MSTN基因siRNA表达载体的构建和鉴定

目的构建能沉默MSTN基因的小干扰RNA表达载体，并鉴定它沉默肌母细胞MSTN基因的效率。方法合成3对发夹小干扰RNA模板寡核苷酸链，退火后插入pSileneer载体．构建成可沉默MSTN基因的小干扰RNA表达载体，通过酶切和测序鉴定构建的小干扰RNA表达载体。将小干扰RNA表达载体转染肌母细胞，用实时荧光定量RT—PCR和Western印迹检测转染的肌母细胞myostatin的表达水平。结果酶切和测序证实3个小干扰RNA表达载体构建正确，实时荧光定量RT—PCR显示所构建的3个小干扰RNA表达载体对肌母细胞MSTN基因的干扰率分别为43．6％、47．7％和81．6％，它们的干扰效果被Western印迹所证实。结论干扰率为81．6％的小干扰RNA表达载体为构建成功的小干扰RNA表达载体，。岜可用作MSTN基因的功能研究和肌病治疗的分子研究。

Construction and Identification of MSTN Gene siRNA Expression Vector

Objective To construct MSTN gene siRNA expression vectors, and to analyze the efficacy of siRNA expression vectors for silencing the MSTN gene expression in myoblasts. Methods Three pairs of complementary oligonucleotides for each siRNA target site of MSTN gene were syn- thesized, annealed, and ligated into the pSilencer vector, respectively. The MSTN siRNA expression vectors were identified by restriction enzyme digestion and sequencing. Myoblasts were transfected with the MSTN siRNA expression vectors. Myostatin expression was determined by real-time quantitative RT-PCR and Western blot. Results Real-time quantitative RT-PCR showed that the established siRNA expression vectors silenced the MSTN gene in transfected myoblasts at the rate of 43.6%, 47.7%, and 81.6%, respectively. The silencing efficacy of the MSTN gene was testified by Western blot. Conclusion The MSTN siRNA expression vector with 81.6% interfering efficacy of interfering may provide a tool for further study on MSTN gene and its application in myopathy therapy.