突变的人铜、锌超氧化物歧化酶基因的克隆、测序及表达

Cloning, Sequencing and Expressing of Human Copper, Zinc-Superoxide Dismutase Mutant cDNA

目的：通过基因工程的方法将ｒｈＣｕ，Ｚｎ－ＳＯＤ ｃＤＮＡ基因改造以得到更加稳定的酶。方法：以本实验室构建的 ｒｈＣｕ，Ｚｎ－ＳＯＤ ｃＤＮＡ为模板，利用含有突变核苷酸的引物进行 ＰＣＲ扩增，将正确测定的含 ｒｈＣｕ， Ｚｎ－ＳＯＤ突变（ｒｈＣｕ，Ｚｎ－ＭＳＯＤ）基因的重组质粒重组到表达载体ＰＥＴ－２２ｂ（＋）中，重组质粒在大肠杆菌ＢＬ２１（ＤＥ３）中表达ｒｈＣｕ，Ｚｎ－ＭＳＯＤ。结果：表达产物占菌体总蛋白的３８％，具有特异性ＳＯＤ酶活性。结论：从基因突变的角度改善酶的性能不仅具有理论意义，又有一定的实用价值。

Purpose: The aim was to obtain more stable enzyme to modify rhCu, Zn-SOD cDNA by genetic engineering. Methods: The cDNA encoding human copper, zinc-superoxide dismutase mutant was amplified from human copper,zinc-superoxide dismutase cDNA which was already constructed at our lab by PCR. The sequence of the cloned gene was determined. RhCu, Zn-MSOD cDNA was then ligated into PET- 22b (+). rhCu,Zn- MSOD was induced by IPTG and expressed in BL21 (DE3). Results: The special protein expressed accounts for 38% of the total protein of the bacteria and has special activity. Conclusion: The improvment of enzymatic property by gene mutation not only enriches the content of applied enzymology, but also possesses the theoretical and practical value.