| Summary: | 碩士 === 國立成功大學 === 微生物暨免疫學研究所 === 89 === DNA microarray technology allows scientists to address simple description about global gene expressions inside cells. In particular, it offers a sensitive and fast way to elucidate potential host genes whose expressions can assist or defend the invasion of a pathogen. The goal of the entire research project is to identify host genes that regulate enterovirus 71 (EV71) and herpes simplex virus type 1 (HSV-1) infections using cDNA microarray technology. Throughout these experiments, I have found that the expression of many genes, including egr-1, cd44, vdup1, GST and PML, changed during the infections of EV71 strain 4643 and/or HSV-1 strain kos in a neuroblastoma cell line SK-N-SH, suggesting that they are involved in the infection processes. The change of gene expressions was confirmed by RT-PCR. Among the identified genes, egr-1 (early growth response protein 1), whose transcriptional activity has been associated to proliferation and differentiation, was found to be induced similarly in SK-N-SH cells infected with EV71 or HSV-1, indicating that egr-1 plays a significant role in a common regulatory pathway for virus transmission. We therefore selected this gene for further characterization on the promoter activities regulated by EV71 or HSV-1. For this, I constructed the serial deleted egr-1 promoters, as follows: pEgr-1/-712 is the pGL3 luciferase-based plasmid containing the full-length egr-1 promoter (-712 to +6) cloned before the luciferase gene, whereas pEgr-1/-550, pEgr-1/-402, pEgr-1/-239, and pEgr-1/-129 were pGL3 inserted with the respective egr-1 promoter fragments from -550, -402, -239,or -129, to +6. These promoter constructs were then transfected into SK-N-SH cells to assay the activities of the egr-1 promoter regulated by viral infections. The result of the luciferase assay showed that the egr-1 promoter region between -402 ~ -550 as the most important region for basal expression of the protein in the cell line. The cis-regulatory element(s) in the egr-1 promoter that was responding to the induction by EV71 infection was located between -402 ~ -550, whereas the region regulated by HSV-1 was located further upstream of -550 ~ -712. In parallel, I investigated the physiological functions of the proteins including EGR-1, CD44, VDUP1, and PML-1, whose gene expressions were altered, in virus infections by using antisense technology. When some of these gene expressions were blocked, the virus proliferation would be limited. The results showed that the production of EV71 or HSV-1 was respectively decreased by he blockage of gene expression of cd44 or egr-1. My experiments have gained an insight into the molecular regulatory mechanism of virus infection mediated by these genes, which may further be used for research and development of anti-viral drugs.
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