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.
出处
《临床检验杂志》
CAS
CSCD
北大核心
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)
关键词
载脂蛋白A5
表达载体
apolipoprotein AS
expression vector
