致病性钩端螺旋体遗传操作体系的建立

Leptospirosis is a worldwide-spread zoonosis caused by pathogenic Leptospira. Albeit the genomic studies have provided a good basis for understanding the Leptospira and leptospirosis, the lack of efficient genetic manipulation tools severely blocks the progress of the research into the molecular pathogenic mechanism of this pathogen. Previous studies have shown that extrachromosomal genetic materials may exist in Leptospria. Actually, saprophytic Leptospira can be infected by Leptopira-specific phages, which can replicate inside as plasmids. Furthermore, a genome island LaiGI was discovered in pathogenic Leptospira Lai, which may either integrate into chromosome or replicate as a plasmid. In our previous study, we identified another three replicons lcp1, lcp2 and lcp3 in 56609 apart from two chromosomes based on sequencing results and bioinformatics analysis also revealed that the replicons were phage related. In this study, we intend to characterize prophage within pathogenic Leptospira, meanwhile, to construct gene complementation and over-expression vectors for wide spectrum Leptospira, which will certainly provide more tools for the study of Leptospira pathogenic mechanisms.

钩端螺旋体病是由致病性钩端螺旋体引起的全球性人畜共患病。虽然基因组研究为钩体和钩体病研究创造了极好的基础，但由于缺乏有效的遗传操作手段，致病性钩体致病机制的研究进展缓慢。已有研究表明，钩体基因组可能存在染色体外遗传物质，非致病性钩体能被钩体特异性噬菌体感染，并以质粒形式存在于染色体外随宿主基因组复制；而在致病性钩体赖株中又存在基因岛可游离又可整合入染色体中。我们前期的研究结果发现，致病性钩体菌株56609的基因组中，可能存在3个染色体外的复制子lcp1、lcp2和lcp3，其编码基因序列与噬菌体相关且具备质粒特性。本课题将在验证这些复制子的基础上，进一步研究其与染色体复制的关系，探索其噬菌体来源的可能性；并在此基础上，进一步构建可用于更宽致病性钩体血清型谱的回补载体和基因过表达载体，从而为阐明致病性钩体的致病机制提供更多的研究方法和手段。

成功完成了问号钩端螺旋体流感伤寒群临海型56609菌株的全基因组测序并获得了全基因组序列。通过生物信息学及分子生物学方法证实了56609菌株染色体外存在3个独立复制的单拷贝大质粒。通过丝裂霉素C诱导、电镜技术观察及RT-PCR、Real-Time PCR 等方法鉴定出噬菌体，并初步确定噬菌体基因组序列。基于已知的双曲钩体穿梭载体（L. biflex-E. coli ）pGKBLe24序列，扩增56609染色体外质粒的复制起始区域（rep及parAB基因）序列替换双曲穿梭载体复制起始域，并转化Patoc I株、问号钩体56601株、56606株、56610株、Manilae WT 及哥本哈根菌株，通过验证转化子序列证明了本课题中构建的致病性钩体穿梭载体的可行性。