加州鲈卵泡抑素cDNA的克隆、分析和原核表达

卵泡抑素是TGF-β超家族中一些成员的抑制蛋白，具有促肌肉生长的作用。采用RT-PCR和RACE技术从加州鲈成鱼卵巢总RNA中扩增得到卵泡抑素cDNA，序列分析结果表明，加州鲈卵泡抑素cDNA全长1444bp，包括开放阅读框966bp，5¢非编码区82bp，3¢非编码区359bp。该基因编码321个氨基酸，其中信号肽31个氨基酸，成熟肽290个氨基酸，成熟蛋白由四个功能区组成，分别为：N-domain、Domain Ⅰ、Domain Ⅱ和Domain Ⅲ，其中N-domain具有与TGF-β超家族中一些成员特异性结合的结构，可抑制这些蛋白发挥作用。将加州鲈卵泡抑素氨基酸序列与红鳍东方鲀、草鱼、斑马鱼、大西洋鲑和斑点叉尾鮰比较，同源性分别为97％、89％、88％、88％和70％，其中N-domain氨基酸序列的保守性更高一些，与红鳍东方鲀、草鱼、斑马鱼、大西洋鲑、斑点叉尾鮰、非洲爪蟾、人、猪、大鼠、鸡的相比较，同源性为75%～100％。为获得重组卵泡抑素蛋白，将成熟肽cDNA插入表达载体pET-32a(+)，转化大肠杆菌，IPTG诱导表达，SDS-PAGE检测，结果检测到一分子量约52kD的特异蛋白带，与预期大小一致，Western blot检测到表达产物表明成功获得了卵泡抑素的融合蛋白。加州鲈卵泡抑素cDNA序列和重组蛋白的获得为进一步研究鱼类卵泡抑素的促肌肉生长奠定了基础。

Cloning and Analysis of Largemouth Bass (Micropterus salmoides) Follistatin cDNA and Its Expression in Escherichia coli

Follistatin inhibits the biological action of several TGF-β family members and promotes muscle growth. In this research largemouth bass Follistatin cDNA was isolated. First, total RNA was isolated from ovary of adult female largemouth bass, and then used to clone the Follistatin cDNA by using RT-PCR and rapid cDNA end amplification. The sequence analysis results showed that the full-length of largemouth bass Follistatin was 1444 bp with an open reading frame(ORF) of 966 bp. The deduced ORF amino acid sequence contained a putative signal peptide of 31 amino acids and a mature peptide of 290 amino acids that was composed of four domains including N-domain, DomainⅠ, Domain Ⅱand Domain Ⅲ. Comparing Follistatin mature peptide of largemouth bass with those of torafugu, grass carp, zebrafish, Atlantic salmon and channel catfish, the results showed that the amino acid homology is 97％, 89％, 88％, 88％ and 70％, respectively. Follistatin protein binds with several TGF-β family members through its N-domain. Comparing Follistatin N-domain of largemouth bass with those of torafugu, grass carp, zebrafish, Atlantic salmon and channel catfish, African clawed toad, human, pig, rat and chicken, the results showed that the amino acid homology is 75%~100％. It was clear that the sequences of N-domain showed more conservation than those of mature peptide, which suggested that it was highly restricted in the process of evolution and its functional importance. In order to gain Follistatin fusion protein, Follistatin mature peptide cDNA was inserted into pET-32a(+) vector, which was transformed into BL21 and induced by IPTG. Expression products were detected by SDS-PAGE and confirmed by Western blotting analysis, which showed that the molecule weight of Follistatin fusion protein was about 52kD. This study will benefit further researching of Follistatin promoting muscle growth.