人类精子VDAC2基因启动子在小鼠GC-2spd细胞的克隆和报告基因载体构建

Clones and the construction of luciferase report vector of human sperm VDAC2 promoter in GC-2spd cell lines

目的线粒体电压依赖性阴离子通道蛋白2(Voltage-dependent anion channel 2,VDAC2)是VDAC重要的家族成员,参与机体众多的生理活动和病理疾病的发生发展。本次研究目的主要是确定和验证人VDAC2基因启动子的活性,为进一步探究它的调控机制奠定基础。方法通过从正常人射出的精子中提取基因组DNA。利用软件预测启动子区,我们针对性设计三对引物并正确的扩增出来。将验证正确的启动子序列结合到质粒PDS psiCHECK-2中,转染到小鼠GC-2细胞中去。转染48h后,通过双荧光素酶报告基因系统检测预测片段的启动子活性。结果 VADC2基因上游-2000^+1000bp具有相对较高的活性。结论这是首次对人类精子中VDAC2基因启动子的确定和活性验证,为后续研究VDAC2基因在精子的发生发展以及弱精子症方面提供新的研究思路。

Objective Voltage-dependent anion channel 2 （VDAC2） is an important member of VDAC family, and involves in many physiological activities and pathological changes in diseases. This study is mainly to identify and analyze the active of promoter of human VDAC2 gene for further investigation on its regulatory mechanism. Methods Genomic DNA from normal human ejaculated sperm was extracted. The promoter region was correctly amplified using three pairs of primer. PDSA0isCHECK-2 carried the correct promoter sequence was transfected into GC-2spd cell line. 48h after cell transfection, a Dual-Luciferase Reporter Assay Kit was used to detect the activity of VDAC2 gene promoter constructs. Results It showed that the -2000-＋1000bp located at upstream of VDAC2 had the highest activity. Conehtsion This study first identify and analyze the activity of promoter region of VDA C2 in human sperm.