CDNA SEQUENCE-ANALYSIS OF A 29-KDA CYSTEINE-RICH SURFACE-ANTIGEN OF PATHOGENIC ENTAMOEBA-HISTOLYTICA
CDNA SEQUENCE-ANALYSIS OF A 29-KDA CYSTEINE-RICH SURFACE-ANTIGEN OF PATHOGENIC ENTAMOEBA-HISTOLYTICA
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DOI：
10.1073/pnas.87.16.6358
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发表时间：
1990-08-01
影响因子：
11.1
通讯作者：
STAMM, WE
中科院分区：
文献类型：
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作者：
TORIAN, BE;FLORES, BM;STAMM, WE
为了克隆和鉴定溶组织内阿米巴滋养体的表面抗原，从聚（U）-琼脂糖选择的内阿米巴滋养体RNA构建了一个. t11 cDNA文库。用兔抗E.溶组织血清一个700碱基对的cDNA插入分离和核苷酸序列进行了测定。推导的氨基酸序列的cDNA揭示了一个富含半胱氨酸的蛋白质。DNA杂交结果表明，该基因为E.由于cDNA探针能与4株溶组织大肠杆菌的DNA发生特异性反应，溶组织阿米巴，但不与来自侵袭内阿米巴、卡氏阿米巴或阴道毛滴虫的DNA反应。将插入片段亚克隆到表达载体pGEX-1中，并以与谷胱甘肽S-转移酶C末端融合的形式表达蛋白。用纯化的融合蛋白制备了22株单克隆抗体（mAb）和1株小鼠多克隆抗血清。融合蛋白的溶组织部分。一个29 kDa的蛋白质被确定为表面抗原时，单克隆抗体被用来免疫沉淀代谢35 S标记的活滋养体的抗原。抗原的表面定位通过来自表面125 I标记的完整滋养体的29-kDa蛋白质的mAb免疫沉淀以及通过在4 ℃进行的间接免疫荧光测定中mAb与活滋养体的反应来证实。用单克隆抗体进行的免疫印迹表明，该抗原存在于四个供试的无菌分离株上。单克隆抗体识别一些但不是所有测试的临床分离株上的29-kDa天然抗原上的表位。
A .lambda.t11 cDNA library was constructed from poly (U)-Sepharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically 35S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-125I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4.degree.C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.