无抗性标记的人白血病抑制因子打靶载体的构建及表达  

Construction and Expression of Marker-free Targeting Vector of Human Leukemia Inhibitory Factor

作　　者：王韵斐[1] 陈秋菊[1] 崔易虹[1] 杨明明[1] 曹斌云[1] 

机构地区：[1]西北农林科技大学动物科技学院,羊分子育种实验室,杨凌712100

出　　处：《农业生物技术学报》2011年第5期960-966,共7页Journal of Agricultural Biotechnology

基　　金：国家高科技研究发展计划(863)项目(No.2007AA10Z167);陕西省13115重大科技创新专项(No.2009ZDKG-1a);国家科技支撑计划(No.2011BAD28B05-3)共同资助

摘　　要：为了实现人白血病抑制因子基因(hLIF)在奶山羊乳腺上皮细胞中的稳定表达,本实验利用条件重组元件LoxP序列、山羊β-酪蛋白5'和3'同源臂、正负向筛选标记基因和hLIF基因构建hLIF基因打靶载体。脂质体法转染奶山羊(capra hircus)乳腺上皮细胞,进行遗传霉素(G418)和丙氧鸟苷(GANC)筛选获得同源重组的阳性克隆,并通过PCR、实时定量PCR和Western blot检测阳性克隆中hLIF基因的表达情况。酶切和测序分析结果显示,实验成功构建了hLIF基因打靶载体pLoxP-NT53L。阳性克隆经PCR、实时定量PCR和Western blot均检测到了目的条带。表明打靶载体pLoxP-NT53L已经成功整合至山羊β-酪蛋白基因座中,并能在奶山羊乳腺上皮细胞中特异性表达hLIF蛋白。为无抗性标记的转hLIF基因奶山羊生产提供了打靶载体。In order to realize the stable expression of human leukemia inhibitory factor in goat mammary cells, in this study, we used the recombination cassette LoxP sequence, goat β-casein 5＇ and 3＇ homologous arms, positive and negative selection marker cassette to get targeting vector of human leukemia inhibitory factor, and homologous recombination positive monoclonal was obtained by transfection of the vector into goat（Capra hircas） mammary cells with LipofectamineTM 2000 conferred resistance to geneticin （G418） and resistance to ganciclovir （GANC） in the cells, then we verify the hLIF expression by PCR, RT-PCR and Western blot. The restriction and sequencing analysis result showed that the targeting vector pLoxP-NT53L was constructed successfully, and the target band, demonstrated the targeting vector pLoxP-NT53L was integrated into the β-casein locus and the goat mammary cells could express protein specificity. The results indicate that the maker-free targeting vector of human leukemia inhibitory is very useful for produce marker-free dairy goat of transfer hLIF gene.

关 键 词：人白血病抑制因子 山羊β-酪蛋白 同源重组 乳腺生物反应器 

分 类 号：Q513.2[生物学—生物化学]