Goddard J M, Caput D, Williams S R, Martin D W
Proc Natl Acad Sci U S A. 1983 Jul;80(14):4281-5. doi: 10.1073/pnas.80.14.4281.
We have obtained cDNA clones that contain the entire coding region of the human purine-nucleoside phosphorylase (PNP; EC 2.4.2.1) mRNA. The cDNA sequences were generated by reverse transcription of PNP-enriched mRNA obtained by immunoadsorption of HeLa cell polyribosomes with monospecific antibody to human PNP. cDNA molecules that were close in length to PNP mRNA were separated by agarose gel electrophoresis and inserted into the Pst I site of the plasmid pBR322. Plasmid DNA from the pooled clones was used to transform PNP-deficient Escherichia coli cells, and those transformants that phenotypically expressed PNP were isolated on selective media. The presence of human PNP in the selected bacterial cells was detected by immunoprecipitation with human PNP antibody.
我们已获得包含人嘌呤核苷磷酸化酶（PNP；EC 2.4.2.1）mRNA完整编码区的cDNA克隆。这些cDNA序列是通过用抗人PNP单特异性抗体免疫吸附HeLa细胞多核糖体获得的富含PNP的mRNA进行逆转录产生的。长度与PNP mRNA相近的cDNA分子通过琼脂糖凝胶电泳分离，并插入质粒pBR322的Pst I位点。来自混合克隆的质粒DNA用于转化缺乏PNP的大肠杆菌细胞，在选择性培养基上分离出那些表型表达PNP的转化体。用抗人PNP抗体进行免疫沉淀检测所选细菌细胞中是否存在人PNP。
