Development of an antibody-basd assay for determination of baculovirus titers in 10 hours

摘要

The baculovirus expression system has been used to produce large amounts of biologically active proteins by infecting insect cells with a recombinant baculovirus expressing the target protein.For an efficient expression of the target ptotein.For an efficient expression of the target protein,it is necessary toinfect insect cells with an adequate amount of virus.However,current methods are time-consuming and either have technical difficulties or are limited as a result of virus expression mechanism using a reporter gene.A novel method is developed to yield virus titersin 10h that is easy to perform using 96-well plates and applicable to both any Autographa californica nucleopoly hyderovirus and Bombyx mori nucleopolyhed rovirus (BmNPV)-based recombinant baculovirus.This assay sues an antibody to a DNA-binding proteinto detect the infected cells via immunostaining.The titer is determined by counting foci produced as a result of was shortened considerably because infected cells expressed viral antigens at the post-infection time of 4h.Therefore,10h was enough to estimate the virus titer including virus infectiontime,insect cell culture,and estimationof virus titer.Titers determined using this immunological assay are comparable,both in value and validity,to those otained using a traditional method,provided that the stocks have titers avove 10~3 pfu/mL.