一种用于大规模蛋白质表达的杆状病毒滴度快速测定方法(英文)OA

The baculovirus expression vector system(BEVS)is widely used for expressing target proteins and requires efficient baculovirus titration to ensure the output.Traditional plaque assay-assisted titration,while reliable,is labor intensive and time consuming.Here,we introduce an improved quantitative real-time polymerase chain reaction(qPCR)-based method for determining baculovirus titers.Briefly,first,target gene-specific primers are designed to produce a 180-bp PCR product of known molecular weight from the target gene cloned into the viral DNA.The PCR products,denoted as the control DNA,are purified and quantified.Second,the measured mass concentration is converted to the molar concentration,which is then converted to the number of molecule per unit volume using the Avogadro constant.Next,the control DNA was 10×serially diluted and used as the template for qPCR.The qPCR CT value and log10 value of the molecule number of each 10×serial dilution are plotted to generate the standard curve.Finally,the number of virus particle(i.e.,titer)of the test viral DNA is inferred from its qPCR CT value by comparison with the standard curve.Our method eliminates the need for a pretitrated viral stock,reducing variability caused by storage conditions.This advancement is particularly beneficial for large-scale production of the target protein using BEVS.