MISⅡR启动子的克隆及其组织特异性鉴定  

Cloning and determination of the tissue-specific activity of mullerian-inhibiting substance type Ⅱ receptor promoter

作　　者：雷俊川[1] 刘忠湘[1] 赵亚[1] 韩骅[2] 

机构地区：[1]第四军医大学基础部微生物学与病原生物学教研室,陕西西安710032 [2]第四军医大学基础部医学遗传学与发育生物学教研室

出　　处：《中国病原生物学杂志》2009年第11期823-826,共4页Journal of Pathogen Biology

摘　　要：目的克隆苗勒氏管抑制物Ⅱ型受体启动子(mullerian inhibiting substance typeⅡreceptor promoter,MISⅡRpr)并鉴定其在卵巢器官中的特异性表达活性。方法利用PCR从C57BL/6小鼠基因组中扩增苗勒氏管抑制物Ⅱ型受体启动子并将其克隆入载体pMD18-T,测序正确后将其亚克隆入pGL3-Basic荧光素酶报告基因载体。将重组质粒pGL3-Basic/MISⅡRpr分别转染卵巢癌细胞系SKOV3、HT-8910,宫颈癌细胞系Hela、Si Ha,绿猴肾细胞系COS-7,乳腺癌细胞系MDA-MB-231和结肠癌细胞系HT-29,通过双荧光素酶检测系统检测荧光素酶在上述细胞系中的活性,以确定克隆的苗勒氏管抑制物Ⅱ型受体启动子是否具有卵巢特异性活性。结果构建的pGL3-Basic/MISⅡRpr质粒用SacⅠ和EcoRⅠ双酶切,片段大小分别为600 bp和1 000 bp,证明MISⅡRpr插入正确。重组质粒转染卵巢细胞系SK-OV3、HT-8910,荧光素酶高表达;对照细胞HT-29等转染后,荧光素酶低表达。结论克隆得到的MISⅡRpr具有明显的卵巢特异性活性。可以利用MISⅡRpr进行相关基因的卵巢特异性表达。Objective To clone the mullerian-inhibiting substance type Ⅱ receptor promoter（MISⅡRpr） and determine the ovary-specific activity of MISⅡRpr.Methods The MISⅡRpr was amplified from the C57BL/6 mice genome using PCR and cloned into the vector pMD18-T.After sequencing,the MISⅡRpr was subcloned upstream of the luciferase-encoding sequence in the pGL3-Basic plasmid.The recombinant plasmid pGL3-Basic/MISⅡRpr was transfected into different cell lines including SKOV3,HT-8910,Hela,SiHa,COS-7,MDA-MB-231,and HT-29.Dual luciferase activity was determined by a dual lueiferase report （DLR） assay system. Results The MISⅡRpr was correctly amplified and has activity highly specific to the ovaries. Conclusion The MISⅡRpr can be used to determine ovary-specific gene expression.

关 键 词：苗勒氏管抑制物Ⅱ型受体启动子 (MISⅡRpr) 组织特异性 荧光素酶 

分 类 号：R347.91[医药卫生—基础医学]