鲢生长激素cDNA表达载体构建及在大肠杆菌中的表达  

Construction of expression vector of growth hormone cDNA from Hypophthalmichthys molitrix and its expression in Escherichia coli

作　　者：陈松林[1] Y.Hong M.Schartl 

机构地区：[1]中国水产科学研究院黄海水产研究所,农业部海洋渔业资源可持续利用重点开放实验室,山东青岛,266071 [2]Biocenter of University Wuerzburg,Wuerzburg 97074

出　　处：《水产学报》2002年第3期201-205,共5页Journal of Fisheries of China

基　　金：农业部"九五"重点渔业科技项目 (渔 95 -B -96 -0 7-0 3)

摘　　要：应用RT—PCR技术从白鲢垂体mRNA中扩增出GH全长cDNA片段 ,长度 1.2kb。将 1.2kb白鲢GHcDNA克隆到载体质粒pBluescriptKSII +(pBSKSII +)中 ,构建了pBS -scGHcDNA克隆 ;通过含有NdeI酶切位点的特异引物扩增出不含信号肽序列的cDNA片段 ,将其重组到pRSET5b载体质粒中 ,构建了白鲢GHcDNA表达质粒pRSET -scGH。将此表达质粒转化BL2 1(DE3)大肠杆菌 ,经IPTG诱导后 ,在转化的大肠杆菌中检测到GH蛋白的存在。Western印迹表明该蛋白带与草鱼GH单克隆抗体具有强烈的免疫反应。Total RNA was isolated from silver carp (Hypophthalmichthys molitrix) pituitaries. The cDNA fragment of 1.2 kb encoding growth hormone (GH)was amplified by reverse transcription polymerase chain reaction (RT-PCR) using total RNA as template. The amplified cDNA was inserted at the EcoRI site in vector pBluescipt KSII+ and resulted in plasmid pBS-scGHcDNA. GH cDNA fragment of 1 kb without signal peptide sequence was amplified using pBS-scGHcDNA as template and introduced to expression vector pRSET5b, generating scGH expression plasmid pRSET5b-scGH. The bacteria BL21(DE3)plys were transfected with pRSET5b-scGH. The expression of scGH peptide of 22kD in the transfected BL21(DE3)plys was detected by PAGE. Western blotting demonstrated that the recombinant scGH could specially react with monoclonal antibody to grass carp (Ctenopharyngodon idellus ) GH.

关 键 词：鲢 生长激素 CDNA 表达载体 大肠杆菌 

分 类 号：S961.6[农业科学—水产养殖] S965.113[农业科学—水产科学]