新型抗肿瘤抗生素C1027生物合成相关基因克隆的研究

Study of biosynthesis gene cloning of novel antitumor antibiotic C1027

Ｃ１０２７是由球孢链霉菌（Ｓｔｒｅｐｔｏｍｙｃｅｓｇｌｏｂｉｓｐａｒｕｓ）Ｃ１０２７产生的一种新型大分子肽类抗肿瘤抗生素。以ｐＩＪ７０２为载体，变青链霉菌（Ｓ．ｌｉｖｉｄａｎｓ）ＴＫ５４为宿主，建立球孢链霉菌Ｃ１０２７总ＤＮＡ的随机克隆文库。以Ｃ１０２７阻断变株ＡＦ４０和ＡＦ４４为互补对象，分别与随机克隆文库中的重组菌株进行共培养，经过抗菌和抗瘤活性检测，发现重组菌株Ｎｏ．２８与阻断变株ＡＦ４４，重组菌株Ｎｏ．７４、Ｎｏ．１０１与阻断变株ＡＦ４０分别进行共培养能恢复变株的生物活性，并且ＳＤＳ－ＰＡＧＥ结果显示：变株辅基蛋白的分泌得以恢复，证明重组质粒ｐＩＪ２８、ｐＩＪ７４和ｐＩＪ１０１所含插入片段与Ｃ１０２７生物合成相关。重组菌株Ｎｏ．７４和阻断变株ＡＦ４０的共培养产物经纯化以后。

C1027 is a new antitumor antibiotic produced by Streptomyces globisporus C1027. Digested total DNA fragments of S.globisporus C1027 were inserted in plasmid pIJ702 and then transformed into S.lividans TK54. Recombinants were co cultured with C1027 block mutant AF40 and AF44 respectively. According to results of antimicrobial assay and spermatogonial asssy, recombinant No.74 and No.101 could complement with block mutant AF40, and recombinant No.28 complement with block mutant AF44. SDS PAGE analysis indicated that secretion of apoprotein could be restored after co culture. Purified product from co culture supernatant of recombinant No.74 and block mutant AF40 was verified to be C1027 by UV spectrum. These experiments showed that recombinant plasmid pIJF74, pIJF101 and pIJF28 contain genes involved in C1027 biosynthesis.