摘要
为探讨裂殖壶菌烯合酶基因的克隆,以及在大肠杆菌中的表达。采用简并引物与RACE相结合的方法从裂殖壶菌(Schizochytrium limacinum)中克隆出鲨烯合酶基因。分析表明该基因的cDNA全长包括1672个核苷酸,编码一个含446个氨基酸的开放阅读框;在裂殖壶菌中以单拷贝的形式存在。同源分析显示裂殖壶菌鲨烯合酶的氨基酸序列中含有5个保守基序。将裂殖壶菌鲨烯合酶的cDNA序列连接到原核表达载体pGEX-4T-3上构建融合表达载体,并在大肠杆菌BL21中进行IPTG诱导表达,SDS-PAGE及Western blotting分析结果显示,诱导表达出的融合蛋白主要以包涵体的形式存在,其相对分子质量与预期相符;且融合蛋白具有一定的免疫原性。
A full length cDNA of squalene synthase （SQS） gene was isolated from Schizochytrium limacinum using homologybased cloning and RACE methods. Molecular biological analysis showed that the full length cDNA was of 1672 bp,with a 1338 bp open reading frame encoding 446 amino acids,and the SQS gene was a single copy gene in Schizochytrium limacinum. Homology analysis showed that Schizochytrium limacinum SQS cDNA had five consensus regions. The SQS cDNA was constructed into the prokaryotic expression vector pGEX-4T-3 and expressed in E.coli BL21. SDS-PAGE and Western blotting analyses showed that the recombinant protein was mainly in the form of inclusion body,of which molecular weight was as expected,and had good immunogenicity.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2010年第19期263-267,共5页
Food Science
基金
鲁东大学校基金项目(LY20063307)
鲁东大学学科建设经费资助项目
关键词
裂殖壶菌
鲨烯合酶
CDNA克隆
原核表达
Schizochytrium limacinum
squalene synthase
cDNA cloning
prokaryotic expression
