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.
出处
《水产学报》
CAS
CSCD
北大核心
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)
关键词
鲢
生长激素
CDNA
表达载体
大肠杆菌
Hypophthalmichthys molitrix
growth hormone cDNA
expression vector
Escherichia coli
