人β-地中海贫血CD41-42型突变基因慢病毒载体的构建和293T的转染

Construction of the human β-thalassemic CD41-42 gene lentiviral expression vector and virus packaging

目的:构建介导人β-地中海贫血CD41-42突变基因为目的基因的慢病毒载体,为基因治疗的细胞模型和转基因小鼠的建立提供基础。方法:抽提β-地中海贫血CD41-42突变纯合子患者的DNA,设计合成相应特异性引物,PCR扩增CD41-42突变基因,加上SphI和NotI两个酶切位点进行T载体克隆,通过筛选获得重组质粒。用限制性内切酶将目的基因片段和LV5慢病毒载体连接,转入感受态细胞,筛选获得慢病毒表达载体LV5-βCD41-42,并进行测序。将慢病毒载体转染293T细胞,包装病毒,测定病毒浓度。结果:通过PCR获得长度为2128bp带有SphI和NotI序列的目的基因。pUC57载体和慢病毒表达载体LV5连接,慢病毒表达载体LV5-βCD41-42构建成功,序列正确。结论:成功构建并包装含β-地中海贫血CD41-42突变基因的人慢病毒。

Objective： To contract containing β-thalassemic CD41 -42 gene lentiviral vector LV5 -βCD41 -42 and make virus particles packaging. Methods： Polymerase chain reaction method was used to obtain the target gene and SphI, NotI two restriction sites were added, after T vector cloning, the gene was transformed into competent ceils. The gene fragment and LV5 vector cloning, the gene was transformed into competent cells by means of enzyme digestion. The lentiviral expression vector LV5 -βCD41 - 42 was obtained after screening followed by sequencing. The lentiviral vector was used to transfect 293T cells and package virus, and virus titers were determined. Results ： A length of 2 128 bp with SphI and NotI target gene sequences was obtained by PCR. The pUC57 vector was connected to the lentiviral vector LV5. Conclusion： Constructed lentiviral expression vector LV5 - βCD41 -42 is corresponded to the expected. The lentiviral particles are successfully packaged.