江浙蝮蛇毒碱性磷脂酶A2的溶血位点

用聚合酶链式反应 (PCR)对江浙蝮蛇毒碱性磷脂酶A2 基因进行点突变 ,使对应 34位氨基酸残基由Arg分别突变为Glu和Gln ,将其基因克隆至温敏表达载体pBLMVL2 ,并在大肠杆菌RR1中成功诱导表达 ,表达产物以包涵体的形式存在。对包涵体进行变复性后 ,用凝胶过滤的方法纯化。对纯化后的产物进行酶活力测定及溶血活性测定。实验结果表明 ,突变后的表达产物维持原有磷脂酶A2 活性 ,且溶血活性显著降低 ,表明磷脂酶A2 的34位碱性氨基酸残基在溶血过程具有重要作用

The Hemolytic Site of the Basic Phospholipase A_2 from Agkistrodon halys Pallas

The gene of the basic phospholipase A 2 from Agkistrodon halys Pallas (BPLA 2 )was mutated site-directedly by polymerase chain reaction (PCR) and the residue Arg 34 of the encloding protein was substituted by Glu and Gln, respectively. The mutant gene has been cloned into the expression vector pBLMVL2 and has been expressed in E.coli RR1 effectively. The protein was produced as insoluble inclusion bodies. After partial purification, the inclusion bodies were denatured and renatured into active form, and the renatured recombinant protein was purified by gel-filtration. The expression product has the same enzymatic activity as the denatured-refolded BPLA 2 and its hemolytic activity dropped distinctly, which suggest that the basic residue Arg 34 of BPLA 2 is a crucial amino acid residue during the process of hemolysis.