摘要
通过PCR扩增得到组织型纤溶酶原激活剂突变体Reteplase (rPA)基因片断 ,将该基因片断插入到含有AOX1启动子和α分泌信号肽序列载体pPIC9K中 ,构建了重组表达质粒pPIC9K rPA ,转化PichiapastorisGS115宿主菌 .通过比较转化子在MM和MD平板上的生长状况 ,筛选His+ Muts 表型转化子 .并在 2 .0mg mLG4 18平板筛选得到多拷贝转化子GR10 ,GR11.摇瓶培养 ,甲醇诱导外源基因表达 ,表达产物经SDS PAGE和Westernblot分析 ,结果表明重组蛋白分子量约 39ku ,能与特异性单克隆抗体发生免疫反应 ;体外气泡法测定rPA活性 ,结果显示重组蛋白具有较好的纤溶活性 .通过正交试验 ,优化发酵条件 ,表达活性最高为 10 5 0IU mL .
The cDNA coding region of Reteplase was amplified with a PCR and cloned into a downstream of α-secreting signal peptide and AOX1 promoter of a pPIC9K expression vector system to construct the pPIC9K/rPA plasmid. This plasmid was then transformed into a Pichia pastoris GS115 strain, and G418-resistant colonies were selected. Transformants with high copy number, GR10 and GR11, were obtained on 2.0?mg/mL G418 plates. The His +Mut s colonies were further obtained by comparison of the growth state on MM and MD plates. Upon induction of methanol, the recombinant rPA protein was expressed in the P. pastoris GS115 strain. The results obtained from SDS-PAGE analysis revealed a protein with a relative molecular weight of 39ku. The results obtained from Western blot hybridization assay indicate a specific reaction with a mouse monoclonal antibody against the rPA protein. The recombinant protein exhibit a high fibrin-dissolving activity by an in vitro Air-bubble method. The optimum fermentation condition of GR10 is achieved by a method of orthogonal experiment and the maximum activity of rPA is 1050?IU/mL.
出处
《山东大学学报（理学版）》
CAS
CSCD
北大核心
2004年第5期107-111,共5页
Journal of Shandong University(Natural Science)
