Finnemann S C, Marmorstein A D, Neill J M, Rodriguez-Boulan E
Department of Ophthalmology, Margaret M. Dyson Vision Research Institute, Cornell University Medical College, New York, NY 10021, USA.
Invest Ophthalmol Vis Sci. 1997 Oct;38(11):2366-74.
To identify the retinal pigment epithelium (RPE) surface antigen recognized by the monoclonal antibody RET-PE2.
A lambda bacteriophage complementary DNA (cDNA) expression library, representing the rat RPE cell line RPE-J, was constructed and screened with the RET-PE2 monoclonal antibody. Transient transfections of the RET-PE2 cDNA, immunofluorescence stainings of tissue sections or cultured cells, and Western blot analyses of tissue and cell detergent extracts served to prove that the protein resulting from expression of the cDNA is the RET-PE2 antigen.
Three independent cDNAs were cloned that shared overlapping sequences. Sequence alignment with EMBL database entries revealed identity to the published cDNA of CE-9/OX-47, a member of the immunoglobulin superfamily. One of the clones encoded the entire open reading frame of CE-9. The expression pattern of the RET-PE2 antigen matched that of CE-9, which is widely expressed. Chinese hamster ovary cells transiently transfected with the RET-PE2 cDNA produced a membrane-localized protein that was recognized by RET-PE2 and CE-9 antibodies.
The antibody RET-PE2 recognizes the CE-9/OX47 gene product, a transmembrane protein of the immunoglobulin superfamily. Contrary to results reported earlier, RET-PE2 immunoreactivity is widely distributed among different rat tissues--kidney, liver, and testis. In epithelia other than the adult RPE, it is confined to the basolateral plasma membrane. Its apical polarization in the RPE of adult rats supports earlier findings that some proteins that are basolateral in other epithelia exhibit reversed polarity in the RPE.
鉴定单克隆抗体RET - PE2所识别的视网膜色素上皮（RPE）表面抗原。
构建了一个代表大鼠RPE细胞系RPE - J的λ噬菌体互补DNA（cDNA）表达文库，并用RET - PE2单克隆抗体进行筛选。对RET - PE2 cDNA进行瞬时转染、对组织切片或培养细胞进行免疫荧光染色以及对组织和细胞去污剂提取物进行蛋白质印迹分析，以证明由该cDNA表达产生的蛋白质就是RET - PE2抗原。
克隆出了三个具有重叠序列的独立cDNA。与EMBL数据库条目进行序列比对后发现，其与免疫球蛋白超家族成员CE - 9/OX - 47的已发表cDNA相同。其中一个克隆编码了CE - 9的完整开放阅读框。RET - PE2抗原的表达模式与广泛表达的CE - 9的表达模式相匹配。用RET - PE2 cDNA瞬时转染的中国仓鼠卵巢细胞产生了一种膜定位蛋白，该蛋白能被RET - PE2和CE - 9抗体识别。
抗体RET - PE2识别CE - 9/OX47基因产物，一种免疫球蛋白超家族的跨膜蛋白。与早期报道的结果相反，RET - PE2免疫反应性广泛分布于不同的大鼠组织——肾脏、肝脏和睾丸。在成年RPE以外的上皮细胞中，它局限于基底外侧质膜。其在成年大鼠RPE中的顶端极化支持了早期的研究发现，即在其他上皮细胞中位于基底外侧的一些蛋白质在RPE中呈现相反的极性。
