辣根过氧化物酶同功酶C基因克隆及其在大肠杆菌中的表达

无论从应用还是从理论研究角度，辣根过氧化物酶（ＨＲＰ）是一种非常重要的酶．ＨＲＰ基因克隆与表达将有利于更深入研究ＨＲＰ的结构与功能．利用反转录ＰＣＲ从天然植物辣根中分离和克隆编码辣根过氧化物酶同功酶Ｃ（ＨＲＰ－Ｃ）一个ｃＤＮＡ，并测定其序列．结果发现，从基因推导出的氨基酸序列与Ｗｅｌｉｎｄｅｒ报道的辣根过氧化物酶序列有９０．６％的同源性．将该基因连接到表达载体ｐＥＴ－２４ｂ上，利用抗ＨＲＰ多克隆抗体进行Ｗｅｓｔｅｒｎｂｌｏｔ，检测有少量目标产物表达．在诱导表达过程中，没有发现细菌生长受抑制或受明显的毒害

Cloning of a cDNA Encoding Horseradish Peroxidase Isozyme C (HRP C) and Its Expression in E.coli

Horseradish peroxidase (HRP) is of great importance from the theoretical and pratical points of view.The cloning and expression HRP gene will provide much of the information on its machanisms and function.A cDNA encoding a mature horseradish peroxidase isozyme C had been cloned and the sequence was analysed.The deduced amino acid sequence was homologous by 90 6% to the HRP amino acid sequence reported by Welinder.However,only six potential N glycosylation sites were found in the deduced amino acid sequence.In oder to express the cDNA efficiently in E.coli ,it was constructed into pET 24b.Western blot analysis showed that small amount of target protein was recognised by polyclonal antibodies against native HRP.No influence of induction on the growth of the expressed strain was observed.