CAD基因原核表达载体的构建及表达产物的活性鉴定

Construction of caspase activated Dnase(CAD) prokaryotic expression vector and identification of expressive product activity

目的构建pCool-GST-ICAD/CAD的表达载体,并在大肠杆菌BL21(DE3)中表达具有生物学活性的CAD核酸酶。方法用PCR扩增CAD基因,将扩增产物克隆入pCool-GST-ICAD载体中构建出pCool-GST-ICAD/CAD表达载体。经酶切和电泳鉴定正确后,在大肠杆菌BL21(DE3)中诱导表达,表达产物经亲和层析、离子交换层析及凝胶过滤等方法分离纯化,最后用SDS-PAGE和DNA降解实验进行鉴定。结果构建了pCool-GST-ICAD/CAD原核表达载体,重组载体转化后表达出毫克级水平的GST-ICAD/CAD蛋白复合体。经分离纯化后得到纯度很好的CAD-ICAD蛋白复合体,在SDS-PAGE电泳上呈现清晰的两条蛋白带。经DNA降解实验证明纯化所得的CAD蛋白具有非特异性降解DNA的核酸酶作用。结论成功制备了具有生物学活性的CAD核酸酶,为进一步研究细胞凋亡的作用机制提供了有效的制剂。

Objective To construct the prokaryotie-expression plasmid pCool-GST-ICAD/CAD expressing the CAD protein with biological function in E. coli Bl21 （DE3） strain. Methods The full-length cDNA of CAD gene was amplified by PCR and inserted into the pCool-GST-ICAD to eonstruct the pCool-GST- ICAD/CAD prokaryotie-expression vector. The plasmid was identified and transformed into BL21 for expression. The GST-ICAD/CAD protein was purified using the GST affinity column, ion exchange and the gel filtration. Protein purity was checked by SDS-PAGE. CAD activity was determined with DNA fragmentation test. Results The pCool-GST-ICAD/CAD prokaryotie-expression vector was constructed and confirmed by restriction enzyme cutting analysis. Some mg level amount of GST-ICAD/CAD protein complex was expressed. After GST affinity column, ion exchange and the gel filtration purification, the purified ICAD/ CAD protein complex was obtained, which two clear protein bands could be seen in SDS-PAGE. Furthermore, the purified CAD protein indeed possessed nonspecifie Dnase activity in the DNA fragmentation test. Conclusion The full-length CAD gene with biological activity is successfully cloned and expressed in E. coli BL21. The purified CAD protein provides a new tool for apoptosis research or a possible target for clinic use.