人lrp-cDNA全长编码区的克隆及其在大肠杆菌中的表达

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.

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

AIM: To clone, 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.