Objective.To construct recombinant BCG again st leptospirosis.Methods.We amplified the entire open readin g frame of the OmpL1gene from the genome of the leptospire serovar Lai strain 017.Two recombin ant plasmids pBQ...Objective.To construct recombinant BCG again st leptospirosis.Methods.We amplified the entire open readin g frame of the OmpL1gene from the genome of the leptospire serovar Lai strain 017.Two recombin ant plasmids pBQ1and pBQ2were constructed by oriented ligation based on the E.coli-BCG shuttle plasmids pMV261and pMV361respectively.The recombinant plasmids were transformed into BCG by electroporation.The rBCGs bearing pBQ1and pBQ2were induced by high temperature of 45℃.Results.The expressed product,a 35kD prote in was detected by SDS-PAGE.The resu lt indicates that pBQ1and pBQ2can express OmpL1in rBCG.Conclusion.The technical methods in this study may help detect the immunogenicity a nd immunoprotection of OmpL1and develop more safe,highl y effective rBCG bearing leptospira l antigen with long-lasting protection.展开更多
Diagnosis of leptospirosis in Colombia is based on clinical history and serological testing. However, disease symptoms are nonspecific and there is no uniform criteria regarding the qualifications considered positive....Diagnosis of leptospirosis in Colombia is based on clinical history and serological testing. However, disease symptoms are nonspecific and there is no uniform criteria regarding the qualifications considered positive. Therefore, it is important to identify and characterize genes associated with pathogenicity in native strains for the development of new diagnostic tests and vaccine production. The aim of this study was to identify the ompL1 and lipL32 genes in Leptospira strains isolated from urine samples of cattle. Sixteen strains were obtained from urine samples and, DNA was isolated to perform two Polymerase Chain Reaction (PCR) tests which identified lipL32 and ompL1 genes. As positive control, a reference strain of L. interrogans was used. L. biflexa and Escherichia coli strains were used as a negative control. In 100% of the samples were identified amplicons of 960 bp and 423 bp corresponding to ompL1 and lipL32 genes respectively. Thus, the pathogenic property and conservation of genes in the isolated strains were confirmed. This study is presented as a contribution to the diagnosis of leptospirosis to use these genes as molecular markers of infection. The results of this study might provide clues for future clinical, epidemiological and molecular research leading to implement new diagnostic strategies and to expand knowledge of the pathophysiology of a disease of public health impact on human and animal.展开更多
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 fiel...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 lipoproreins with structural gene variation. Sequence similarity analysis revealed that these protein sequences, namely OmpL1, LipL32 and LipL41, showed no more homologies to outer membrane lipoproteins of non-pathogenic Leptospira and other closely related Spirochetes, but showed a strong identity within L. interrogans, suggesting intra-specific phylogenetic lineages that might be originated from a common pathogenic leptospiral origin. Moreover, the ompL1 gene showed more antigenic variation than lipL32 and lipL41 due to less conservation in secondary structural evolution within closely related species. Phylogenetically, ompL1 and lipL4l of these strains gave a considerable proximity to L. weilii and L. santaro- sai. The ompI,1 gene of L. interrogans clustered distinctly from other pathogenic and non-pathogenic leptospiral species. The diversity of ompL genes has been an- alyzed and it envisaged that sequence-specific variations at antigenic determinant sites would result in slow evolutionary changes along with new serovar origination within closely related species. Thus, a crucial work on effective recombinant vaccine development and engineered antibodies will hopefully meet to solve the therapeutic challenges.展开更多
文摘Objective.To construct recombinant BCG again st leptospirosis.Methods.We amplified the entire open readin g frame of the OmpL1gene from the genome of the leptospire serovar Lai strain 017.Two recombin ant plasmids pBQ1and pBQ2were constructed by oriented ligation based on the E.coli-BCG shuttle plasmids pMV261and pMV361respectively.The recombinant plasmids were transformed into BCG by electroporation.The rBCGs bearing pBQ1and pBQ2were induced by high temperature of 45℃.Results.The expressed product,a 35kD prote in was detected by SDS-PAGE.The resu lt indicates that pBQ1and pBQ2can express OmpL1in rBCG.Conclusion.The technical methods in this study may help detect the immunogenicity a nd immunoprotection of OmpL1and develop more safe,highl y effective rBCG bearing leptospira l antigen with long-lasting protection.
文摘Diagnosis of leptospirosis in Colombia is based on clinical history and serological testing. However, disease symptoms are nonspecific and there is no uniform criteria regarding the qualifications considered positive. Therefore, it is important to identify and characterize genes associated with pathogenicity in native strains for the development of new diagnostic tests and vaccine production. The aim of this study was to identify the ompL1 and lipL32 genes in Leptospira strains isolated from urine samples of cattle. Sixteen strains were obtained from urine samples and, DNA was isolated to perform two Polymerase Chain Reaction (PCR) tests which identified lipL32 and ompL1 genes. As positive control, a reference strain of L. interrogans was used. L. biflexa and Escherichia coli strains were used as a negative control. In 100% of the samples were identified amplicons of 960 bp and 423 bp corresponding to ompL1 and lipL32 genes respectively. Thus, the pathogenic property and conservation of genes in the isolated strains were confirmed. This study is presented as a contribution to the diagnosis of leptospirosis to use these genes as molecular markers of infection. The results of this study might provide clues for future clinical, epidemiological and molecular research leading to implement new diagnostic strategies and to expand knowledge of the pathophysiology of a disease of public health impact on human and animal.
基金supported by grants from the Department of Science and Technology,Government of India (Sanction order No. SR/FT/L-47/2006)
文摘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 lipoproreins with structural gene variation. Sequence similarity analysis revealed that these protein sequences, namely OmpL1, LipL32 and LipL41, showed no more homologies to outer membrane lipoproteins of non-pathogenic Leptospira and other closely related Spirochetes, but showed a strong identity within L. interrogans, suggesting intra-specific phylogenetic lineages that might be originated from a common pathogenic leptospiral origin. Moreover, the ompL1 gene showed more antigenic variation than lipL32 and lipL41 due to less conservation in secondary structural evolution within closely related species. Phylogenetically, ompL1 and lipL4l of these strains gave a considerable proximity to L. weilii and L. santaro- sai. The ompI,1 gene of L. interrogans clustered distinctly from other pathogenic and non-pathogenic leptospiral species. The diversity of ompL genes has been an- alyzed and it envisaged that sequence-specific variations at antigenic determinant sites would result in slow evolutionary changes along with new serovar origination within closely related species. Thus, a crucial work on effective recombinant vaccine development and engineered antibodies will hopefully meet to solve the therapeutic challenges.
