-mac-rophages were better than HL-60-ATRA neutrophils in killing intracellular Leptospira (P<0. 05). More phagolysosomes were formed in THP-1-PMA mononuclear-macrophages than in HL-60-ATRA neutrophils ( P<0. 05). Conclusions Human mononuclear-macrophages but not neutrophils act as major phagocytes that play an important role in phagocytizing and killing Leptospira during infection. Less fusion of the phagosomes with lysosomes may be responsible for the lower Leptospira-killing ability of neutrophils.%目的 了解单核-巨噬细胞和中性粒细胞抗问号钩端螺旋体(简称钩体)感染能力及吞噬溶酶体形成是否存在差异.方法 人THP-1单核细胞和HL-60细胞分别用佛波酯(PMA)和全反式维甲酸(ATRA)预处理,使其分化为单核-巨噬细胞和中性粒细胞.采用激光共聚焦显微镜法检测THP-1-PMA单核-巨噬细胞和HL-60-ATRA中性粒细胞吞噬问号钩体能力.采用透射电镜法检测胞内钩体形态.采用激光共聚焦显微镜法和荧光分光光度法检测胞内问号钩体活力和死亡率.采用激光共聚焦显微镜法检测不同吞噬细胞内吞噬溶酶体形成率.结果 THP-1-PMA单核-巨噬细胞和HL-60-ATRA中性粒细胞均能吞噬问号钩体,但前者吞噬问号钩体能力显著强于后者(P<0.05).胞内问号钩体都以吞噬泡的形式存在于上述两种吞噬细胞内.THP-1-PMA单核-巨噬细胞杀灭胞内问号钩体能力显著强于HL-60-ATRA中性粒细胞(P<0.05).THP-1-PMA单核-巨噬细胞内问号钩体与溶酶体共定位率显著高于相应的HL-60-ATRA中性粒细胞(P<0.05).结论 单核-巨噬细胞而非中性粒细胞作为主要的吞噬细胞在吞噬、杀灭入侵问号钩体过程中起着重要作用.中性粒细胞内较低的吞噬溶酶体形成率可能是导致其杀灭问号钩体能力较低的原因.

摘要： 目的 确定问号钩端螺旋体(简称钩体)赖株AhpC蛋白的过氧化物还原酶活性及其在问号钩体感染宿主细胞过程中是否具有抵抗氧化应激的功能.方法 构建问号钩体黄疸出血群赖型赖株ahpC基因原核表达系统.Ni-NTA亲和层析法提纯目的重组蛋白rAhpC,检测其酶学活性和保护DNA不被氧化的功能.用定点突变方法验证AhpC的过氧化半胱氨酸和还原性半胱氨酸.经不同浓度的Conoidin A抑制问号钩体过氧化物还原酶活性后,比较抑制前后构体内的活性氧自由基(reactive oxygen species,ROS)水平和钩体存活率的变化.结果 所构建的原核表达系统能有效表达rAhpC.rAhpC具有过氧化物还原酶的活性,其催化反应依赖于硫氧还蛋白和硫氧还蛋白还原酶系统.AhpC分子中含有的两个半胱氨酸对维持酶的活性起着至关重要的作用,其中Cys47是AhpC的过氧化半胱氨酸,而Cys167是还原性半胱氨酸.AhpC能够保护质粒DNA免遭过氧化氢的氧化损伤.经不同浓度的Conoidin A抑制问号钩体的过氧化物还原酶的活性后,钩体在巨噬细胞内的存活率明显下降,并且呈现出剂量依赖的效应,表明钩体存活率的降低与钩体的过氧化物还原酶活性丧失从而无法有效降解构体内活性氧水平有着密切的关系.结论 问号钩体的AhpC具有过氧化物还原酶的活性,能抵抗宿主细胞引起的氧化损伤,与钩体在巨噬细胞内的存活有关.%Objective To analyze the enzymatic properties of alkyl hydroperoxide reductase sub-unit C (AhpC) from Leptospira interrogans (L.interrogans) and to elucidate its physiological roles in host-pathogen interactions in macrophages during Leptospira infection. Methods A prokaryotic expression system for ahpC gene of L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was established to ex-press the recombinant AhpC(rAhpC). After purified by Ni-NTA affinity chromatography,the enzymatic ac-tivity of the rAhpC and its role in protecting DNA from oxidation were analyzed. The importance of each cys-teine in its molecule was evaluated through site-directed mutation. L.interrogans strains were pretreated with or without Conoidin A, a covalent inhibitor of peroxiredoxin, and then were used to infect macrophages. Changes in oxidative status in leptospires and survival rates of L.interrogans strains were analyzed by fluores-cence-activated cell sorting and colony counting method. Results The rAhpC was successfully expressed in the established prokaryotic expression system. It had peroxiredoxin activity that was able to catalyze the re-duction of hydrogen peroxide. Its ability of reducing hydrogen peroxide depended on the thioredoxin/thiore-doxin reductase system. Cys47 (a peroxidatic cysteine) and Cys167 (a resolving cysteine) were critical to maintaining the enzymatic activity of AhpC. AhpC could protect DNA from hydrogen peroxide induced-oxida-tive damage. When L.interrogans strains were pretreated with Conoidin A,the oxidative status in leptospires was elevated and the survival of L.interrogans in macrophages was significantly reduced in a dose-dependent manner. Conclusion The AhpC of L.interrogans is a thioredoxin-dependent peroxiredoxin that plays an im-portant role in protecting L.interrogans against oxidative stress in macrophages.

摘要： Objective To investigate the influences of Leptospira interrogans (L.interrogans) in-fection on the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion mole-cule-1 (VCAM-1) on endothelial cells. Methods Expression of ICAM-1 and VCAM-1 at mRNA level was detected by reverse transcription-polymerase chain reaction (RT-PCR) after infecting human umbilical vein endothelial cells (HUVEC) with L.interrogans strain Lai. Silver staining was used to detect leptospires in lung,liver and kidney tissues of L.interrogans-infected C3H/HeJ mice. Expression of ICAM-1 and VCAM-1 in lung,liver and kidney tissues of L.interrogans-infected mice was measured with immunohistochemistry. Results L.interrogans infection increased the expression of ICAM-1 and VCAM-1 on HUVEC(P<0.05). Moreover,the expression of VCAM-1 at mRNA level was significantly higher than that of ICAM-1 (P<0.05). Silver-stained leptospires could be found in lung,liver and kidney tissues of L.interrogans-infected C3H/HeJ mice. Results of the immunohistochemical examination showed that increased expression of both ICAM-1 and VCAM-1 could be detected in ling,liver and kidney tissues of L.interrogans-infected mice,and the VCAM-1 level was significantly higher than that of ICAM-1 in every tissue sample(P<0.05). Conclu-sion L.interrogans infection could induce the expression of ICAM-1 and VCAM-1 on endothelial cells and increase the expression of VCAM-1 to a level significantly higher than that of ICAM-1, which mediated the infiltration of specific inflammatory cells to the site of infection.%目的 了解问号钩端螺旋体(简称钩体)感染对内皮细胞表面细胞间黏附分子-1 (ICAM-1)和血管细胞黏附分子-1(VCAM-1)表达的影响及差异.方法 采用问号钩体赖株体外感染人脐静脉内皮细胞(HUVEC)后运用real-time RT-PCR技术检测细胞ICAM-1和VCAM-1 mRNA表达水平.采用组织镀银染色法检测问号钩体感染C3H/HeJ小鼠后肺、肝、肾组织内钩体侵袭情况.采用免疫组化法检测小鼠肺、肝、肾组织中 ICAM-1和 VCAM-1表达的差异.结果 问号钩体感染HUVEC后细胞ICAM-1和VCAM-1 mRNA表达水平与感染前相比均显著升高(P<0.05),且VCAM-1 mRNA表达水平显著高于ICAM-1(P<0.05).组织镀银染色结果显示在感染C3H/HeJ小鼠肺、肝、肾组织内均出现入侵钩体.免疫组化结果显示,问号钩体感染小鼠后,肺、肝、肾组织内均出现ICAM-1和VCAM-1表达升高,但VCAM-1的表达量显著高于ICAM-1(P<0.05).结论 问号钩体感染可激活内皮细胞ICAM-1和VCAM-1的表达,且VCAM-1的表达量显著高于ICAM-1,从而介导特异性炎症细胞浸润至感染部位.

摘要： 目的 了解问号钩端螺旋体(简称钩体)感染过程中单核-巨噬细胞和中性粒细胞浸润的差异及其机制.方法 采用问号钩体赖株感染C3H/HeJ小鼠后运用HE染色法检测小鼠肺、肝、肾组织病理变化.采用免疫组化法检测肺、肝、肾组织中外周血来源的CD11b阳性单核-巨噬细胞和Ly6G阳性中性粒细胞浸润的差异.采用趋化因子抗体芯片检测问号钩体感染小鼠血清中单核-巨噬细胞和中性粒细胞趋化因子表达水平变化.结果 问号钩体感染C3H/HeJ小鼠后,肺、肝、肾组织出现典型的钩体病病理变化,如炎性细胞浸润、肺出血、肝细胞坏死、肾充血等.免疫组化结果显示,问号钩体感染小鼠后,肺、肝、肾组织中出现大量外周血来源的单核-巨噬细胞浸润,而在以上组织中只检测到少量中性粒细胞浸润.小鼠趋化因子抗体芯片检测结果显示,问号钩体感染小鼠血清中单核-巨噬细胞趋化因子(I-309、MCP-1、MCP-5、MIP-1α和RANTES)表达水平与正常小鼠相比显著升高(P0.05).结论 问号钩体感染过程中,单核-巨噬细胞而非中性粒细胞作为主要的浸润吞噬细胞在清除入侵问号钩体时起着重要作用.%Objective To understand the differences between infiltration of mononuclear-macro-phages and neutrophils during Leptospira interrogans (L.interrogans) infection and the underlying mecha-nisms. Methods Histological changes in mouse lung, liver and kidney tissues were detected using hema-toxylin and eosin (HE) staining following infection of C3H/HeJ mice with L.interrogans serovar Lai strain 56601. Infiltration of peripheral blood-derived CD11b+mononuclear-macrophages and Ly6G+neutrophils in lung,liver and kidney tissues collected form L.interrogans-infected C3H/HeJ mice was detected with immu-nohistochemistry. Levels of mononuclear-macrophage chemokines and neutrophil chemokines in serum sam-ples of L.interrogans-infected mice were detected with chemokine detection microarray. Results Lung,liv-er and kidney tissue samples collected from L. interrogans-infected C3H/HeJ mice presented typical his-topathological changes of leptospirosis, such as inflammatory cell infiltration in these tissues, pulmonary hemorrhage,extensive hepatocyte necrosis and serious nephrohemia. Results of immunohistochemical stai-ning showed that a large number of peripheral blood-derived CD11b+mononuclear-macrophages were presen-ted in lung,liver and kidney tissues of L.interrogans-infected mice, but few neutrophils could be found in these tissues. The mouse chemokine detection microarray confirmed that the levels of mononuclear-macro-phage chemokines (I-309,MCP-1,MCP-5,MIP-1α and RANTES) in serum samples of L.interrogans-in-fected C3H/HeJ mice were significantly increased during infection (P0.05). Conclu-sion Mononuclear-macrophages rather than neutrophils are the major infiltrating phagocytes during L.inter-rogans infection and play a crucial role in the elimination of Leptospira invasion.

摘要： 目的：构建问号钩端螺旋体(简称钩体)lipL32/1-lipL41/1融合基因及其原核表达系统，鉴定表达产物的免疫原性，了解钩体野生株lipL32和lipL41基因携带和表达情况及钩体病人血清抗体水平。rn 方法：采用连接引物PCR构建lipL32/1-lipL41/1融合基因，常规方法构建其原核表达系统。rn 采用SDS-PAGE检测目的重组蛋白rLipL32/1-rLipL41/1表达情况。采用Western blot鉴定rLipL32/1-rLipL41/1的免疫原性。采用PCR和MAT分别检测97株问号钩体野生株lipL32和lipL41基因及其表达情况。采用ELISA检测228例钩体病人血清lipL32和lipL41基因产物的抗体。rn 结果：与报道的相关序列比较，lipL32/1-lipL41/1融合基因核苷酸和氨基酸序列相似性分别为99.9％和99.8％。目的重组蛋白rLipL32/1-rLipL41/1表达产量约为细菌总蛋白的10％，主要以包涵体形式存在。rn rLipL32/1和rLipL41/1兔抗血清均能与rLipL32/1-rLipL41/1结合。97.9％(95/97)和87.6％(85/97)问号钩体野生株分别有lipL32和lipL41基因。95.9％(93/97)和84.5％(82/97)问号钩体野生株分别与rLipL32s和rLipL41s兔抗血清出现效价范围为1:4～1:128的MAT阳性结果。分别有94.7％～97.4％和78.5％～84.6％病人血清rLipL32s和rLipL41s抗体阳性。rn 结论：本研究成功地构建了lipL32/1-lipL41/1融合基因及其原核表达系统，所表达的产物具有良好的免疫原性。lipL32和lipL41是广泛存在于不同血清群问号钩体并有较高表达率的基因，且其不同基因型的表达产物有广泛的抗原和抗体交叉。rLipL32/1-rLipL41/1具有成为钩体属特异性基因工程疫苗抗原的良好前景。

摘要： 目的：构建ltB/ctB-ompL1/1融合基因及其原核表达系统，鉴定表达产物的免疫和佐剂活性，检测问号钩端螺旋体(简称钩体)野生株ompL1基因的携带、表达情况和钩体病人血清特异性抗体。rn 方法：采用连接引物PCR构建ltB-ompL1/1和ctB-ompL1/1融合基因，常规方法构建其原核表达系统。采用SDS-PAGE检测目的重组蛋白rLTB-rOmpL1/1和rCTB-rOmpL1/1表达情况。采用Western Blot和GM1-ELISA分别检测上述目的重组蛋白的免疫原性和佐剂活性。采用PCR和MAT分别检测97株问号钩体野生株ompL1基因及其表达情况。采用ELISA检测228例钩体病人血清ompL1基因产物的抗体。rn 结果：与报道的相关序列比较，ltB-ompL1/1和ctB-ompL1/1融合基因核苷酸和氨基酸序列相似性分别为99.7％-99.9％和99.5％-100％。rLTB-rOmpL1/1和rCTB-rOmpL1/1表达产量均约为细菌总蛋白的10％，主要以包涵体形式存在。rLTB-rOmpL1/1和rCTB-rOmpL1/1均分别能与rOmpL1/1兔抗血清和牛GM1结合。89.7％(87/97)问号钩体野生株含有ompL1基因，87.6％(85/97)问号钩体野生株分别与rOmpL1/1和rOmpL1/2兔抗血清出现效价范围为1：4～1：256的MAT阳性结果。86.8％(198/228)和88.6％(202/228)的病人血清标本分别rOmpL1/1和rOmpL1/2抗体阳性。rn 结论：本文成功地构建了ltB-ompL1/1和ctB-ompL1/1融合基因及其原核表达系统。所表达的rLTB-rOmpL1/1和rCTB-rOmpL1/1融合蛋白有良好的免疫原性和佐剂活性。ompL1是不同问号钩体血清群中广泛存在和高频率表达的基因，且其不同基因型的表达产物有广泛的抗原和抗体交叉。rLTB-rOmpL1/1和rCTB-rOmpL1/1具有成为问号钩体属特异性疫苗抗原的良好前景。

摘要： 本发明公开了一种问号赖型钩端螺旋体基因损失率分析方法，首先，通过对问号赖型钩端螺旋体进行比对挑出复制基因，再分别计算出每对复制基因的每个同义位点和每个非同义位点的替代数目，最后包括利用性质构造微分方程使用回归分析进行函数拟合，了解了复制基因进化过程中选择约束力的变化情况，并引入以S为分歧时间的概念，推断了复制基因的复制基因损失率。

摘要： 本发明公开了一种问号赖型钩端螺旋体复制基因挑选方法，包括以下步骤：1)获得问号赖型钩端螺旋体；2)挑选复制基因及进化距离信息提取。本发明也可推广到其他基因、基因组的提取及分析。

摘要： 本发明公开了一种犬型钩端螺旋体、黄疸出血型钩端螺旋体、犬冠状病毒三联灭活疫苗。本发明采用我国分离的犬型钩端螺旋体、黄疸出血型钩端螺旋体和犬冠状病毒流行株经灭活后加入佐剂研制而成。本发明涉及病原体的分离培养、鉴定、灭活方法、生产工艺、疫苗保存和免疫方法。犬三联灭活疫苗用于预防犬型钩端螺旋体病、黄疸出血型钩端螺旋体病和犬冠状病毒病，免疫犬后可产生较高的抗体，对强毒攻击具有很好的保护作用。钩端螺旋体为人畜共患传染病，犬为人类的伴侣动物，宠物犬与人类密切接触，因此，该三联灭活疫苗具有较好的社会效益和重要的公共卫生意义。

摘要： 本发明公开了一种抗钩端螺旋体的单克隆抗体及钩端螺旋体的检测方法。56601－MAb对钩体黄疸出血群赖型有特异性免疫反应，56602－MAb对钩体爪哇型有特异性免疫反应，56606－MAb对钩体秋季型有特异性免疫反应，56609－MAb对钩体流感伤寒群临6型有特异性免疫反应，56610－MAb对钩体七日热型有特异性免疫反应，而5种单克隆抗体均与其他血清型的钩体没有免疫反应。本发明提供的抗5种致病血清型钩体的5种单克隆抗体，对准确、快速分型、抗原的纯化及用于临床病人与动物钩体病的诊断均具有重要意义。本发明提供的以单抗为基础的TAS－ELISA检测诊断方法具有快速、方便、灵敏正确、特异性好、结果可肉眼观察等优点，有利于其的推广应用。

摘要： 提供四种抗原性制备物,每种均包含一种不同的钩端螺旋体蛋白,所述蛋白可免疫学性用于钩端螺旋体所致钩端螺旋体病的疫苗中。本发明还提供编码这四种蛋白质的多核苷酸以及可与这四种蛋白质结合的抗体以及用于诊断钩端螺旋体病。

摘要： 本发明公开了一种问号赖型钩端螺旋体基因损失率分析方法，首先，通过对问号赖型钩端螺旋体进行比对挑出复制基因，再分别计算出每对复制基因的每个同义位点和每个非同义位点的替代数目，最后包括利用性质构造微分方程使用回归分析进行函数拟合，了解了复制基因进化过程中选择约束力的变化情况，并引入以S为分歧时间的概念，推断了复制基因的复制基因损失率。

摘要： 本发明公开了一种问号赖型钩端螺旋体复制基因挑选方法，包括以下步骤：1)获得问号赖型钩端螺旋体；2)挑选复制基因及进化距离信息提取。本发明也可推广到其他基因、基因组的提取及分析。

摘要： 本发明涉及医药生物技术领域，具体涉及一种问号钩端螺旋体DNA疫苗及其构建方法和应用。问号钩端螺旋体可经皮肤或黏膜迅速侵入人和多种动物体内引起感染，人类感染后引起以发热、黄疸、出血为主要症状的钩体病，是一种世界分布广、严重威胁人类健康和畜牧业生产的、常见的人兽共患病，中国是钩体病主要流行国家之一。本发明提供了一种问号钩端螺旋体DNA疫苗，该DNA疫苗由真核表达载体和问号钩端螺旋体LB018基因组成，所述的LB018基因的核苷酸序列如SEQ ID NO：1所示。本发明能有效地诱发仔猪的免疫应答反应，说明其对动物的问号钩端螺旋体感染有一定免疫保护作用；且生产工艺简单，成本低廉，理化性质稳定、便于储存和运输。

摘要： 本发明公开了通过构建问号钩端螺旋体LipL21、LipL32、groEL和loa22蛋白的优势T‑和B‑细胞T‑B联合多价抗原表位融合基因及其原核表达系统，表达由抗原表位组成的多抗原肽（multiple antigenic peptide，MAP）与高分子核心载体人工交联制备MAP疫苗。本发明制备的MAP疫苗免疫效果好、成本较低，能有效诱导细胞免疫，减少了疫苗毒副作用，提高了安全性，增强了免疫针对性。

摘要： 本发明公开了一种TaqMan探针法检测问号钩端螺旋体金属蛋白酶基因的试剂盒，包括用于问号钩端螺旋体金属蛋白酶基因检测的特异性引物对LA2582‑F和LA2582‑R、荧光探针LA2582‑P和阳性质控品。探针的两端分别进行修饰，5’端标记报告荧光素或荧光染料，3’端标记淬灭基团。阳性质控品为含有问号钩端螺旋体金属蛋白酶靶基因LA2582序列重组pET42a质粒溶液。本发明试剂盒具有快速准确、高特异性、高效率的特点，可以实现对问号钩端螺旋体金属蛋白酶基因的定性定量检测，具有广泛的临床检测应用价值和流行病学调查应用价值。