鸡卵清蛋白基因调控序列的克隆与载体构建

Cloning and vector construction of chicken ovalbumin gene regulatory sequences

卵清蛋白(ovalbumin)基因在鸡基因组中只有一对等位基因,却能每天合成分泌多达2 g的蛋白,占据卵白蛋白质的50%以上,是外源基因载体表达调控构件的首选。该研究从输卵管特异性启动子方面着手,通过对卵清蛋白基因启动子的筛选优化,找出启动子增强因子的位置以及组织特异性区域。将卵清蛋白基因上游-922^-2 073和-2 801^-3 100区域平均分成12个长度约为150 bp的序列,分别插入到-921^+38序列的上游,成功构建12个系列表达载体,为进一步筛选短缩版优化启动子提供材料;卵清蛋白基因第一内含子区域截断成300 bp左右的迷你内含子序列,成功构建8个迷你内含子系列载体,为筛选优化的迷你内含子提供必要的材料;成功分离鸡输卵管上皮细胞并优化电转染条件,通过荧光素酶活性检测初步筛选出具有最强活性重组质粒pGL4-UP-1412和内含子重组质粒pGL4-mini-intron-3,同时推断出若干包含增强子序列区域。

There was only one allele of ovalbumin gene in the chicken genome,but it could synthesize and secrete 2g protein per day,which were accounting for more than 50% of the albumin protein and became the preferred choice in the regulation of exogenous gene expression. This study aimed to identify promoter enhancer and tissue-specific regional location factor by screening the optimization of ovalbumin gene promoter. The upstream-922 ～-2 073 and-2 801 ～-3 100 of ovalbumin promoter were divided into 12 regional which average sequence length were about150 bp,and inserted into the upper reaches of-921 ～ ＋ 38 sequences,those 12 series successfully constructed expression vectors provided materials for further optimization promoter with a shortened version. The first intron region of ovalbumin promoter was truncated around 300 bp of mini intron sequences,and successfully constructed 8 mini intron series of vectors. We also successfully separated chicken oviduct epithelial cells and optimized the electricity transfection conditions. In this study,the promoter region with strongest activity pGL4-UP-1412 and pGL4-mini-intron-3 were screened through detected luciferase activity of the initial screening recombinant plasmid and intron re-combinant,while inferred several regions containing enhancer sequence.