绵羊MSTN基因慢病毒表达载体的构建及其对成肌细胞的分化作用

【目的】构建新疆细毛羊MSTN基因慢病毒表达载体，研究其在细毛羊成肌细胞分化中的作用，并探讨其作用机制。【方法】采用RT-PCR技术，扩增MSTN编码区序列，克隆入plex-mcs慢病毒表达载体，构建plex-MSTN慢病毒表达载体，并进行酶切及测序鉴定。将plex-MSTN包装成plex-MSTN慢病毒。用酶消化法分离培养新疆细毛羊成肌细胞，慢病毒感染制备MSTN过表达成肌细胞系，通过马血清诱导分化，Western blotting检测分化细胞MSTN基因的表达，免疫荧光分析分化肌管的融合率及肌管直径，Realtime RT-PCR检测分化相关基因的表达。【结果】 克隆的新疆细毛羊MSTN基因编码区全长序列（1 128 bp）与NCBI上公布的序列99.9%同源，仅在外显子2上存在单碱基突变；Western blotting检测结果显示，MSTN过表达成肌细胞过表达MSTN蛋白；免疫荧光检测表明，MSTN过表达成肌细胞的肌管融合率与肌管直径分别为5.69%和12.35 μm，显著低于非转化成肌细胞（10.21%和18.5 μm）（P<0.05）；Realtime RT-PCR结果显示，MSTN过表达成肌细胞中的Myogenin、p21、MyoD-基因表达显著下调，Smad3基因表达极显著上调（P<0.01）。【结论】 成功构建了细毛羊MSTN基因慢病毒表达载体，MSTN基因对细毛羊成肌细胞的分化具有显著的抑制作用，确定了MSTN基因与Myogenin、p21、MyoD及Smad3基因在成肌细胞分化过程中的调控关系。

Construction of lentiviral vector for sheep MSTN gene and its effects on ovine myoblast differentiation

【Objective】 The study was to construct the lentiviral expression vector for MSTN gene of Xinjiang Merino Sheep and investigated the influence and mechanism of MSTN gene on ovine myoblast differentiation.【Method】 The coding region of MSTN gene was amplified by RT-PCR and cloned into lentiviral expression vector plex-mcs.The constructed vector was identifed by restrictive enzyme and sequencing.Primary ovine myoblasts of Xinjiang Merino Sheep was cultured by enzyme digestion method,overexpression of MSTN cell line by lentiviral transfection primary ovine myoblast was prepared.Through inducing differentiation of horse serum,we used Western blotting to detect the MSTN protein expression in differentiated ovine myoblast,immunofluorescence staining to determine the myogenic fusion index and myotube diameter and Realtime RT-PCR to assay the related genes expression in differentiation condition.【Result】 Cloned MSTN gene(1 128 bp CDs sequence) of Xinjiang Merino Sheep showed 99.9% nucleotide homology with sequence reported in NCBI and existed a single base mutation in exon 2.Western blotting detected the MSTN protein stable expression in overexpression ovine myblast cell line.Immunofluorescence staining determined that the myogenic fusion index and myotube diameter of overexpression ovine myblasts respectively were 5.69% and 12.35 μm,significantly lower than 10.21% and 18.5 μm of non-transfected cells(P<0.05).The Realtime RT-PCR assay indicated that MSTN obviously down-regulated the expression of MyoD,Myogenin and p21 genes,but up-regulated Smad3 expression.【Conclusion】 Lentiviral expression vector for MSTN gene was successfully constructed,ovine MSTN inhibits differentiation of ovine myoblasts,and ascertained the relationship of expression regulation between MyoD,Myogenin,p21,Smad3 genes and MSTN gene under differentiation condition.