绵羊Follistatin基因表达及其结构域的功能分析

Ovine Follistatin gene expression and functional analysis of follistatin domains

摘要:

为研究羊Follistatin基因的功能，提取了绵羊卵巢总RNA，通过RT-PCR方法获得羊Follistatin cDNA的完整开放阅读框 (1 038 bp)。去除信号肽序列后与原核表达载体pET41a连接，构建重组表达质粒pFSsig?，经大肠杆菌诱导表达获得FS sig?蛋白 (66 kDa)。通过RT-PCR克隆了包含N端和结构域1的Follistatin突变体 (FS N+D1)，将FS N+D1片段插入慢病毒载体 (pLEX-MCS) 构建了pFS-N+D1慢病毒重组表达质粒。在293T细胞中进行慢病毒的包装，再感染绵羊肌肉原代细胞，得到稳定表达FS N+D1的肌肉细胞系，通过细胞生长曲线结果显示稳定表达FS N+D1的肌肉细胞明显比正常肌肉细胞增殖快，且差异极显著 (P＜0.01)，表明绵羊FS N+D1结构域有促进肌肉细胞生长的功能。

Abstract:

In order to study ovine follistatin function, we amplified the total of 1 038 base pair of ovine complete follistatin cDNA and cloned into pGEM-T vector by RT-PCR from ovine ovary RNA. After removal of the signal peptide it was subcloned into the pET41a to construct the prokaryotic expression vector, named pFSsig?. SDS-PAGE and Western blotting identified the 66 kDa product of the expression of follistatin cDNA. Based on the complete CDS sequence, we cloned follistatin N-terminal domain and domain 1 with PCR and inserted into pLEX-MCS lentiviral vector, named pFS-N+D1. After package and passage of lentivirus in 293T cells, and then infected sheep primary muscle cells (SPMC). The expression of FS N+D1 in SPMC was assayed by Western blotting. The cell growth curve of the infected SPMC and noninfected control cells displayed that FS N+D1 stablly transfected SPMC proliferated significantly faster than the control cells (P<0.01). Our data inferred that ovine FS N+D1 domain had the function to stimulate sheep muscle cell growth.