尼罗罗非鱼绿色荧光蛋白/GH融合表达载体的构建及其在NIH293T细胞中的表达  

Construction of pEGFP/GH Expression Vector of Nile Tilapia and Expression of GH/EGFP in NIH293T Cells

作　　者：王茂元[1] 张平 邹芝英[3] 李大宇[3] 杨弘[3] 

机构地区：[1]福建省淡水水产研究所,福州350002 [2]睢宁县水产技术指导站,江苏睢宁221200 [3]中国水产科学研究院淡水渔业研究中心农业部淡水鱼类遗传育种和养殖生物学重点开放实验室,江苏无锡214081

出　　处：《中国农学通报》2011年第7期385-389,共5页Chinese Agricultural Science Bulletin

基　　金：国家科技支撑计划专题(2006BAD01A1202);2007年公益性行业(农业)科研专项(nyhyzx07-044);"国家重点基础研究发展计划973计划"项目(2004CB117401);国家罗非鱼产业技术体系(nycytx-48-14)

摘　　要：从尼罗罗非鱼(Oreochromis niloticus)脑垂体中分离其总RNA,经反转录合成为第一条cDNA链,再以第一条cDNA为模板,经过RT-PCR,克隆得到全长785bp的生长激素基因,序列分析表明:GH开放阅读框为615bp,共编码204个氨基酸;其中含有17个氨基酸的信号肽和187个氨基酸的成熟GH序列。同时将其插入到载体启动子下游,与增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因融合,重组质粒经菌液PCR、限制性内切酶酶切和测序进行鉴定。通过脂质体转染分别将pEGFP-GH重组表达载体、pEGFP-N1转到293T细胞中,以检测尼罗罗非鱼GH基因表达的活性。结果显示,阳性对照组pEGFP-N1,有荧光表达的细胞占90%以上;pEGFP-GH可见绿色荧光,但是有荧光表达的细胞较少,强度较弱。上述试验现象表明,所获得的尼罗罗非鱼GH基因能够在真核细胞中表达,因此具有较好的表达活性。Total RNA was isolated from Nile tilapia pituitary gland.Then we cloned the cDNA encoding growth hormone from the Nile tilapia pituitary by Reverse Transcription-Polymerase Chain Reaction （RT-PCR）,sequence of 785 bp.The result of sequence analysis and restriction map showed that an open reading frame of tilapia growth hormone gene contains 615 base pairs which code for a polypeptide of 204 amino acids including 17 amino acids of the signal peptide and 187 amino acids of the mature growth hormone.Nucleotide sequence and amino acid sequence of tilapia growth hormone gene were as same as Rentier-Delrue ＇ s tilapia GH cDNA in code region.The objective fragment was inserted into the downstream of the carrier promoter and combined with the report gene EGFP.The combinant plasmid was verified by PCR of the bacterium liquid,restriction enzyme digestion and gene sequencing.The pEGFP-GH recombinant was transfected into 293T cells by lipofection respectively.The results showed that the expressing fluorescent cells were more than 90% by pEGFP-N1;but the pEGFP-GH was weaker intensity.The phenomena showed that the Nile tilapia GH gene has good activity in eukaryotic cells.

关 键 词：尼罗罗非鱼 生长激素基因 荧光蛋白 转染 

分 类 号：Q786[生物学—分子生物学]