To evaluate how the efficacy of DNA inocutation affects the ability to raise protective immunity against Leptospira. [WT5”BX]Methods.[WT5”BZ] A pair of oligonucleotide primers were designed to amplify the endoflagel...To evaluate how the efficacy of DNA inocutation affects the ability to raise protective immunity against Leptospira. [WT5”BX]Methods.[WT5”BZ] A pair of oligonucleotide primers were designed to amplify the endoflagellar gene of L. interrogans sensu stricto serovar lai. An approximately 840bp fragment was generated with PCR and inserted into VR1012, a plasmid DNA expression vector, after the fragment and VR1012 were digested respectively with EcoRV and Sal I. A recombinant plasmid designated as VR1012+flaB2 was obtained. The vector, VR1012 consits of a pUC18 backbone with the cytomegalovirus(CMV) IE1 enhancer, promoter, and intron A, transcription regulatory elements and the BGH polyadenylation sequences driving the expressing of leptospiral endoflagellar gene of L. interrogans sensu stricto serovar lai. Plasmid encoding leptospiral endoflagellin gene was injected into quadriceps of NZW rabbits. [WT5”BX]Results.[WT5”BZ]This resulted in the generation of specific leptospiral antibody with high ELISA titer (1:32768) in the rabbits. Immuno/protection was performed in guinea pigs without adjuvant. The group“VR1012+flaB2” showed higher survival rate(90%,9/10 animals),compared with the group “VR1012 lack flaB2” and the group “normal saline”. [WT5”BX]Conclusion.[WT5”BZ]The technique of DNA vaccine has potential advantages over certain other vaccine preparation technologies. However whether DNA vaccine will be useful for vaccine development remains to be tested.展开更多
A pair of oligonucleotide primers were designed to amplify the endoflagella gene of L. Interrogans serovar lai. An approximately 840 bp fragment was generated with PCR and inserted into plasmid pUC8,after the fragment...A pair of oligonucleotide primers were designed to amplify the endoflagella gene of L. Interrogans serovar lai. An approximately 840 bp fragment was generated with PCR and inserted into plasmid pUC8,after the fragment and pUC8 were digested respectively with BamHI and PstI. A recombinant plasmid (designated as pLF1 ) was obtained. SDS-PAGE analysis indicated that a 33 kDa was expressed in E. Coli JM103 harboring pLF1 and the expression level of the protein was 11 % of the total bacterial soluble proteins. Western blot analysis showed that the protein band could be recognized by the antiserum against the endoflegella (Axial filament ) of Leptospira interrt,gans serover lai. Nucleotide sequence data showed an open reading frame encoding 282 aminoacids residues,corresponding to a protein of molecular weight 33. 6kDa. Comparison of the deduced endoflagellar subunit protein (flaB) amino acid sequence with flagellins from other bacteria revealed a high level of identity with the Trehoema pallid um flaB proteins. Immunization/protection experiment was performed on the model of BALB/C mice and showed that there was higher survival rate in the group JM103-pLE1 than in the group JM103-pUC8.展开更多
基金This project was supported by a research grant from theNational Natural Science Foundation of China(No.399706 67)
文摘To evaluate how the efficacy of DNA inocutation affects the ability to raise protective immunity against Leptospira. [WT5”BX]Methods.[WT5”BZ] A pair of oligonucleotide primers were designed to amplify the endoflagellar gene of L. interrogans sensu stricto serovar lai. An approximately 840bp fragment was generated with PCR and inserted into VR1012, a plasmid DNA expression vector, after the fragment and VR1012 were digested respectively with EcoRV and Sal I. A recombinant plasmid designated as VR1012+flaB2 was obtained. The vector, VR1012 consits of a pUC18 backbone with the cytomegalovirus(CMV) IE1 enhancer, promoter, and intron A, transcription regulatory elements and the BGH polyadenylation sequences driving the expressing of leptospiral endoflagellar gene of L. interrogans sensu stricto serovar lai. Plasmid encoding leptospiral endoflagellin gene was injected into quadriceps of NZW rabbits. [WT5”BX]Results.[WT5”BZ]This resulted in the generation of specific leptospiral antibody with high ELISA titer (1:32768) in the rabbits. Immuno/protection was performed in guinea pigs without adjuvant. The group“VR1012+flaB2” showed higher survival rate(90%,9/10 animals),compared with the group “VR1012 lack flaB2” and the group “normal saline”. [WT5”BX]Conclusion.[WT5”BZ]The technique of DNA vaccine has potential advantages over certain other vaccine preparation technologies. However whether DNA vaccine will be useful for vaccine development remains to be tested.
文摘A pair of oligonucleotide primers were designed to amplify the endoflagella gene of L. Interrogans serovar lai. An approximately 840 bp fragment was generated with PCR and inserted into plasmid pUC8,after the fragment and pUC8 were digested respectively with BamHI and PstI. A recombinant plasmid (designated as pLF1 ) was obtained. SDS-PAGE analysis indicated that a 33 kDa was expressed in E. Coli JM103 harboring pLF1 and the expression level of the protein was 11 % of the total bacterial soluble proteins. Western blot analysis showed that the protein band could be recognized by the antiserum against the endoflegella (Axial filament ) of Leptospira interrt,gans serover lai. Nucleotide sequence data showed an open reading frame encoding 282 aminoacids residues,corresponding to a protein of molecular weight 33. 6kDa. Comparison of the deduced endoflagellar subunit protein (flaB) amino acid sequence with flagellins from other bacteria revealed a high level of identity with the Trehoema pallid um flaB proteins. Immunization/protection experiment was performed on the model of BALB/C mice and showed that there was higher survival rate in the group JM103-pLE1 than in the group JM103-pUC8.
