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在乙脑病毒SA14-14-2株复制子载体pPartial△prM/E中克隆入DV2(Dengue virus serotype 2)的prM/E基因,构建乙脑/登革2型嵌合体克隆。将嵌合体克隆线性化后体外转录,获得的RNA转染BHK-21细胞,5～7d可观察到CPE。收获病毒上清液分别感染BHK-21细胞及C6/36细胞。接种于C6/36细胞中的嵌合病毒可使细胞出现CPE,RT-PCR、间接免疫荧光和Western blot检测显示:获得的嵌合病毒具有预期嵌合性核酸并能表达DV2的包膜蛋白,但不能在BHK-21细胞中传代培养。成功构建的乙脑/登革2型感染性克隆为进一步研究登革病毒疫苗奠定了基础。
The prM / E gene of DV2 (Dengue virus serotype 2) was cloned into the replicon vector pPartialΔ prM / E of JE virus SA14-14-2 to construct JE / Dengue type 2 chimera clone. After chimera cloning was linearized and transcribed in vitro, the obtained RNA was transfected into BHK-21 cells and CPE was observed from 5 to 7 days. The virus supernatant was harvested to infect BHK-21 cells and C6 / 36 cells, respectively. The results of RT-PCR, indirect immunofluorescence and Western blot showed that the obtained chimeric virus had the expected chimeric nucleic acid and expressed the envelope protein of DV2, But can not be subcultured in BHK-21 cells. The successfully constructed JE / dengue 2 infectious clone laid the foundation for further research on dengue virus vaccine.