摘要
采用 PCR技术人工合成了一段长约 1 75 bp的人表皮生长因子 ( h EGF)的基因片段 .为了便于克隆 ,在该片段的 5′端设计了 Pst 的酶切位点及与 p US1 86载体 signal sequence相连接的碱基部分 ,在 3′端设计了 Hind 的酶切位点及终止密码子 .经 DNA测序分析 ,合成的片段与已发表的 h EGF在序列上完全一致 ;之后将其克隆至枯草杆菌分泌型质粒载体p US1 86上 ,构建重组载体 p USE并转化一株枯草杆菌突变菌株 BS992 0感受态细胞 ;以 PCR法快速筛选重组菌落 ,RIA检测结果表明 BS992 0阳性转化子能够表达和分泌 h
A gene coding for human epidermal growth factor (hEGF) was synthesized by PCR technology. The 175 bp synthesized DNA duplex consists of structural gene encoding hEGF, restriction site PstI at 5' end, the base pair linked to the signal sequence of B.subtilis secretive vector-pUS186 and Hind III at 3' end. The DNA sequencing result revealed that the sequence of the synthesized fragment is totally identical with the published hEGF. It was then cloned into pUS186 and constructed the recombinant secretive vector-pUSE. The resultant plasmid was introduced into BS9920 (one mutant of B.subtilis). The recombinant colonies were identified by PCR screening and the result of RIA showed that the BS9920 transformants could express and secrete hEGF.
出处
《大连理工大学学报》
EI
CAS
CSCD
北大核心
2002年第1期47-50,共4页
Journal of Dalian University of Technology
