学术
人lrp-cDNA全长编码区的克隆及其在大肠杆菌中的表达
宋庆贺 柴玉波 陈苏民 陈南春 黄勇 代忠明
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
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，将其克隆人原核表达载体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。[著者文摘]
文章出处:
《第四军医大学学报》-2006年27卷5期 -385-388页
栏目信息:
分 类 号:
文献标识码:
A
文章编号:
1000-2790（2006）05-0385-04
Cloning of full-length coding region of human lipopolysaccharide responsed gene cDNA and its expression in E. coli
SONG Qing-He, CHAI Yu-Bo, CHEN Su-Min, CHEN Nan-Chun, HUANG Yong, DAI Zhong-Ming （Department of Biochemistry and Molecular Biology, School of Basic Medicine, Fourth Military Medical University, Xi＇an 710032, China）
Abstract:
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.[著者文摘]
Key words:
lipopolysaccharide; responsed gene; reverse tran-scriptase-polymerase chain reaction ; gene expres-sion
基金资助:
国家自然科学基金（30170361）
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