人lrp-cDNA全长编码区的克隆及其在大肠杆菌中的表达  

Cloning of full-length coding region of human lipopolysaccharide responsed gene cDNA and its expression in E.coli

作　　者：宋庆贺[1] 柴玉波[1] 陈苏民[1] 陈南春[1] 黄勇[1] 代忠明[1] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710033

出　　处：《第四军医大学学报》2006年第5期385-388,共4页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金(30170361)

摘　　要：目的:扩增人lrp-cDNA全长编码区、并在大肠杆菌中表达和鉴定.方法:提取脂多糖刺激后HEK293细胞的总RNA,通过RT-PCR的方法扩增出全长人lrp序列,将其克隆入原核表达载体pcTAT后转化大肠杆菌诱导表达,做SDS-PAGE分析;并用免疫印迹法鉴定6His-TAT-LRG融合蛋白表达.结果:测序结果表明,获得了全长人lrp-cDNA全长编码区,其序列与GenBank已经公布的不完全一致;SDS-PAGE分析表明,6His-TAT-Lrp融合蛋白在大肠杆菌中成功表达,表达量约占菌体总蛋白的17%;免疫印迹法鉴定显示,该融合蛋白可与相应抗血清产生阳性反应.结论:得到人lrp-cDNA全长编码区序列,并成功表达,为人lrp功能的深入研究奠定了基础.AIM： To done, express and identify full-length human lipopolysaccharide （ LPS ） responsed gene （ lrp ） -cDNA coding sequence. METHODS： Total RNA was extracted from LPS-stimulated human embryonic kidney cells （ HEK293 ） and the full-length human lrp-cDNA sequence was obtained by RT-PCR. The lrp-cDNA coding sequence was cloned into pcTAT fusion expression vector, then transferred into E. coli BL21 and induced to express with IPTG. The expressed fusion protein （6His-TAT- Lrp） was analyzed by SDS-PAGE and Western blot. RESULTS： DNA sequencing result showed that the lrp-cDNA coding sequence we cloned was not exactly consistent with that issued by GenBank. SDS-PAGE analysis demonstrated that the 6His-TAT-Lrp fusion protein was expressed successfully in E. coli. The fused protein band amounted to 17% of the total bacteria protein and the expressed protein reacted with antisera. CONCLUSION： Human lrp-cDNA full-length coding sequence is successfully cloned and expressed, which offers a basis for further research of lrp function.

关 键 词：脂多糖 应答基因 逆转录聚合酶链反应 基因表达 

分 类 号：Q78[生物学—分子生物学]