奶牛载脂蛋白B100基因克隆及原核表达

摘要

本研究根据GenBank已收录奶牛ApoB100基因序列,设计特异性引物获得目的基因.经pGM-T载体克隆,对重组质粒进行BamHⅠ和EcoRⅠ双酶切鉴定并测序,序列同源性达100％;将目的基因连接到pET-28a表达载体中,提取质粒,转化到Rosetta(DE3)中,筛选的阳性克隆经IPTG诱导.SDS-PAGE初步分析表明,成功获得分子质量为35 ku的蛋白质；Western blotting结果呈阳性,表明通过本试验成功获得了目的蛋白.%Specific primers were designed according to ApoB100 gene sequence reported in GenBank in this study. Cloning was carried out by the pGM-T vector, recombined plasmid DNA was cut by BamH Ⅰ and EcoR Ⅰ enzymes and then sequencing. The recombinant expression vector pET~28a-ApoB100 was constructed with target gene and pET-28a vector, and transformed into Roseua(DE3) ,then induced by IPTG. The expression product was identified by SDS-PAGE and Western blotting. The results showed that the homology of the cloned ApoB100 gene was 100% to that reported in GenBank. SDS-PAGE analysis showed that the molecular weight of recombinant protein pET~28a-ApoB100 was 35 ku. Western blotting positive results showed that the experiment successfully obtained the target protein.