Effect Of Radix Isatidis Preparation On Nucleoprotein Gene Of Influenza Virus

Objective:To study the different preparations of Isatis on nucleoprotein (NP) gene of influenza virus in vitro,we recombinant DNA plasmid pcDNA3.1 (+) / NP transfected Hela cell. This finding provides a novel therapy to anti-influenza A virus.Methods:The Radix decoction and Isatis Agglutinin were separately decocted and concentrated by boiled leaching and organic solvent immersion method. Isatis injection was purchased. Different concentrations of every drug were added respectively into cultured medium of Hela cells. In the Hela cells culture experiment in vitro, the cytotoxic influence of different concentrations diluted at multiple proportions was studied to determine the highest non-toxic concentration. The next diluted concentraion of the hightest non-toxic concentration was choosed to be the experiment concentration. Expression vector pcDNA3.1 (+) / NP was transfected to Hela cells by the liposome-mediated for 48h. Indirect immunofluorescence and colloidal gold immunochromatography assay were used for determining the expression of NP gene. Experiment group consist of Group 1: the Isatis injection; Group 2: the Radix decoction; Group 3: the Isatis agglutinin; Group 4: the plasmid transfection; control Group: Drug control, cells control, empty vector control. With indirect immunofluorescence and colloidal gold immunochromatography, it was studied the expression of NP gene.Results1. Respectively, the Isatis test concentrations were: Isatis injection 1.95mg/ml, Radix decoction 2.60mg/ml, Isatis Agglutinin 0.78mg/ml.2. Colloidal gold immunochromatography determined the expression of NP gene on Hela cells. And strong green fluorescent by indirect immunofluorescence to assess the expression of NP gene in Hela cells.3. Use of Colloidal gold immunochromatography, Group 1, Group 2 and Group4 appeared two red bands on the test paper card as the positive results, others the negative results. And a strong green fluorescent was appeared in Group 1, Group 2 and Group 4 by indirect immunofluorescence to assess the expression of NP gene in Hela cells, no others.Conclusion1. Immunofluorescence and colloidal gold chromatography confirmed successful trasfect ion NP expression in Hela cells.2. Isatis Agglutinin had apparent inhibition effect to expression of NP gene, neither of Isatis injection and water decoction.This study proved that Isatis Agglutinin had apparent inhibition effect to NP gene expression in vitro,and it may provide some certain experience basis for the therapy of clinical anti—influenza A virus.