To determine the pathogenic potential of the leptospiral flagella-associated proteins, the genes flhA and flhB2 encoding the biosynthesis of flagella of leptospira interrogans serogroup icterohaemor- rhagiae serovar l...To determine the pathogenic potential of the leptospiral flagella-associated proteins, the genes flhA and flhB2 encoding the biosynthesis of flagella of leptospira interrogans serogroup icterohaemor- rhagiae serovar lai stain 56601 were cloned and their prokaryotic expression systems were constructed. It was demonstrated that the cloned flhA and flhB2 genes had 2118 bp in length and showed 100% and 99.9% of homologies in their nucleotide sequences and 100% and 98.8% of homologies in their putative amino acid sequences respectively, in comparison with those of previously reported. The prokaryotic expression systems under the induction with IPTG could efficiently express the target proteins rFlhA and rFlhB2 with the outputs of approximate 10% of the total bacterial proteins. Based on the sequences of the cloned genes flhA and flhB2, the structural features in associated with pathogenesis and the functions of the target proteins were analyzed with bioinformatics softwares, in which the FlhA was found to have 7 major transmembrane helices, while the FlhB2 had 5 ones. The conserved domains in the FlhA showed high similarity to those of the FHIPEP of the other bacterial FlhA and EscV families, but the conserved domains in the FlhB2 were similar to those of bac-export-2 and EscU families, EscV and EscU families being the protein products of the type IR secretion system in association with pathogenesis. The FlhA and FlhB2 also contained protein kinase C （PKC） and protein tyrosine kinase （PTK） phosphorylation sites, indicating that PKC and PTK of host cells were involved in the internalization and intracellular proliferation in the pathogenesis of microorganisms. All these data leads to a conclusion that the flhA and flhB2 genes of L. interrogans are relatively conserved and their gene products have great potential in the pathogenesis of this organism.展开更多
目的 构建钩端螺旋体外膜脂蛋白 L ip L41基因的重组原核表达质粒 ,为进一步制备以 L ip L41为目的基因的亚单位疫苗提供实验依据。方法 根据钩端螺旋体 L.kirscneri RM5 2株外膜脂蛋白 L ip L41基因序列设计引物 ,以赖型钩端螺旋体 0...目的 构建钩端螺旋体外膜脂蛋白 L ip L41基因的重组原核表达质粒 ,为进一步制备以 L ip L41为目的基因的亚单位疫苗提供实验依据。方法 根据钩端螺旋体 L.kirscneri RM5 2株外膜脂蛋白 L ip L41基因序列设计引物 ,以赖型钩端螺旋体 0 17株基因组 DNA为模板 ,进行 PCR扩增并 DNA测序 ,以质粒 p GEX1λT为载体 ,插入 L ip L41基因片段构建重组原核表达质粒 ,并检测 L ip L41的表达。结果 测序结果示所得片段为 L ip L41的编码序列 ,酶切及 PCR分析证实重组质粒构建成功 ,SDS- PAGE和 Western blotting分析重组质粒可高效表达蛋白质 L ip L41。结论 L ip L41基因的重组原核表达质粒构建成功 。展开更多
文摘To determine the pathogenic potential of the leptospiral flagella-associated proteins, the genes flhA and flhB2 encoding the biosynthesis of flagella of leptospira interrogans serogroup icterohaemor- rhagiae serovar lai stain 56601 were cloned and their prokaryotic expression systems were constructed. It was demonstrated that the cloned flhA and flhB2 genes had 2118 bp in length and showed 100% and 99.9% of homologies in their nucleotide sequences and 100% and 98.8% of homologies in their putative amino acid sequences respectively, in comparison with those of previously reported. The prokaryotic expression systems under the induction with IPTG could efficiently express the target proteins rFlhA and rFlhB2 with the outputs of approximate 10% of the total bacterial proteins. Based on the sequences of the cloned genes flhA and flhB2, the structural features in associated with pathogenesis and the functions of the target proteins were analyzed with bioinformatics softwares, in which the FlhA was found to have 7 major transmembrane helices, while the FlhB2 had 5 ones. The conserved domains in the FlhA showed high similarity to those of the FHIPEP of the other bacterial FlhA and EscV families, but the conserved domains in the FlhB2 were similar to those of bac-export-2 and EscU families, EscV and EscU families being the protein products of the type IR secretion system in association with pathogenesis. The FlhA and FlhB2 also contained protein kinase C （PKC） and protein tyrosine kinase （PTK） phosphorylation sites, indicating that PKC and PTK of host cells were involved in the internalization and intracellular proliferation in the pathogenesis of microorganisms. All these data leads to a conclusion that the flhA and flhB2 genes of L. interrogans are relatively conserved and their gene products have great potential in the pathogenesis of this organism.
文摘目的 构建钩端螺旋体外膜脂蛋白 L ip L41基因的重组原核表达质粒 ,为进一步制备以 L ip L41为目的基因的亚单位疫苗提供实验依据。方法 根据钩端螺旋体 L.kirscneri RM5 2株外膜脂蛋白 L ip L41基因序列设计引物 ,以赖型钩端螺旋体 0 17株基因组 DNA为模板 ,进行 PCR扩增并 DNA测序 ,以质粒 p GEX1λT为载体 ,插入 L ip L41基因片段构建重组原核表达质粒 ,并检测 L ip L41的表达。结果 测序结果示所得片段为 L ip L41的编码序列 ,酶切及 PCR分析证实重组质粒构建成功 ,SDS- PAGE和 Western blotting分析重组质粒可高效表达蛋白质 L ip L41。结论 L ip L41基因的重组原核表达质粒构建成功 。
