綿羊Follistatin基因表達及其結構域的功能分析

張寧，張雪梅，劉明軍，譚立新

1 新疆維吾爾自治區動物生物技術重點開放實驗室，烏魯木齊 830000

2 新疆畜牧科學院 農業部草食家畜繁育生物技術重點開放實驗室，烏魯木齊 830000

動物及獸醫生物技術

綿羊Follistatin基因表達及其結構域的功能分析

張寧1,2，張雪梅1,2，劉明軍1,2，譚立新1,2

1 新疆維吾爾自治區動物生物技術重點開放實驗室，烏魯木齊 830000

2 新疆畜牧科學院 農業部草食家畜繁育生物技術重點開放實驗室，烏魯木齊 830000

為研究羊Follistatin基因的功能，提取了綿羊卵巢總RNA，通過RT-PCR方法獲得羊Follistatin cDNA的完整開放閱讀框 (1 038 bp)。去除信號肽序列后與原核表達載體pET41a連接，構建重組表達質粒pFSsig?，經大腸桿菌誘導表達獲得FS sig?蛋白 (66 kDa)。通過RT-PCR克隆了包含N端和結構域1的Follistatin突變體 (FS N+D1)，將FS N+D1片段插入慢病毒載體 (pLEX-MCS) 構建了pFS-N+D1慢病毒重組表達質粒。在293T細胞中進行慢病毒的包裝，再感染綿羊肌肉原代細胞，得到穩定表達FS N+D1的肌肉細胞系，通過細胞生長曲線結果顯示穩定表達FS N+D1的肌肉細胞明顯比正常肌肉細胞增殖快，且差異極顯著 (P＜0.01)，表明綿羊FS N+D1結構域有促進肌肉細胞生長的功能。

綿羊Follistatin基因，原核表達，慢病毒，細胞生長曲線

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.

Keywords:ovineFollistatingene, prokaryotes expression, lentivirus, cell growth curve

卵泡抑素 (Follistatin，FS) 是1987年自卵泡液 中分離的一種前垂體細胞分泌的對卵泡刺激素(Follicle stimulating hormone，FSH) 起拮抗作用的單鏈多肽，富含半胱氨酸，又名垂體促卵泡素抑制蛋白 (FsH-suppressing protein，FsP)，隨后發現FS能與激活素 (Activin) 結合，對卵母細胞的成熟和卵泡的發育[1]、精子的生成[2]、胚胎發育與分化、催產素的釋放等起著重要的調節作用。FS基因敲除小鼠研究表明FS基因的缺失能引起肌肉、皮膚等生長發育的異常[3]。……