乙酰肝素酶大亚基在毕赤酵母中的表达

Expression of Heparanase C-terminal Subunit in Pichia pastoris

目的构建乙酰肝素酶(HPA)大亚基片段真核表达质粒,并在毕赤酵母中表达重组蛋白。方法根据GenBank中登录的HPA大亚基基因序列设计引物,PCR方法扩增肝素酶基因,构建毕赤酵母真核表达质粒pPICZαA-HPA。电击穿孔法转化感受态毕赤酵母SDM1168,斑点免疫印迹法筛选重组菌株,PCR法鉴定阳性克隆。甲醇诱导表达重组蛋白,Western blot鉴定表达产物。结果构建的重组真核表达质粒经双酶切及测序鉴定证明构建正确;斑点免疫印迹法筛选出的9个单菌落经PCR扩增,均可见1161bp的目的基因条带;表达产物经Western blot分析,具有良好的反应原性,表达量约为5μg/L。结论已成功构建了HPA大亚基片段真核表达质粒pPICZαA-HPA,并在毕赤酵母SDM1168中分泌表达了HPA大亚基。

Objective To construct a eukaryotic expression vector for heparanase（HPA）C-terminal subunit fragment and express in Pichia pastoris.Methods Primers were designed according to the HPA C-terminal subunit gene sequence reported in GenBank for amplification of target gene fragment by PCR,based on which a recombinant plasmid pPICZαA-HPA was constructed and transformed to competent P.pastoris SDM1168 by electroporation.The recombinants were screened by dot blot,identified by PCR and induced by methanol.The expressed product was identified by Western blot.Results Both restriction analysis and sequencing proved that recombinant plasmid pPICZαA-HPA was constructed correctly.Nine recombinants were screened by dot blot,from which the target gene fragments each at a length of 1 161 bp were amplified by PCR.Western blot showed good reactogenicity of expressed product,and the expression level reached about 5μg/L.Conclusion The eukaryotic expression vector pPICZαA-HPA for HPA C-terminal subunit was successfully constructed and expressed in a secretory form in P.pastoris SDM1168.