.1 Human Lectin-like Oxidized Low-density Lipoprotein Receptor-1 Expression In Pichia Pastoris And Its Receptor Antagonist Screening Model For The Establishment Of The Preliminary Study Of The Two Taxadiene Synthase Gene Expression In Aspergillus Niger,

Oxidized low-density lipoprotein (oxLDL) plays an important role in the pathogenesis of atherosclerosis by eliciting endothelial dysfunction or activation. Recently, a novel endothelial oxLDL receptor, lectin-like oxLDL receptor-1 (LOX-1) has been identified. LOX-1 is uniquely expressed in the endothelial cells of large arteries, it binds, internalizes and proteolytically degrades oxLDL . Many evidences from in vivo and in vitro suggested that LOX-1 mediates broad bioactivities. It is believed that LOX-1 is a key molecule in the generation of atherosclerosis and might be as a target for novel anti-atherosclerotic drugs. In this paper, the recombinant human LOX-1 was successfully cloned and expressed in the heterologous host Pichia pastoris. The expressed protein exhibited the natural binding activity to its ligand ox-LDL and was used to set up a high throughput screening (HTS) model for the screening of LOX-1 antagonists.The human leukemic monocyte cell line THP-1 cells were incubated with 50 μM histamine for 3h at 37℃ in RPMI 1640 medium prior to RNA isolation. Total RNA was isolated from the treated cells using TRIzol reagent (Invitrogen, USA) and standard protocols as recommended. The encoding sequence of human LOX-1 was amplified by RT-PCR with the total RNA as a template. Amplicons were cloned into pMD18-T vector and the latter was introduced into E. coli strain DH5a. DNA sequencing and BLAST results indicated that the cDNA was homologous to the reported sequence in GenBank (accession number NM₀02543) with 100% identity. The human LOX-1 cDNA was inserted downstream of the a-signal peptide encoding sequence in pPIC9K, resulting in the recombinant plasmid pPIC9K-HL. After linearized by the restriction enzyme SacI, the recombinant plasmid was transformed into P. pastoris GS115 with PEG 1000.The His⁺Mut⁺ transformants were screened by using MD medium and the antibiotic G418. After identified by PCR, the transformants with correct phenotype were fermented for 4⁵ days with BMMC medium, induced by 0.8^l% methanol. The culture filtrate was analyzed by SDS-PAGE and a distinctive band, with a...