CAJ | 학술논문

目的构建乙脑病毒SA14-14-2株复制子载体,为进一步研究以乙脑病毒为载体的新型疫苗奠定基础.方法 (1)构建了两个乙脑病毒复制子:一个为完全缺失PrM/E基因(命名为Full △prM/E Replicon);一个保留E基因C端213 bp(命名为Partial △prM/E Replicon),并代之以多克隆位点.(2)将复制子RNA转染BHK-21细胞,于24、48、72、96 h采用Real-time PCR验证复制子的自主复制能力.(3)于复制子多克隆位点处插入YFP报告基因,并将含报告基因的复制子RNA转染BHK-21细胞,采用荧光显微镜观察及流式细胞仪检测验证YFP的表达.结果 (1)两个复制子RNA转染BHK-21细胞后,RNA有随时间增加而不断增多的趋势.(2)含报告基因的复制子RNA转染BHK-21细胞后,荧光信号持续增强,表达YFP的阳性细胞率也逐渐增多.结论构建的乙脑病毒SA14-14-2株复制子载体Full △prM/E Replicon及Partial △prM/E Replicon具有自主复制能力及对外源蛋白的表达能力.
목적 구건을뇌병독SA14-14-2주복제자재체,위진일보연구이을뇌병독위재체적신형역묘전정기출.방법 (1)구건료량개을뇌병독복제자:일개위완전결실PrM/E기인(명명위Full △prM/E Replicon);일개보류E기인C단213 bp(명명위Partial △prM/E Replicon),병대지이다극륭위점.(2)장복제자RNA전염BHK-21세포,우24、48、72、96 h채용Real-time PCR험증복제자적자주복제능력.(3)우복제자다극륭위점처삽입YFP보고기인,병장함보고기인적복제자RNA전염BHK-21세포,채용형광현미경관찰급류식세포의검측험증YFP적표체.결과 (1)량개복제자RNA전염BHK-21세포후,RNA유수시간증가이불단증다적추세.(2)함보고기인적복제자RNA전염BHK-21세포후,형광신호지속증강,표체YFP적양성세포솔야축점증다.결론 구건적을뇌병독SA14-14-2주복제자재체Full △prM/E Replicon급Partial △prM/E Replicon구유자주복제능력급대외원단백적표체능력.
Objective In order to lay the groundwork for studying the novel vaccine Identified.Methods (1)Two replicons were constructed.One's prM/E gene was deleted completely(Full △prM/E Replicon),the other's prM/E gene was deleted partially(213 bp of C terminal of E gene was reserved;PaaiM △prM/E Replicon),and the deleted parts Was replaced as the MCS.(2)Replicons RNA were which will use the JEV as the vector,replicon vectors of JEV was constructed and transfected into BHK-21 cell.After 24,48,72,96 h,method of real-time PCR was used to identify Replicons'replication ability.(3)YFP gene was inserted into the MCS of those two replicons.Their RNA was transfected into BHK-21 cell.Expression of YFP was tested by the fluorescence microscopy and flow cytometer.Results (1)After the two replicons RNA were transfected into BHK-21 cell,as time went by,the quantity of RNA inereased.(2)After RNA of the replicons with YFP were transfected into BHK-21cell,increasing trend of fluorescent signal and rate of YFP positive cell was observed and tested.Conclusion Full △prM/E Replicon and Partial △prM/E Replicon have the ability to duplicate itself and express the foreign protein.