蚊浓核病毒介导的人工内含子在蚊体内外的初步应用  

Preliminary Application of a Mosquito Densovirus-Mediated Artificial Intron in vitro and in vivo of Mosquito

作　　者：王衍海[1] 赖植发 顾金保[3] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052 [2]深圳福田区疾病预防控制中心,深圳518040 [3]南方医科大学公共卫生与热带医学学院,广州510515

出　　处：《病毒学报》2010年第5期379-384,共6页Chinese Journal of Virology

基　　金：国家自然科学基金(No.30771871)

摘　　要：根据人β-球蛋白基因第一内含子5′-端供位序列与免疫球蛋白重链可变区基因3′-端受位序列构建人工内含子,将人工内含子插入重组蚊浓核病毒质粒载体p7NS1-GFP中GFP融合表达的病毒非结构蛋白NS1的编码框内,构建成载体p7NS1-Intron-GFP,与辅助载体pUCA共转染蚊C6/36细胞系,荧光显微镜下观察细胞内GFP的表达情况。纯化、回收共转染后形成重组病毒与野生病毒,共感染白纹伊蚊二龄幼虫并观察幼虫体内GFP的表达情况。结果在C6/36细胞与蚊幼虫体内均观察到GFP的高效表达,证实人工内含子无论在蚊细胞系内还是在活体幼虫内均可正常行使其自我剪切的功能,未影响到下游蛋白GFP的表达。本研究为人工内含子在蚊虫细胞内的应用奠定基础,为蚊虫及其病原体相关基因工程技术提供新的策略。An artificial intron consisting of the 5′-donor site（from the first intron of the human β-globin gene） and the 3′-acceptor site（from the intron of an immunoglobulin gene heavy chain variable region） was obtained with a splice overlap extension PCR and was then inserted in frame into the coding sequence of nostructural protein NS1 gene fused to GFP gene in a recombinant mosquito densovirus plasmid p7NS1-GFP.The constructed plasmid was named as p7NS1-Intron-GFP.The plasmid p7NS1-Intron-GFP was cotransfected with the helper plasmid pUCA into C6/36 cells,then the packaged recombinant and wild type viruses were purified and recovered.The second-instars of Aedes albopictus larvae were exposed to recombinant and wild type virus mixed stock.The high level GFP expression in C6/36 cells and larvae was observed under fluorescence microscope,indicating that the inserted artificial intron exerted its normal function in self-splicing both in vitro and in vivo.This study laid a foundation for application of an artificial intron in insect cells and development of new strategy for genetic engineering technology of mosqtuito and its pathogens.

关 键 词：人工内含子 蚊浓核病毒 白纹伊蚊 

分 类 号：R373.3[医药卫生—病原生物学] Q78[医药卫生—基础医学]