摘要
目的构建DcR3反义RNA表达载体PVAXⅠasDcR3,并研究其对肝癌细胞中DcR3蛋白表达的影响。方法从肝癌组织中提取总RNA,RTPCR法克隆出DcR3基因片段,将其与PMD18T载体连接,经BamHⅠ、XholⅠ双酶切TADcR3及PVAXⅠ()真核表达载体后,使用T4DNA连接酶连接,构建反义DcR3表达载体。转染肝癌细胞株SMMC7721。Westernblot法检测DcR3蛋白表达。结果用RTPCR方法从肝癌组织中扩增出DcR3片段,并经测序证实。重组质粒经酶切鉴定正确,转染肝癌细胞株SMMC7721后,经Westernblot证实DcR3蛋白表达较对照组下降。结论成功构建反义RNA表达载体PVAXⅠasDcR3,构建的反义表达载体PVAXⅠasDcR3具有阻断DcR3基因表达的效应。
Objective ： To obtain recombinant expressive vector PVAX Ⅰ -as-DcR3 ,and study Its effect on DcR3 protein in hepatoma cell line SMMC7721. Methods： Extracted total RNA from hepatoma tissue,DcR3 gene was cloned with RT-POR and liBated with PMD18-T vector. Both TA-DcR3 and PVAX Ⅰ （-） expression vector were excised using BamH Ⅰ ,Xhol Ⅰ, then DcR3 gene was liBated into the same site of PVAX Ⅰ （-） excised by BamH Ⅰ and Xhol Ⅰ In antlsense direction.Hepatoma cell line SMMC7721 was transfected by PVAX Ⅰ-as -DcR3 expression vector. Western blot was performed to observe protein expression of DcR3. Results ： DcR3 gene was replicated from hepatoma tissue with RT-POR and products were confirmed corresponding to DcR3 gene sequence, furtherly the recombinant PVAX Ⅰ-as-DcR3 was verified by excision of BamH Ⅰ ,XholⅠ and electrophoresis. Comparing with contrast group, protein expression of DcR3 was demonstrated declining by Western blot in hepatoma cellular line SMMO7721 transfected with PVAX Ⅰ -as-DcR3. Coneluslon ：PVAX Ⅰ -as-DcR3 was successfully constructed and showed tissue specificity and blocking function of DcR3 expression.
出处
《中国现代普通外科进展》
CAS
2005年第1期17-19,共3页
Chinese Journal of Current Advances in General Surgery
关键词
基因
DcR3
RNA
反义
基因转移
水平
基因表达
癌
肝细胞
Gene, DcR3 RNA, antisense Gene transfer, horizotal Gene expression Carclnoma, hapatocellular
