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Molecular Cloning, Sequence Analysis and Prokaryotic Expression of Ovine Activin Receptor Type IIB(ActRIIB) Gene 被引量:1

绵羊激活素受体IIB(ActRIIB)基因的克隆、序列分析及原核表达
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摘要 Objective] This study aimed to clone ovine activin receptor type llB (Ac-tRIIB) gene, construct the prokaryotic expression vector and express the target gene in vitro, thus providing basis for further function verification. [Method] The template cDNA which was reversely transcribed from total RNA of sheep liver tissue, was subjected to polymerase chain reaction (PCR) using specific primers of ActRIIB. The ful-length cDNA of ovine ActRIIB was obtained by pMD18-T cloning and sequencing for bioinformatics analysis. Ovine ActRIIB encoding sequence was subcloned into prokaryotic expression vector pET41a with restriction sites BamHl/Notl, and then transformed into BL21 (DE3). The induced products by lPTG were analyzed with SDS-PAGE and Western Blot. [Result] The amplified ful-length cDNA of ovine Ac-tRllB gene was 1 564 bp in length (Genbank accession number: JX422071.1) with an open reading frame of 1 539 bp, encoding 512 amino acides. Ovine ActRllB shared the highest homology (99.6%) with bovine ActRllB. ActRllB had highly ho-mologous C-terminal domains and belonged to the TGFβ family. After prokaryotic expression, an approximately 92 kD His-tagged ActRllB recombinant protein was obtained, which was consistent with the excepted result. [Conclusion] Cloning and successful expression of ovine ActRIIB laid solid foundation for further investigation of its biological function. [目的]旨在克隆绵羊激活素受体IIB(ActRIIB)基因,并构建原核表达载体进行体外表达,为进一步验证功能奠定基础。[方法]以绵羊肝脏组织为材料,以提取总RNA反转录得到的cDNA为模板,利用同源序列克隆技术,对绵羊ActRIIB基因的cDNA全长进行克隆,并对其进行生物信息学分析。再根据克隆序列设计引物,将其与原核表达载体pET41a连接,构建重组表达质粒pET41a-ActRIIB,经IPTG诱导后进行表达鉴定。[结果]扩增获得绵羊ActRIIB基因cDNA全长1 564 bp(Genbank登陆号为:JX422071.1),最大开放阅读框为1 539 bp,共编码512个氨基酸;其氨基酸序列与牛的同源性最高(99.6%),ActRIIB的C末端区域高度同源且属于TGFβ家族。原核诱导表达获得符合预期大小(约92 kD)并带有组氨酸标签序列的ActRIIB重组蛋白。[结论]绵羊ActRIIB基因的克隆和表达为进一步研究其生物学功能奠定了基础。
出处 《Agricultural Science & Technology》 CAS 2014年第10期1644-1648,共5页 农业科学与技术(英文版)
基金 Supported by Natural Science Foundation of Xinjiang Uygur Autonomous Region(2012211B54)~~
关键词 SHEEP ActRIIB gene Sequence analysis Prokaryotic expression 绵羊 激活素受体 IIB基因 序列分析 原核表达
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