斑节对虾酚氧化酶原在大肠杆菌中的表达

Expression and Purification of Prophenoloxidase of the Black Tiger Shrimp(Penaurs Monodon)in E.coli

将斑节对虾酚氧化酶原(prophenoloxidase,proPO)基因克隆进pET28a(+),在大肠杆菌BL21菌株中诱导表达,SDS-PAGE和Western-blotting分析均能检测到一条分子量为79.4kDa的特异性条带,与推导的融合蛋白理论分子量82.4kDa基本相符;包涵体变性后对经Ni-NTA agarose纯化的变性液进行复性,表现出0.3851U/mg.min的PO活性;不同条件下的诱导表达结果显示,18℃时诱导培养能检测到目的蛋白的可溶性表达,经诱导9h后可溶性表达比例达到最高,约占菌体可溶性蛋白总量的5.56%;可溶性表达的目的蛋白纯化后经胰酶消化,可检测到分子量为60kDa、42.7kDa的特异性条带,并表现出0.4249U/mg.min的PO活性.

The prophenoloxidase gene of the black tiger shrimp （Penaurs monodon） was cloned into pET28a（＋） vector and expressed in E.coli BL21. A special band of 79.4kDa was detected using SDS-PAGE and Western blot, which matched the theoretical molecular mass（82.4kDa） of the fusion protein on the whole. The obtained inclusion body was denatured, purified with nickel-resin column and renatured, resulting in the enzyme activity of 0.3851 U/ mg.min. The transformed E.coli BL21 were induced under different conditions. The soluble recombinant protein could be detected only when bacteria were induced at 18 ℃, and the expression ratio became the highest at the 9th hour post-induction, which reached 5.56% of the total soluble protein in E.coli. The soluble recombinant protein purified was digested with trpisin, resulting in the special bands of 60kDa and 44.5kDa ws detected using SDS-PAGE,and the enzyme activity of 0.4249 U/mg.min was observed in the solution of digested soluble recombinant protein.