摘要
从小龙虾(Procambarus clarkii:pc)的肌肉组织中提取其总RNA,设计特异性引物,通过反转录聚合酶链反应(RT-PCR)克隆出编码小龙虾钙结合蛋白(sarcoplasmic calcium-binding protein:SCP)基因片段,扩增片段为582bp.该序列与已报道的几类虾SCP序列比对结果表明:在氨基酸水平上同源性为81%～96%.将SCP基因与pET-28a载体连接,构建了原核表达载体pET-28a-pcSCP重组质粒,转化E.coliBL21(DE3),经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,SDS-PAGE结果表明:表达蛋白约为22kDa,与预期的相对分子质量大小相符.并用Ni2+亲和层析柱对重组变应原进行纯化,得到了纯度较高的pcSCP.研究重组蛋白pcSCP的获得,为pcSCP变应原性及相关的研究奠定基础.
The gene of Procambarus clarkii sarcoplasmic calcium-binding protein （pcSCP） was amplified by RT-PCR using gene specific primers from total RNA extracted from Procambarus clarkia muscle.The 582 bp amplified DNA fragment encoded a protein sequence with identity of 81% to 96% with several published shrimp SCP in amino acids.The gene was inserted into pET-28a expression vector and the resulting pET-28a-pcSCP was transformed into E.coli BL21（DE3）.The recombinant protein was highly expressed in DE3 with a molecular weight about 22 kDa upon induction of IPTG and purified by 6-His-tag purification system.The study paves the way to allergenicity study of pcSCP.
出处
《江西师范大学学报（自然科学版）》
CAS
北大核心
2010年第6期567-571,共5页
Journal of Jiangxi Normal University(Natural Science Edition)
基金
国家自然科学基金(30871752)
深圳出入境检验检疫局科技计划(SZ2008105)
深圳市深港创新圈资助项目
关键词
小龙虾
钙结合蛋白
基因表达
Procambarus clarkii
sarcoplasmic calcium-binding protein
expression
