南移养殖刺参(Apostichopus japonicus)原肌球蛋白基因的原核表达研究

PROKARYOTIC EXPRESSION OF TROPOMYOSIN GENE FROM SOUTH CULTURED SEA CUCUMBER APOSTICHOPUS JAPONICUS

本研究克隆和表达了刺参原肌球蛋白(Tropomyosin,TRP)基因,进一步研究刺参再生过程中重要分子的功能。结果表明,TRP基因序列总长为1203bp,5′非翻译区为105bp,3′非翻译区为240bp,该序列包含一个855bp的开放阅读框(openreading frame,ORF),编码284个氨基酸,分子量为33.27kDa,等电点为4.56。利用Escherichiacoli对TRP进行了体外重组表达,在1mmol/LIPTG和37℃条件下诱导,能产生分子质量约为38kDa的重组蛋白,Westernblot证明重组TRP与鼠抗刺参原肌球蛋白的多克隆抗体能特异性结合。

Regeneration is a vital physiological process in Apostichopusjaponicus, and was considered to a good model for organ and tissues culture in vitro. In order to further understand the function of some key molecules in this process, the tropomyosin gene ofA. japonicus was cloned and expressed. The full-length of tropomyosin gene was 1203bp including a 105bp of 5＇ untranslated region （UTR）, a 240bp of 3＇ UTR and a 855bp of open reading frame （ORF） encoding a polypep- tide of 284 amino acids. The estimated molecular mass of tropomyosin gene is 33.27kDa, and the theoretical isoelectric point is 4.56. The recombinant pET-Trp protein was successfully expressed in E. coli. The recombinant protein pET-Trp had clearly visible band in 38kDa. The polyclonal antibody could specifically bind to the recombinant TRP by Western blot