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[绵羊卵泡抑素基因表达及卵泡抑素结构域的功能分析]

[Ovine Follistatin gene expression and functional analysis of follistatin domains].

作者信息

Zhang Ning, Zhang Xuemei, Liu Mingjun, Tan Lixin

机构信息

Key Laboratory of Animal Biotechnology of Xinjiang, Urumqi 830000, China.

出版信息

Sheng Wu Gong Cheng Xue Bao. 2010 Aug;26(8):1050-6.

Abstract

In order to study ovine follistatin function, we amplified the total of 1038 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.

摘要

为了研究绵羊卵泡抑素的功能,我们通过逆转录聚合酶链反应(RT-PCR)从绵羊卵巢RNA中扩增出1038个碱基对的绵羊完整卵泡抑素cDNA,并克隆到pGEM-T载体中。去除信号肽后,将其亚克隆到pET41a中构建原核表达载体,命名为pFSsig-。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质免疫印迹法(Western blotting)鉴定出卵泡抑素cDNA表达的66 kDa产物。基于完整的编码序列(CDS),我们通过聚合酶链反应(PCR)克隆了卵泡抑素N端结构域和结构域1,并将其插入pLEX-MCS慢病毒载体中,命名为pFS-N+D1。慢病毒在293T细胞中包装和传代后,感染绵羊原代肌肉细胞(SPMC)。通过蛋白质免疫印迹法检测SPMC中FS N+D1的表达。感染的SPMC和未感染的对照细胞的细胞生长曲线显示,稳定转染FS N+D1的SPMC增殖明显快于对照细胞(P < 0.01)。我们的数据推断,绵羊FS N+D1结构域具有刺激绵羊肌肉细胞生长的功能。

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