Construction Of The Herpes Simplex Virus 1 ICP0 Eukaryotic Expression Vector And Its Effects Of Macrophage Secrete Activity

Objectives: To construct the eukaryotic expression vector of herpes simplex virus 1 ICP0(HSV-1 ICP0) and test its expression in macrophage cells. Then study the effects of GFP-ICP0 on LPS induced chang of macrophage secrete activity.Methods: Restriction enzyme digest PT7-110 , the fragment of ICP0 was subcloned into eukaryotic expression vector pEGFP-C1 , then the recombined vector pEGFP/ICP0 was transfected into macrophage. The expression of GFP-ICPO was analyzed by fluorescent microscope and Western blotting. TNF-a and IL-1β ELISA kits were respectively used to determine the effects of GFP-ICP0 protein transient expression on cytokines secretion induced by lipopolysaccharide of macrophage. Macrophages were stimulated with lipopolysaccharide to assay the production of NO with Griess Reagents.Results: ①The recombinant pEGFP/ICP0 was digested by Smal, a approximate 2300bp DNA fragment can be seen in agrose electrophoresis, it indicated that ICP0 fragment was already subcloned into pEGFP-Cl plasmid, namely, the eukaryotic expression vector pEGFP/ICP0 has been constructed successfully, pEGFP/ICP0 was transfected into macrophage, fluorescent microscope and western blotting identified specially the expression of GFP-ICP0 protein, result showed GFP-ICP0 can be expressed in macrophage. ② The secretory volume of cytokines from GFP-ICP0-stimulated macrophage was determined by ELISA, the results from analysis of variance showed as following: there is a remarkable difference between pEGFP/ICP0 group and lipopolysaccharide control group and pEGFP-C1 control group, but no remarkable difference between pEGFP-C1 control group and lipopoly — saccharide control group, all of the results indicated transient expression of...