乙脑病毒SA14-14-2株复制子载体的构建及鉴定  

Identification and construction of replicon vectors of Japanese encephalits virus（ strain SA14-14-2）

作　　者：韦艳[1] 李川[1] 陆鹏[1] 于建石[1] 李建东[1] 刘琴芝[1] 张全福[1] 李德新[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室,北京100052

出　　处：《中华实验和临床病毒学杂志》2009年第6期418-420,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金项目（30770087）.

摘　　要：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 △prMJE Replicon）；一个保留E基因C端213bp（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、96h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。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 Replicon）, and the deleted parts was replaced as the MCS. terminal of E gene was reserved; Partial △prM/E （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 increased. （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.

关 键 词：脑炎病毒 日本 复制子 杂合子检测 

分 类 号：R3[医药卫生—基础医学]