Gantt J S, Key J L
Mol Gen Genet. 1986 Feb;202(2):186-93. doi: 10.1007/BF00331635.
A pea leaf cDNA library was constructed in the expression vector lambda gt11 and screened with antisera raised against proteins extracted from 30S and 50S ribosomal subunits and 70S ribosomes prepared from isolated pea chloroplasts. Six recombinant phage were identified that encoded fusion proteins containing plastid ribosomal protein antigenic determinants. Phage-induced cell lysate proteins, containing the fusion proteins, were bound to nitrocellulose membranes and used as affinity matrices to prepare monospecific antibodies. These antibodies were then used to identify by Western blotting which plastid ribosomal protein shared antigenic determinants with the fusion proteins. cDNA inserts from the antigen-producing phage were used to hybrid-select complementary mRNAs. The cell-free translation products of these mRNAs were added to a pea chloroplast in vitro transport system and imported proteins analyzed by two-dimensional gel electrophoresis. The imported proteins comigrated with the plastid ribosomal proteins that were identified as being antigenically related to the fusion proteins produced by the corresponding recombinant phage. The imported proteins were 3,500-5,500 daltons smaller than their precursors.
构建了一个豌豆叶片cDNA文库，该文库置于表达载体λgt11中，并使用针对从分离的豌豆叶绿体中制备的30S和50S核糖体亚基以及70S核糖体提取的蛋白质产生的抗血清进行筛选。鉴定出六个重组噬菌体，它们编码含有质体核糖体蛋白抗原决定簇的融合蛋白。含有融合蛋白的噬菌体诱导的细胞裂解物蛋白与硝酸纤维素膜结合，并用作亲和基质来制备单特异性抗体。然后使用这些抗体通过蛋白质印迹法鉴定哪些质体核糖体蛋白与融合蛋白具有共同的抗原决定簇。来自产生抗原的噬菌体的cDNA插入片段用于杂交选择互补mRNA。将这些mRNA的无细胞翻译产物添加到豌豆叶绿体体外转运系统中，并通过二维凝胶电泳分析导入的蛋白质。导入的蛋白质与质体核糖体蛋白迁移到同一位置，这些质体核糖体蛋白被鉴定为与相应重组噬菌体产生的融合蛋白具有抗原相关性。导入的蛋白质比其前体小3500 - 5500道尔顿。
