问号钩端螺旋体基因组DNA芯片的研制

目的 研制并初步评估问号钩端螺旋体(简称钩体)赖型赖株的基因组DNA芯片。方法 利用Primegens引物设计软件筛选出问号钩体赖型赖株全基因组中的特异性基因进行引物设计。对成功设计出相应引物的3 290个基因用聚合酶链反应方法进行扩增,以纯化后的产物点样制备芯片。并用双色荧光杂交策略对芯片质量进行了初步平估。结果 共获得3 290个基因产物用于点样。参考株自身杂交实验结果表明:该芯片有较高的点一致性、信噪比和较低的假阳性率。结论 成功制备了包含问号钩体赖型赖株3 290个目的基因的基因组DNA芯片,并可用于基于该芯片的问号钩体比较基因组学的研究。     

问号钩端螺旋体中一个新的OmpA家族蛋白抗原性及保守性研究

目的克隆表达和鉴定问号钩端螺旋体(L.interrogans)黄疸出血群赖型赖株中一个新的外膜蛋白(Omp)A家族基因LA0301,研究LA0301编码蛋白的抗原性和在15个钩端螺旋体(简称钩体)血清群代表株中的保守性,探讨其在疫苗研究中的意义。方法生物信息学软件分析预测LA0301的特征。构建原核表达重组体pQE31-LA0301,经IPTG诱导后用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及蛋白质印迹法(Western blot)鉴定表达情况。用表达的重组蛋白免疫BALB/c小鼠,Western印迹检测其免疫反应性和在不同血清型钩体中的保守性。酶联免疫吸附试验(ELISA)和Western印迹检测兔抗钩体全菌血清中的LA0301编码蛋白的抗体。结果生物信息学预测结果显示,LA0301具有OmpA家族的结构域。克隆表达了重组质粒pQE31-LA0301,重组蛋白能刺激BALB/c小鼠产生特异性抗体,效价为1:32000。在兔抗钩体全菌血清中检测到特异的LA0301蛋白抗体,并在15个血清群的代表株钩体中均可检测到LA0301蛋白。结论LA0301蛋白是问号钩体中一个新的OmpA家族蛋白,具有良好的抗原性和保守性,并且能在钩体感染的过程中刺激机体产生相应的抗体。为进一步研究钩体新型疫苗候选基因奠定了基础。     

问号钩端螺旋体疫苗候选基因LB061的抗原性和保守性研究

目的 克隆表达和鉴定问号钩端螺旋体黄疸出血群赖型赖株中疫苗候选基因LB061,研究LB061的免疫原性和在不同血清型钩端螺旋体菌中的保守性。方法 生物信息学软件分析预测LB061的特征。构建原核表达质粒pQE31-LB061,经IPTG诱导后用SDS-PAGE及Western印迹法鉴定表达情况。用表达的重组蛋白免疫BALB/c小鼠,Western印迹法检测其抗原性和在不同血清型钩端螺旋体中的保守性。Western印迹法检测钩端螺旋体全菌兔抗血清中的LB061抗体。结果 生物信息学预测结果显示,LB061含有DUF839家族结构域。成功克隆了重组质粒pQE31-LB061,表达的重组蛋白能刺激BALB/c小鼠产生抗体(效价为1∶32000),并能与相应抗体反应,具有良好的抗原性。在16株不同血清型的钩端螺旋体中均可检测到LB061蛋白的表达,并在钩端螺旋体赖株全菌兔抗血清中检测到其抗体。结论 LB061蛋白可以作为外膜蛋白刺激宿主免疫系统产生抗体,具有良好的抗原性和保守性。本研究为其作为疫苗候选基因的研究奠定了基础。     

钩端螺旋体LA_1100蛋白膜定位研究

目的 研究LA_1100蛋白在钩端螺旋体中的膜定位并分析其在中国流行血清群中的保守性.方法 利用生物信息学软件对LA_1100的二级及三级结构进行分析,以Triton X-114抽提分离钩体细胞的各个组分,Westernblot及FACS方法验证LA_1100在钩端螺旋体中的膜定位.Western Blot和PCR在蛋白水平及核酸水平检测了其在13个中国流行血清群代表株中的保守性.结果 LA_1100的二级结构显示具有α-螺旋及β-折叠结构,进一步分析表明,此蛋白具有跨膜区.LA_1100单体结构具有典型的TolC结构域,三级结构模拟显示三聚体可形成孔状结构.膜定位显示,该蛋白定位于外膜.该基因的保守性分析结果表明,在中国13个流行代表株中有12株均检出该基因或该蛋白的特征性条带.结论 LA_1100为具有TolC结构域可以形成孔状结构的钩端螺旋体外膜蛋白,在中国流行株钩端螺旋体中保守.由于TolC蛋白与病原菌分泌致病因子密切相关,推测此蛋白可能与钩体感染宿主时粘附及致病相关,进一步对此蛋白进行研究有利于揭示钩体的致病机制.     

基于响应面分析方法的钩端螺旋体三联属特异性蛋白抗原原核表达条件的优化

脂蛋白LipL32和LipL21及穿膜蛋白OMPL1是问号钩端螺旋体(简称钩体)属特异性表面抗原,可作为通用型钩体基因工程疫苗抗原.[目的]在我们的研究中,为了获得使得重组人工融合基因lipL32/1-lipL21-ompL1/2获得较高的表达水平,我们优化了重组宿主大肠杆菌的表达条件.[方法]我们采用了基于中心复合设计(CCD)的响应面分析方法(RSM).影响重组大肠杆菌生长的主要因素包pH、诱导剂IPTG浓度、诱导前培养实践、诱导时间以及诱导温度.响应面分析方法可以从这些因素中找到适合目的蛋白表达的最优表达条件.[结果]我们的结果显示在pH7.9、0.20mmol/LIPTG、诱导前培养时间2.5 h、诱导时间5.83 h、诱导后温度31℃条件下,可获得目的重组蛋白37.78 mg/L的最大表达量.[结论]实验结果表明采用基于中心复合设计的响应面分析方法能够较吹靥岣吣康牟锏牟?利用统计方法不但可以减少试验的次数,而且还能够从影响蛋白的因素中找出最重要的影响因素,因此该方法不仅对重组蛋白表达条件的优化有用,而且是一种很好的筛选方法.     

不同毒力钩端螺旋体引起THP-1细胞中细胞因子表达的研究

本文旨在通过观察不同毒力钩端螺旋体激活人单核巨噬细胞株THP-1中细胞因子信号途径的差异,探讨天然免疫在钩端螺旋体病发病机制中的作用.将3种不同毒力的钩端螺旋体--赖型有毒株Lai株、赖型减毒株IPAV株及非致病性Patoc型Patoc 1株与诱导后的细胞相互作用,在作用0、2、12、24、48 h 后收集标本,应用实...     

GST-Ccd1融合蛋白的表达、纯化及多克隆抗体制备

目的：利用大肠杆菌DH5α表达GST—Ccd1融合蛋白,并用亲和层析分离纯化,进行动物免疫制备多克隆抗体。方法：利用本室构建好的pGEX-5X-1-Ccd1-N原核表达重组质粒,转化大肠杆菌DH5α,经IPTG诱导表达,在大肠杆菌表达系统中获得可溶性表达。经谷胱甘肽Sepharose 4B介质填充的层析柱分离纯化蛋白,制备抗原免疫动物,得到Ccd1的兔源多克隆抗体。结果：ELISA结果显示血清抗体效价可以达到1∶40 000。免疫组化分析表明自制的抗体能特异性与Ccd1蛋白相互作用,可以用于实验分析。结论：制备了效价高特异性良好的抗Ccd1多克隆抗体,经实验验证获得的抗体能够满足针对Ccd1的免疫印迹和免疫组化检测的实验要求,为今后深入研究Ccd1表达的组织分布、细胞内定位及其生物学功能提供了有用的实验工具。     

Characterization of cheW genes of Leptospira interrogans and their effects in Escherichia coli

The motility and chemotaxis systems are critical for the virulence of leptospires. In this study, the phylogenetic profiles method was used to predict the interaction of chemotaxis proteins. It was shown that CheW1 links to CheA1, CheY, CheB and CheW2, CheW3 links to CheA2, MCP （LA2426）, CheB3 and CheD1; and CheW2 links only to CheW1. The similarity analysis demonstrated that CheW2 of Leptospira interrogans strain Lai had poor homology with Chew of Escherichia coli in the region of residues 30-50. In order to verify the function of these proteins, the putative cheW genes were cloned into pQE31 vector and expressed in wild-type E. coli strain RP437 or chew defective strain RP4606. The swarming results indicated that CheW1 and CheW3 could restore swarming of RP4606 while CheW2 could not. Overexpression of CheW1 and CheW3 in RP437 inhibited the swarming of RP437, whereas the inhibitory effect of CheW2 was much lower. Therefore, we presumed that CheW1 and CheW3 might have the function of CheW while CheW2 does not. The existence of multiple copies of chemotaxis homologue genes suggested that L. interrogans strain Lai might have a more complex chemosensory pathway.     

Whole-genome analysis of Leptospira interrogans to identify potential vaccine candidates against leptospirosis

Leptospirosis is an important global human and veterinary health problem. Humans can be infected by exposure to chronically infected animals and their environment. An important focus of the current leptospiral research is the identification of outer membrane proteins (OMPs). Due to their location, leptospiral OMPs are likely to be relevant in host-pathogen interactions, hence their potential ability to stimulate heterologous immunity. The existing whole-genome sequence of Leptospira interrogans serovar Copenhageni offers a unique opportunity to search for cell surface proteins. Predicted genes encoding potential surface proteins were amplified from genomic DNA by PCR methodology and cloned into an Escherichia coli expression system. The partially purified recombinant proteins were probed by Western blotting with sera from human patients diagnosed with leptospirosis. Sixteen proteins, out of a hundred tested, were recognized by antibodies present in human sera. Four of these proteins were conserved among eight serovars of L. interrogans and absent in the non-pathogenic Leptospira biflexa. These proteins might be useful for the diagnosis of the disease as well as potential vaccine candidates.     

Leptospira interrogans and Leptospira peptidoglycans induce the release of tumor necrosis factor a from human monocytes

Abstract Elevated plasma concentrations of the cytokine tumor necrosis factor α (TNFα) have been observed in patients affected by leptospirosis. In this study we found that a preparation of peptidoglycan of Leptospira interrogans , serovar copenhageni , was able to induce the release of TNFa from peripheral blood mononuclear cells. TNFa induction occurred in a dose dependent manner and was not affected by the endotoxin inhibitor polymixin B. This is the first report on induction of TNFa release by a peptidoglycan of spirochetes. Our findings are consistent with existing clinical data and provide a potential mechanism for TNFa production.     

Evolutionary Implication of Outer Membrane Lipoprotein-Encoding Genes ompL1,lipL32 and lipL41 of Pathogenic Leptospira Species

Leptospirosis is recognized as the most widespread zoonosis with a global distribution. In this study, the antigenic variation in Leptospira interrogans and Leptospira borgpetersenii isolated from human urine and field rat kidney was preliminarily confirmed by microscopic agglutination test using monoclonal antibodies, and was further subjected to amplification and identification of outer membrane lipoproteins with structural gene variation. Sequence similarity analysis revealed that these protein sequences...     

A novel leptospiral protein increases ICAM-1 and E-selectin expression in human umbilical vein endothelial cells

It has been reported previously that activation of vascular endothelium by outer membrane proteins of the spirochetes Borrelia sp. and Treponema sp. resulted in enhanced expression of endothelial cell adhesion molecules. To investigate the role of leptospiral proteins in this process, a predicted lipoprotein encoded by the gene LIC10365 was selected, which belongs to a paralogous family that presents a domain of unknown function, DUF1565. The LIC10365 gene was cloned and the protein expressed in Escherichia coli C43 (DE3) strain using the vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and was used to assess its ability to activate cultured human umbilical vein endothelial cells. The rLIC10365 activated endothelium in such a manner that E-selectin and intercellular adhesion molecule 1 (ICAM-1) became upregulated in a dose-dependent fashion. The LIC10365-encoded protein was identified in vivo in the renal tubules of animal during experimental infection with Leptospira interrogans. Collectively, these results implicate the LIC10365-coding protein of L. interrogans as a potential effector molecule in the promotion of a host inflammatory response. This is the first report of a leptospiral protein capable of up-regulating the expression of endothelial cell adhesion molecules ICAM-1 and E-selectin.     

Mammalian cell entry (Mce) protein of Leptospira interrogans binds extracellular matrix components,plasminogen and β2 integrin

A severe re‐emergingzoonosis, leptospirosis, is caused by pathogenic spirochetes of the genus Leptospira. Several studies have identified leptospiral surface proteins with the ability to bind ECM and plasma components, which could mediate adhesion and invasion through the hosts. It has been shown that Mce of pathogenic Leptospira spp. is an RGD (Arg‐Gly‐Asp)‐motif‐dependent virulence factor, responsible for infection of cells and animals. In the present article, we decided to further study the repertoire of the Mce activities in leptospiral biological properties. We report that the recombinant Mce is a broad‐spectrum ECM‐binding protein, capable of interacting with laminin, cellular and plasma fibronectin and collagen IV. Dose­–r­esponse interaction was observed for all the components, fulfilling ligand­–receptor requirements. Mce is a PLG binding protein capable to recruit this component from NHS, generating PLA in the presence of PLG activator. Binding of Mce was also observed with the leukocyte cell receptors αLβ2 [(CD11a/CD18)‐LFA‐1] and αMβ2 [(CD11b/CD18)‐Mac‐1], suggesting the involvement of this protein in the host immune response. Indeed, virulent Leptospira L1‐130 was capable of binding both integrins, whereas culture‐attenuated M‐20 strain only bind to αMβ2 [(CD11b/CD18)‐Mac‐1]. To the best of our knowledge, this is the first work to describe that Mce surface protein could mediate the attachment of Leptospira interrogans to human cell receptors αLβ2(CD11a/CD18) and αMβ2(CD11b/CD18).