作　　者： ; ; ; ; ; ;

机构地区： 广东海洋大学农学院

出　　处： 《广东海洋大学学报》 2017年第1期95-100,共6页

摘　　要： 为研究热应激蛋白70(HSP70)在巴马香猪(Sus scrofa domestica)耐热中的作用,利用NCBI公布的猪Hsp70基因序列设计引物,用RT-PCR克隆巴马香猪Hsp70基因。核酸序列分析表明,巴马香猪Hsp70基因开放阅读框(ORF)长1 932 bp,编码643个氨基酸。该蛋白分子质量为71.1 ku,等电点(PI)为5.88,与野猪、人、牛、羊、犬和鸡的同源性分别为99.2%、82.8%、83.2%、83.6%、82.4%和77.7%。该蛋白非叶绿体转运肽和线粒体导肽,亦非信号肽分泌通道,存在多个明显的疏水区,其中397位疏水性最强,达2.211。将该基因的ORF与真核表达载体pc DNA 3.1/V5-HisTOPO连接构建真核表达载体,并转染IPEC-J2细胞,荧光定量PCR及Western blot检测可见其在基因和蛋白水平的高量表达。 In order to elucidate the role of heat stress protein 70 (HSP70) in heat resistance performance of Bama miniature pigs (Sus scrofa domestica), Hsp70 gene was cloned from spleen of Bama miniature pig by reverse transcription polymerase chain reaction (RT-PCR). The results showed that Hsp70 gene consisted of 1 932 bp, and coded 643 amino acids. The molecular weight of HSP70 protein was 71.1 ku and the isoelectric point was 5.88. The Hsp70 gene sequence had a high homology compared with thatof Sus scrofa (99.2%), Homo sapiens (82.8%), Bos taurus (83.2%), Capra hircus (83.6%), Canis lupus familiaris (82.4%) and Gallus gallus (77.7%). The protein is not a chloroplast transit peptide and mitochondrial peptide, also not a signal peptide secretion channel. There are many hydrophobic domains in HSP70 of Bama miniature pigs, and the most lyophobic point was located at AA397 (2.211).After connecting the open read frame (ORF) of the gene with pcDNA 3.1/V5-His TOPO for a eukaryotic expression vector of HSP70 and transfecting it into intestinal porcine epithelial cell (IPEC-J2), HSP70 was highly expressed in IPEC-J2 cells detected by fluorogenic quantitative PCR(qRT-PCR) and western blot.

关 键 词： 巴马香猪 基因 克隆 真核表达

领　　域： [生物学]