pPIC9K/Ang-1毕氏酵母表达载体的构建及体外表达

Construction and expression of recombinant Pichia pastoris for pPIC9K/angiopoietin-1

目的构建毕氏酵母表达载体pPIC9K/Ang-1,并进行体外表达研究。方法从PGEM-T/Ang-1载体中切取目的片段Ang-1,利用定向克隆到毕氏酵母表达载体pPIC9K中,测序正确后,pPIC9K/Ang-1载体经SalⅠ酶切线形化,以电转化法导入GS115毕氏酵母菌株中,经YPD平板G418抗性筛选后获得Ang-1表达阳性菌株;用SDS-PAGE测定其分子量,用Western Blotting检测发酵上清中Ang-1的抗原性和表达量。结果成功切取1.5 kb的Ang-1片段,成功构建pPIC9K/Ang-1毕氏酵母表达载体,Blast序列分析与GenBank中AY121504一致。酶切线性化的质粒成功转入GS115毕氏酵母中,诱导表达后SDS-PAGE显示重组人Ang-1分子量约66 kD,Western Blotting证实为人Ang-1。结论成功构建pPIC9K/Ang-1毕氏酵母表达载体,成功获得稳定分泌Ang-1重组蛋白的基因工程菌株。

Objective To construct a pPIC9K/Ang-1 yeast expression vector and assay Ang-1 expression in vitro.Methods The fragment of Ang-1 was cut,cloned into Pichia pastoris expression vector and then sequenced.pPIC9K/Ang-1 was linearized by SalⅠenzyme,and guided into GS115 Pichia pastoris after being transformed by electricity,then filtrated through YPD flat G418.After that,the positive Pichia pastoris of Ang-1 was attained.The molecular weight of Ang-1 was measured by SDS-page.The antigen and the quantity of expression of Ang-1 in fermental supernatant was measured by Western Blotting.Results Pichia pastoris expression vector pPIC9K/Ang-1 was constructured successfully.Blast sequence analysis was in accordance with AY121504 in Genbank.SDS-PAGE indicated that the molecular weight of reconstructed Ang-1 was 66 kd.Conclusions Pichia pastoris expression vector pPIC9K/Ang-1 can be constructed successfully,and transformed strain secreting Ang-1 protein steadily can be attained.