赖型钩端螺旋体重组DNApCX7的特异性鉴定和限制性内切酶谱分析

本实验将赖型钩端螺旋体（简称钩体）ＤＮＡ基因库的克隆ｐＣＸ７制备成￣（３２）Ｐ－重组ＤＮＡ探针，对８个不同血清群的１７株问号状钩体、双曲钩体ＰａｔｏｃⅠ株以及细螺旋体３０５５株ＤＮＡ进行打点杂交；同时用１５种ＤＮＡ片断进行限制性内切酶谱分析。结果表明，该重组ＤＮＡ具有问号状钩体种（Ｓｐｅｃｉｅｓ）特异性，但与不同问号状钩体之间的同源性程度有差别；限制性内切酶谱分析发现ｐＣＸ７重组ＤＮＡ片段长约１．７ｋｂ，具有１个Ｂｇ１Ⅱ识别位点和３个ＢｓｔＢ１识别位点。     

我国主要致病钩端螺旋体DNA与OmpL1基因同源性的研究

用￣（３２）Ｐ标记的ＯｍｐＬ１基因片段与我国１８株钩体进行Ｓｏｕｔｈｅｒｎ杂交分析，此片段与非致病钩体ＰａｔｏｃＩ株、伊利尼细丝体３０５５株无杂交信号；问号钩体中，除爪哇群、塔拉索夫群、曼耗群和明尼群的４株钩体外，黄疸出血群、犬群、拜伦群、致热群、秋季群、澳洲群、波摩那群、流感伤寒群、七日热群、巴达维亚群、赛罗群各群钩体参考株ＤＮＡ均有杂交信号。     

钩端螺旋体16SrRNA逆转录聚合酶链反应

用ＳｉＯ＿２－高盐吸附法提取钩体ＲＮＡ，以问号状钩体１６ＳｒＲＮＡ基因引物对问号状钩体ｌａｉ型Ｌａｉ株和双曲钩体ｐａｔｏｃ型ＰａｔｏｃⅠ株的ＲＮＡ进行逆转录聚合酶链反应扩增。结果表明，采用本法对ＲＮＡ和ＤＮＡ同时检测时，在琼脂糖凝胶电泳中，可使肉眼检测水平提高约１００倍。     

我国主要致病钩端螺旋体DNA与OmpL1基因同源性的研究

用＾３２Ｐ标记的ＯｍｐＬ１基因片段与我国１８株钩体进行Ｓｏｕｔｈｅｒｎ杂交分析。此片段与非致病钩体ＰａｔｏｃＩ株、伊利尼细丝体３０５５株无杂交信号；问号钩体中，除爪哇群、塔拉索夫群、曼耗群和明尼群的４株钩 体外，黄疸出血群、犬群、拜伦群、致热群、澳洲群、波摩那群、流感伤寒群、七日热群、巴达维亚群、赛罗群各群钩体参考株ＤＮＡ均有杂交信号。     

赖型钩端螺旋体基因文库的构建及重组质粒pDC38的初步研究

应用质粒载体ｐＵＣ１８购建了黄疸出血群赖型钧体的基因文库，重组率８６％，重组子约１２０００个。以ＯｍｐＬ１基因片断作探针从该基因文库中筛出１０个阳性克隆，其中重组质粒ｐＤＣ３８与７型８株致病钩体（黄疸出血群赖型、大型、致热型、秋季型、澳大利亚型、波摩那型、流感伤寒群临海型）ＤＮＡ杂交有杂交信号；与非致病的双曲钩体、细丝体，大肠杆菌和２型问号钩体（爪哇型、拜伦型）ＤＮＡ无杂交信号。     

保护性MbE_4B_7G_5对问号状钩体疏水外膜蛋白OmpL_(39)的免疫印迹分析

选用具有高效价、特异性的黄疸出血群保护性单克隆抗体（ＭｃＡｂ）Ｅ４Ｂ７Ｇ５，用Ｗｅｓｔｅｒｎ－ｂｌｏｔｉｎｇ，对ＴｒｉｔｏｎＸ－１１４（ＴＸ－１１４）抽提的６株问号状钩体（０１７、６０１、６０３、６０９、６２０、２４５株）的疏水外膜蛋白进行分析鉴定。结果表明，提纯的０１７、６０１、６０３、６０９株问号状钩体３９ｋｄ疏水外膜蛋白（ＯＭＰ）有明显的免疫反应，而６２０、２４５株此带区无明显的免疫识别。这一结果提示：用ＭｂＥ４Ｂ７Ｇ５筛选问号状钩体保护性抗原，分离、纯化３９ｋｄ的疏水外膜蛋白（ＯｍｐＬ３９）在构建钩体基因工程疫苗中有重要的研究价值。     

赖型017株钩体疏水外膜蛋白及其免疫特征的研究

采用低浓度非离子型去污剂ＴｒｉｔｏｎＸ－１１４（ＴＸ－１１４）后提和分离问号状钩端螺旋体（钩体）黄疸出血群赖型０１７株（０１７），选择性地将钩体外膜蛋白分离为ＴＸ－１１４疏水相和亲水相两个部分。疏水相外膜蛋白含５条主要蛋白区带，其分子量分别为６６ｋｄ、３９ｋｄ、３５ｋｄ、２７ｋｄ和６ｋｄ，，ａｄｋｈｗｃｙ ３９ｋｄ外膜蛋白区带可特异地分别被抗全０１７血清，抗０１７外膜血清和抗０１７保护性单克隆抗体     

保护性Mb E4B7G5对问号状钩体疏水外膜蛋白OmPL39的免疫…

选用具有高效价、特异性的黄疸出血群保护性单克隆抗体（ＭｃＡｂ）Ｅ４Ｂ７Ｇ５，用Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ，对Ｔｒｉｔｏｎ Ｘ－１４４（ＴＸ－１４４）抽提的６株问号状钩体（０１７、６０１、６０３、６０９株）的疏水外膜蛋白进行分析鉴定。结果表明，提纯的０１７、６０１、６０３、６０９株问号状钩体３９ｋｇ疏水外膜蛋白（ＯＭＰ）有明显的免疫反应，而６２０、２４５株此带区无明显的免疫识别。这一结果提示：用     

赖型钩体重组质粒pDJH_2亚克隆及其在大肠杆菌中的高效表达

为了探讨ｐＤＪＨ２的１．９ｋｂ０１７株钩体ＤＮＡ片段可否作为钩体重组疫苗广谱保护性抗原的候选，采用重组ＤＮＡ技术，将ｐＤＪＨ２的１．９ｋｂＤＮＡ片段分别与ｐＴ７－７，ｐＲＳＥＴ系列重组，转化入大肠杆菌进行亚克隆，ＩＰＴＧ诱导表达。结果显示：阳性亚克隆ｐＤＪｔ．ｐＤＪｒＢ１能在大肠杆菌中高效表达；对其进行ＳＤＳ－ＰＡＧＥ分析，可见６８ｋｄ和２３ｋｄ处出现新带，与钩体０１７株外膜同分子量蛋白带位置一致，免疫印迹（特异性抗０１７株外膜抗血清）在６８ｋｄ和２３ｋｄ处分别有印迹；用ｐＤＪｔ亚克隆重组质粒主动免疫豚鼠，可抵抗强毒力株攻击，表现出免疫保护作用。本实验结果提示：（１）ｐＤＪＨ２１．９ｋｂ重组ＤＮＡ片段能以不同质粒为载体，在不同大肠杆菌株中高效表达；（２）该重组ＤＮＡ片段表达产物——６８ｋｄ和２３ｋｄ蛋白，可能是赖型钩体０１７株外膜的保护性抗原。     

赖型017株钩体疏水外膜蛋白及其免疫特征的研究

采用低浓度非离子型去污剂ＴｒｉｔｏｎＸ－１１４（ＴＸ－１１４）抽提和分离问号状钩端螺旋体（钩体）黄疸出血群赖型０１７株（０１７），选择性地将钩体外膜蛋白分离为ＴＸ－１１４疏水相和亲水相两个部分。疏水相外膜蛋白含５条主要蛋白区带，其分子量分别为６６ｋｄ、３９ｋｄ、３５ｋｄ、２７ｋｄ和１６ｋｄ，其中仅３９ｋｄ外膜蛋白区带可特异地分别被抗全０１７血清、抗０１７外膜血清和抗０１７保护性单克隆抗体Ｅ４Ｂ７Ｇ５所识别。本研究结果表明经ＴＸ－１１４抽提分离的３９ｋｄ疏水外膜蛋白为０１７钩体稳定的免疫原，在研究钩体保护性抗原和构建基因工程疫苗中有十分重要的研究价值。     

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.     

赖型钩端螺旋体基因库的构建及致病性钩端螺旋体DNA同源序列的克隆

作者应用质粒载体pUC9构建了肺弥漫性出血型构端螺旋体(钩体)病的主要病原——赖型钩体的基因库,并从中筛选出与致病钩体DNA同源的重组质粒pCX7和pCX9。重组质粒pCX9可识别致病钩体017株DNA1.7kb片段、601株9.0kb片段及610株30.0kb片段,而不与非致病的双曲钩体Patoc I株和illini细螺旋体DNA杂交。本研究结果进一步表明:致病钩体与非致病钩体DNA的同源性很低;致病钩体间DNA同源性较高。     

Homology study of leptospires by molecular hybridization]

Nick translation and random primer labelling method were applied to prepare three genomic DNA probes from Leptospira interrogans strain 017, Leptospira biflexa strain Patoc I and Leptonema illini strain 3055, and then hybridized with DNA of 17 strains leptospires from different genus, species, serogroup and serovar. The results showed no homology between Leptospira and Leptonema, and only a low degree of homology between L. interrogans and L. biflexa but it showed a high degree of homology among L. interrogans. The study also proved the possibility to establish a DNA probe prepared from a single leptospira strain to detect different serovars.     

钩端螺旋体重组DNA探针对致病性黄疸出血群钩体DNA的杂交识别

Two recombinant DNA probes-PLIpso1 (15kb) and PLIEc34 (4kb), derived from Leptospira serovaricterohaemorrhagiae genomic libraries, were applied for the hybridization and identification of 13 strains of Leptospira in serogroup icterohaemorrhagiae. Difference in hybridization signal in combination with the banding pattern provide a good way for identification of serovars and strains. The recombinant DNA, specific to L. serogroup icterohaemorrhagiae, hybridized with a limited number of DNA fragments which had been digested by several restriction endonucleases. The less complex banding pattern and higher sensitivity facilitate characterization of various serovars and strains in serogroup icterohaemorrhagiae. In general the DNA patterns recognized by both probes have extensive genomic homology in same serovar (but still can distinguish strains in same serovar by some unique bands) and apparent difference in various serovars (especially serovars naam, nanxi and honghe). The results indicated that Southern blotting with recombinant DNA probe might provide tools for identification, characterization and analysis 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。     

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.     

钩端螺旋体重组DNA探针对致病性黄疸出血群钩体DNA的杂交识别

作者从建立的黄疸出血群钩端螺旋体(下称钩体)DNA基因库中筛选出重组DNA克隆PLIps01(15kb)和PLIEc34(4kb)制备成~(32)P放射性探针。以该二探针对致病性黄疸出血群所属血清型的13个菌株钩体DNA进行Southern印迹分析,结果表明,该二DNA探什对黄疸出血群各株钩体DNA具有强烈的特异性结合,能识别出简洁的DNA带型,并能区分不同血清型以至菌株的DNA带型差别。作者认为,钩体DNA重组探什结合Southern印迹分析是一种灵敏而特异的检测分析方法,可作为诊断、鉴定和分析钩体的工具。     

应用重组DNA探针检测不同毒力钩端螺旋体

应用质粒载体ｐＵＣ１８构建赖型钩端螺旋体０１７株的基因库。筛选出重组质粒ｐＤＪ６和ｐＤＪ８，插入片段分别是１．９ｋｂ和２．２ｋｂ。地高辛随机引物标记１．９ｋｂ片段制备探针，对５个血清型６株钩体ＤＮＡ进行杂交。结果显示，重组探针与致病钩体ＤＮＡ出现明显杂交信号，与非致病钩体，人白细胞ＤＮＡ及大肠杆菌ＪＭ１０３株ＤＮＡ杂交信号不明显。此特异性重组探针可作为鉴定、识别致病性钩体和进行钩体属、种分类的一种手段。     

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.