摘要
目的:构建水蛭素毕赤酵母表达载体,获得重组蛋白在毕赤酵母中的分泌型表达.方法:通过PCR扩增获得水蛭素HV2,HV2-N47K基因,将序列正确的基因序列插入酵母表达载体pPIC9α分泌信号下游,再亚克隆入高拷贝载体pPIC9K,构建表达质粒pPIC9K-HV2,pPIC9K-HV2-N47K.重组质粒转化宿主菌GSM1168,G418抗性筛选高拷贝的稳定表达菌株,经甲醇诱导表达并对表达产物进行鉴定.结果:成功构建了水蛭素毕赤酵母表达载体,获得水蛭素在毕赤酵母中的分泌型表达,表达产物具有抗凝血酶活性.结论:水蛭素在毕赤酵母中获得分泌型表达,为研究新药奠定了基础.
AIM： To construct hirudin expression vectors and obtain its secretory expression in Pichia pastoris （ P. pastoris ）. METHODS： Hirudin HV2 and HV2-N47K genes were amplified by PCR and the correct genes were fused to pPIC9 ot factor signal and subcloned into pPIC9K secretory expression vector. The constructs were transformed into P. pastoris strain GSM1168, stable multicopy integrants were screened on medium containing increasing concentrations of G418. Then the screened-out clones were induced by methanol to express hirudin proteins. The supernatants of expression products were identified by SDS-PAGE and Western blot analysis, and its antithrombotie activity was determined too. RESYLTS： The expression vectors were successfully constructed, hirudins were expressed in supematants of P. pastoris, and the expression products possessed antithrombotie activity. CONCLUSION ： Hirudins can be expressed secretorily in P. pastoris, which offers a basis for newdrug researches.
出处
《第四军医大学学报》
北大核心
2006年第8期673-676,共4页
Journal of the Fourth Military Medical University
基金
国家自然科学基金资助(30200359)
