人珠蛋白MAR序列的克隆与表达载体pCAT-MAR的构建

目的克隆人β-珠蛋白核基质结合区(matrix attachment regions,MAR),构建包含MAR及报告基因CAT的哺乳动物载体pCAT-MAR。方法酚/氯仿抽提、乙醇沉淀提取人基因组DNA,根据GenBank报道的序列设计引物PCR扩增人β-MAR,琼脂糖凝胶电泳鉴定,测序,软件分析其序列特征。限制酶酶切,连接至pCAT3-control载体上构建pCAT-MAR载体。结果琼脂糖凝胶电泳PCR扩增出770bp条带,序列和报道的序列相似性为99.9%,克隆的DNA片段具备典型的MAR特征。酶切及琼脂糖凝胶电泳证明所构建的pCAT-MAR载体正确。结论PCR克隆了人-β珠蛋白MAR序列,成功构建了包含MAR的表达载体pCAT-MAR。

Cloning of the human β-globin MAR and construction of the pCAT-MAR expression vector

Objective To clone the human β-globin matrix attachment region(MAR) and construct the mammalian animal expression vector pCAT-MAR, which contains the MAR and CAT reporter gene.Methods The human genomic DNA was extracted through phenol/chloroform and precipitated with ethanol, followed the MAR was amplified through PCR using the primers designed according to the GenBank sequence. After identified by agarose gel electrophoresis, sequenced and analyzed by the software, the PCR products were cut with restriction enzymes and ligated into the pCAT3-control vector to construct the pCAT-MAR vector. Results About 770bp band appeared in the agarose gel electrophoresis, the similarly compared with the published MAR sequence was 99.9%, the DNA fragment has the MAR typical features. The pCAT-MAR vector was demonstrated to be right through digestion with the corresponding enzymes and agarose gel electrophoresis. Conclusion The humanβ-globin MAR is cloned through PCR, and the expression vector pCAT-MAR containing the MAR is successfully constructed.