红鳍东方鲀Sirt1基因的克隆及其原核表达

Cloning and prokaryotic expression of Sirt1gene from torafugu Takifugu rubripes

【目的】克隆红鳍东方鲀Sirtuin1(Sirt1)基因编码阅读框(ORF),并对其进行原核表达。【方法】采集红鳍东方鲀脂肪组织,提取其总RNA,采用RT-PCR方法扩增和克隆红鳍东方鲀Sirt1基因的ORF,构建其原核表达载体pET32a/Sirt1,并在大肠杆菌Rosetta(DE3)中进行表达。【结果】红鳍东方鲀Sirt1基因ORF区由2 070个核苷酸组成,编码689个氨基酸。根据红鳍东方鲀Sirt1ORF核苷酸序列推测的氨基酸序列与其他物种进行比对,发现其与罗非鱼(Oreochromis niloticus)、非洲齿鲤(Nothobranchius furzeri)、科恩氏假鳃鳉(Nothobranchius kuhntae)、斑马鱼(Danio rerio)、人(Homo sapiens)和小鼠(Mus musculus)的同源性分别为82%,82%,81%,66%,72%和69%;成功构建了重组质粒pET32a/Sirt1,用IPTG进行诱导表达,SDS-PAGE电泳结果显示,在约105ku处有特异性的蛋白条带出现。【结论】克隆得到红鳍东方鲀Sirt1基因的ORF序列,并成功对其进行了原核表达。

【Objective】 The study aimed to clone and analyse Sirtuin1（Sirt1）gene in torafugu,then to study the prodaryotic expression of Sirt1.【Method】 The ORF of torafugu Takifugu rubripes Sirt1 gene was amplified from total RNA of torafugu adipose tissue by reverse transcription-polymerase chain reaction（RT-PCR）.The prokaryotic expreion vector pET32a/Sirt1 was constructed and expressed in E.coli Rosetta（DE3）.【Result】 The full-length open reading frame of torafugu Sirt1 cDNA consisted of 2 070 bp and encoded a polypeptide of 689 amino acids.The amino acid identities of Takifugu rubripes Sirt1 to Oreochromis niloticus Sirt1,Nothobranchius furzeri Sirt1,Nothobranchius kuhntae Sirt1,Danio rerio Sirt1,Homo sapiens Sirt1 and Mus musculus Sirt1 were 82%,82%,81%,66%,72% and 69%,respectively.The prokaryotic expression system of recombined vector pET32a/Sirt1 was construted successfully.SDS-PAGE analysis showed that the expressed protein accumulated as inclusion bodies and the molecular weight of expressed fusion protein was 105 ku.【Conclusion】 The ORF of torafugu Takifugu rubripes Sirt1 gene was cloned and expressed in E.coli successfully.