钩端螺旋体外膜蛋白研究现状

<正>钩端螺旋体病(leptospirosis,简称钩体病),是由致病性钩端螺旋体(简称钩体)引起的钩端螺旋体病(钩体病),是一种全球性自然疫源性疾病,广泛分布在世界各地。我国是受钩体病危害十分严重的国家,目前已发现有18个血清群75个血清     

钩端螺旋体病检测技术的研究进展

钩端螺旋体病临床症状复杂、多样,早期容易误诊,实验室诊断在该病的防治工作中极为重要。目前应用的实验诊断方法较多,有病原学检测、血清学检测、分子生物学检测等,本文对目前应用的检测技术作一综述。     

问号状钩端螺旋体外膜单克隆抗体凝集反应特性的研究

Three McAb were produced against an outer envelope preparation from Leptospira, interrogans, serovar Lai by fusion of SP2/0 myeloma cells with immune BALB/c mice spleen cells. The fusion rate was 96% and the antibody positive rate was 50%. One of the hybridomas, E4B11C9, reacted with 13 of the 13 serovars of the Icterohaemorrhagiae serogroup in microscopic agglutination test (MAT) but did not react with the 18 representative serovars of L. interrogans and L. biflexa serovar patoc and Leptonema illini. For all non-reactive serovars the MAT titres were greater than 1:25. The McAb, E4B7G5, reacted similarly with all serovars except smithi and tonkini. E4B7D4 reacted also similarly with all serovars except serovars birkini, ndambari, bogvere, smithi and tonkini. Therefore, 3 McAb showed serogroup specificity and partial serogroup specificity by agglutination. The agglutination titres were high and hybridomas were stable, so it might be useful in providing a simple, rapid method for the classification and identification of clinical isolates such as pathogenic L. interrogans in place of the complicated and time-consuming conventional methods.     

以钩端螺旋体重组蛋白为包被抗原的间接ELISA检测试剂盒的研制与性能评测

目的·研制适用于检测钩端螺旋体(钩体)特异性抗体的间接酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)检测试剂盒,并对其性能进行评估.方法·选择我国主要致病性钩体流行株中高度保守的外膜蛋白进行原核表达,将获得的重组蛋白纯化后作为包被抗原行质谱鉴定,并对其抗原性进行检测....     

四株钩端螺旋体外膜单克隆抗体的建立,鉴定及对外膜蛋白抗原...

Four McAbs (1A7E7, 2F9D4, 2G3H5, 1A7H12) against outer membrane antigens of L. interrogans serovar Lai strain 017 were produced by hybridoma technique. McAb 1A7E7 was identified to be IgG2a, the rest IgG1 by immunodiffusion. Outer membrane antigens of three pathogenic leptospiral strains (017, 601, 156) and two non-pathogenic leptospiral strains (Patoc I, 3055) were analysed by SDS- polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with McAbs. It was found that McAb 1A7E7 recognized specifically the 42kd antigenic band of strain 017; McAb 2F9D4 the 42kd antigenic bands of strain 017, 601, 156, and McAbs 2G3H5 and 1A7H12 the 31 kd antigenic bands of 017, 601, 156. Further study of antigenic properties possessed by pathogenic leptospira may provide some new clues to look for specific diagnostic and protective antigens.     

黄疸出血群赖型017株钩端螺旋体外膜单克隆抗体免疫...

BALB/c mice were immunized intraperitoneally with outer envelopes of serogroup icterohaemorrhagiae lai serovar strain 017 leptospires. Monoclonal antibodies against outer envelopes (IgG, agglutinating titre 1:25,600) were produced by hybridoma technique. The monoclonal antibodies ascites (diluted 1:100) 1 ml administered intraperitoneally 1 hour before the intraperitoneal injection of 2 x 10(8) leptospires of strain 017 and the subsequent daily administration of McAb in similar doses for five days protected 80% of guinea pigs. Survival rates of three control groups which received physiological saline, ascites of BALB/c mouse myeloma cell lines SP2/0, and monoclonal antibodies against Pseudomonas aeruginosa in place of monoclonal antibodies against outer envelopes of strain 017 leptospires were 10%, 20% and 10% respectively. When killed 20 days after challenge, guinea pigs of experiment group were normal at autopsy. Old pulmonary haemorrhage were present in the animals of three control groups. Passive immunoprotection experiments have demonstrated immunoprotection of monoclonal antibodies against outer envelopes of strain 017 leptospires. It will be valuable for separating protective antigen fraction of outer envelopes and studying new vaccine of leptospira.     

钩端螺旋体外膜抗原基因OMPL1在卡介苗中的克隆和初步表达

采用ＰＣＲ技术直接从致病性钩端螺旋体整形０１７菌株基因组中扩增钩体外膜蛋白基因ＯｍｐＬ１，并克隆到大肠杆菌－卡介苗穿梭质粒载体ＰＨ６００２中，重组质粒经电转化导入卡介苗。玉点杂交筛选重组卡介苗，再通过免疫印迹对其表达进行初步研究。     

研究钩端螺旋体外膜脂蛋白Loa22的免疫原性

目的 研究钩端螺旋体外膜脂蛋白Loa22的免疫原性.方法 取健康豚鼠12只,分蛋白免疫组和PBS(磷酸盐缓冲液)对照组,每组6只.蛋白免疫组以Loa22重组蛋白50 μg加等量完全弗氏佐剂乳化一免,Loa22重组蛋白配以不完全弗氏佐剂乳化二免和三免;对照组注射PBS与佐剂混合物.分别检测血清中抗体和T淋巴细胞增殖水平.结果 在末次免疫后2周时血清抗体效价和T淋巴细胞增殖与对照组相比,均显著升高(P<0.05).结论 Loa22重组蛋白有较好的免疫原性.     

艾灸对钩端螺旋体OmpL39免疫保护力的促进作用

选用３９ＫＤａ的钩端螺旋体外膜蛋白（ＯｍｐＬ３９）免疫豚鼠并施予艾灸,研究艾灸对ＯｍｐＬ３９免疫保护力的影响。结果表明：灸加ＯｍｐＬ３９都有合格的免疫力,但前者的特异性抗体效价比后者高,提示艾灸通过扶助正气对ＯｍｐＬ３９的免疫保护力有促进作用。     

波摩那型钩端螺旋体外膜蛋白LipL32基因片段的克隆表达

目的　构建L3 2_pGEX_5x_2重组质粒 ,诱导表达重组钩端螺旋体外膜脂蛋白LipL3 2。方法　PCR获取编码LipL3 2的基因片段 ,构建重组克隆载体和表达载体 ,转化受体菌 ,诱导表达重组LipL3 2蛋白。将重组LipL3 2蛋白和钩体抗血清进行Western_blot。结果　扩增出约 750bp的LipL3 2成熟蛋白基因 ,LipL3 2基因插入pGEX_5x_2表达载体 ,表达产物谷胱甘肽S_转移酶 (GST ,2 6× 10 3)与LipL3 2蛋白的融合蛋白的相对分子质量约为 53× 10 3 ,与预期大小一致。Western_blot显示重组LipL3 2蛋白能与钩体抗血清特异结合。结论　LipL3 2蛋白能在大肠埃希菌中表达 ,重组LipL3 2蛋白具有免疫反应性     

赖型钩端螺旋体017株外膜蛋白LipL32基因重组质粒的构建及其细胞毒性的研究

目的构建赖型钩端螺旋体(钩体)017株外膜蛋白LipL32基因的重组质粒,并研究其细胞毒性。方法从钩体017株全基因组中用PCR方法扩增出目的基因,双酶切构建重组质粒,转化大肠杆菌,诱导表达LipL32蛋白,将表达的目的蛋白免疫新西兰大白兔,制备多克隆抗体,Western Blotting鉴定其免疫原性。将目的蛋白纯化、复性后作用于ECV304细胞,通过检测细胞的乳酸脱氢酶(LDH)、NO释放量研究其细胞毒性。结果扩增出816bp的LipL32基因,重组质粒经双酶切、PCR鉴定、测序均表明重组载体构建成功。经IPTG诱导表达的融合蛋白相对分子质量约52×103,主要以包涵体的形式表达,经免疫动物制备得到多克隆抗体,ELISA检测效价达1:32000,Western Blotting显示在目的蛋白位置处有特异性阳性条带。经过LipL32蛋白作用的ECV304细胞LDH、NO释放量和对照组比较有明显升高。结论成功构建LipL32基因重组质粒,该质粒能在大肠杆菌中表达,表达的目的蛋白对细胞有一定的毒性效应。     

SDS－PAGE鉴定霍乱弧菌不同菌株外膜蛋白成分

霍乱弧菌５６９Ｂ等菌株用ＥＤＴＡ－溶菌酶法提取其外膜蛋白，以高速离心法纯化并以聚丙烯酰胺凝胶电泳分离其主要蛋白区带。经ＳＤＳ－ＰＡＧＥ及免疫电泳等方法鉴定，表明不同菌株的外膜蛋白均显示一主要区带，其分子量为２５ＫＤａ，免疫双扩试验表明不同霍乱弧菌菌株的外膜上都有一个共同的蛋白抗原。     

ELISA法和免疫印迹法检测抗ENA抗体的比较

目的：将目前国内新开展的两种检测ＥＮＡ技术,即免疫印迹法（ＩＢＴ）和酶免疫法（ＥＬＩＳＡ）进行对比、分析。方法：用 ＩＢＴ法及 ＥＬＩＳＡ法同时检测 １３０例各种结缔组织病、非结缔组织病人及健康人血清中的抗ＥＮＡ抗体。结果：２０例健康人,两法均为阴性;８０例结缔组织病患者,两法检出抗 Ｓｍ抗体的符合率为 ９１．３％,检测抗 ＲＮＰ抗体的符合率为 ８２．５％,以上抗体两法的敏感性无显著差异（Ｐ＞０．０５）。检测抗ＳＳＡ抗体,ＩＢＴ法检出率低。抗ＳＳＢ抗体检出率两法亦无显著差异（Ｐ＞０．０５）,符合率为８７．５０％,在１２例ＳＳ的检测中符合率比较低,为５０％。结论：两种方法在检测抗Ｓｍ、ＲＮＰ、ＳＳＢ抗体时,敏感性无显著差异,ＥＬＩＳＡ法检测抗ＳＳＡ抗体优于ＩＢＴ法。上述两种方法在临床应用于抗ＥＮＡ抗体的检测中于可互相验证和补充。     

抗沙眼衣原体主要外膜蛋白种特异性单克隆抗体

用人工合成沙眼衣原体主要外膜蛋白（ＭＯＭＰ）氨基端１／４肽链免疫８周龄雄性ＢＡＬＢ／ｃ小鼠３只，第１４天用Ｌ１／４４０／Ｂｕ原体加强免疫，第２４天将免疫反应良好的１只小鼠脾细胞与ＮＳ－１瘤细胞融合。用１／４ＭＯＭＰ和Ｌ１原体抗原包被的聚丙乙烯板检测上清中抗体，淘汰与鹦鹉热衣原体种（ＥＡＥ株）、肺炎衣原体种（ＡＴＣＣＶＲ１３１０株）以及正常组织培养细胞（ＢＧＭＫ）有交叉反应的克隆，最后得到４株抗沙眼衣原体特异性单克隆抗体（ＭＡｂｓ），其抗体属ＩｓＧ１和ＩｇＧ２ａ亚类。微量免疫荧光试验（ｍｉｃｒｏ－ＩＦ）发现４株ＭＡｂｓ均与本实验室制备的沙眼衣原体Ｌ１、Ｌ２、Ａ、Ｂ、Ｃ、Ｅ原体及Ｌ２包涵体抗原发生反应，并与沙眼衣原体所有１５个标准血清型结合，其腹水ＭＡｂｓ滴度≥１：１２８００。免疫印迹试验显示４株ＭＡｂｓ均与沙眼衣原体分子量为４００００的ＭＯＭＰ反应。     

布鲁氏菌外膜蛋白OMP31基因的克隆、表达和免疫学特性的初步研究

目的研究布鲁氏菌外膜蛋白OMP31的克隆、测序和表达,并对其进行初步的血清学鉴定。方法从布鲁氏菌基因组中获得OMP31基因连接入PMD-18T克隆质粒并测序,克隆入融合表达载体PGEX-4T-1,构建重组质粒PGEX-4T-1/OMP31,在大肠杆菌中将该蛋白表达。用Western-blot分析重组表达的GST-OMP31的免疫学特性。结果成功构建了PGEX-4T-1/OMP31原核表达载体并在大肠杆菌中表达了OMP31基因,布鲁氏菌免疫动物血清能特异性识别所表达的蛋白。结论成功表达了布鲁氏菌外膜蛋白OMP31基因,它可以与布鲁氏菌免疫血清产生特异性结合反应,为之后的抗原性以及免疫原性研究打下良好的基础。     

人血小板膜糖蛋白功能测定的酶联免疫分析方法的建立

目的建立测定血小板膜糖蛋白（GP）功能的酶联免疫分析（ELISA）方法 ,并对其灵敏度、批内和批间差异进行方法学考核。方法以抗血小板CD62P单抗SZ51和抗血小板膜糖蛋白Ⅱb/Ⅲa（GPⅡb/Ⅲa）单抗7E3分别包板,加入经ADP诱导的或未诱导的正常人富含血小板血浆（PRP）,以生物素标记的抗GPIb单抗SZ2（Biotin-SZ2）反应后,加入亲和素标记的辣根过氧化物酶（Avdin-HRP）检测,邻苯二胺（OPD）显色后,酶标仪读取吸光度（A）值。对其灵敏度及批内、批间差异作方法学评估,应用该方法对抑制性单抗SZ-21和阿司匹林抑制血小板活化的功能效果进行测定,并与流式细胞术（FCM）测定结果进行比较。结果该方法可检测血小板计数低达6.25×109/L（SZ51包被板）和3.13×109/L（7E3包被板）的标本,批内和批间变异系数均小于10%。测得50名健康正常人ADP诱导的（未诱导的）血小板的CD62P和GPⅡb/Ⅲa的A值分别为0.68±0.21（0.18±0.09）和0.87±0.29（0.44±0.14）。ELISA测定结果表明,SZ21和阿司匹林可明显抑制ADP诱导的血小板功能。流式细胞仪测定ADP诱导的（未诱导的）人血小板表面CD62P和GPⅡb/Ⅲa的阳性细胞百分率分别为（60.1±7.12）%[（2.56±0.61）%]和（86.32±17.82）%[（20.25±14.56）%],其批内和批间变异系数均〈10%。结论该方法可以测定血小板功能活化剂或抑制剂对血小板功能的影响,可以测定血栓性疾病或血栓形成相关性疾病引起的血小板活化程度。     

尿调蛋白酶联免疫吸附试验检测方法的建立及其在IgA肾病中的应用

目的:建立尿尿调蛋白的酶联免疫吸附试验(enzyme-linked immunosorbent assays,ELISA)检测方法,并对IgA肾病患者的尿尿调蛋白水平监测进行进一步验证。方法:以多克隆抗体作为包被抗体,单克隆抗体作为检测抗体,建立尿调蛋白的快速双抗夹心检测方法,检测其精确性及重复性,随机选取55例尿液标本,同时以商品化试剂盒与本实验方法进行检测,比较166例IgA肾病患者和正常人的尿尿调蛋白水平。结果:获得的标准曲线为0.78～12.5μg/L,实验室内变异系数为7.5%,实验室间变异系数为7.9%。55例尿液标本的商品化试剂盒与本实验方法结果比对,相关系数为r=0.615,P<0.001;166例IgA肾病患者的尿尿调蛋白/尿肌酐比值低于正常人。结论:尿尿调蛋白的ELISA检测方法灵敏,重复性较好,可运用于大样本的人群检测,IgA肾病患者的尿尿调蛋白分泌低于正常人。     

Expression of Human Sperm Membrane Protein in the Recombinant Salmonella Typhimurium Vaccine

A 550 bp cDNA fragment of HSD-Ⅰ coding for an extracellular domain of hu-man sperm membrane protein(hSMP-1)was ligated with an Adapter containing the universal stop codon,and the ligated fragment cDNA was then cloned into the MAS of pUC19.The desired plasmid with correct open reading frame was obtained, and was cut with EcoR Ⅰ.The insert was purified and then cloned into the two asd^ Salmonella expression vectors(the low copy number plasmid-pYA292 and the high copy number plasmid-pYA3137).The recombinant plasmid containing the insert with the correct orientation was selected by restriction enzyme digestion analysis. The recom-binant plasmids were transferred into the non-pathogenic Salmonella typhimurium X4550,which was deletion of the △cya, △crp and △asd genes.Western-blot analysis of the whole cell lysate of the two recombinants of S.typhimurium showed a predomi-nant protein band at 21 KD,which reacted with the anti-hSMP-1 antiserum. The re-sult indicated that two recombinants of S.typhimurium containing the 550 bp cDNA of HSD-Ⅰ were constructed and the characteristics of their growth in vitro were deter-mined. They may be used as new potential mucosalanti fertility.     

金葡菌、葡聚糖和大肠杆菌外膜蛋白诱导IgAN模型病理学分析

研究疾病模型的建立,遵循的原则是与所研究的疾病从临床指标到病理指标必须相一致,而且还不能给其他组织和器官造成损伤。本研究是从多数研究者曾使用的模型和在前辈模型的基础上有所创新的模型进行了对比研究,提出了金葡菌诱导的IgAN模型是当今最理想的IgAN模型。