摘要
为了建立敏感和特异的鉴别致病性钩体菌株的方法，用ＡＢＩ自动测序仪对问号钩体（Ｌ．ｉｎｔｅｒｒｏｇａｎｓｓｅｎｓｕｓｔｒｉｃｔｏ）种特异探针ＤＮＡ重组质粒ｐＣＸ７，１．７ｋｂＰｓｔⅠ部分插入片段进行了核苷酸序列测定。结果：该部分插入片段ｂｐ总数为１～５０１ｂｐ，其中可读框（ＯＲＦ）１个，序列为８７～３９３，共３０６ｂｐ。ＯＲＦ除去一些核苷酸不能确定外（图中以Ｎ表示），绝大多数在一个ＯＲＦ。计算机检索有关同源序列１８个，较高序列类似性５个，均在４１％以下。经查ＥＭＢＬＤＮＡ序列数据库未见类似序列。由此提示，以ｐＣＸ７作为探针可用于钩体菌株鉴定。
The etiological agents of leptospirosis are the pathogenic leptospires( L.interrogans sensu lato) which can be divided into 223 serovars organized into 23 serogroups. The serovar remains the basic taxon, but serotyping may now be accomplished and recognized by acceptable methods. Complementary molecular approaches are being used extensively to assess genetic relatedness amongst leptospires with restriction endonuclese analysis (REA), pulse field gel electrophoresis (PFGE) and DNA DNA hybridization as well established tools. However, the method is cumbersome and unsuitable for routine application. To develop a sensitive and specific method for identification of pathogenic leptospires, a genomic library of L.interrogans sensu stricto serovar lai was constructed with the plasmid vector pUC9. A recombinant plasmid, designated pCX7 which has homologous fragment of pathogenic leptospires was screened from the bank. pCX7 could recognize pathogenic leptospiral DNA fragment 1 7kb of strain 017 without cross hybridization to nonpathogenic leptospiral DNA. Inserted fragment of pCX7 DNA sequencing was performed by Dr. Yan Zhengxin (Max Plank Institut fur Biology, Tubingen, Germ any). Insert fragment was cloned into pBluescript and sequenced by using ABI(Applied Bio. Systems, Model 373A). Nucleotide sequences were analyzed by Dr. Xiao Jianguo(Texas University Medical School and School of Public Health, Center for Infectious Diseases) using a suit of computer program(NIH). One open reading frame of 306 nucleotids were identified. There were identifiable initiation codons, terminators, pribnow box and sextama box within the sequenced regions. These results further confirmed that the little homology between L.interrogans sensu strito and L. borgpeterseni serovar javanica, L.inadai serovar ranarun and serovar manhao (L.genomospecies 2), L.biflexa serovar patoc, L.illini. pCX7 DNA probe could provide a base for identification and classification of leptospires.
出处
《华西医科大学学报》
CSCD
1998年第4期349-354,共6页
Journal of West China University of Medical Sciences
基金
国家自然科学基金
