绿色荧光蛋白在IGF2b基因慢病毒载体感染鲤受精卵中的标记效果  

Flag results of GFP in fertilized eggs infected by lentiviral vector contained common carp(Cyprinus carpioL.) IGF2b

作　　者：苏胜彦[1,2] 陈为先[1] 马佳璐[1] 张勇[1] 胡鹏晨[1] 闻剑旻[1] 张成锋[2] 董在杰[1,2,3] 袁新华[1,2] 徐跑[1,2] 

机构地区：[1]南京农业大学无锡渔业学院,江苏无锡214081 [2]中国水产科学研究院淡水渔业研究中心农业部淡水渔业种质资源利用重点实验室,江苏无锡214081 [3]南京农业大学动物科技学院,江苏南京210095

出　　处：《上海海洋大学学报》2012年第3期331-336,共6页Journal of Shanghai Ocean University

基　　金：现代农业产业技术体系建设专项(CARS-46);国家“十一五”科技支撑计划项目(2006BAD01A1208);中央级公益性科研院所基本科研业务费专项(2009JBFB01);江苏省自然科学基金(BK2010146)

摘　　要：以水母绿色荧光蛋白基因为模板进行PCR扩增得到目的基因(Green fluorescence protein,GFP),然后加上酶切位点BamHI和NheI,构建pLenti6.3-IRES-EGFP载体,转染DH5α感受态细胞进行菌落PCR,取阳性进行酶切鉴定,再取呈阳性的质粒进行测序,使用浓度为1μg/μL的质粒与慢病毒表达载体进行连接,通过荧光显微镜观察到绿色荧光,表明本实验获得的GFP和慢病毒载体整合成功;用此转染293T细胞,通过荧光显微镜同样检测到了绿色荧光。使用建鲤的组织提取RNA,然后按照Fermentas公司的M-MLV操作说明书进行反转录,得到IGF2b基因后加酶切位点进行扩增,将IGF2b整合到用GFP作为标记基因的慢病毒载体上,再以此转染建鲤未分裂的受精卵,48 h后通过荧光显微镜也观察到了绿色荧光蛋白的表达。试验表明绿色荧光蛋白在IGF2b基因慢病毒载体感染鲤受精卵中的标记是成功的。这些结果为基于含有GFP慢病毒转基因鱼育种技术的开发奠定了基础。The vector pLenti6.3-IRES-EGFP was established using cloned jellyfish GFP gene and connected Barn HI and Nhe I restrictionsites. Then, the vector was approved by transfecting the DH52 competent cells, restriction enzyme digestion and sequencing. After that, 1 p,g/p,L plasmid was connected with lentiviral vector. To explore the GFP marked effect flag on lentiviral vector mediated transgenetic fish production, this article observes the expression of GFP using fluorescence microscope after infecting 293 T cells by lentiviral vectors which contained only GFP and contained both GFP and fuctional gene at cell levels; at vivo level, the expression of GFP during the developmented fertilized fingers of Jian carp which were infected by lentiviral vector containing the GFP and IGF2b . The results showed that： the green fluorescence was found in 293T cells infected by both GFP contained vector and GFP-IGF2b contained vector. After 48h, the expression of GFP was also observed when lentiviral vector（Lenti-IGF2b-IRES-EGFP） was infecting the undivided fertilized eggs from bright field and dark field. So, the GFP can be used successfully to flag the integrated effect through observing the green fluorescence in vivo when the undivided fertilized egg was infected by Lenti- IGF2b-IRES-EGFP. These illustrated that this study may lay a solid foundation for developing the transgenetic fish breeding based on the lentiviral vector contained GFP.

关 键 词：绿色荧光蛋白 慢病毒载体 IGF2b 标记效果 

分 类 号：S917[农业科学—水产科学]