光敏生物素和32P标记DNA打点杂交检测钩端螺旋体

Photobiotin- and 32P-labelled DNA probes of L. interrogans sv. lai strain 017 were produced and the DNA and leptospires were dotted on the NC filter and hybridized. The results showed that photobiotin and 32P-labelled DNA probes could detect DNAs of homology. The smallest amount of DNA that could be detected with the probes were 5pg and 1pg, and the smallest numbers of pathogenic leptospires were 5 x 10(3) and 10(3), respectively. Nonpathogenic leptospires. L. biflexa sv. patoc strain Patoc 1, L. illini strain 3055, Escherichia coli, Bacillus aerogenes capsulatus, and Salmonella anatis, could not be detected by the probes. The study demonstrates that leptospires DNA probe could be produced by labelling the DNA with photobiotin. It has higher sensitivity and specificity and can be used for detection of leptospires in the field and clinic.     

问号钩体（狭义）种特异探针重组质粒pCX7插入片段核苷酸序 …

为了建立敏感特异的鉴别致病性钩体菌侏的方法，用ＡＢＩ自动测序仪对问号钩体（Ｌ．ｉｎｔｅｒｒｏｇａｎｓｓｅｎｓｕｓｔｒｉｃｔｏ）种异探针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１个，序列为８７－３９３，共３０６ｂｐ。     

赖型钩湍螺旋体基因库的构建及致病性钩端螺旋体DNA同源序列...

A gene bank of the main pathogen of pulmonary diffuse haemorrhage type leptospirosis (PDH), L. interrogans serovar lai strain 017, was first constructed with plasmid vector pUC9, which contained 610 recombinant clones and laid the foundation for further investigation of molecular characteristics of leptospires with strong virulence. Recombinant plasmids which have homological sequences of pathogenic leptospires were screened from the gene bank. A recombinant plasmid, designated pCX7, could detect 1.7 kb fragment of strain 017, 9.0 kb of strain 601 and 30.0 kb of strain 610 respectively without cross hybridization with nonvirulent leptospires such as L. biflexa strain Patoc I and Leptonema illini. pCX9, another recombinant plasmid, could detect 1.9 kb fragment of strain 017 and had no hybridization with other pathogenic or nonpathogenic leptospires. The results showed that the degree of homology between pathogenic and non-pathogenic leptospires was very low and the degree of homology was very high among the pathogenic leptospires.     

应用分子杂交对钩端螺旋体DNA同源性的研究

Homology of leptospires from different genus, different serogroups were studied with molecular hybridization. Leptospiral DNAs were extracted and purified with phenolchloroform-isoamylalcohol method. Alpha 32P-dCTP was used to label DNA from L. interrogans serogroup icterohaemorrhagiae serovar lai strain 017 as a DNA probe, and hybridized with DNAs of 2 genus, 5 serogroups of leptospires represented by 6 strains on NC filter. Four serogroups of pathogenic leptospires which caused endemic disease in Sichuan Province were also detected by the probe. The results showed that L. interrogans serogroup icterohaemorrhagiae, serogroup autumnalis and serogroup hebdomadis had a high degree of homology while there was a low degree of homology between L. interrogans and L. biflexa and Leptonema illini. Four major serogroups of pathogenic leptospires in Sichuan Province, with their high degree of homology, could be detected by a radiolabelled probe from serogroup icterohaemorrhagiae. Hybridization may be used as a tool for diagnosis of leptospirosis in human beings and animals.     

IDENTIFICATION OF PATHOGENIC LEPTOSPIRES BY RECOMBINANT DNA PROBES

Early diagnosis of leptospirosis of pulmonary diffuse bernorrhage type (PDH) is of crucial importance in saving patients. To develop a sensitive and specific method for diagnvsis, a genonlic library of the main pathogen of PDH, L. interogans serovar lai strath 017, was constructed with the plasmid vector pUC9. Recmbinant plasmids which have hornologotLq fragments of pathogenic inptospires were screened from the bank. A recombinant plasmid.designated pCX7, could detect 1. 7 kb fragment of strain 017. 9. 0 kb of strain 601 and 30. 0 kb of strain Hebdo-maclis, respectively, without cross hybridization with nonpathogcnic leptospires such as L. biflexa strain Patoc 1 and Leptonema illini. The recombinant plasmid pCX7 could detect pathogenic leptospires which are the main pathogens endemic to Sichuan Province.     

用分子杂交技术分析不同毒力钩端螺旋体基因组DAN同源性

Nick translation labelling technique was applied to the preparation of two 32P labelled genomic DNA probes from L. interrogans serovar Lai strain 017 and Leptonema illini strain 3055, respectively. Dot-blotting and Southern-blotting with DNA of five strains leptospira from different genus and species were performed. The results showed that certain differences among the L. interrogans, L. biflexa and Leptonema illini could be detected by endonuclease assay. L. interrogans strain 017 with L. biflexa strain Patoc I and Leptonema illini 3055 strain exhibited very little homology. L. interrogans strains 017, 601, and 245 from different serogroup and serovar had a high degree of homology. Leptonema illini strain 3055 showed little homology with L. interrogans and L. biflexa. Therefore DNA hybridization may be used as a tool for the identification and classification of leptospira.     

应用分子杂交对钩端螺旋体DNA同源性的研究

作者以~(32)P标记的黄疸出血群赖型017株钩体DNA为探针,分别与2个属5个血清群的6株钩体DNA进行分子杂交。结果表明,黄疸出血群赖型017株与同群、型的56601株同源性较高,与秋季群(56606株)、七日热群(56610株)次之,与双曲钩体patoc型Patoc Ⅰ株、细螺旋体属illini细螺旋体同源性很低;~(32)P标记的钩体DNA探针能检测四川地区流行的致病性钩体。     

赖型钩体pCX7探针Southern杂交分析不同血清群型钩体DNA

用赖型０１７株钩体ＤＮＡ基因库中筛选出的重组克隆ｐＣＸ７制备重组质粒，再以￣（３２）Ｐ标记为探针，对１１个血清群的１８株问号钩体、双曲钩体ＰａｔｏｃＩ株和细螺旋体ｉｌｌｉｎｉ３０５５株ＤＮＡＳｏｕｔｈ－ｅｒｎ杂交。放射自显影结果显示：双曲钩体ＰａｔｏｃＩ株和细螺旋体ｉｌｌｉｎｉ３０５５株ＤＮＡ未获杂交阳性区带；ｊａｖａｎｉｃａ（爪哇）、ｍａｎｈａｏ２（曼耗２）和ｒａｎａｒｕｍ（蛙）三个低毒力血清型的问号状钩体ＤＮＡ亦为阴性；１５株问号状钩体ＤＮＡ均出现了杂交信号，而且不同群型钩体ＤＮＡ杂交带型明显不同，赖型钩体０１７株（强毒株）与６０１株（弱毒株）杂交带型也有细微差别。作者认为，以ｐＣＸ７探针进行Ｓｏｕｔｈｅｒｎ杂交可望作为钩体分类和鉴定的工具或参考。     

用分子杂交技术分析不同毒力钩端螺旋体基因组DAN同源性

用~(32)P标记钩端螺旋体(钩体)基因组DNA,以Dot-blot和Southern-blot杂交法分析5株钩体DNA同源性。结果表明:问号钩体黄疸出血群赖型017株和601株及七日热群七日热型245株与双曲钩体patoc型Patoc I株、细螺旋体illini型3055株同源程度极低;3株问号钩体有不同程度同源;illini型3055株与各株同源性均低。     

赖型钩体DNA重组质粒rpDJt及其表达蛋白P68对豚鼠的免疫？…

为了进一步鉴定赖型钩体ＤＮＡ重组质粒ｒｐＤＪｔ及其纯化表达蛋白质Ｐ６８例的免疫保护作用，采用随机、以盲和对照法对５０只豚鼠进行了３次和６个部位主动免疫接种，并于接种后３０天以强毒力、大剂量活钩体攻击。结果：Ｐ６８组存活率１００％（７／７）；ｒｐＤＪｔ组存活率７７％（１０／１３）；ｐＴ７－７组（无重组质粒）存活率２５％（３／１０）；全钩灭活疫苗组存活率９３％（１３／１４）。作者认为该质粒的表达蛋白质