水蛭素在毕赤酵母中的分泌表达_医学论文.docVIP

水蛭素在毕赤酵母中的分泌表达_医学论文 水蛭素在毕赤酵母中的分泌表达_医学论文 作者：龙铟，刘家云，刘莉，王宗仁 【关键词】 毕赤酵母 Secretory expression of hirudins in Pichia pastoris 　　【Abstract】 AIM: To construct hirudin expression vectors and obtain its secretory expression in Pichia pastoris(P. pastoris). METHODS: Hirudin HV2 and HV2N47K genes were amplified by PCR and the correct genes were fused to pPIC9 α 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 screenedout clones were induced by methanol to express hirudin proteins. The supernatants of expression products were identified by SDSPAGE and Western blot analysis, and its antithrombotic activity was determined too. RESULTS: The expression vectors were successfully constructed, hirudins were expressed in supernatants of P. pastoris, and the expression products possessed antithrombotic activity. CONCLUSION: Hirudins can be expressed secretorily in P. pastoris, which offers a basis for newdrug researches.　　【Keywords】 hirudin Pichia secretory expression 　　【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, HV2N47K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pPIC9KHV2, pPIC9KHV2N47K. 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. 　　【关键词】 水蛭素；毕赤酵母；分泌表达 　　0引言 　　水蛭素(hirudin)是从水蛭（Hirudo medicinalis）中分离出的抗凝血活性成分，是凝血酶的天然抑制剂［1］，具有高效的抗血栓形成作用和非常广阔的应用前景［2-3］. 但是天然水蛭素来源匮乏，限制了其研究和临床应用，我们以水蛭素HV2, HV2N47K的氨基酸序列为对象，通过PCR扩增得到了目的基因，克隆入毕赤酵母(Pichia Pastoris)表达载体pPIC9K中，构建了水蛭素酵母分泌型表达载体，转化SMD1168毕赤酵母细胞并进行诱导表达，获得了具有抗凝血酶活性的表达产物，为水蛭素的研究及临床应用奠定了基础. 　　1材料和方法 　　1.1材料质粒pPIC9, pPIC9K, Top10F菌株及毕赤酵母菌SMD1168由本实验室保存. TaKaRa Taq酶，限制性内切酶（EcoRI, XhoI, BamHI, Sa1I），T4 DNA连接酶均购自宝生物公司. 发色底物Chromozym TH及ECL化学发光检测试剂盒购自Roche公司，鼠抗hirudin单抗(mAb) 购自AntibodyShop公司，酵母基因组DNA纯化试剂盒购自上海华舜生物工程公司. 电泳仪、电转仪均为BioRad公司产品.