链脲佐菌素实验性糖尿病研究——Ⅰ.链脲佐菌素诱发大鼠糖尿病模型

本文报道用链脲佐菌索给Wistar大鼠腹腔一次注射,建立了实验性糖尿病模型。实验动物血糖反应呈三时相变化:初期高血糖相(2小时),低血糖相(6～12小时)和糖尿病血糖相(24小时～12周)。为进一步研究糖尿病发病机理、并发症、治疗实验和筛选新的抗糖尿病药物等提供了一个较好模型。所建立的模型具有用药量小,方法简便,血糖反应敏感等优点。     

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。     

钩端螺旋体澳洲群群特异性McAb的建立、鉴定及群特异性抗原检测

作者成功地获得4株分泌抗澳洲群钩瑞螺旋体的单克隆抗体(McAb)杂交瘤。其中2E_1株能与所选澳洲群的11个型的钩体都发生凝集反应,而不与澳洲群外的20群22个型的钩体、非致病性Patot Ⅰ株钩体和伊利尼细螺旋体3055株发生凝集反应。由此表明,2E_1 McAb具有澳洲群钩体的群特异性。用SDS-PAGE、免疫印迹法对澳洲型620株、赖型601株、七日热型156株、Patoc Ⅰ株及3055株外膜蛋白中McAb识别的抗原成分进行检测,结果2E_1 McAb能特异地识别澳洲型620株34kd外膜蛋白区带。其余3株McAb具有澳洲群钩体部分群特异性。     

抗黄疸出血群赖型017株钩端螺旋体单克隆抗体杂交瘤细胞株的筛选和检定

用黄疸出血群赖型017株钩端螺旋体免疫BALB/c/小鼠的脾细胞与骨髓瘤细胞系SP2/0融合,经克隆化获得分泌抗赖型017株钩端螺旋体的单克隆抗体的杂交瘤细胞株,用酶联免疫吸附试验和间接免疫荧光抗体法,证实该株杂交瘤细胞所分泌的是特异性抗体,注入同系小鼠腹腔可诱生合高效价单克隆抗体的腹水,与杂交瘤细胞培养上清效价相比,增高800倍。单克隆抗体亚类检定结果为IgM及IgG_(2b)。     

一种钩端螺旋体DNA疫苗的免疫原性和免疫保护性研究

为了研究赖型钩体内鞭毛蛋白基因在动物体内的表达及其免疫保护性,采用ＰＣＲ扩增内鞭毛蛋白编码基因,以表达质粒ＶＲ１０１２为载体,构建了含有钩体的内鞭毛基因的重组质粒。在新西兰大白兔四头肌内注射该重组质粒ＤＮＡ５００μｇ／只,重复３次,每次间隔３周,于每次注射前和末次注射后３周,检测血清抗赖型钩体抗体效价。结果显示：在第１次加强免疫注射后３周,试验组血清抗体效价显著增高。豚鼠免疫保护试验表明,该ＤＮＡ重组质粒有明显免疫保护作用,在无佐剂条件下试验组存活率为９０％（９／１０只）。本研究为钩体核酸疫苗研制奠定了基础。     

问号赖型钩端螺旋体重组质粒pDL121的初步研究

钩端螺旋体病的传染和流行常给人类生命及畜牧业生产造成严重危害。我国曾发生过数十次大规模钩端螺旋体病流行。作为我国两个流行菌型之一的问号赖型钩端螺旋体在生物学特性和致病性有其独特性，但其遗传学特性和致病机制尚不完全清楚。为了探讨问号赖型钩端螺旋体抗原基因的特点，应用六种常见内切酶（ＢａｍＨＩ，ＢｇｌⅡ，ＥｃｏＲＩ，ＨｉｎｄⅢ，ＰｓｔⅠ，ＰｖｕⅡ）对从问号赖型钩端螺旋体０１７株的基因文库中筛选出来的重组质粒ｐＤＬ１２１外源基因进行酶谱分析，同时用Ｄｉｇｏｘｉｎ标记的ｐＤＬ１２１外源基因片段作探针对不…     

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.     

不同血清群型钩端螺旋体DNA的限制性内切酶谱分析

用7种限制性内切酶分析了不同血清群型钩端螺旋体DNA间的遗传差异。结果表明:CfoI和Eco RI的消化酶谱分辨效果较好,各血清型钩体间多存在明显差异,但同血清型的不同株之间酶谱基本相同。     

钩端螺旋体七日热群部分群特异McAb的建立与鉴定

Hebdomadis is one of the major serogroups found in China. In Sichuan, this serogroup appears to have a close relationship with local outbreak of leptospirosis. BALB/c mice were immunized intrasplenically with outer envelope of serogroup Hendomadis serovar hebdomadis strain 245. Spleen cells were fused with SP2/0 myeloma cells, two monoclonal antibodies Af2 and Bb2 were produced by hybridoma technique. McAb Af2 and McAb Bb2 were identified to be IgG1 and IgG3 by immunodiffusion respectively. Specificities of these two McAbs were determined by MAT; both reacted to 8 serovars (hebdomadis, nona, kambale, kremastos, worsfoldi, jules, maruborincana) of Hebdomadis serogroup. The agglutination titres of McAb Af2 and McAb Bb2 were 1:640-1:2,500,000 and 1:320-1:2,500,000, respectively. The two McAbs did not agglutinate with serovar kabura of Hebdomadis serogroup, they did not agglutinate with 11 serovars of Sejroe serogroup, 4 serovars of Mini serogroup, 18 representive serovars of L. interrogans in 18 serogroups, L. biflexa strain Patoc I and Leptonema illini. So it was found that McAb Af2 and McAb Bb2 showed partial serogroup specificity for Hebdomadis by agglutination.     

黄疸出血群赖型017株钩端螺旋体外膜单克隆抗体免疫保护作用的探讨

作者采用黄疸出血群赖型017株钩端螺旋体(钩体)外膜免疫BALB/c小鼠,通过细胞融合等技术,获得单克隆抗体E4B7D5。亚类鉴定为IgG_1,凝集效价为1:25 600。被动免疫保护试验证明:将单克隆抗体E4B7D5腹水稀释100倍,给实验组豚鼠每只每天腹腔注射1ml,连续6天,能使豚鼠在接受同型017株钩体2ml(每毫升1×10~3条)的攻击后,存活率达80％,且存活20天的豚鼠处死解剖,未见任何病变;3个对照组的存活率分别为10％,20％和10％,该三组仅有4只存活20天,经处死解剖,其中 3只发现较多的陈旧性肺出血。作者认为,单克隆抗体E4B7D5对同型017株钩体有免疫保护作用。