Zeitlin P L, Crawford I, Lu L, Woel S, Cohen M E, Donowitz M, Montrose M H, Hamosh A, Cutting G R, Gruenert D
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):344-7. doi: 10.1073/pnas.89.1.344.
The gene responsible for the lethal disorder cystic fibrosis encodes a 1480-amino acid glycoprotein, CFTR. Using polyclonal antibodies directed against separate phosphorylation sites in the pre-nucleotide-binding fold (exon 9) and the R domain (exon 13), we have identified a 165-kDa protein in Xenopus laevis oocytes injected with recombinant CFTR cRNA transcribed from the full-length CFTR plasmid pBQ4.7. A protein of the same mobility was also detected with Western blotting techniques in whole cell extracts of cells that express CFTR mRNA (T84, FHTE, HT-29), including biopsied human nasal and bronchial tissue. Immunodetectable 165-kDa protein was concentrated in the apical membrane fraction of ileal villus tissue. We also report that the 165-kDa protein levels can be modulated pharmacologically, and these levels are appropriately correlated with second-messenger-regulated Cl- efflux. Thus, native or recombinant CFTR can be recognized by these anti-CFTR peptide polyclonal antibodies.
导致致命性疾病囊性纤维化的基因编码一种含1480个氨基酸的糖蛋白——囊性纤维化跨膜传导调节因子（CFTR）。我们使用针对核苷酸结合前折叠区（第9外显子）和R结构域（第13外显子）中不同磷酸化位点的多克隆抗体，在注射了从全长CFTR质粒pBQ4.7转录而来的重组CFTR cRNA的非洲爪蟾卵母细胞中鉴定出一种165 kDa的蛋白质。在表达CFTR mRNA的细胞（T84、FHTE、HT - 29）的全细胞提取物中，包括活检的人鼻和支气管组织，通过蛋白质印迹技术也检测到了迁移率相同的蛋白质。免疫可检测到的165 kDa蛋白质集中在回肠绒毛组织的顶端膜部分。我们还报告说，165 kDa蛋白质的水平可以通过药理学方法进行调节，并且这些水平与第二信使调节的氯离子外流适当相关。因此，这些抗CFTR肽多克隆抗体可以识别天然或重组的CFTR。
