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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 inhibitory substance type Ⅱ receptor promoter (MISⅡRpr) and identify its specific expression activity in ovarian organs. Methods The promoter of type II receptor of Müllerian tube inhibitor was amplified from the genome of C57BL / 6 mouse by PCR and cloned into vector pMD18-T. After sequencing, it was subcloned into pGL3-Basic luciferase reporter Gene vector. The recombinant plasmid pGL3-Basic / MISⅡRpr was transfected into ovarian cancer cell lines SKOV3, HT-8910, cervical cancer cell line Hela, Si Ha, green monkey kidney cell line COS-7, breast cancer cell line MDA- The cancer cell line HT-29 was tested for luciferase activity in the above cell line by dual luciferase assay to determine whether the cloned Müllerian tube inhibitor type II receptor promoter has ovarian-specific activity. Results The pGL3-Basic / MISⅡRpr plasmid was digested with SacⅠ and EcoRⅠ, and the fragments were 600 bp and 1 000 bp, respectively. This proved that MISⅡRpr was inserted correctly. The recombinant plasmids were transfected into ovarian cell lines SK-OV3, HT-8910 and luciferase. The transfection of control cells HT-29 and so on, the expression of luciferase was low. Conclusion The cloned MISⅡRpr has obvious ovarian specific activity. MIS II Rpr can be used for ovary-specific expression of related genes.