大鲵铁蛋白重链FTH基因的克隆、鉴定及表达分析  

CLONING, IDENTIFICATION AND EXPRESSION OF FERRITIN HEAVY CHAIN FROM CHINESE GIANT SALAMANDERS, ANDRIAS DAVIDIANUS

作　　者：杨辉[1,2] 李锋刚[1,2] 蓝青景 胡伟[1] 刘小林[1] 王立新[1,2] 

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100 [2]陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《水生生物学报》2014年第1期27-34,共8页Acta Hydrobiologica Sinica

基　　金：陕西省科技攻关(2012K01-18);西北农林科技大学基本科研业务费专项(QN2011062);西北农林科技大学后稷学者支撑计划(Z11021007)资助

摘　　要：从大鲵皮肤cDNA文库中,筛选出大鲵铁蛋白重链AdFTH的cDNA序列,其全长为864 bp,开放阅读框为531 bp,编码176个氨基酸,5′和3′非翻译编码区(Untranslated region,UTR)分别为120 bp和214 bp,预测蛋白质的分子量为20.6 kD,理论等电点PI为5.41,预测蛋白无信号肽及跨膜结构,在5′-UTR的22—51 bp处有一个特殊的铁反应元件(Iron response element,IRE)。同源性和系统进化分析表明,铁蛋白重链在进化上有着较高的保守性。实时定量PCR结果表明,大鲵铁蛋白重链mRNA在各个组织中广泛表达,其中肝脏的表达量高于其他8种组织,表明肝脏是大鲵主要的参与铁储存代谢的器官。成功构建了大鲵铁蛋白重链的重组表达载体pET32a-AdFTH,利用大肠杆菌Escherichia coli BL21表达系统和Ni2+亲和层析方法,获得了纯度较高的重组大鲵铁蛋白,并证实重组大鲵铁蛋白(Recombinant AdFTH protein,rAdFTH)具有氧化和吸收铁离子的功能,为进一步制备AdFTH抗体了解其在大鲵体内的多种作用机制打下坚实的基础。Ferritin is an important iron storage protein in organism with the function of detoxification, anti-inflammatory and anti-stress. In this study, the full length cDNA ofAndrias davidianus ferritin heavy chain （AdFTH） was isolated from the constructed skin cDNA library, and it consists of 864 bp including an open reading frame （ORF） of 531 bp, a 5′-terminal untranslated region （UTR） of 120 bp, and a 3′-UTR of 214 bp. The predicted molecular weight is 20.6 kD and the theoretical isoelectric point is 5.41 with no amino-terminal signal peptide and transmembrance do-main. A complete iron-responsive element （IRE） locates at the 5′-UTR corresponding to the nucleotide sequence at the positions of the 22-51 bp. The homology and phylogenetic analysis of FTH among other animals indicated that it is an evolutionarily conserved gene. The results of quantitative real time PCR demonstrated thatAdFTH was ubiquitously expressed, and the highest level ofadFTH was observed in the liver from total 9 tissues, which may support that the liver is the major organ for the storage and metabolism of iron inAndrias davidianus. Additionally, a recombinant ex-pression vector pET32a-AdFTH was constructed, and the recombinant protein was purified using the expression system inE. coli BL21 （DE3） pLysS and Ni2＋-chelating chromatography. The purified recombinant AdFTH protein （rAdFTH） promoted iron oxidation and iron uptakein vitro, suggesting that the rAdFTH protein may be used to produce the monoclonal antibodies and provide a foundation for further investigation of the physiological function of AdFTH.

关 键 词：大鲵 铁蛋白重链 序列分析 原核表达 组织分布 

分 类 号：Q513[生物学—生物化学]