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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 family member of VDAC and participates in many physiological activities and pathogenesis of the body. The purpose of this study is to determine and verify the activity of human VDAC2 gene promoter, and lay a foundation for further exploration of its regulatory mechanism. Methods Genomic DNA was extracted from sperm emitted from normal subjects. Using software to predict the promoter region, we targeted three pairs of primers and amplified them correctly. The verified correct promoter sequence was incorporated into the plasmid PDS psiCHECK-2 and transfected into mouse GC-2 cells. Forty-eight hours after transfection, the promoter activity of the predicted fragment was detected by the dual luciferase reporter system. The results of VADC2 gene upstream -2000 ~ +1000 bp has a relatively high activity. Conclusion This is the first validation of the promoter of VDAC2 gene in human spermatozoa and provides new research ideas for the follow-up study of VDAC2 gene in the development of spermatogenesis and asthenospermia.