CPP32 cDNA的克隆及其表达和活性检测

Cloning and Functional Expression of CPP32 cDNA in E.coli.

ＣＰＰ３２在细胞编程死亡调控中起重要的作用．利用ＣＰＰ３２专一性引物，用ＲＴＰＣＲ方法从人鼻咽癌ＣＮＥ细胞中扩增出约８３０ｂｐ的片段，经序列分析证明与已发表的ＣＰＰ３２序列完全一致．将扩增出的编码人全长ＣＰＰ３２的ｃＤＮＡ片段克隆入ｐＧＥＸ２Ｔ中，转化大肠杆菌ＤＨ５α．转化菌经诱导表达出较高含量的ＧＳＴＣＰＰ３２融合蛋白．进一步研究显示，细菌中表达的ＣＰＰ３２蛋白能自我切割，而且能裂解体外翻译的ＰＡＲＰ，从而证明其具有生物活性．

CPP32 has been recently reported to be involved in the early process of programmed cell death. To further study CPP32 and its regulation in the cell, a 830 bp cDNA was cloned by RTPCR from CNE cells encoding the full length human CPP32 protein and high level expression was achieved in E.coli by using GST expression system. The results showed that the bacterially expressed CPP32 protein is autocleaved and capable of cleaving in vitrotranslated PARP, thus is fully functional.