汉森酵母表达载体的构建和人血管生成抑制素基因的表达

Construction of Integrative Expression Vector and Expression of Human Angiostatin in Hansenula polymorpha

汉森酵母 (H .polymorpha)是一类能以甲醇为唯一碳源和能源的甲基营养酵母 ,具有高表达外源基因、易于高密度发酵和产业化的特点。应用PCR技术扩增汉森酵母甲醇氧化酶 (MethanoloxidaseMOX)基因启动子和转录终止序列 ,并与汉森酵母Leu基因 (Hpleu2 )和人血管生成抑制素基因一起重组进大肠杆菌质粒pSP72 ,构建了整合型表达载体pSMA1 7,采用LiAc法将pSMA1 7转入汉森酵母A1 6 (leu) ,筛选出阳性转化子H .polymorphaA1 6 (pSMA1 7)。转化子在YPGE培养基中培养至对数生长后期 ,用甲醇进行诱导表达。ELISA和SDS PAGE分析结果证明人血管生成抑制素已获表达 ,表达产物分泌至培养基中。Westernblot结果显示重组的人血管生成抑制素能与抗人纤溶酶原抗血清特异结合 。

Hansenula polymorpha is a methylotrpphic yeast which can grow on methanol as the sole source of carbon and energy and is characterized by its easy manipulation in industry. The construction of an integrative expression vector and the expression of angiostatin in H.polymorpha have been described.The methanol oxidase(MOX) gene promoter and terminator of H.polymorpha was amplified by PCR and recombined with its leu gene and angiostatin gene on plasmid pSP72 resulting in the integrative vector pSMA17 which was then transformed into H.polymorpha A16(leu-).The positive transformant HpA16(pSMA17) was screened on SC medium without leucine.HpA16(pSMA17) was grown on YPGE medium to late log phase and induced with methanol.ELISA and SDS-PAGE results show that angiostatin was expressed and the expressed product secreted into the medium at 50mg/L fermentation broth(in the flasks).Western blot analysis Indicates that the recombinant angiostatin specifically reacts with antiserum against human plasminogen.