在大肠杆菌中分泌表达水蛭素基因的初步研究

Preliminary Study on Secretory Expression of Biologically Active Hirudin Ⅲ in E.coli

为实现水蛭素 (HV3)基因的分泌表达 ,设计合成了两对 (四条 )寡核苷酸链 ,退火、拼连成 L-门冬酰胺酶信号肽简并基因 ,同时利用密码子的简并性在该基因 3′端基因内引入了一个Nhe I限制酶切位点。此外 ,用 PCR技术在 HV3基因 5′端引入 Nhe I限制酶切位点 ,利用该酶切位点将信号肽基因与 HV3基因进行连接且保持原有阅读框架不变 ,构建了重组质粒 p UCSH,测序结果表明 ,融合基因核苷酸序列与设计要求完全一致 ,将该融合基因插入 p KK2 33- 2中构建成HV3分泌表达质粒 p KSH,该质粒在大肠杆菌中表达后 ,表达产物分泌至细胞膜间质中 ,膜间质蛋白抽提液抗凝血酶生物活性为 1 0～ 1 4 ATU/

For the secretory manufacture of HV3,a DNA fragment coding for the E.coli L ASPsⅡ signal peptide was assembled from four chemically synthesized oligonucleotides.A single NheⅠ restriction site was designed at the 3′ end of the signal sequence by using the degeneracy of codons.HV3 gene was joined to the signal sequence and the hybrid gene was placed under the control of the tac promotor in a recombinant plasmid pKSH.HV3 was expressed and secreted into the persplasm of E.coli, and the yield of biologically active HV3 was 10~14 ATU/ml culture.