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Abstract #22049 Published in IGR 10-4
Construction of eukaryotic expression vector for pathogenic gene MYOC of primary open-angle glaucoma and its expression in COS-7 cells
Wang J-W; Liu J-J; Zhou W-YChinese Journal of Biologicals 2008; 21: 675-678
OBJECTIVE: To construct the eukaryotic expression vector for pathogenic gene MYOC of primary open-angle glaucoma (POAG) and express in COS-7 cells. METHODS: MYOC cDNA was amplified from human eye tissue (comes limbus) by RT-PCR, purified and cloned into vector pGEM-T, then subcloned to eukaryotic expression vector pEGFP-N3. Transfect COS-7 cells with the constructed recombinant plasmid pEGFP-N3-MYOC, observe the expression of MYOC protein under fluorescent microscope and identify the expressed product by Western blot. RESULTS: Both restriction analysis and DNA sequencing proved that recombinant plasmid pEGFP-N3-MYOC was constructed correctly. Fluorescent microscopy showed that MYOC protein was expressed and located in cytoplasm, while green fluorescent protein (GFP) was distributed in the whole cells. Western blot revealed that expressed MYOC protein could be secreted to the extracellular region. CONCLUSION: The eukaryotic expression vector for MYOC gene was successfully constructed and expressed in COS-7 cells, which laid a foundation of further study on pathogenic mechanism of POAG. LA: Chinese
Dr. J.-W. Wang. Department of Ophthalmology, First Hospital of Harbin Medical University, Harbin 150001, China
