赖型钩端螺旋体毒力株新基因差异片段筛选和核苷酸序列分析

Screening and sequencing of the novel differential gene in leptospira serovar lai

目的 :筛选致病钩端螺旋体赖型 0 1 7株毒力相关基因DNA差异片段 ,并进行核苷酸及基因信息学分析。方法 :提取钩端螺旋体 0 1 7株基因组DNA ,根据抑制消减杂交 (SSH)筛选的 0 1 7株特有的 1 53bp核苷酸短片段 ,采用盒式连接半巢式PCR技术 ,扩增相邻未知序列 ,进行序列测定、分析及同源性检索 ,并进行蛋白二级结构预测。结果 :克隆获得 580bp核苷酸长度的基因片段 ,包含 4个重叠开放阅读框架 (ORF) ,在氨基酸水平上与A型化脓性链球菌 ,肺炎链球菌保守的假想蛋白高度同源。被GenBank收录 ,收录号为AF495587。结论 :本研究筛选并分析了可能是钩体毒力相关的基因片段 ,为进一步探讨该基因的生物学功能及阐明赖型钩端螺旋体致病分子机制打下了基础。

AIM: To amplify and analyze the differential DNA fragment between pathogenic leptospira serovar lai and nonpathagenic leptospira serovar patoc I. METHODS: The previously subtractive DNA fragment only existing in leptospira serovar Lai was amplified by cassette ligation and semi-nested PCR.The obtained gene was sequenced and searched homologically. In addition, the deduced amino acid was analyzed and the secondary stracture of protein was predicted. RESULTS: The 580 bp DNA fragment, which deposited in GenBank (AF495587), was cloned, and four overlapping open reading frames (ORF) was contained. The high homology with conserved hypothetical protein streptococcus pyogenes was found. CONCLUSION: This study lays foundation for deeply exploring biological actions of new gene and pathogenic mechanism of leptospira serovar lai.