fat-1基因密码子优化及在家兔胎儿成纤维细胞中的初步表达  

Codon optimization of fat-1 gene and its expression profiles in rabbit fetal fibroblast cells

作　　者：周红梅[1] 王颖[1] 张立凡[1] 华绪川[1] 王争光[1] 徐宁迎[1] 郭晓令[1] 

机构地区：[1]浙江大学动物科学学院,浙江杭州310029

出　　处：《江苏农业学报》2011年第2期329-336,共8页Jiangsu Journal of Agricultural Sciences

基　　金：国家"863"计划项目(2007AA10Z1A1)

摘　　要：根据家兔基因密码子使用偏好性,对引自GenBank的秀丽隐杆线虫fat-1基因编码序列进行密码子优化,通过全基因合成的方法获得优化后的fat-1基因（命名为opfat-1）;从秀丽隐杆线虫中RT-PCR扩增获得fat-1。将扩增的fat-1和合成的opfat-1基因分别构建到绿色荧光蛋白真核表达载体pEGFP-C1中。采用脂质体Lipo-fectamineTM转染法将fat-1和opfat-1基因转入家兔胎儿成纤维细胞（Rabbit fetal fibroblast cells,rFFCs）中。对比不同DNA和转染试剂用量以及细胞暴露于DNA、转染试剂中作用时间探讨影响rFFCs转染效率的参数,经过优化,最优的rFFCs转染条件为：LipofectamineTM用量2~3μl,DNA量0.8~1.0μg,LipofectamineTM与DNA、细胞共培养时间为8 h。通过绿色荧光标记和RT-PCR检测转染细胞中融合蛋白和目的基因的表达。密码子优化的opfat-1与fat-1相比,体外瞬时表达效率有极为显著的提高;G418抗性筛选获得转基因单克隆细胞,RT-PCR初步验证fat-1和opfat-1基因均在家兔胎儿成纤维细胞中稳定转录表达。Based on the biased codon usage of rabbit,Caenorhabditis elegans fat-1 coding sequence referred from GenBank was optimized and synthesized,and fat-1 coding sequence was amplified from C.elegans by RT-PCR.The optimized fat-1（named as opfat-1） and the amplified fat-1 coding sequence were cloned into green fluorescent protein expression vector pEGFP-C1 and transfected to rabbit fetal fibroblast cells（rFFCs） using LipofectamineTM,respectively.The impact factors were compared on transfection efficiency,including DNA,liposome dose as well as exposure time of the cells to DNA-liposome complexes.The optimized transfection condition was 2-3 μl LipofectamineTM and 0.8-1.0 μg DNA for 8 h transfection.The expressions of fusion protein and target genes were verified by GFP marker and RT-PCR assay.Transfection efficiency of opfat-1 was significantly increased compared with the wild type gene（fat-1 coding sequence）.The monoclonal cell lines were obtained by G418 screening.Initial experiments demonstrated fat-1 coding sequence and opfat-1 gene were both transcripted steadily in the rabbit fetal fibroblast cells by RT-PCR.

关 键 词：fat-1基因 密码子优化 转染 家兔胎儿成纤维细胞 

分 类 号：S829.1[农业科学—畜牧学]