摘要
利用PCR扩增技术,从泥蚶的cDNA文库中克隆出了精氨酸激酶(arginine kinase)基因,并进行了原核表达。精氨酸激酶是调节能量代谢的重要酶类,在无脊椎动物体内能量平衡过程中起重要作用。结果表明,泥蚶的精氨酸激酶全长1601bp,包括5′非翻译区(5′-UTR)序列332bp,3′非翻译区(3′-UTR)序列246bp、1023bp的开放阅读框序列(ORF),编码340个氨基酸,具有精氨酸激酶典型的酶活性部位氨基酸序列(CPTNLGT)。构建了原核表达质粒AK-pET-28a(+)转化表达菌株,经IPTG诱导表达后,获得了与预期的分子量大小一致的表达产物。利用该目的蛋白制备了泥蚶精氨酸激酶的特异性多克隆抗体,经Western blot证明该蛋白为精氨酸激酶。
In vertebrate, arginine kinase is a phosphotransferase that plays a critical role in maintaining equilibrium in energy metabolism. We isolated full-length cDNA of arginine kinase from Tegillarca granosa. It is 1601 bp long, including 203 bp of 5＇ untranslated region （5＇-UTR） sequence and 438 bp of 3＇ untranslated region （3＇-UTR） sequence. It encodes 319 amino acids with arginine kinase activity in part of the amino acid sequence （CPTNLGT）. And the resulting product was then cloned into the expression vector. The prokaryotic expression plasmid AK-Pet-28a（＋） containing the intact ORF was constructed and the target protein was induced with IPTG. The overexpressed protein was with the expected size. This study provided information further built on the foundation in understanding the function of arginine kinase in energy metabolism and host defense.
出处
《海洋与湖沼》
CAS
CSCD
北大核心
2011年第1期119-123,共5页
Oceanologia Et Limnologia Sinica
基金
国家自然科学基金资助项目
30771665号
长江学者和创新团队发展计划资助项目
2007-2009
浙江省科技厅重点科研社会发展项目
2005C23083号
关键词
泥蚶
精氨酸激酶
基因克隆与表达
多克隆抗体
Tegillarca granosa, Arginine kinase, Genic cloning and expression, Polyclonal antibody
