钩端螺旋体LA_1100蛋白膜定位研究

目的 研究LA_1100蛋白在钩端螺旋体中的膜定位并分析其在中国流行血清群中的保守性.方法 利用生物信息学软件对LA_1100的二级及三级结构进行分析,以Triton X-114抽提分离钩体细胞的各个组分,Westernblot及FACS方法验证LA_1100在钩端螺旋体中的膜定位.Western Blot和PCR在蛋白水平及核酸水平检测了其在13个中国流行血清群代表株中的保守性.结果 LA_1100的二级结构显示具有α-螺旋及β-折叠结构,进一步分析表明,此蛋白具有跨膜区.LA_1100单体结构具有典型的TolC结构域,三级结构模拟显示三聚体可形成孔状结构.膜定位显示,该蛋白定位于外膜.该基因的保守性分析结果表明,在中国13个流行代表株中有12株均检出该基因或该蛋白的特征性条带.结论 LA_1100为具有TolC结构域可以形成孔状结构的钩端螺旋体外膜蛋白,在中国流行株钩端螺旋体中保守.由于TolC蛋白与病原菌分泌致病因子密切相关,推测此蛋白可能与钩体感染宿主时粘附及致病相关,进一步对此蛋白进行研究有利于揭示钩体的致病机制.

Study on cellular location of protein LA1100 in Leptospira

Objective To investigate the cellular location and conservation of LA₁100 in Leptospira.Methods The characteristics and the three-dimensional structure of the protein were analyzed by several bioinformation softwares.Triton X-114 fractionation and Fluorescence-activated cell sorter were used to verify the location of LA₁100.Western blot and PCR were performed to study the conservation of LA₁100 among 13 Leptospira strains prevalent in China.Results LA₁100 protein had shown α-helices and β-fold with its secondary structure.The built structure of the protein LA₁100 was an assembly of a trimeric single pore channel.By means of Triton X-114 fractionation and Fluorescence-activated cell sorter,it showed that LA₁100 was an outer membrane protein.Among the 13 Leptospira strains prevalent in China,there were 12 with LA₁100 genes or showing positive results by both Western blot and PCR.Conclusion LA₁100 is the surface-exposed pore-forming outer membrane protein of Leptospira and conserved among Leptospira strains prevalent in China.These findings indicate that LA₁100 may be associated with adhesion and virulence of L.interrogans.