摘要
选用大肠杆菌偏爱的密码子,用计算机辅助设计、合成长度分别为59 、59 及60 碱基的三段寡核苷酸。以中段寡核苷酸为模板,两端寡核苷酸为引物,经PCR 合成完整的hPTH(134) 基因,并克隆到pUC18 载体中。对克隆基因进行序列分析,结果与设计的完全一致。将该基因与表达载体pKA 连接构建表达质粒pKAT,转化大肠杆菌JM105 细胞,SDSPAGE 表明hPTH(134) 融合蛋白获得了表达,并且酸水解后融合蛋白被裂解为大小两个片段。
The human parathyroid hormone (PTH) N terminal 34 peptide gene has been synthesized and cloned. Computer progamming was applied to the design of optimized segments for the synthesis of the hPTH(1 34) gene using preferred codons for expression in E.coli .Three segments with 59,59 and 60 base long were synthesized while W1 was homologous with W2 and W2 complementary to W3.The entire gene of 140bp was obtained by PCR and cloned into pUC18.Recombiant plasmid was transformed into host E.coli JM105 and positive clonies were screened. The DNA sequence of the cloned hPTH(1 34) gene was proved to be identical with designed DNA sequence . The hPTH(1 34) gene was inserted into expression vector pKA which contains L AnsB gene .The recombiant plasmid pKAT was used to transform E.coli JM105. After IPTG induced 2h, SDS PAGE proved that fusion protein was expressed in JM105. Then, fusion protein was cleaved by acid successfully.
出处
《中国药科大学学报》
CAS
CSCD
北大核心
1999年第3期216-219,共4页
Journal of China Pharmaceutical University
基金
国家自然科学基金!编号:39870175
江苏省自然科学基金资助项目!编号:BK95092309 。