论文部分内容阅读
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。
Objective To study the membrane localization of LA_1100 protein in Leptospira and analyze its conservatism in Chinese epidemic serogroups. Methods The secondary and tertiary structures of LA_1100 were analyzed by bioinformatics software. The components of leptospira were extracted by Triton X-114. The localization of LA_1100 in Leptospira was validated by Western blot and FACS. Western Blot and PCR at protein and nucleic acid levels were tested for their conservatism in 13 representative Chinese isolates of serogroups. Results The secondary structure of LA_1100 showed a-helical and β-sheet structure. Further analysis showed that this protein has a transmembrane domain. LA_1100 monomer structure has a typical TolC domain, tertiary structure simulation shows that the trimer can form a pore structure. The membrane localization shows that the protein is located on the outer membrane. Conservative analysis of the gene showed that 12 of the 13 representative strains in China were found to be characteristic of the gene or the protein. Conclusion LA_1100 is a Leptospira outer membrane protein with a TolC domain that can form a pore structure and is conserved in the Chinese endemic strain Leptospira. Since TolC protein is closely related to the pathogenicity of pathogenic bacteria, it is speculated that this protein may be related to the adhesion and pathogenicity of host infected with leptospira, further study of this protein is helpful to reveal the pathogenic mechanism of leptospira.