摘要
本研究以近期报道的高生物学活性、高代谢稳定性的牛生长激素释放因子类似物 (Ile2 ,Ala15,Leu2 7)bGRF(1- 2 9) -NH2 的氨基酸序列为模板 ,选用大肠杆菌高效表达所偏爱的密码子设计出该基因的全序列。采用化学合成法合成两个 3′末端有 19碱基互补的 80碱基的寡核苷酸片段。并互为模板和引物 ,通过酶促延伸合成完整的bGRF类似物基因 ,克隆于 pT7BlueT Vector中 ,通过蓝 /白筛选和酶切分析确定了阳性克隆。然后将目的基因从 pT7BlueT Vector克隆于pUC18载体中 ,以便多拷贝基因的制备。通过测序证明我们获得了目的克隆。将目的基因首尾串联 ,制备得到的 16拷贝基因在温控启动子PRPL
According to the genetic codon bias of E.coli,the full sequence of bGRF analogue gene was deduced from the amino acid sequence of the recently reported bGRF analogue with high bioactivity and metabolic stability.To gain bGRF analogue gene,two 80bp oligonucleotide fragments complementary at their 3’termini were synthesized. The two fragments annealed and the recessed 3’termini were filled by the extension reaction of Taq DNA Polymerase.The resulted fragment (full length gene)was ligated to pT 7 Blue T vector and positive clones were selected by blue/white screening and restriction pattern analysis. The bGRF gene was sequenced and ligated into the form of direct tandem repeats to produce multicopy gene to increase the gene expression efficiency and the stability of the translation products.A 16 copy gene was gained and induced to express in E.coli under the driver of P RP L promoters regulated by the temperature sensitive repressors.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2000年第4期296-300,共5页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
山东省科委资助项目
关键词
bGRF类似物
表达
基因克隆
bGRF analogue gene
Chemosynthesis
Cloning
Expression
