鸭FST基因编码区克隆、序列分析及其在大肠杆菌中的表达  

Molecular Cloning and Analysis of Duck Follistatin Gene CDS and Its Expression in Escherichia coli

作　　者：刘贺贺[1] 金海波[1] 王继文[1] 曹伟[1] 韩春春[1] 李亮[1] 许峰[1] 

机构地区：[1]四川农业大学畜禽品种资源发掘与利用重点实验室,雅安625014

出　　处：《畜牧兽医学报》2011年第2期190-195,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金："十一五"国家科技支撑计划重点项目(2008BADB2B08);四川省科技厅应用基础项目(2008JY0099);四川省教育厅重点项目(07ZA056)

摘　　要：本研究旨在获取鸭卵泡抑素(FST)外源蛋白以及进一步深入了解其在肌肉发育和繁殖上的作用,采用RT-PCR方法从鸭卵巢中克隆鸭FST基因CDS序列,并将编码FST成熟蛋白的核酸片段连入原核表达载体pET-32a(+)中,转化大肠杆菌(Escherichiacoli)并采用IPTG进行诱导,诱导后的蛋白经SDS-PAGE分析后用蛋白免疫技术鉴定。序列分析表明,FST基因编码区序列为1 032 bp,编码343个氨基酸,其中信号肽由28个氨基酸组成,成熟蛋白具有4个保守结构域:N-domain、DomainⅠ、DomainⅡ和DomainⅢ,其中DomainⅠ与鼠DomainⅠ结构较相似,DomainⅡ和DomainⅢ差异很大。SDS-PAGE电泳结果显示表达出的融合蛋白为52 ku,免疫鉴定结果呈阳性。结果表明,FST基因在3个功能结构域上存在差异,推测DomainⅠ主要与其肌肉发育有关,而DomainⅡ、DomainⅢ与鸭繁殖关系密切。本研究成功获取了鸭卵泡抑素蛋白,为进一步研究卵泡抑素在鸭肌肉发育以及繁殖中的功能奠定了基础。In order to gain the duck FST exogenous protein and to elucidate about its role in muscle development and reproduction,the total RNA was extracted from adult female duck and the FST coding domain sequence was cloned using RT-PCR method.The duck FST mature peptide cDNA was inserted into pET-32a（＋） vector,and transformed into Escherichia coli Rosetta,and then it was induced by IPTG,after that,the recombinant expression products were detected by SDS-PAGE and confirmed by Western blotting analysis.The results of sequence analysis showed that the full duck FST coding domain sequence was 1 032 bp,encoding 343 AA,and the deduced CDS amino acid sequence contains a 28 AA putative signal peptide.The FST amino acid sequence of duck was composed of four domains,including the N-domain,Domain Ⅰ,Domain Ⅱ and Domain Ⅲ.The follistatin Domain Ⅰ in FST was conservative between the duck and mouse,whereas there were many variations between DomainⅡand Domain Ⅲ.The results of SDS-PAGE showed that the molecule weight of induced protein was about 52 ku and display a positive reaction by Western blotting analysis.These results indicated that the three follistatin domains in duck have different roles,and it can be concluded that Domain Ⅰ is mainly related to muscle development,and the Domain Ⅱ and Domain Ⅲ are important for the animal reproduction.The duck FST protein has been gained successfully and these results will be benefit for further researching the effects of FST on promoting muscle growth and egg production of duck in the future.

关 键 词：鸭卵泡抑素 CDS 分子克隆 原核表达 

分 类 号：Q751[生物学—分子生物学] S834[农业科学—畜牧学]