Huang L S, Bock S C, Feinstein S I, Breslow J L
Proc Natl Acad Sci U S A. 1985 Oct;82(20):6825-9. doi: 10.1073/pnas.82.20.6825.
An expression library made in plasmids pUC8 and pUC9 with mRNA derived from the human hepatoma cell line HepG2 was screened with a rabbit antiserum to human low density lipoprotein (LDL). Approximately 12,000 clones were screened and five positives were identified. The cDNA inserts were all 1500-1600 base pairs in length. The insert from one clone, pB8, was isolated, labeled by nicktranslation, and found to cross-hybridize strongly with the other four cDNA clones. The pB8 clone produces a fusion protein of approximately equal to 37.5 kDa that reacts in electrophoretic transfer blot analysis with rabbit anti-human LDL. This reactivity can be abolished by pretreatment of the antiserum with purified human LDL, p = 1.025 - 1.050 g/ml. A pB8-derived probe was used to demonstrate that apolipoprotein B (apo B) mRNA is present in HepG2 cells and liver extracts but not in HeLa cells or extracts from small intestine, heart, aorta, spleen, brain, skeletal muscle, lung, kidney, or ovary. RNA transfer blot analysis revealed that HepG2 cell apo B mRNA was approximately equal to 22 kilobases in length. These cDNA clones should allow the isolation of the apo B gene and ultimately the elucidation of the primary structure of this protein.
用源自人肝癌细胞系HepG2的mRNA构建于质粒pUC8和pUC9中的表达文库，用兔抗人低密度脂蛋白（LDL）抗血清进行筛选。筛选了约12,000个克隆，鉴定出5个阳性克隆。cDNA插入片段长度均为1500 - 1600个碱基对。从一个克隆pB8中分离出插入片段，通过缺口平移进行标记，发现它与其他四个cDNA克隆强烈交叉杂交。pB8克隆产生一种约37.5 kDa的融合蛋白，在电泳转移印迹分析中与兔抗人LDL发生反应。用纯化的人LDL（p = 1.025 - 1.050 g/ml）预处理抗血清可消除这种反应性。用源自pB8的探针证明载脂蛋白B（apo B）mRNA存在于HepG2细胞和肝提取物中，但不存在于HeLa细胞或小肠、心脏、主动脉、脾脏、大脑、骨骼肌、肺、肾脏或卵巢的提取物中。RNA转移印迹分析显示HepG2细胞apo B mRNA长度约为22千碱基。这些cDNA克隆应有助于分离apo B基因，并最终阐明该蛋白的一级结构。
