线粒体相关途径在问号钩端螺旋体诱导巨噬细胞凋亡中的作用以及致病性钩端螺旋体OmpL1特点的研究
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摘要
研究目的
在以往的研究中发现,致病性问号钩端螺旋体(简称钩体)可以在体外诱导小鼠单核巨噬细胞系J774A.1细胞凋亡,但是其具体的凋亡机制还未阐明。本研究的目的是探讨线粒体相关途径在问号钩体诱导巨噬细胞凋亡中的作用。
材料与方法
(1)建立致病性问号钩体黄疸出血群赖型赖株体外诱导巨噬细胞凋亡的模型:钩体分别与小鼠腹腔巨噬细胞(MPMs),小鼠单核巨噬样细胞系J774A.1细胞,人单核样细胞系THP-1细胞,共同孵育。采用AnnexinV-FITC和PI双染法,通过流式细胞术检测钩体感染后巨噬细胞凋亡率。(2)透射电镜观察诱导凋亡的巨噬细胞超微结构改变,尤其是线粒体改变。(3)应用活性检测试剂盒测定钩体感染后的巨噬细胞的caspase-8,caspase-9活性变化;非特异性Caspase阻断剂对凋亡的影响;JC-1染色后流式细胞仪和荧光显微镜检测线粒体膜电位(MPT)变化,应用荧光探针DCFH-DA检测细胞内的活性氧(ROS)水平。(4)Western blot分别检测诱导钩体感染后的巨噬细胞在感染不同时间后的线粒体或胞浆成份中的细胞色素C,EndoG,AIF和Smac的表达,同时应用免疫荧光染色法检测相应蛋白的转位。线粒体膜通透性转运孔阻断剂CsA预处理细胞后,对凋亡及线粒体释放蛋白的影响。(5)应用基因芯片技术检测钩体感染后的巨噬细胞的凋亡相关基因Bcl-2家族的mRNA表达水平变化,对表达发生变化的及代表性Bcl-2家族基因的应用Real-time PCR再次确认mRNA水平变化。
结果
(1)成功建立致病性问号钩体黄疸出血群赖型赖株体外诱导巨噬细胞凋亡的模型,显示问号钩体赖株可诱导3种巨噬细胞均出现不同程度的细胞凋亡,细胞凋亡率在一定范围内,随着感染时间延长和感染菌量的增加而升高。但是感染菌量过高时,细胞凋亡率开始下降,同时细胞坏死率升高。(2)透射电镜结果显示:感染问号钩体后,3种巨噬细胞均出现细胞凋亡的特征,例如染色质浓缩和边缘化,新月形核,细胞空泡化,细胞线粒体肿胀和嵴消失,但是3种被感染巨噬细胞的形态改变之间无明显差异。(3)问号钩体赖株感染3种巨噬细胞后,均可检测到caspase-8活性明显升高,caspase-9活性无明显升高;但是非特异性caspase抑制剂不能完全阻断细胞凋亡。被钩体感染后,3种巨噬细胞线粒体膜电位均随着感染时间的延长而逐渐下降。其中,MPMs的MPT下降幅度大于J774A.1和THP-1细胞。此外,与正常对照细胞相比,被钩体感染的3种巨噬细胞均出现胞内活性氧(ROS)水平升高。(4)Western Blot显示,在问号钩体赖株诱导3种巨噬细胞凋亡的过程中,在MPMs和J774A.1细胞中检测到AIF和EndoG从线粒体释放到胞浆,在THP-1细胞中检测到AIF从线粒体释放到胞浆;但是未检测到细胞色素C和Smac从线粒体释放到胞浆。同时,免疫荧光染色证实钩体感染后AIF或EndoG从线粒体转位到细胞核,从而诱导细胞凋亡的发生。MPT阻断剂可以部分性阻断细胞凋亡,完全阻断AIF或EndoG的释放。(5)线粒体相关的凋亡调节蛋白,Bcl-2家族的mRNA表达水平检测结果显示:钩体感染后3种巨噬细胞后,Bcl-2家族抗凋亡基因Bcl-2,Bcl-XL均无明显变化,但是促凋亡基因Bid,Bik1,Bnip3,Bcor,Bcl-211和Bclaf1在MPMs和J774A.1细胞中表达明显上调,促凋亡基因Bid,Bik1,Bnip3,Bcl-211和Bclaf1在THP-1细胞中小幅上调,其他促凋亡基因无明显变化。
结论
(1)致病性的问号钩体黄疸出血群赖型赖株可以诱导巨噬细胞凋亡。
(2)问号钩体赖株通过caspase依赖的caspase-8途径和线粒体相关的非caspase依赖途径诱导巨噬细胞凋亡。
(3)Bcl-2家族在问号钩体赖株诱导细胞凋亡的过程中,一些促凋亡基因mRNA转录水平上调,可能参与凋亡的调控,具体机制有待进一步研究。
研究目的
研究中国流行的致病性钩端螺旋体(简称钩体)的ompL1基因的特点和分布,OmpL1蛋白的交叉免疫反应性和免疫保护作用。
材料与方法
将致病性钩体,包括15株中国参考标准株和163株临床分离株,分别提取基因组DNA,PCR法扩增ompL1基因,测序并对核苷酸序列进行比对和分子进化树分析,根据分析结果对ompL1基因进行分型。然后,通过构建原核表达系统,表达并收获重组OmpL1蛋白(rOmpL1);以纯化的人OmpL1蛋白免疫家兔后获得抗rOmpL1的多克隆抗体。采用Western blot和ELISA法鉴定表达的rOmpL1蛋白的免疫原性和免疫反应性。应用显微镜凝集试验(MAT)检测兔抗rOmpL1抗体的交叉凝集效价,同时应用钩体细胞粘附实验检测此抗体阻断致病钩体赖型赖株的细胞粘附作用。最后,以rOmpL1免疫豚鼠后,检测对不同ompL1基因型别的致病性钩体感染的免疫保护作用。
结果
全部被检测钩体菌株的基因组DNA都能扩增出ompL1基因。根据钩体标准株的ompL1基因核苷酸序列比对和序列特征的分析结果,将扩增出的ompL1基因分成3型,即ompL1/1,ompL1/2和ompL1/3。通过原核系统表达的3种rOmpL1蛋白与钩体病人血清可以发生免疫反应。MAT试验中,兔抗rOmpL1的3种抗体与不同ompL基因型的钩体菌株之间可以产生广泛和明显的交叉凝集反应。同时,使用rOmpL1蛋白作为包被抗原的ELISA法,进一步验证了3种rOmpL1蛋白与钩体病病人血清样本之间同样可以发生交叉免疫反应。3种兔抗rOmpL1抗血清均能抑制致病性问号钩体赖型赖株粘附J774A.1细胞。此外,3种rOmpL1蛋白免疫豚鼠后,对来自不同ompL1基因类型的致病性钩体的感染具有交叉免疫保护作用。
结论
中国流行的致病性钩体的ompL1基因可以分成ompL1/1,ompL1/2和ompL1/3三型。外膜蛋白OmpL1是一种具有交叉免疫保护作用的属特异性抗原,可以用于通用新疫苗的研究和对钩端螺旋体病诊断。
AIM AND GROUND
Our previous study found that Leptospira infection could induce monocyte-macrophage-like cells(J774A.1) apoptosis,but the specific mechanism of apoptosis has not been clarified.The purpose of this study is to investigate mitochondria-related pathway of L.interrogans-induced macrophages apoptosis.
MATERIAL AND METHODS
(1) Establish pathogenic leptospires infected macrophages apoptosis model in vitro: murine peritoneal macrophages(MPMs),murine monocytic macrophage-like cells (J774A.1) and human monocyte-like cells(THP-1) were infected with L.interrogans serovar Lai strain Lai with different MOIs for different times,respectively.Detect apoptosis of the macrophages by Armexin V-FITC/PI double staining method;analyze apoptosis rates by flow cytometry(2) Detect morphological change and the mitochondria impairment by transmission electron microscopy of leptospiral induced apoptotic macrophages.(3) Caspase-8 and caspase-9 activity was measured with assay kits.To detect mitochondrial potential with JC-1 staining by flow cytometry and fluorescence microscopy,simultaneously detect cells of reactive oxygen species(ROS) levels by DCFH-DA fluorescent probe.(4) Expression of cytochrome C,Smac,EndoG and AIF in macrophages were determined in the mitochondrial and cytosol component after infection by Western Blot,and translocation of AIF and EndoG were examined by immunofluorescence staining.(5) Apoptosis-related Bcl-2 family mRNA expression levels was detected by microarray and confirmed by real-time PCR.
RESULTS
(1) Pathogenic L.interrogans serogroup Lai strain Lai could induce apoptosis of MPMs,J774A.1 and THP-1 cells with a time-and dose-dependent manner.However, with the increase of MOI,apoptosis ratio firstly rose,and then declined,while cell necrosis ratio increased.(2) Transmission Electron Microscopy results showed that all three types macrophages demonstrated apoptotic morphological changes,such as chromatin condensation and marginalization and cell vacuolization,mitochondria swelling and disappearance of mitochondrial ridges.(3) The activity of caspase-8 of the L.interrogans strain Lai-infected macrophages increased,but activity of caspase-9 did not increase obviously.However,pan-caspase inhibitors could not completely block macrophages apoptosis.Mitochondrial membrane potential(MPT) in infected macrophages declined gradually as infected time;while the cells showed a visible color change under the fluorescence microscope.At the same time,reactive oxygen species (ROS) levels increased in the infected macrophages.(4) The results of Western Blot showed that it was not detectable of cytochrome C and Smac release from mitochondria to cytoplasm in the L.interrogans strain Lai induced-apoptotic macrophages.However, pro-apoptotic proteins,AIF and EndoG,releasing from mitchondria to cytosol was detectable in the infected MPMs and J774A.1 cells,and AIF releasing was detectable in THP-1 cells.Results of immunofluorescent staining confirmed that AIF and/or EndoG translocation from mitochondria to the nucleus,thereby inducing cell apoptosis.MPT blocker could completely block the release of AIF or EndoG,and partly blocked the macrophages apoptosis.(5) The results of microarray and real-time PCR showed that Bcl-2 family mRNA levels changed in the L.interrogans strain Lai induced apoptotic macrophages.Pro-apoptotic genes(Bid,Bik1,Bnip3,Bcor,Bcl-211 and Bclafl) were significantly up-regulated in the infected MPMs and J774A.1 cells,pro-apoptotic genes (Bid,Bik1,Bnip3,Bcl-211 and Bclafl) were lightly up-regulated in the infected THP-1 cells,other proapoptotic genes were unchanged.
CONCLUSIONS
(1) L.interrogans serovar Lai strain Lai induced apoptosis and necrosis in macrophages in vitro;apoptosis rates in primary macrophages was higher than that of continuous cell lines,apoptosis rates in murine macrophage was higher than people originate macrophages.The leptospires induced apoptosis in a certain range was time-and dose-dependent.
(2) L.interrogans strain Lai-induced macrophages apoptosis via caspase-8 dependent pathway and mitochondrial related caspase-independent pathway.
(3) In the process of Leptospiral stain Lai-induced macrophages apoptosis,mRNA expression level of some pro-apoptotic genes of Bcl-2 family were up-regulated,which may be involved in the process of apoptosis.
AIM AND GROUND
The usefulness of available vaccine and serological tests for leptospirosis is limited by the low cross-reactivity of antigens from numerous serovars of pathogenic Leptospira spp.Identification of genus-specific protein antigens(GP-Ag) of Leptospira would be important for development of universal vaccines and serodiagnostic methods. OmpL1,a transmembrane porin of pathogenic leptospires,was identified as a possible GP-Ag,but its sequence diversity and immune cross-reactivity among different serovars of pathogenic leptospires remains largely unknown.
MATERIAL AND METHODS
(1) Amplification and sequencing the ompL1 gene in all 15 official Chinese standard strains and 163 clinical isolated strains of pathogenic leptospires.(2) Molecular phylogeny analysis and gene-typing of ompL1 genes,phylogenetic analysis and secondary structure prediction of OmpL1 protein.(3) Prokaryotic expression and identification of recombinant OmpL1(rOmpL1) proteins,then preparation of rabbit antisera against rOmpL1 proteins.(4) Detect cross-immunoagglutination among the antisera against(rOmpL1) and leptospiral strains belonging to different ompL1 gene types by microscopic agglutination test(MAT).Then detect cross-immunoreactions by using the OmpL1 proteins as the coated antigens in serum samples from 385 leptospirosis patients by ELISAs.(5) The effects of the antisera against rOmpL1 proteins to inhibit L.interrogans strain Lai from adhering to J774A.1 cells.(6) Detect cross-immunoprotection of each rOmpL1 by challenging immunized guinea pigs with lethal dose leptospires from different ompL1 gene types.
RESULTS
PCR analysis demonstrated that the ompL1 gene is expressed in all 15 official Chinese standard strains as well as 163 clinical strains of pathogenic leptospires isolated in China.In the standard strains,the ompL1 gene could be divided into three groups (ompL1/1,ompL1/2 and ompL1/3) according to their sequence identities.Immune electron microscopy demonstrated that all products of the different gene types of ompL1 are located on the surface of leptospires.The microscopic agglutination test revealed extensive yet distinct cross-immunoagglutination among the antisera against recombinant OmpL1(rOmpL1) and leptospiral strains belonging to different ompL1 gene types.ELISAs using the OmpL1 proteins as the coated antigens in serum samples from 385 leptospirosis patients further verified these cross-immunoreactions.All the antisera against rOmpL1 proteins could inhibit L.interrogans strain Lai from adhering to J774A.1 cells.Furthermore,immunization of guinea pigs with each of the rOmpL1 proteins could cause cross-immunoprotection against lethal challenge with leptospires from different ompL1 gene types.
CONCLUSION
Three types of the ompL1 gene are present in pathogenic leptospires in China. OmpL1 is an immunoprotective GP-Ag which should be considered in the design of new universal vaccines and serodiagnostic methods against leptospirosis.
在以往的研究中发现,致病性问号钩端螺旋体(简称钩体)可以在体外诱导小鼠单核巨噬细胞系J774A.1细胞凋亡,但是其具体的凋亡机制还未阐明。本研究的目的是探讨线粒体相关途径在问号钩体诱导巨噬细胞凋亡中的作用。
材料与方法
(1)建立致病性问号钩体黄疸出血群赖型赖株体外诱导巨噬细胞凋亡的模型:钩体分别与小鼠腹腔巨噬细胞(MPMs),小鼠单核巨噬样细胞系J774A.1细胞,人单核样细胞系THP-1细胞,共同孵育。采用AnnexinV-FITC和PI双染法,通过流式细胞术检测钩体感染后巨噬细胞凋亡率。(2)透射电镜观察诱导凋亡的巨噬细胞超微结构改变,尤其是线粒体改变。(3)应用活性检测试剂盒测定钩体感染后的巨噬细胞的caspase-8,caspase-9活性变化;非特异性Caspase阻断剂对凋亡的影响;JC-1染色后流式细胞仪和荧光显微镜检测线粒体膜电位(MPT)变化,应用荧光探针DCFH-DA检测细胞内的活性氧(ROS)水平。(4)Western blot分别检测诱导钩体感染后的巨噬细胞在感染不同时间后的线粒体或胞浆成份中的细胞色素C,EndoG,AIF和Smac的表达,同时应用免疫荧光染色法检测相应蛋白的转位。线粒体膜通透性转运孔阻断剂CsA预处理细胞后,对凋亡及线粒体释放蛋白的影响。(5)应用基因芯片技术检测钩体感染后的巨噬细胞的凋亡相关基因Bcl-2家族的mRNA表达水平变化,对表达发生变化的及代表性Bcl-2家族基因的应用Real-time PCR再次确认mRNA水平变化。
结果
(1)成功建立致病性问号钩体黄疸出血群赖型赖株体外诱导巨噬细胞凋亡的模型,显示问号钩体赖株可诱导3种巨噬细胞均出现不同程度的细胞凋亡,细胞凋亡率在一定范围内,随着感染时间延长和感染菌量的增加而升高。但是感染菌量过高时,细胞凋亡率开始下降,同时细胞坏死率升高。(2)透射电镜结果显示:感染问号钩体后,3种巨噬细胞均出现细胞凋亡的特征,例如染色质浓缩和边缘化,新月形核,细胞空泡化,细胞线粒体肿胀和嵴消失,但是3种被感染巨噬细胞的形态改变之间无明显差异。(3)问号钩体赖株感染3种巨噬细胞后,均可检测到caspase-8活性明显升高,caspase-9活性无明显升高;但是非特异性caspase抑制剂不能完全阻断细胞凋亡。被钩体感染后,3种巨噬细胞线粒体膜电位均随着感染时间的延长而逐渐下降。其中,MPMs的MPT下降幅度大于J774A.1和THP-1细胞。此外,与正常对照细胞相比,被钩体感染的3种巨噬细胞均出现胞内活性氧(ROS)水平升高。(4)Western Blot显示,在问号钩体赖株诱导3种巨噬细胞凋亡的过程中,在MPMs和J774A.1细胞中检测到AIF和EndoG从线粒体释放到胞浆,在THP-1细胞中检测到AIF从线粒体释放到胞浆;但是未检测到细胞色素C和Smac从线粒体释放到胞浆。同时,免疫荧光染色证实钩体感染后AIF或EndoG从线粒体转位到细胞核,从而诱导细胞凋亡的发生。MPT阻断剂可以部分性阻断细胞凋亡,完全阻断AIF或EndoG的释放。(5)线粒体相关的凋亡调节蛋白,Bcl-2家族的mRNA表达水平检测结果显示:钩体感染后3种巨噬细胞后,Bcl-2家族抗凋亡基因Bcl-2,Bcl-XL均无明显变化,但是促凋亡基因Bid,Bik1,Bnip3,Bcor,Bcl-211和Bclaf1在MPMs和J774A.1细胞中表达明显上调,促凋亡基因Bid,Bik1,Bnip3,Bcl-211和Bclaf1在THP-1细胞中小幅上调,其他促凋亡基因无明显变化。
结论
(1)致病性的问号钩体黄疸出血群赖型赖株可以诱导巨噬细胞凋亡。
(2)问号钩体赖株通过caspase依赖的caspase-8途径和线粒体相关的非caspase依赖途径诱导巨噬细胞凋亡。
(3)Bcl-2家族在问号钩体赖株诱导细胞凋亡的过程中,一些促凋亡基因mRNA转录水平上调,可能参与凋亡的调控,具体机制有待进一步研究。
研究目的
研究中国流行的致病性钩端螺旋体(简称钩体)的ompL1基因的特点和分布,OmpL1蛋白的交叉免疫反应性和免疫保护作用。
材料与方法
将致病性钩体,包括15株中国参考标准株和163株临床分离株,分别提取基因组DNA,PCR法扩增ompL1基因,测序并对核苷酸序列进行比对和分子进化树分析,根据分析结果对ompL1基因进行分型。然后,通过构建原核表达系统,表达并收获重组OmpL1蛋白(rOmpL1);以纯化的人OmpL1蛋白免疫家兔后获得抗rOmpL1的多克隆抗体。采用Western blot和ELISA法鉴定表达的rOmpL1蛋白的免疫原性和免疫反应性。应用显微镜凝集试验(MAT)检测兔抗rOmpL1抗体的交叉凝集效价,同时应用钩体细胞粘附实验检测此抗体阻断致病钩体赖型赖株的细胞粘附作用。最后,以rOmpL1免疫豚鼠后,检测对不同ompL1基因型别的致病性钩体感染的免疫保护作用。
结果
全部被检测钩体菌株的基因组DNA都能扩增出ompL1基因。根据钩体标准株的ompL1基因核苷酸序列比对和序列特征的分析结果,将扩增出的ompL1基因分成3型,即ompL1/1,ompL1/2和ompL1/3。通过原核系统表达的3种rOmpL1蛋白与钩体病人血清可以发生免疫反应。MAT试验中,兔抗rOmpL1的3种抗体与不同ompL基因型的钩体菌株之间可以产生广泛和明显的交叉凝集反应。同时,使用rOmpL1蛋白作为包被抗原的ELISA法,进一步验证了3种rOmpL1蛋白与钩体病病人血清样本之间同样可以发生交叉免疫反应。3种兔抗rOmpL1抗血清均能抑制致病性问号钩体赖型赖株粘附J774A.1细胞。此外,3种rOmpL1蛋白免疫豚鼠后,对来自不同ompL1基因类型的致病性钩体的感染具有交叉免疫保护作用。
结论
中国流行的致病性钩体的ompL1基因可以分成ompL1/1,ompL1/2和ompL1/3三型。外膜蛋白OmpL1是一种具有交叉免疫保护作用的属特异性抗原,可以用于通用新疫苗的研究和对钩端螺旋体病诊断。
AIM AND GROUND
Our previous study found that Leptospira infection could induce monocyte-macrophage-like cells(J774A.1) apoptosis,but the specific mechanism of apoptosis has not been clarified.The purpose of this study is to investigate mitochondria-related pathway of L.interrogans-induced macrophages apoptosis.
MATERIAL AND METHODS
(1) Establish pathogenic leptospires infected macrophages apoptosis model in vitro: murine peritoneal macrophages(MPMs),murine monocytic macrophage-like cells (J774A.1) and human monocyte-like cells(THP-1) were infected with L.interrogans serovar Lai strain Lai with different MOIs for different times,respectively.Detect apoptosis of the macrophages by Armexin V-FITC/PI double staining method;analyze apoptosis rates by flow cytometry(2) Detect morphological change and the mitochondria impairment by transmission electron microscopy of leptospiral induced apoptotic macrophages.(3) Caspase-8 and caspase-9 activity was measured with assay kits.To detect mitochondrial potential with JC-1 staining by flow cytometry and fluorescence microscopy,simultaneously detect cells of reactive oxygen species(ROS) levels by DCFH-DA fluorescent probe.(4) Expression of cytochrome C,Smac,EndoG and AIF in macrophages were determined in the mitochondrial and cytosol component after infection by Western Blot,and translocation of AIF and EndoG were examined by immunofluorescence staining.(5) Apoptosis-related Bcl-2 family mRNA expression levels was detected by microarray and confirmed by real-time PCR.
RESULTS
(1) Pathogenic L.interrogans serogroup Lai strain Lai could induce apoptosis of MPMs,J774A.1 and THP-1 cells with a time-and dose-dependent manner.However, with the increase of MOI,apoptosis ratio firstly rose,and then declined,while cell necrosis ratio increased.(2) Transmission Electron Microscopy results showed that all three types macrophages demonstrated apoptotic morphological changes,such as chromatin condensation and marginalization and cell vacuolization,mitochondria swelling and disappearance of mitochondrial ridges.(3) The activity of caspase-8 of the L.interrogans strain Lai-infected macrophages increased,but activity of caspase-9 did not increase obviously.However,pan-caspase inhibitors could not completely block macrophages apoptosis.Mitochondrial membrane potential(MPT) in infected macrophages declined gradually as infected time;while the cells showed a visible color change under the fluorescence microscope.At the same time,reactive oxygen species (ROS) levels increased in the infected macrophages.(4) The results of Western Blot showed that it was not detectable of cytochrome C and Smac release from mitochondria to cytoplasm in the L.interrogans strain Lai induced-apoptotic macrophages.However, pro-apoptotic proteins,AIF and EndoG,releasing from mitchondria to cytosol was detectable in the infected MPMs and J774A.1 cells,and AIF releasing was detectable in THP-1 cells.Results of immunofluorescent staining confirmed that AIF and/or EndoG translocation from mitochondria to the nucleus,thereby inducing cell apoptosis.MPT blocker could completely block the release of AIF or EndoG,and partly blocked the macrophages apoptosis.(5) The results of microarray and real-time PCR showed that Bcl-2 family mRNA levels changed in the L.interrogans strain Lai induced apoptotic macrophages.Pro-apoptotic genes(Bid,Bik1,Bnip3,Bcor,Bcl-211 and Bclafl) were significantly up-regulated in the infected MPMs and J774A.1 cells,pro-apoptotic genes (Bid,Bik1,Bnip3,Bcl-211 and Bclafl) were lightly up-regulated in the infected THP-1 cells,other proapoptotic genes were unchanged.
CONCLUSIONS
(1) L.interrogans serovar Lai strain Lai induced apoptosis and necrosis in macrophages in vitro;apoptosis rates in primary macrophages was higher than that of continuous cell lines,apoptosis rates in murine macrophage was higher than people originate macrophages.The leptospires induced apoptosis in a certain range was time-and dose-dependent.
(2) L.interrogans strain Lai-induced macrophages apoptosis via caspase-8 dependent pathway and mitochondrial related caspase-independent pathway.
(3) In the process of Leptospiral stain Lai-induced macrophages apoptosis,mRNA expression level of some pro-apoptotic genes of Bcl-2 family were up-regulated,which may be involved in the process of apoptosis.
AIM AND GROUND
The usefulness of available vaccine and serological tests for leptospirosis is limited by the low cross-reactivity of antigens from numerous serovars of pathogenic Leptospira spp.Identification of genus-specific protein antigens(GP-Ag) of Leptospira would be important for development of universal vaccines and serodiagnostic methods. OmpL1,a transmembrane porin of pathogenic leptospires,was identified as a possible GP-Ag,but its sequence diversity and immune cross-reactivity among different serovars of pathogenic leptospires remains largely unknown.
MATERIAL AND METHODS
(1) Amplification and sequencing the ompL1 gene in all 15 official Chinese standard strains and 163 clinical isolated strains of pathogenic leptospires.(2) Molecular phylogeny analysis and gene-typing of ompL1 genes,phylogenetic analysis and secondary structure prediction of OmpL1 protein.(3) Prokaryotic expression and identification of recombinant OmpL1(rOmpL1) proteins,then preparation of rabbit antisera against rOmpL1 proteins.(4) Detect cross-immunoagglutination among the antisera against(rOmpL1) and leptospiral strains belonging to different ompL1 gene types by microscopic agglutination test(MAT).Then detect cross-immunoreactions by using the OmpL1 proteins as the coated antigens in serum samples from 385 leptospirosis patients by ELISAs.(5) The effects of the antisera against rOmpL1 proteins to inhibit L.interrogans strain Lai from adhering to J774A.1 cells.(6) Detect cross-immunoprotection of each rOmpL1 by challenging immunized guinea pigs with lethal dose leptospires from different ompL1 gene types.
RESULTS
PCR analysis demonstrated that the ompL1 gene is expressed in all 15 official Chinese standard strains as well as 163 clinical strains of pathogenic leptospires isolated in China.In the standard strains,the ompL1 gene could be divided into three groups (ompL1/1,ompL1/2 and ompL1/3) according to their sequence identities.Immune electron microscopy demonstrated that all products of the different gene types of ompL1 are located on the surface of leptospires.The microscopic agglutination test revealed extensive yet distinct cross-immunoagglutination among the antisera against recombinant OmpL1(rOmpL1) and leptospiral strains belonging to different ompL1 gene types.ELISAs using the OmpL1 proteins as the coated antigens in serum samples from 385 leptospirosis patients further verified these cross-immunoreactions.All the antisera against rOmpL1 proteins could inhibit L.interrogans strain Lai from adhering to J774A.1 cells.Furthermore,immunization of guinea pigs with each of the rOmpL1 proteins could cause cross-immunoprotection against lethal challenge with leptospires from different ompL1 gene types.
CONCLUSION
Three types of the ompL1 gene are present in pathogenic leptospires in China. OmpL1 is an immunoprotective GP-Ag which should be considered in the design of new universal vaccines and serodiagnostic methods against leptospirosis.
引文
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