摘要
目的构建能同时表达轮状病毒vp2、vp6、vp7基因的重组家蚕杆状病毒,并在BmN细胞中进行初步表达。方法利用MA104细胞繁殖人A组轮状病毒,RT-PCR扩增病毒结构蛋白vp2、vp6、vp7基因,连接至转移载体pFBDM和pUCDM中,同时引入含IE1启动子的绿色荧光蛋白基因利于检测感染和表达量,再分别通过Tn7及Cre-LoxP重组构建重组病毒。为方便检测基因的表达,在VP7的C-末端添加6-组氨酸(6-His)标签,可通过ELISA等方法快速检测目的基因表达。结果扩增获得了轮状病毒三个结构蛋白基因,构建了能高效组装轮状病毒样颗粒的重组表达载体,ELISA检测VP7得到了表达,在BmN细胞中观察到病毒样颗粒。结论成功构建杆状病毒多基因的表达系统,为生产多亚基蛋白复合物,研究大分子结构提供借鉴。
Objectives To construct recombinant baculoviruses co-expressing three structural genes vp2, vp6 and vp7 of rotavirus, and assemble rotavirus-like particles （VLPs） in BmN cells. Methods Human group A rotavius was cultivated in MA104 cells, and the RNA was extracted and the three genes were obtained by RT-PCR. The PCR products were inserted into the transfer vectors pFBDM and pUCDM, respectively. A enhanced green fluorescent protein gene （egfp） driven by IE1 promoter was introduced into pFBDM to investigate the efficiency of infection. The expression baculoviruse was constructed by Tn7 and Cre-LoxP recombinant and transfected into BmN cells. The gene expression was determined by detecting 6-His tag fused into VP7 C-terminus, and the assembled VLPs were observed by transmission electron micrography. Results Three genes of rotavirus were cloned and BmMultiBac was constructed. The genes were expressed and the rotavirus-like particles assembled in BmN cells successfully as verified by ELISA and electron microscope. Conclusion We have successfully constructed the recombinant baculovirus co-expressing the 3 structural genes of rotavirus, which provide the basis for producing protein complex containing multiple subunits and investigation of the structure of the macromolecules.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2010年第7期1491-1495,共5页
Journal of Southern Medical University
基金
国家自然科学基金(30700750)
广东省自然科学基金(7006695)
华南农业大学校长基金(2008X002)
作者简介
龙虎（1984-），男，硕士生，电话：020—85280366，E-mail：maclong@yahoo．cn，主要研究方向：分子病毒学；
姚伦广（1974-），男，博士，副教授，电话：0377-63525085，E-mail：lunguangyao@163．com，主要研究方向：分子病毒学。姚伦广为共同第一作者
通讯作者：孙京臣（1971-），男，博士，副教授，电话：020-85280366，E-mail：cyfz@scau．edu．cn，主要研究方向：病毒分子生物学和结构生物学
