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Construction of caspase activated Dnase (CAD) prokaryotic expression vector and identification of expressive product activity
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- Author:
- No author available
- Journal Title:
- JOURNAL OF MODERN CLINICAL MEDICAL BIOENGINEERING
- Issue:
- 4
- DOI:
- 10.3760/cma.j.issn.1008-634X.2006.04.017
- Key Word:
- CAD基因;原核表达;DNA降解实验
Abstract： 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.
- [1]于永发,张莹,樊丽超.大鼠脑缺血再灌注后半暗带CAD基因的表达改变[J].2004,(3).
- [2]Kyogoku Y,Sakahira H,Uegaki K,etc.Structure of the CAD domain of caspase-activated DNase and interaction with the CAD domain of its inhibitor.[J].2000,297(5).
- [3]Enari,M,Sakahira,H,Yokoyama,H,etc.A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD (see comments) (published erratum appears in Nature 1998 May 28; 393(6683):396)[J].1998,391(6662).
- [4]Wati MR,Sprengart ML,Janicke RU,etc.Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis.[J].1998,273(16).
- [5]Sole C,Comella JX,Dolcet X,etc.The contribution of apoptosis-inducing factor, caspase-activated DNase, and inhibitor of caspase-activated DNase to the nuclear phenotype and DNA degradation during apoptosis[J].2005,280(42).
- [6]Kothakota, Srinivas,Azuma, Toshifumi.Caspase-3-generated fragment of gelsolin: Effector of morphological change in apoptosis.[J].1997,278(5336).