人胰岛素基因的人工合成及转化银耳的研究  

Synthesis of Human Insulin Gene and Transformation into Tremella fuciformis

作　　者：谢宝贵[1] 卢启泉[1] 饶永斌[1] 孙淑静[1] 郑金贵 

机构地区：[1]福建农林大学,福州350002

出　　处：《食用菌学报》2007年第2期1-14,共14页Acta Edulis Fungi

基　　金：国家自然科学基金项目"银耳生物反应器基础研究"(No30471210);福建省卫生教育联合攻关计划项目"银耳生物反应器生产人胰岛素的研究"(NoWKL2005-2-009)的部分研究内容

摘　　要：用PCR法合成人胰岛素基因,克隆到pUC18载体上,经测序证实其碱基序列与人胰岛素基因的序列完全一致。本实验还构建了银耳表达载体,由限制性内切酶介导(Restriction enzy me-mediated DNA Integration,REMI)转化银耳芽孢。随机挑取21个抗性菌落,转管繁殖2代后检测其GUS活性,实验结果:18个菌株阳性,3个菌株阴性。从这21个菌株中选取10个菌株,提取染色体DNA,用人胰岛素基因、GUS基因和Tnos序列的特异引物进行PCR,结果表明,这10菌株都能扩增出相应长度的特异片段,证明了它们是人胰岛素基因转化子。A human insulin gene was synthesized using PCR-based methodology, cloned into plasmid pUC18, and the sequence of the inserted fragment was confirmed. A Tremella fuciformis expression vector was constructed and used to transform T. fuciformis yeast-like conidia employing Restriction Enzyme-Mediated DNA Integration （REMI）. A total of 21 antibiotic resistant colonies were selected at random of which 18 tested positive for β-glucuronidase （GUS） activity. Genomic DNA was extracted from 10 of the 21 positive transformants and PCR performed using genomic DNA as the template and specific primers for amplifying the human insulin gene, the GUS gene and the Tnos sequence. PCR data showed that the expected fragment was amplified from all 10 isolates demonstrating that they were true human insulin gene transformants.

关 键 词：人胰岛素 基因合成 基因转化 银耳 芽孢 

分 类 号：S567.34[农业科学—中草药栽培] TQ460.72[农业科学—作物学]