赖型钩体pD C 38核酸序列测定及其与流感伤寒型钩体外膜蛋白基因om pL 1核酸序列的类似性匹配  

Alignment of DNA Sequences of ompL1 Genes of Insert Fragment of Recombinant Plasmid, pDC38 of L. interrogans serovar lai and L. kirschneri

作　　者：戴保民[1] 陈庄 DavidA.Haake 游自立[1] 方之茂[1] 

机构地区：[1]华西医科大学基础医学院钩端螺旋体病研究室,成都610041 [2]UniversityofCaliforniaLosAngels,SchoolofMedicine,U.S.A

出　　处：《华西医科大学学报》1999年第3期236-240,共5页Journal of West China University of Medical Sciences

基　　金：卫生部科研基金;高校博士学科点专项基金

摘　　要：为了探讨赖型钩体 ｐ Ｄ Ｃ３８ 插入片段基因组 Ｄ Ｎ Ａ 编码、位置与ｏｍ ｐ Ｌ１ 基因的关系，作者采用在流感伤寒型钩体外膜蛋白基因ｏｍ ｐ Ｌ１ 首尾区段设计一对引物，以 ｐ Ｄ Ｃ３８ 插入片段作模板进行 Ｐ Ｃ Ｒ 扩增、测定和类似性匹配（ａｌｉｇｎｍ ｅｎｔ）。结果发现：ｐ Ｄ Ｃ３８ 含有 ２．７ｋｂ 和３．０ｋｂ 两个插入片段，３．０ｋｂ 片段含有 １ 个ｏｍ ｐ Ｌ１ 基因全拷贝。结论：类似性匹配表明，ｐ Ｄ Ｃ３８ 和ｏｍ ｐ Ｌ１ 相同碱基８６４ 个（９０％ ），碱基变异（不配对）９６（１０％ ）个， ｐ Ｄ Ｃ３８ 可用于钩体诊断、分类、鉴定、疫苗和发病机理的研究。In a previous study a genomic library of L.interrogans serovar lai was constructed by the present authors. Hybridization analysis (In situ, dot blot, Southern blot) with the DNA fragment containing OmpL1 (α 32 P labeled) was performed. One of positive clones designated pDC38, was analyzed with 9 restriction enzymes ( EcoRI, Bam HI, Hind Ш, Bgl, XbalI, ScaI, KpnI, PstI, DraⅡ). DNA hybridization was applied to analyze the homology of the recombinant fragment of ompL1 with the DNA of 18 strains of L.interrogans. The results showed that the homology of fragments of ompL1 were present in pathogenic Leptospira strains, but they were not in the non pathogenic Leptospira biflexa strain Patoc I, Leptonema illini strain 3055. Therefore, Dr. David A. Haake performed the sequencing of pDC38. The results showed that pDC38 contained two inserts of 2.7kb and 3 0kb. The 3.0 insert contained a complete copy of the ompL1 gene. Dr. David A. Haake amplified the ompL1 gene using PCR primers specific for the ends of the gene and cloned the amplicon into pBluescript KS for sequencing. The alignment of complete DNA sequences of ompL1 of L. kirschneri and L.interrogans serovar lai showed the similarity of nucleotide sequences was 90%, variation was 10%. The derived amino acid sequence showed there was a high degree of amino acid sequence homology with ompL1 of L. kirschneri. These findings indicated that ompL1 gene was one of the important outer membrane protein genes in the L. interrogans serovar lai and using the probe pDC38 might provide a good tool for classification and identification of Leptospira.

关 键 词：钩端螺旋体病 重组质粒 外膜蛋白基因 DNA 

分 类 号：R514.402[医药卫生—内科学]