Huang Ying, Liu Shan, Yang Peng, Wang Chao, Du Yun, Sun Zhiwei, Yu Weiyuan
Laboratory for Protein Engineering, Institute ofBiotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
Sheng Wu Gong Cheng Xue Bao. 2010 Aug;26(8):1088-94.
To optimize a self-replicate Japanese enciphalitis virus (JEV) replicon, and to make it as an efficient vector to express the heterologous protein, we constructed three JEV replicons by PCR-based shortening the length of capsid genes. The vectors remained full or part of C gene, based on the JEV replicon pCTCJEV. Lac Z was selected as the reporter gene to verify the self-replicate ability of these DNA-based replicons. While transfected into the cell lines CME-4, which continuously expressing the JEV structure proteins C-prM-E, the JEV replicons pCMW-2M-1LACZ, pCMW-2M-3LACZ, which remained the first 23aa and 68aa of C protein, can express the reporter protein as the same level as pCMW-2M-LACZ with the full-length C protein. These results illustrated that the JEV replicon vector with 69-nt of the C gene can retain the self-replicate ability, and provide valuable tools to construct a possible vector for a long-lasting JEV RNA virus expression system.
为优化一种自我复制的日本脑炎病毒（JEV）复制子，并使其成为表达异源蛋白的高效载体，我们通过基于PCR缩短衣壳基因长度构建了三种JEV复制子。基于JEV复制子pCTCJEV，这些载体保留了完整或部分C基因。选择Lac Z作为报告基因来验证这些基于DNA的复制子的自我复制能力。当转染到持续表达JEV结构蛋白C-prM-E的细胞系CME-4中时，保留C蛋白前23个氨基酸和68个氨基酸的JEV复制子pCMW-2M-1LACZ、pCMW-2M-3LACZ能够与具有全长C蛋白的pCMW-2M-LACZ以相同水平表达报告蛋白。这些结果表明，具有69个核苷酸C基因的JEV复制子载体能够保留自我复制能力，并为构建用于持久JEV RNA病毒表达系统的可能载体提供了有价值的工具。
