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

文献类型： 外文期刊

第一作者： Zheng, Xuchen

作者： Zheng, Xuchen;Tong, Wu;Liu, Fei;Liang, Chao;Gao, Fei;Li, Guoxin;Tong, Guangzhi;Zheng, Hao;Tong, Guangzhi;Zheng, Hao

作者机构：

关键词： Japanese encephalitis virus;Genetic instability;Prokaryotic promoter

期刊名称：VIRUS GENES （ 影响因子：2.332； 五年影响因子：2.0 ）

ISSN：

年卷期：

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收录情况： SCI

摘要： 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 A degrees C. However, the 5'-untranslated region and half of the 3'-E gene could cause the instability of the 2913-nt cDNA at 37 A degrees 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 A degrees C, and the activity declined markedly at 25 A degrees 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.

分类号： R37

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