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

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1 μg/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。

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

The vector pLenti6.3-IRES-EGFP was established using cloned jellyfish GFP gene and connected Bam HI and NheⅠ restrictionsites.Then,the vector was approved by transfecting the DH52 competent cells,restriction enzyme digestion and sequencing.After that,1 μg/μL 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.