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。     

聚合酶链反应检测致病性钩端螺旋体微量DNA的研究

作者通过对黄疸出血群钩端螺旋体DNA的分子克隆和筛选,获得了两个重组DNA片段,其中一个具有广泛针对各群致病性构体的特性,另一个仅对黄疸出血群钩体起杂交反应。经DNA序列分析和限制性谱测定,分别合成两对聚合酶链反应寡核苷酸引物——引物B1、2和引物B3、4。以该引物对各群、型钩体微量DNA(0.1 ng)行聚合酶链扩增反应,引物B1、2能引导扩增全部致病性钩体DNA,引物B3、4则特异性地扩增黄疸出血群钩体DNA,非致病性钩体和其他微生物DNA均无扩增反应。结果表明,聚合酶链反应是一种灵敏度极高、针对性很强的DNA扩增技术,可用于钩体病早期诊断和流行病学分类鉴定。     

用分子杂交技术对17株钩端螺旋体基因组DNA同源性的研究

采用缺口平移及随机引物标记法制备出问号状钩体017株、双曲钩体Patoc Ⅰ株和细螺旋体illini型3055株3株不同属种钩体基因组DNA的~(32)P标记探针,用之分别与构端螺旋体属和细螺旋体属的5个血清群17株钩体DNA进行打点杂交。结果发现:此两属钩体间基因组DNA同源性极低;属内不同种间有较低同源性;问号状钩体中不同血清型之间有较高的同源性;赖型017株钩体DNA与不同群型的致病性钩体均有较高的同源性,表明该探针可作为广泛识别各致病性钩体的通用探针。     

地高辛配基标记DNA打点杂交和原位杂交检测钩端螺旋体

应用一种新的非放射标记物地高辛配基(DIG)制备黄疸出血群赖型017株钩端螺旋体DNA(017DNA)探针,与~(32)P和光敏生物素标记017DNA探针对照打点杂交检测钩端螺旋体DNA。结果,上述三种探针均能检测到不同群型的问号状钩体,DIG探针的敏感性为0.1pg,~(32)P和光敏生物素探针的敏感性分别为1 pg和10pg。以上探针均不能检测双曲钩体patoc型Patocl株、细螺旋体illini型3065株,与其它致病菌及人白细胞DNA也无明显杂交信号。DIG标记017DNA探针原位杂交检测感染017株钩体的豚鼠系列组织切片和血浆涂片,以对比度极大的着色反应,清晰地显示各组织和血浆中的钩体形态。细螺旋体illini型3055探针和HBV-DNA探针不能检测到上述标本中的钩体.     

光敏生物素和~(32)P标记DNA打点杂交检测钩端螺旋体

应用光敏生物素和~(32)P标记黄疸出血群赖型017株钩端螺旋体DNA(简称017DNA)作为探针,打点杂交检测问号状钩体。结果表明:两种探针均能检测不同群、型的问号状钩体DNA,最小检测量分别为5pg和1pg;与双曲钩体patoc型PatocⅠ株、细螺旋体属illini型3055株及其他致病菌和正常人血清未发现明显杂交信号。提示光敏生物素制备的017DNA探针可以用于检测钩体,也可应用于钩体病的早期诊断和流行病学调查。     

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。     

早期钩体病患者血清钩体DNA聚合酶链反应和地高辛—硷性磷酸酶发光体探针杂交分析

作者从已建立的问号钩体基因文库中筛选合成一对引物G_1G_2,中国四川14份早期钩体病患者血清中的DNA经硫酸氰胍、二氧化硅和蛋白酶K纯化后,在G_1G_2引导下行聚合酶链反应(PCB),并以地高辛标记的同源探针行DNA印迹杂交,全部血清样品均获得确切地阳性结果。用地高辛—硷性磷酸酶发光体标记PCR扩增的同源DNA探针,与16株Yasuda基因组钩体作DNA印迹分析,其中8株存在同源单拷贝序列,它们皆为我国常见和流行的优势致病菌株,本研究结果表明:引物G_1G_2可用于四川钩体PCR检测和分析,PCR常规结合同源DNA探针杂交识别,能提高其敏感性和可靠性;地高辛—硷性磷酸酶发光体配合PCR等方法,可使微量DNA的检测和分析更趋完善。同时,本研究还提供了有价值的钩体DNA同源序列资料。     

几个血清型钩端螺旋体DNA碱基组成的测定

作者用热变性温度法测定了几个血清型钩端螺旋体的DNA碱基组成(G+C mo 1％)。问号钩端螺旋体赖型017株为36.8％,黄疸出血型8244株为35.5％,波摩拿型109株为36.1％,犬型611株为35.2％,双曲钩端螺旋体帕托克型Patoc Ⅰ株39.1％,其中赖型017株的DNA碱基组成属首次报道。     

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。     

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.     

问号赖型钩体与七日热型钩体内鞭毛蛋白基因的克隆及序列分析

目的　筛选钩体核酸疫苗的候选株 ,并从基因水平解释钩体血清型的特异性。方法　通过 PCR方法扩增赖型 0 17株及七日热型 5 6 6 10株钩体的内鞭毛蛋白 (fla B)基因 ;经 T/A快速克隆法将 PCR产物连接于PGEM- T Easy Vector载体。用 α-互补法、PCR法及酶切分析鉴定重组质粒 ,获得了重组质粒 p DHTF0 17和p DHTF6 10 ;双脱氧终止法对两个重组质粒中的克隆化 fla B基因进行测序。结果　两型 fla B基因长 85 2 bp,编码2 83个氨基酸。两个基因的限制性内切酶位点相似 ,含多个常见酶切位点。基因编码的蛋白质预测相对分子质量为31.3× 10 3。结论　几个血清型的 fla B基因同源性达 90 %～ 99%。     

用限制性核酸内切酶分析钩端端螺旋体DNA的研究

For the application of restriction endonuclease analysis in typing and identifying leptospira, we selected some serovars and isolates, and analysed preliminarily their DNA with four restriction enzymes, EcoR I, Bgl II, Hha I, and Hind III. The DNA samples were isolated from the reference strains and isolates as follows: Serovar lai 56601, 017 (the virulent strain for PDH model of guinea pig), Serovar autumnalis 56606, Serovar manhao II 67020, and isolates 87112 and 87369. Each 2 micrograms of DNA was digested with 20mu of restriction enzyme at 37 degrees C for 2h and electrophoresed in 0.8% agarose gel. The gels were stained in ethidium bromide and photographed with UV light. In our experiments, apparently different restriction patterns of serovar lai 017 were observed with four restriction enzymes. Serovar lai, serovar autumnalis and serovar manhao II showed different patterns with EcoR I, especially in high molecular regions. We also observed in serovar lai 017 a distinct 10.5kb band which was obscure in 56601, the reference strain of serovar lai, after EcoR I digestion. The three serovars showed some delicate differences in Hind III restriction pattern. The two isolates from Apodemus agrarius in Sichuan (1987) 87112 and 87369 had patterns identical to those of serovar lai 56601, 017 with EcoR I, and 87112 also had a pattern identical to 56601, 017 with Hind III. Our results indicate that selected three serovars can be identified by analysis of their DNA with EcoR I and Hind III. It is suggested that restriction endonuclease analysis be a good method in typing and identify leptospira and in studying the differences of special DNA molecules.     

大肠杆菌-钩端螺旋体重组穿梭质粒pGKINVA的构建及重组钩体的筛选

目的 构建大肠杆菌-钩体穿梭质粒pGKINVA,并重组于钩体PatocⅠ株,为进一步确定钩体invA基因的功能奠定基础.方法 将invA基因克隆于pGKble24载体,PCR、限制性内切酶双酶切及测序鉴定重组穿梭质粒pGKINVA.质粒pGKINVA经电穿孔法转化无毒钩体PatocⅠ株,并筛选重组钩体菌株.结论 从钩体基因组中克隆出长558 bp的invA基因,定向插入pGKble24构建重组穿梭质粒pGKINVA,抗生素及蓝/白斑筛选和测序鉴定并获得正确的重组质粒后,电穿孔法转化钩体PatocⅠ株,在Korthof固体培养基生长并抗生素筛选、PCR鉴定出重组钩体菌株.结论 成功构建重组大肠杆菌-钩端螺旋体穿梭质粒pGKINVA,并筛选出2个重组钩体菌株,将有助于进一步阐明invA基因在钩体体内中的功能分析.     

人乳头瘤病毒11型的DNA分子克隆

以E coli C300为受体菌,利用人乳头瘤病毒11型DNA与pBR322的重组质粒(pBR322-HPV11DNA),成功地进行了HPV11DNA的转化,经筛选获得61株重组子,随机对15株转化子以碱变性法及清亮裂解法抽提了pBR322-HPV11DNA的重组质粒,纯化后经BamHI酶切及琼脂糖凝胶电泳,以λDNA/HindⅢ作参考分子量测定,初步结果证实存在有分子量为5.5×10~6(约8.1kb)的HPV11DNA。这为HPV11DNA的分子扩增,基因探针的制备及HPV11型量鳞癌的研究提供了方便。     

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.     

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。     

聚合酶链反应检测致病性钩端螺旋体微量DNA的研究

Leptospirosis is a severe zoonosis in the world. The methods for detecting leptospira are not sensitive and specific so far. The problems in early diagnosis and epidemiological identification of Leptospirosis remain unsolved. Two recombinant DNA fragments of serogroup Icterohaemorrhagiae, Leptospira were selected by repeated molecular cloning and screening in this study firstly. One of them can hybridize with the DNA of various serogroups of Leptospira interrogans; the other can only hybridize with the DNA of serogroup Icterohaemorrhagiae. After the nucleotides sequence analysis, from these 2 recombinant DNA fragments, 2 pairs of polymerase chain reaction (PCR) oligonucleotide primers were synthesized, named primer B 1, 2 and primer B 3, 4 PCRs were carried out with these 2 primers for detecting the microquantity (0.1 ng) of various serovars, serogroup of leptospires. All the DNA of Leptospira interrogans can be amplified by primer B 1, 2, and only the DNA of serogroup icterohaemorrhagiae, Leptospira reacted specially with primer B 3, 4. The DNA of non-pathogenetic Leptospira and some other microbes, however, had no amplification at all. This study is first reported at home and abroad. The results demonstrate that PCR is a very sensitive and specific technique of DNA amplification, which can be used as a powerful tool in the early diagnosis and epidemiological identification of leptospirosis.