Villa-Komaroff L, Efstratiadis A, Broome S, Lomedico P, Tizard R, Naber S P, Chick W L, Gilbert W
Proc Natl Acad Sci U S A. 1978 Aug;75(8):3727-31. doi: 10.1073/pnas.75.8.3727.
We have cloned double-stranded cDNA copies of a rat preproinsulin messenger RNA in Escherichia coli chi1776, using the unique Pst endonuclease site of plasmid pBR322 that lies in the region encoding amino acids 181-182 of penicillinase. This site was reconstructed by inserting the cDNA with an oligo(dG)-oligo(dC) joining procedure. One of the clones expresses a fused protein bearing both insulin and penicillinase antigenic determinants. The DNA sequence of this plasmid shows that the insulin region is read in phase; a stretch of six glycine residues connects the alanine at position 182 of penicillinase to the fourth amino acid, glutamine, of rat proinsulin.
我们利用质粒pBR322位于青霉素酶氨基酸181 - 182编码区域的独特Pst核酸内切酶位点，在大肠杆菌chi1776中克隆了大鼠胰岛素原信使核糖核酸的双链互补脱氧核糖核酸拷贝。通过寡聚（dG）-寡聚（dC）连接程序插入互补脱氧核糖核酸来重建该位点。其中一个克隆表达了一种带有胰岛素和青霉素酶抗原决定簇的融合蛋白。该质粒的脱氧核糖核酸序列表明胰岛素区域是按相位读取的；一段六个甘氨酸残基的序列将青霉素酶第182位的丙氨酸与大鼠胰岛素原的第四个氨基酸谷氨酰胺相连。
