论文部分内容阅读
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 green fluorescence protein (GFP) was amplified by PCR using the jellyfish GFP gene as a template, and then the pLenti6.3-IRES-EGFP vector was constructed by inserting the restriction sites BamHI and NheI into DH5α competent cells The colony PCR was carried out and the positive plasmids were identified by restriction enzyme digestion. The positive plasmids were sequenced. The plasmids were ligated with the lentivirus expression vector at a concentration of 1 μg / μL and the green fluorescence was observed by fluorescence microscope. And lentiviral vector integration success; use this transfection 293T cells, also detected by fluorescence microscopy of green fluorescence. RNA was extracted from Jian carp tissues and then reverse transcribed according to the M-MLV operating instructions of Fermentas. The IGF2b gene was amplified by restriction enzyme digestion, and IGF2b was integrated into lentiviral vector using GFP as a marker gene , And then transfected Jian carp unsplit egg, after 48 h by fluorescence microscopy also observed the expression of green fluorescent protein. Experiments show that green fluorescent protein in IGF2b gene lentivirus vector infected zygote fertilization eggs were successful. These results lay the foundation for the development of breeding techniques based on GFP lentivirus-containing fish.