Objective:Pulmonary haemorrhage is an increasing cause of death in leptospirosis patients.However,molecular mechanism underlying pathologies in this organ is not clearly understood.It has been shown that sodium transp...Objective:Pulmonary haemorrhage is an increasing cause of death in leptospirosis patients.However,molecular mechanism underlying pathologies in this organ is not clearly understood.It has been shown that sodium transport was disturbed following Leptospira infection.LipL21 is the second abundant outer membrane protein found only in pathogenic Leptospira.Its expression in vivo has been shown which suggests that this protein may be involved in survival in hosts or pathogenesis.However,the expression of this protein in host organs and its role in lung pathology has not been demonstrated.In this study we demonstrated the expression of LipL21 in lungs of hamsters infected with pathogenic Leptospira.Methods:Lung tissues were collected from Golden Syrian hamsters injected with Leptospira interrogans serovar Pyrogenes at days 3,5 and 7 post-infection.Four hamsters were used for each time point.Lungs from non-infected hamsters were collected as a control group.LipL21 mRNA expression in lung tissues was investigated by reverse transcription and nested PCR.Results:LipL21 mRNA expression was detected in all lung tissues from hamsters infected with pathogenic Leptospira.No PCR product was detected when tissues from non-infected hamsters were investigated.Conclusion:Our data demonstrated that LipL21 is expressed in lungs of hamsters infected with pathogenic Leptospira.Additional experiments such as quantitation and localization of LipL21 expression in lungs will provide further information whether this protein is involved in pathogenesis.展开更多
In this study, the full lipL21 gene fragment encoding outer membrane protein LipL21 was cloned from L. interrogans serovar Lai and inserted into eukaryotic expression vector pcDNA3.1（＋）. The guinea pigs were immuni...In this study, the full lipL21 gene fragment encoding outer membrane protein LipL21 was cloned from L. interrogans serovar Lai and inserted into eukaryotic expression vector pcDNA3.1（＋）. The guinea pigs were immunized with pcDNA3.1（＋）-lipL21, pcDNA3.1（＋） or PBS. Six weeks after the second immunization, the splenocytes were isolated to detect their proliferative ability by lymphocyte transformation experiments. In addition, microscopic agglutination test was used for quantitative detection of specific antibodies. The rest guinea pigs were challenged intraperitoneally with L. interogans sorevar Lai. Then, protective effect was evaluated on the basis of survival and histopathological lesions in the kidneys, lungs, and liver. The lipL21 gene was successfully expressed in COS-7 cells through recombinant pcDNA3.1（＋）-lipL21. The titer of specific antibodies substantially increased, and the stimulation index of splenocytes increased significantly. Hence, the pcDNA3.1（＋）-lipL21 could protect the immunized guinea pigs from homotypic Leptospira infection. Furthermore, no obvious pathologic changes were observed in the pcDNA3.1（＋）-lipL21 immunized guinea pigs. The results showed that the protective effect with pathogenic strains of Leptospira was shared by LipL21 mediated through a plasmid vector. Consequently, these results indicated that the lipL21 DNA vaccine was a promising candidate for the prevention of leptospirosis.展开更多
基金This work was supported by the National Natural Science Foun-dation of China ( No 30370071 & 30670102)the NationalHigh Technology Research and Development Program of China( No 2006AA02Z1760)Shanghai Leading Academic Disci-pline Project (No T0206)
文摘目的选取致病性钩端螺旋体(简称钩体)中高度保守,同时是钩体外膜蛋白中含量最多的两个脂蛋白LipL32和LipL21构建成融合基因DNA疫苗pVAX1/LipL21-LipL32 ,观察在BALB/c小鼠中重组DNA疫苗诱导免疫应答反应的能力。方法采用连接引物PCR构建融合基因LipL21-LipL32 ,并将其插入真核表达载体构成重组DNA疫苗pVAX1/Li-pL21-LipL32 ,脂质体转染人胚肾细胞( HEK293细胞)后Western Blot验证重组DNA疫苗在真核细胞中的表达,并将其肌注BALB/c小鼠,用显微凝集试验( MAT)检测所产生的特异性抗体与问号钩体的凝集效价,用IL-10和TNF-β细胞因子试剂盒检测体液免疫和细胞免疫应答水平。结果 Western Blot分析显示重组DNA疫苗pVAX1/LipL21-LipL32在HEK293细胞中得到表达,小鼠动物实验结果显示重组DNA疫苗能有效地诱导机体的体液免疫和细胞免疫应答。结论成功构建钩体融合基因LipL21-LipL32重组DNA疫苗,所表达的融合蛋白能诱导特异的免疫应答反应,为进一步研究和发展新型的钩体病疫苗提供了实验基础。
基金supported by National Research Council of Thailand and Rachadapiseksompoj Grant, Faculty of Medicine,Chulalongkorn University, Thailand.
文摘Objective:Pulmonary haemorrhage is an increasing cause of death in leptospirosis patients.However,molecular mechanism underlying pathologies in this organ is not clearly understood.It has been shown that sodium transport was disturbed following Leptospira infection.LipL21 is the second abundant outer membrane protein found only in pathogenic Leptospira.Its expression in vivo has been shown which suggests that this protein may be involved in survival in hosts or pathogenesis.However,the expression of this protein in host organs and its role in lung pathology has not been demonstrated.In this study we demonstrated the expression of LipL21 in lungs of hamsters infected with pathogenic Leptospira.Methods:Lung tissues were collected from Golden Syrian hamsters injected with Leptospira interrogans serovar Pyrogenes at days 3,5 and 7 post-infection.Four hamsters were used for each time point.Lungs from non-infected hamsters were collected as a control group.LipL21 mRNA expression in lung tissues was investigated by reverse transcription and nested PCR.Results:LipL21 mRNA expression was detected in all lung tissues from hamsters infected with pathogenic Leptospira.No PCR product was detected when tissues from non-infected hamsters were investigated.Conclusion:Our data demonstrated that LipL21 is expressed in lungs of hamsters infected with pathogenic Leptospira.Additional experiments such as quantitation and localization of LipL21 expression in lungs will provide further information whether this protein is involved in pathogenesis.
基金grants from the Hunan Provincial Education Department (06B088)the Hunan Provincial Health Department (2004B165)
文摘In this study, the full lipL21 gene fragment encoding outer membrane protein LipL21 was cloned from L. interrogans serovar Lai and inserted into eukaryotic expression vector pcDNA3.1（＋）. The guinea pigs were immunized with pcDNA3.1（＋）-lipL21, pcDNA3.1（＋） or PBS. Six weeks after the second immunization, the splenocytes were isolated to detect their proliferative ability by lymphocyte transformation experiments. In addition, microscopic agglutination test was used for quantitative detection of specific antibodies. The rest guinea pigs were challenged intraperitoneally with L. interogans sorevar Lai. Then, protective effect was evaluated on the basis of survival and histopathological lesions in the kidneys, lungs, and liver. The lipL21 gene was successfully expressed in COS-7 cells through recombinant pcDNA3.1（＋）-lipL21. The titer of specific antibodies substantially increased, and the stimulation index of splenocytes increased significantly. Hence, the pcDNA3.1（＋）-lipL21 could protect the immunized guinea pigs from homotypic Leptospira infection. Furthermore, no obvious pathologic changes were observed in the pcDNA3.1（＋）-lipL21 immunized guinea pigs. The results showed that the protective effect with pathogenic strains of Leptospira was shared by LipL21 mediated through a plasmid vector. Consequently, these results indicated that the lipL21 DNA vaccine was a promising candidate for the prevention of leptospirosis.
