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

作　　者：张宁[1,2] 张雪梅[1,2] 刘明军[1,2] 谭立新[1,2] 

机构地区：[1]新疆维吾尔自治区动物生物技术重点开放实验室,乌鲁木齐830000 [2]新疆畜牧科学院农业部草食家畜繁育生物技术重点开放实验室,乌鲁木齐830000

出　　处：《生物工程学报》2010年第8期1050-1056,共7页Chinese Journal of Biotechnology

基　　金：新疆维吾尔自治区高技术研究发展计划(No.200611106)资助~~

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FollistatincDNA的完整开放阅读框(1038bp)。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FSsig-蛋白(66kDa)。通过RT-PCR克隆了包含N端和结构域1的Follistatin突变体(FSN+D1),将FSN+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FSN+D1的肌肉细胞系,通过细胞生长曲线结果显示稳定表达FSN+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FSN+D1结构域有促进肌肉细胞生长的功能。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.

关 键 词：绵羊Follistatin基因 原核表达 慢病毒 细胞生长曲线 

分 类 号：S826[农业科学—畜牧学] Q78[农业科学—畜牧兽医]