Molecular Cloning Of OAS Gene Promoter And Construction Of PGL3-Basic Eukaryotic Expression Vector Under OAS Promoter And A Scientific Analysis To Its Activity In L02 Expressing The HCV Core Protein

Objective:To clone OAS gene promoter and construct pGL3-Basic eukaryotic expression vector under OAS promoter,and then analysis its activity in L02 expressing the HCV core protein.Methods:①Adding restriction enzyme sites HindⅢand SacⅠ,OAS gene promoter region(-159～+82)according to the literature was amplified by polymerase chain reaction.②Right size band was gel received and digested by Sacl and Hindlll enzyme as well as pGL3-Basic vector,Then OAS promoter gene fragment was relegated with linearized pGL3-Basic vector to construct pGL3-Basic eukaryotic expression vector bearing OAS gene promoter(OAS promoter-pGL3-Basic),which was further confirmed by double enzyme digestion.③OAS Promotor-pGL3-Basic vector was transiently transfected into L02 cells which expressed the HCV core protein using liposome transfection reagent and the activity of OAS gene promoter was determined by adding luciferase substrate into transfected cells 72 hours later.Results:①Restriction enzyme analysis and sequencing result showed that the sequence of this gene fragment was identical with OAS promoter gene sequence reported in genbank.②the OAS promoter-pGL3-Basic eukaryotic expression vector was confirmed by restriction enzyme digestion and sequencing.③the activity of luciferase reporter gene was 84±9 in L02 and 443±14 in L02 which expressedthe HCV core protein 72 hours after transfection.The bright of luciferase of the latter is 4.5 times than those of the former.Conclusion:①About 241bp OAS gene fragment was successfully amplified by PCR method and OAS promoter-pGL3-Basic eukaryotic expression vector was successfully constructed.②It is confirmed that HCV core protein can active the OAS promoter.③OAS promoter we constructed can start up the expression of a downstream target gene efficiently and specifically.④This study laid a foundation for gene therapy of hepatitis C.