Birch H E, Nagashima M, Simpson R J, Schreiber G
Biochem Int. 1983 May;6(5):653-61.
Polyadenylated RNA was isolated from acute-phase liver and transcribed into cDNA. After homopolymer tailing this was cloned into the PstI site of pBR322 which was used to transform E. coli RR1 cells. Clones containing cDNA corresponding to acute-phase proteins were identified by differential hybridization with 32P-labelled normal and acute-phase cDNA. A clone synthesizing about 10 ng of alpha 1-acid glycoprotein per E. coli colony was detected using a solid-phase based immunochemical procedure. The identification of the clone was verified by competition assay with authentic alpha 1-acid glycoprotein isolated from serum and by restriction analysis of the cDNA inserted into pBR322.
从急性期肝脏中分离出聚腺苷酸化RNA，并将其转录成cDNA。在进行同聚物加尾后，将其克隆到pBR322的PstI位点，用于转化大肠杆菌RR1细胞。通过与32P标记的正常和急性期cDNA进行差异杂交，鉴定出含有与急性期蛋白相对应的cDNA的克隆。使用基于固相的免疫化学方法检测到一个每个大肠杆菌菌落合成约10 ng α1-酸性糖蛋白的克隆。通过与从血清中分离出的 authentic α1-酸性糖蛋白进行竞争分析以及对插入pBR322的cDNA进行限制性分析，验证了该克隆的鉴定结果。
