黑龙江立克次体054株单克隆抗体对斑点热群立克次体抗原…

本文利用三株黑龙江立克次体０５４株（ＨＬＪ－０５４）单克隆抗体对四株斑点热群立克交体西伯利亚种（Ｒ．ｓ．）２４６株，清河株（ＪＨ－７４），立氏立克次体（Ｒ．ｒ．）Ｒ株及ＨＬＪ－０５４株进行了血清学分析，对其蛋白组成及单抗结合抗原特性等方面进行了研究，结果表明：ＪＨ－７４株与２４６株在上述三个方面完全一致，不同于ＨＬＪ－０５４及Ｒ．ｒ．Ｒ株；ＨＬＪ－０５４株与Ｒ．ｒ．Ｒ株与三株单抗血清学反应高度交叉     

应用不同单克隆抗体和扩增引物快速诊断立克次体的实验研究

用不同特异性的黑龙江斑点热立克次体（Rickettsialheilongjiangii）054株单克隆抗体（McAb）或PCR引物检测立克次体的结果表明：群特异性的mcAb2E1和mcAb4G2用微量免疫间接荧光抗体染法（MIIF）法可检测出文中所用各种斑点热立克次体（SFGR）：立氏（R．rickettsii）、康氏（R．conerii）、小蛛（R．akari）、派克（R．parkeri）、西伯利亚（R．sibirica）、澳大利亚（R．australia）和054株立克次体；F7单抗只能检测出R．rickettsii和054株立克次体。PCR扩增结果表明：引物Rt120和Rr120．2048p—1625n分别对恙虫病立克次体（R．tsutsug－amushi）和SFGR扩增出现388bp和523bp扩增带；Rr190．70p—602n和Rr190．4442—5664n两对引物扩增结果相同，即除R．tsutsugamushi外对Q热（C．burnetii）、SFGR、普氏（R．prowazekii）和莫氏（R．mooserii）均扩增阳性，分别为532bp和1222bp电泳带；引物Rpcs．977p—1258n则可扩增上述所有立克次体，电泳带381bp。这样，可在短期内同时检测和初步鉴定不同立克次体。     

斑点热群立克次体内蒙W-88蛛89102抗原的蛋白免疫印迹分析

蛋白免疫印迹试验表明从内蒙通辽病人旺某血标本分离的斑点热群立克次体W-88株与西伯利亚立克次体(232株及246株)的蛋白图谱完全相同,具有2个分子量为130KD及181KD的主要抗原性多肽及1个反应较弱的分子量为110KD的抗原性多肽,此与其它斑点热群立克次体不同。说明W-88株为西伯利亚立克次体。斑点热群立克次体中西伯利亚立克次体、立氏立克次体、康氏立克次体及派氏立克次体相互间抗原有较强的交叉反应。小蛛立克次体、澳大利亚立克次体与上述立克次体仅有微弱的交叉反应。说明斑点热群立克次体主要抗原分子有相同的决定簇。     

四株斑点热群立克次体DNA同源性的初步研究

用缺口翻译法将斑点热群立克次体西伯利亚 246 株 DNA 和康氏 Simko株DNA标记~(32)P。分别与中国分离株黑龙江 054株和新疆精河株 DNA进行斑点杂交,以对国内两分离株基因组与北亚、康氏基因组同源性进行初步研究。结果显示,新疆精河株 DNA与西伯利亚246株DNA高度同源。与血清分类实验结果相吻合。黑龙江 054株DNA 与西伯利亚 246株 DNA和康氏 Simko 株 DNA的杂交阳性最低稀释度均低于阳性对照100倍以上。提示054株基因组与西伯利亚立克次体、康氏立克次体 Simko 株基因组存在较明显之差别。     

蜱传斑点热(黑龙江立克次体)的抗体调查

<正> 1983年,我们于黑龙江省罗北县与绥芬河市,从三种蜱体内分离得一个斑点热群立克次体新种,暂命名为黑龙江立克次体。为查明该立克次体感染情况,我们采集了黑龙江省罗北县、绥芬河市、东宁县、爱辉县、内蒙东部喜桂图旗及沈阳市健康人血清243份,以斑点热群四个不同种的立克次体:黑龙江立克次体嗜群血蜱株、新疆立克次体精河株、西伯利亚立克次体R.FS127株、小蛛立克次体Kapian株为抗原,应用微量免疫荧光试验进行抗体分型检测。判定标准,≥1∶64为阳性,按滴度高者判别阳性血清的抗体型别。同时以西伯利亚立克次体Barbash株可溶性补结抗原作补体结合试验,滴度≥1∶8为阳性。     

190 kD R.rOmpA基因序列对斑点热群立克次体的检测

目的为探讨190 kD R.rOmpA基因序列对斑点热群立克次体的检测作用.方法从立氏立克次体190 kD外膜蛋白A(R.rOmpA)基因序列设计引物,利用PCR检测的方法,检测了683只蜱类标本和146个鼠类脏器标本,并随机抽取1株森林革蜱阳性扩增产物进行克隆与序列测定.结果从蜱类标本和鼠类脏器标本中检测出了斑点热立克次体DNA片段;所测序列的分型结果表明与前苏联的DnS 14株型别一致,与我国曾经检测出的斑点热立克次体株差异较大.结论190kD外膜蛋白A基因序列可用于斑点热立克次体检测,并可用于斑点热群立克次体基因型或亚型之间的鉴别.     

快速简便纯化立克次体的方法

本文采用差速离心沉淀和高盐乙醚处理方法从立克次体感染鸡胚卵黄囊膜中提取纯化立克次体。纯化品经涂片染色镜检立克次体形态完整,纯度较高。其蛋白回收量普氏立克次体(E株)为0.88mg/克膜,西伯利亚立克次体(Barbash株)为0.39mg/克膜,国内分离株(斑点热立克次体精河株、黑龙江株和内蒙株)为0.34～0.40mg/克膜。纯化的立克次体用于核酸提取、DNA碱基成分的测定和研究全立克次体蛋白组成及抗原多肽构成的分析。     

斑点热群立克次体中国分离株HL-93和HLJ-054亲缘关系的研究

目的　通过斑点热群立克次体 (spottedfevergrouprickettsiae ,SFGR)rOmpA基因片段的序列分析对HL 93和HLJ 0 5 4的亲缘关系进行研究。方法　用PCR分段扩增rOmpA基因重复区后 1 9kb片段 ,PCR产物直接测序 ,用WinstarDNA分析软件包进行同源性比较、进化树分析及酶切位点的分析。　结果　HL 93株和HLJ 0 5 4株核苷酸及推定氨基酸的同源性均在 99%以上 ,在树形图上单独聚为一类。常用酶的酶切位点完全一致。结论　HL 93株和HLJ 0 5 4株为同一种SFGR ,建议均命名为黑龙江立克次体 ,但可能存在株间差异性。     

立氏立克次体蛋白抗原基因的表达和重组蛋白抗原的免疫印迹分析

目的表达立氏立克次体(Rickettsia rickettsii)主要抗原基因,免疫印迹初步分析重组蛋白抗原的抗原性。方法根据立氏立克次体全基因组序列选出主要抗原相关基因,采用PCR从全基因组中扩增抗原相关基因,将扩得的基因片段插入原核表达质粒构建抗原基因重组表达质粒,将重组质粒转化大肠杆菌并诱导转化菌表达重组蛋白抗原。采用立氏立克次体感染的豚鼠血清和斑点热患者血清分别与纯化的重组蛋白抗原做免疫印迹分析。结果经PCR扩增后获得43个目的基因片段,其中36个基因片段在大肠杆菌中高效表达重组蛋白抗原,其中5个蛋白抗原在免疫印迹中与感染豚鼠血清和斑点热患者血清反应均为阳性。结论免疫印迹筛选出5种立氏立克次体重组蛋白抗原,结果提示它们是能够诱导机体产生特异性免疫应答的主要抗原,可作研制斑点热诊断试剂或亚单位疫苗的新候选蛋白抗原。     

斑点热群立克次体新种──虎林－93株的分离和鉴定

本文用鸡胚卵黄囊培养法及实验动物感染法从黑龙江省虎林县境内采集的嗜群血蜱中分离到了一株立克次体。命名为ＨＬ－９３株立克次体。经形态学及血清学试验证实为斑点热群立克次体。分别用微量免疫荧光法、十二烷基硫酸钠－聚丙烯酰胺凝胶不连续电泳法、单克隆抗体免疫酶斑点法、单克隆和多克隆抗体免疫印迹法、聚合酶链反应和限制性片段长度多态性分析法对该分离株进行鉴定并同已知国际标准株、国际及国内参考株进行比较。结果表明;ＨＬ－９３株立克次体无论在抗原性多肽上,还是在基因水平上在斑点热群立克次体中都是独特的。根据目前立克次体分类法,ＨＬ－９３株立克次体可以考虑是斑点热群立克次体的新种。     

Antigenic diversity of Rickettsia conorii.

Analysis of seven strains designated as Rickettsia conorii for reactivity with a panel of 12 monoclonal antibodies to surface-protein epitopes of spotted fever group rickettsiae and by Western immunoblotting with standard serotyping sera revealed remarkable antigenic diversity. Rickettsial strains from France, Morocco, Ethiopia, Kenya, South Africa, India, and the USSR differed from one another in reactivity with at least one and as many as five monoclonal antibodies. Simko and Indian strains were similar to one another and differed substantially from other R. conorii strains. All seven strains reacted with three R. conorii-specific monoclonal antibodies. Western immunoblotting demonstrated a major 120-kD protein and a major 135-kD protein in all strains. The principal differences were the presence of a major undenatured 130-kD protein in all strains except Indian and Simko, which had an analogous protein of 124 kD. Immunodominant antigenically related, heat-denatured protein bands of 170 kD (Malish 7 strain), 175 kD (Manuel strain), and 190 kD (Kenya tick typhus, Indian, and Simko strains) were not detected in the M-1 and Moroccan strains. This antigenic diversity is greater than that previously reported for other spotted fever group rickettsial species, suggesting that R. conorrii is an older species than R. rickettsii with a longer period of time for evolutionary divergence.     

抗隐孢子虫单克隆抗体研制及其特异性的鉴定

目的：制备抗隐孢子虫单克隆抗体（McAb）并对其特异性进行鉴定。方法：用纯化的人源微小隐孢子虫卵囊免疫BALB／c小鼠，杂交瘤技术制备McAb，间接免疫荧光（IFA）和免疫印迹试验分析其特性。结果：制备出Z4C8、Z2B6、Z3D7和Z3D2四株能稳定分泌抗隐孢子虫McAb的杂交瘤细胞株。经鉴定，Z4C8和Z3D7为IgM ，Z3D2和Z2B6为IgG1，轻链均为κ链，与多种肠道病原体及肺孢子虫无交叉反应。除Z2B6在间接免疫荧光试验中识别卵囊壁外，其余均识别隐孢子虫子孢子（CSP）。免疫印迹定位表明，四株McAb分别识别42条CSP抗原中的20．5kDa、60．5kDa、33kDa和95kDa4个条带，其中Z4C8和Z3D7均识别20．5kDa主要抗原带。结论：这四株McAb在识别CSP抗原表位上具有不同的特性，但对隐孢子虫抗原均呈高度特异性反应。     

结核分支杆菌H37Rv株菌体抗原单克隆抗体制备,特性？…

目的 利用单克隆抗体技术分析分支杆菌多肽抗原的共性和个性,建立纯化单克隆抗体的最佳方法。方法 按常规方法制备单克隆抗体。分泌单克隆抗体阳性杂交细胞株的筛选采用ＥＬＩＳＡ法,确认采用免疫印迹法。２４种分支杆菌菌株均生长于改良罗氏培养基上,Ｈ３７Ｒｖ株分别生长于改良罗氏培养基和苏通液体培养基上。单克隆抗体的纯化采用硫酸铵沉淀法、辛酸沉淀法和离子交换法。结果 经筛选获得１４株单克隆抗体杂交细胞瘤。其中,     

The causative agent from a patient with spotted fever group rickettsiosis in Japan on Awaji Island, Hyogo

Misaka strain was isolated as the causative agent from a patient with spotted fever group rickettsiosis in Japan by using nude mice on Awaji Island, Hyogo in September 1988. The nude mice infected with the isolate showed weakness and splenomegaly and died in two or three weeks after the infection. The cyclophosphamide-treated mice infected with the isolate died between four and seven days after the infection. The infected normal mice recovered and acquired immunity. The infected adult male guinea pigs were feverish and showed swelling and redness of the scrotum between two and eight days after the infection, and recovered. The Misaka strain was propagated well in Vero cells in tissue culture. The rickettsial particles were seen as diplobacillary and diplococcal forms growing predominantly in the cytoplasm and occasionally in the nucleus of infected cells. The serological characteristics of the Misaka strain were analyzed by the cross-immunofluorescent antibody method. The Misaka strain, the Katayama strain first isolated in Tokushima in 1987, and the representative strains of spotted fever group rickettsiae in the world; R. rickettsii Smith, R. sibirica 246, R. conorii Moroccan, R. akari MK (Kaplan), R. australis Phillips, R. montana Tick and Thai TT-118 strains were used as antigens. And immune mouse serum samples against the Misaka, Katayama, 246, Phillips and TT-118 strains were used as antisera. The result revealed that these strains showed cross-reaction and share a common antigen of spotted fever group rickettsiae. Furthermore, it became obvious that the Misaka strain and the Katayama strain have the same serotype-specific antigen different from the strains of other spotted fever group rickettsiae using Anti-Katayama monoclonal antibodies.     

抗霍乱弧菌O139型的单克隆抗体免疫印迹分析抗原表位及应用

目的制备霍乱弧菌O139型(V ibrio cholerae O139,VC O139)单克隆抗体后应用免疫印迹技术识别抗原表位特性。方法SDS-PAGE结合W estern b lotting以及APAAP方法。结果霍乱弧菌O139型抗原经还原剂处理,SDS-PAGE 11%~20%梯度胶电泳,转印染色后,能分辨出20余条清晰的蛋白带和脂多糖片状图谱。3株抗霍乱弧菌O139型单抗的抗原表位,主要集中于20~70 kD相对分子质量的蛋白带,其中O139的2株单抗O139-14、O139-15分别与68~50 kD、45~25 kD、20~14 kD对应VC O139的蛋白质抗原表位起反应,而另外一株单抗,O139-92只与65 kD、20kD的抗原表位特异性结合。结论VC O139的O139-14、O139-15 M cAb同对应抗原表位点的结合分布较广,有利于对VC O139抗原的快速、敏感、特异检测。     

堆型艾美球虫子孢子单克隆抗体的制备及鉴定

目的 建立堆型艾美球虫（Eimeria acervulina）子孢子表面抗原的杂交瘤细胞株。 方法 使用纯化的堆型艾美球虫子孢子直接免疫BALB/c小鼠，采用杂交瘤技术建立细胞株，制备单克隆抗体。用ELISA确定单克隆抗体的交叉反应性、相对亲和力、免疫球蛋白的类型和亚类，并对单克隆抗体进行鉴定。 结果 获得4株可稳定分泌单克隆抗体的杂交瘤细胞株，其中Easp-3G3、Easp-5G10为IgG1 类，Easp-3H6为IgG2b 类，Easp-5H4为IgG2a 类。4株单克隆抗体均能与堆型艾美球虫子孢子的抗原蛋白反应，但仅Easp-5H4能与柔嫩艾美球虫子孢子抗原蛋白反应。Easp-3G3和Easp-5G10与另外2种单克隆抗体的识别位点不同，而Easp-3G3与Easp-5G10、Easp-3H6与Easp-5H4的识别位点相近。 结论 制备了4株堆型艾美球虫子孢子单克隆抗体。     

Monoclonal antibodies reacting with Schistosoma japonicum eggs and their target epitopes

Ten monoclonal antibodies (McAbs) raised to Schistosoma japonicum eggs could be assigned using several serological and immunochemical techniques to 3 groups. The McAbs, termed A, B and C-McAbs, apparently recognize carbohydrate epitopes that can be located on the same antigen molecule. The antibodies, generally of IgM isotype, are idiotypically related. They are distinct from another IgM McAb (Group D-McAb) the carbohydrate target epitope of which can also be associated with the epitopes of A, B and C-McAbs. The McAbs produce large vacuolated bleb reactions in the circumoval precipitin test (COPT) and target epitopes have different representations in various life cycle stages such as immature and mature eggs, male and female worms (including S. mansoni). Antigens affinity purified on columns containing A, B, C and D-McAbs stimulate proliferation of T cells from egg-sensitized mice and elicit DTH reactions in such mice. This raises the possibility that the target antigens of these carbohydrate-reactive monoclonal antibodies are immunopathologic and involved in egg-induced granuloma formation.     

Antigenic analysis of Chinese strains of spotted fever group rickettsiae by protein immunoblotting

Protein immunoblotting demonstrated that 6 Chinese strains of spotted fever group (SFG) rickettsiae from northern China had antigenic polypeptides identical to those of Rickettsia siberica (strains 246 and 232), and dissimilar to other SFG rickettsiae. The various species of other SFG rickettsiae exhibited serologically distinct epitopes as well as many cross-reactive epitopes in protein immunoblotting. All SFG rickettsiae examined in this study had major antigenic polypeptides of 113-160 kDa.     

鸡疟原虫雌配子单克隆抗体的制备和鉴定

用鸡疟原虫雌配子免疫ＢＡＬＢ／ｃ小鼠，取免疫鼠脾细胞与ＮＳＩ鼠骨髓瘤细胞融合，筛选出的６株（３Ｈ１、２Ｈ１、１Ｇ１、１２Ｄ１、６Ｅ２、６Ｃ２）杂交瘤细胞，证明能分泌效价高、特异性强的单克隆抗体（ＭｃＡｂ）。亚类鉴定为ＩｇＧ１（６Ｃ２，３Ｈ１）、ＩｇＧ２ａ（１Ｇ１，２Ｈ１）、ＩｇＧ２ｂ（６Ｅ２）、ＩｇＧ（１２Ｄ１），其中２Ｈ１、６Ｅ２、１Ｇ１、１２Ｄ１株ＭｃＡｂ为抗膜抗体，而３Ｈ１和６Ｃ２株ＭｃＡｂ为非抗膜抗体。除１２Ｄ１株ＭｃＡｂ与蓝氏贾第鞭毛虫滋养体有交叉反应外，其它各株ＭｃＡｂ与阴道毛滴虫滋养体、杜氏利什曼原虫前鞭毛体、鼠疟原虫和鸡疟原虫红细胞内期均无交叉反应。