广西眼镜王蛇毒酸性磷脂酶A_2-1的表达

Expression of Acidic Phospholipase A_2-1 from Guangxi Ophiophagus Hannah (King Cobra) Venom

将广西眼镜王蛇毒酸性磷脂酶A2 - 1(APLA2 - 1)基因克隆至表达载体pET2 8a(+) ,转化入大肠杆菌BL2 1,经过诱导 ,SDS -PAGE检测 ,重组质粒pET2 8a-APLA2 - 1在 18KD有一明显的表达条带 ,表达产物APLA2 - 1约占细菌总量 3 0 % ,并以包涵体的形式存在 ,将产物变复性后用SephadexG - 75纯化 ,产物经过SDS -PAGE检测只有单一条带 ,通过Western -blot检测 ,证实纯化产物是酸性磷脂酶A2 .对表达的APLA2 进行酶活性和药理活性的测定 .至此我们在大肠杆菌中表达了广西眼镜王蛇毒APLA2 ,这将为以后对PLA2 的结构与功能的研究打下了良好的基础 .

A cDNA encoding acidic phospholipase A 2-1(APLA 2-1) from Guangxi Ophiophagus Hannah(King Cobra) Venom was inserted into bacterial expression vector pET28a(+) and expressed in E.coli BL21.pET28a-APLA 2-1 was effectively expressed in E.coli BL21.The protein was produced as insoluble inclusion bodies.After partial purification by washing the inclusion bodies with Triton X-100,denaturing and refolding,the renatured recombinant protein was purified by Sephadex G-75.The expressed protein exhibited enzymatic activity and platelet aggregation inhibiting effect.These would provide a good basis for further discussing the relation of structure and function,and the studying of evolution of PLA 2 from snake venom.