人胰岛素基因的合成。VII. 类胰岛素原人DNA的合成、克隆及其在M13噬菌体中的表达。

Synthesis of a human insulin gene. VII. Synthesis of preproinsulin-like human DNA, its cloning and expression in M13 bacteriophage.

作者信息

Georges F, Brousseau R, Michniewicz J, Prefontaine G, Stawinski J, Sung W, Wu R, Narang S A

出版信息

Gene. 1984 Feb;27(2):201-11. doi: 10.1016/0378-1119(84)90141-0.

Abstract

A 74-bp DNA sequence coding for the pre sequence of human preproinsulin and containing EcoRI termini was synthesized by the chemical enzymatic method, joined with previously synthesized proinsulin DNA, and cloned in the M 13mp8 vector. A clone pNB82 -121 was identified by DNA sequence which confirmed the correct orientation of the pre sequence to the proinsulin DNA. The EcoRI site at the junction of pre- and proinsulin DNA was eliminated by removing a triplet ATT using a synthetic 19-mer primer. To simplify preproinsulin isolation and to study its expression in the M 13 system, a 25-bp affinity leader sequence coding for (glu)7 was inserted at the remaining EcoRI site; this put the preproinsulin DNA in a correct reading frame with the AUG initiation codon of beta-galactosidase. Preproinsulin was expressed under lac promoter control as analyzed by a radioimmunoassay (RIA) against C-peptide.

摘要

采用化学酶法合成了一段74个碱基对的DNA序列，该序列编码人胰岛素原前体序列并含有EcoRI末端，将其与先前合成的胰岛素原DNA连接，并克隆到M 13mp8载体中。通过DNA序列鉴定出一个克隆pNB82 - 121，该序列证实了前体序列与胰岛素原DNA的正确方向。使用合成的19聚体引物去除三联体ATT，消除了胰岛素原前体和胰岛素原DNA连接处的EcoRI位点。为了简化胰岛素原前体的分离并研究其在M 13系统中的表达，在剩余的EcoRI位点插入了一段25个碱基对的编码（glu）7的亲和前导序列；这使得胰岛素原前体DNA与β-半乳糖苷酶的AUG起始密码子处于正确的阅读框中。通过针对C肽的放射免疫分析（RIA）分析，胰岛素原前体在lac启动子控制下表达。