重组人载脂蛋白A5原核表达载体的构建及分析鉴定  

Construction of prokaryotic expression vector of recombinant human apolipoprotein A5 and its analysis and identification

作　　者：魏红霞[1,2] 张葵[2] 李雷[2] 邱方[2] 顾光煜[2] 倪军[2] 

机构地区：[1]东南大学临床医学院,南京210008 [2]南京大学医学院附属南京鼓楼医院检验科,南京210008

出　　处：《临床检验杂志》2009年第1期53-56,共4页Chinese Journal of Clinical Laboratory Science

基　　金：南京市科技发展重点项目(ZKM06055)

摘　　要：目的构建重组人载脂蛋白A5(apoA5)原核表达载体,为进一步制备抗apoA5的单/多克隆抗体奠定基础。方法从人肝癌组织中抽提总RNA,以此为模板,逆转录降度PCR扩增载脂蛋白A5编码区基因全长,构建含有目的片段的克隆载体pPCR-ScriptampSK(+)-apoA5及原核表达载体pET16B-apoA5,通过酶切、测序及诱导表达等验证重组质粒的准确性。结果PCR扩增出1.1kb特异性片段,克隆至pPCR-ScriptampSK(+)后测序,结果表明序列同源性与genebank报道一致,重组质粒pET16B-apoA5经双酶切后电泳显示约为5.7kb和1.1kb的片段,酶切图谱与预期一致,诱导表达蛋白的大小与预期一致。结论成功构建了重组人载脂蛋白A5的原核表达载体。Objective To construct a prokaryotic expression vector of recombinant human apolipoprotein A5 （ apoA5 ） for the preparation of specific antibody to apoAS. Methods The total RNA was extracted from human hepatoma tissue as template, and reverse transcription polymerase chain reaction （RT-PCR） was performed to amplify the full length of coding region of ApoA5 gene. The cloning vector pPCR-Script ampSK（ ＋ ）-apoA5 and expression vector pET16B-apoA5 including the objective fragment were constructed, and the cor- rectness of recombinant plasmid was verified by enzyme incision, electrophoresis and induced expression. Results A specific fragment with 1 100 bp was amplified by PCR, and cloned into pPCR-Script-ampSK（ ＋ ）. The homology of sequence was confirmed by compa- ring to GeneBank. The electrophoresis of recombinant plasmid pET16B-apoA5 after enzyme incision displayed 2 DNA fragments with 5 700 bp and 1 100 bp. The magnitude of expressed apoA5 induced by IPTG was as expected. Conclusions The prokaryon expression vector of recombinant human apoA5 was constructed successfully.

关 键 词：载脂蛋白A5 表达载体 

分 类 号：Q789[生物学—分子生物学]