重组人组织型纤溶酶原激活剂衍生物在毕赤酵母中的表达

目的在毕赤酵母中表达重组人组织型纤溶酶原激活剂衍生物(rPAm),并检测rPAm的活性。方法用PCR从质粒pLFrGGI上扩增出目的蛋白(rPAm)基因,再亚克隆到酵母表达载体pMEX9K中,转化到宿主菌GS115,菌落PCR鉴定转化子,重组酵母经甲醇诱导后,通过SDS-PAGE及Western-blot分析鉴定表达产物,再通过层析法纯化目的蛋白,用溶圈法测定目的蛋白的活性。结果表达的重组蛋白分子量约为50kD。纯化后的目的蛋白的纯度可达到95%。Western-blot显示能与抗tPA抗体特异性结合,纯化后rPAm比活性为5.02×105IU/mg,比活性与t-PA相当。结论成功构建了rPAm酵母表达工程菌,建立了对目的蛋白的纯化方法。

The expression of a recombtnant protein of Tissue plasminogen Activator in P. Pastoris

Objective To explore the expression of the recombinant protein of Tissue Plasminogen Activator in P.Pastoris and detect the bioactivity.Methods The target protein (rPAm) gene was amplified from plasmid pLFrGGI by PCR and subcloned to the yeast expression vector pPMEX9K.The resultant Plasmid pPMEX9K-rPAm was linearized and transformed into P.Pastoris GS115.The recombinant Pichia strains were identified by PCR screening and transformants were cultured and induced with methanol.The expression was detected by SDS-PAGE and Western-blot and purified by chromatography.The bioactivity of the product was detected by Fibrin Agarose Plate Assay.Results The target protein was 50kD molecular weight.The purity of target protein could reach 95% after purification.The recombinant protein could be speciticeg recoghised by anti-tPA. The bioactivity of recombinant protein could reach 5.02×105IU/mg,and equaled to t-PA.Conclusion P.Pastoris engineered bacterium for recombinant protein of Tissue Plasminogen Activator is constructed and the method for the purification of target protein is established.