靶向MSTN基因shRNA表达载体的构建与鉴定  

Construction and Identification of shRNA Expression Vector Targeting MSTN Gene

作　　者：王丽俊 钮利喜 Wang Lijun;Niu Lixi(Institute of Biotechnology,Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education,Shanxi University,Shanxi 030006,China)

机构地区：[1]山西大学生物技术研究所、教育部化学生物学与分子工程重点实验室,太原030006

出　　处：《医学研究杂志》2020年第5期165-169,共5页Journal of Medical Research

摘　　要：目的旨在构建靶向MSTN基因的短发夹RNA(shRNA)真核表达载体,并鉴定其在小鼠体内的抑制效果。方法根据MSTN基因的mRNA序列(NM_010834.3)设计siRNA,并挑选出3条可能具有良好干扰效果的siRNA,设计合成shRNA,构建pSIREN-MSTN-shRNA表达载体。通过肌内注射法将载体递送至小鼠体内,利用实时荧光定量PCR和Western blot法检测干扰后小鼠肌肉组织中MSTN的mRNA表达量和蛋白质表达水平。结果测序鉴定表明pSIREN-MSTN-shRNA表达载体构建成功,3个重组质粒均能够明显降低肌肉组织中MSTN的mRNA和蛋白质水平,其中pSIREN-M819表达载体干扰效果最好,与对照组比较,MSTN的mRNA表达量降低了71%,蛋白水平也明显降低。结论成功构建了pSIREN-MSTN-shRNA表达载体,并在小鼠体内能够有效抑制MSTN基因的表达。Objective To construct a short hairpin RNA(shRNA)eukaryotic expression vector targeting the MSTN gene and to identify its inhibitory effect in mice.Methods The siRNA was designed based on the mRNA sequence of MSTN gene(NM_010834.3).Three siRNAs which may have good interference effect were selected and shRNA was designed and synthesized,and the pSIREN-MSTN-shRNA expression vector was constructed.The vector was delivered to mice by intramuscular injection,and the mRNA expression and protein expression levels of MSTN in the muscle tissue of the mice were detected by real-time fluorescent quantitative PCR(qRT-PCR)and Western blotting(WB).Results The sequencing analysis showed that the pSIREN-MSTN-shRNA expression vector was successfully constructed.Three recombinant plasmids could significantly reduce the mRNA and protein levels of MSTN in muscle tissue.The pSIREN-M819 expression vector had the best interference effect.Compared with the control group,MSTN mRNA expression decreased by 71%,and the protein level also decreased significantly.Conclusion The pSIREN-MSTN-shRNA expression vector was successfully constructed and can effectively inhibit the expression of MSTN gene in mice.

关 键 词：抑肌素 RNA干扰 实时荧光定量PCR 免疫印迹 

分 类 号：R746[医药卫生—神经病学与精神病学]