Genetic instability of Japanese encephalitis virus cDNA clones propagated in Escherichia coli

Abstract

The genetic instability of Flavivirus cDNA clones in transformed bacteria is a common phenomenon. Herein, a cDNA fragment of the nucleotide (nt) 1–2913 of the genome of a flavivirus, Japanese encephalitis virus (JEV), was used to investigate factors that caused the instability of cDNA clones. Several cDNA fragments with different 5′- or 3′-termini of the 2913-nt cDNA were obtained by PCR amplification or restriction enzyme digestion and cloned into a pCR-Blunt II-TOPO vector. All the cDNA fragments were stably propagated at 25 °C. However, the 5′-untranslated region and half of the 3′-E gene could cause the instability of the 2913-nt cDNA at 37 °C. The 5′-terminus sequences of the 2913-nt fragment were subjected to testing of the prokaryotic promoter activity by luciferase assay and Western blot. The sequences of 54–120 nt of the JEV genome exhibited high prokaryotic promoter activity at 37 °C, and the activity declined markedly at 25 °C. These findings revealed that the high prokaryotic promoter activity of the 54–120 nt sequences of the JEV genome together with expression of JEV structural genes determined the instability of a JEV cDNA clone. Growth at room temperature may reduce the prokaryotic promoter activity of 5′-sequences of the JEV genome and could represent an effective way to improve the stability of flavivirus cDNA clones in host bacteria.