摘要
根据JEV病毒减毒株SA14-14-2基因组序列,设计覆盖全长的4对重叠引物,以提取的活疫苗病毒RNA为模板,RT-PCR扩增出4个片段,并克隆到质粒载体中,进一步构建两个半端分子克隆,然后将全长cDNA序列克隆到一个新改造的低拷贝质粒载体pBR-kpn中,构建我国流行性乙型脑炎病毒(JEV)基因组全长cDNA克隆。经过体外转录后得到的转录子转染BHK-21细胞,重新获得JEV的恢复病毒,通过生物学特性、分子生物学水平、蛋白水平等几个方面对恢复病毒进行鉴定。结果获得了稳定的全长cDNA克隆,转录子转染BHK-21细胞后,第4天开始出现细胞病变(CPE),第6～7天时CPE为 ,经过Vero细胞进一步放大培养后,间接免疫荧光实验和RT-PCR实验均为阳性。证实了构建的JEV的全长cDNA克隆有感染性,为进一步的研究奠定了基础。
Based on the published se quence of JEV SA_(14)-14-2 from GenB ank,four pairs of primers were des igned for amplifying four overlapp ed sequences of the virus by RT-PC R,and then they were cloned into v ectors.Subclones were ligated into a full-length sequence.A new modified plasmid vector pBR-kpn was constru cted by inserting a kpn restrictio n site and a T7 terminator sequenc e into a low copy plasmid pBR322.T his modified vector was used to cl one the full-length sequence.The f ull-length cDNA clone was transfec ted into BHK-21 cells,CPE was obser ved on day 4.Its infectivity was v erified by observation of CPE,indi rect immunofluorescene assay and R T-PCR.The construction of a stable Japanese encephalitis virus infect ious cDNA clone made the further r easerch more easier.
出处
《病毒学报》
CAS
CSCD
北大核心
2003年第4期313-319,共7页
Chinese Journal of Virology
