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

    SONG Qingª²He, CHAI Yuª²Bo, CHEN Suª²Min, CHEN Nanª²Chun, HUANG Yong, DAI Zhongª²Ming

    Department of Biochemistry and Molecular Biology, School of Basic Medicine, Fourth MilitaryMedical University, Xi¬ðan 710032, China

    ¡¾Abstract¡¿ AIM: To clone, express and identify fullª²length human lipopolysaccharide (LPS) responsed gene (lrp)ª²cDNA coding sequence. METHODS: Total RNA was extracted from LPSª²stimulated human embryonic kidney cells (HEK293) and the fullª²length human lrpª²cDNA sequence was obtained by RTª²PCR. The lrpª²cDNA 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 (6Hisª²TATª²Lrp) was analyzed by SDSª²PAGE and Western blot. RESULTS: DNA sequencing result showed that the lrpª²cDNA coding sequence we cloned was not exactly consistent with that issued by GenBank. SDSª²PAGE analysis demonstrated that the 6Hisª²TATª²Lrp 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 lrpª²cDNA fullª²length coding sequence is successfully cloned and expressed, which offers a basis for further research of lrp function. ......