CAJ | 학술논문

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 enzyme-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.
용PCR법합성인이도소기인,극륭도pUC18재체상,경측서증실기감기서렬여인이도소기인적서렬완전일치.본실험환구건료은이표체재체,유한제성내절매개도(Restriction enzyme-mediated DNA Integration, REMI)전화은이아포.수궤도취21개항성균락,전관번식2대후검측기GUS활성,실험결과:18개균주양성,3개균주음성.종저21개균주중선취10개균주,제취염색체DNA,용인이도소기인、GUS기인화Tnos서렬적특이인물진행PCR,결과표명,저10균주도능확증출상응장도적특이편단,증명료타문시인이도소기인전화자.
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.