Venkatesan S, Elango N, Chanock R M
Proc Natl Acad Sci U S A. 1983 Mar;80(5):1280-4. doi: 10.1073/pnas.80.5.1280.
Cytoplasmic poly(A)-containing RNA from respiratory syncytial virus-infected cells was used as a template to synthesize oligo(dT)-primed cDNAs. Discrete size classes of single-stranded cDNAs, resolved by alkali agarose gel electrophoresis, were used separately to construct double-stranded cDNAs that were subsequently inserted into the plasmid vector pBR322 at the Pst I site by means of oligo(dG)oligo(dC) tailing. After transfection of Escherichia coli, recombinant plasmids were screened mostly by serial rounds of hybrid selection of mRNAs from virus-infected cells and subsequent in vitro translation of the selected mRNAs. Comparative peptide mapping of the translation products with those of authentic virion proteins served to establish the viral origin of the cDNA recombinants. In this manner, four distinct classes of recombinant plasmids were identified. These encode sequences corresponding to those of respiratory syncytial virus nucleocapsid protein, matrix protein, phosphoprotein, and a nonstructural protein.
来自呼吸道合胞病毒感染细胞的含细胞质多聚腺苷酸RNA被用作模板来合成寡聚（dT）引发的cDNA。通过碱性琼脂糖凝胶电泳分离出的不同大小类别的单链cDNA，分别用于构建双链cDNA，随后通过寡聚（dG）-寡聚（dC）加尾将其插入质粒载体pBR322的Pst I位点。在大肠杆菌转染后，主要通过对病毒感染细胞的mRNA进行连续多轮杂交选择以及随后对所选mRNA进行体外翻译来筛选重组质粒。将翻译产物与真实病毒粒子蛋白的产物进行比较肽图谱分析，以确定cDNA重组体的病毒来源。通过这种方式，鉴定出了四类不同的重组质粒。它们编码与呼吸道合胞病毒核衣壳蛋白、基质蛋白、磷蛋白和一种非结构蛋白相对应的序列。
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