1pcDNA3.1-Ercc6l真核表达载体的构建及在P19细胞内的表达

Construction of eukaryotic expression vectors pcDNA3.1-Ercc6l and its expression in P19 cells

目的构建Ercc6l的正、反义真核表达载体,并在P19胚胎癌细胞内瞬时表达,在细胞学水平对基因功能进行初步研究。方法KpnⅠ/XhoⅠ双酶切pGEM-T-Ercc6l获得目的基因Ercc6l,将其克隆到同样经过双酶切的真核表达载体pcDNA3.1(+)和pcDNA3.1(-)Myc His6,重组质粒pcDNA3.1(+/-)-Ercc6l经菌落PCR和双酶切鉴定;脂质体介导法体外瞬时转染正义载体至P19细胞,RT-PCR检测基因在细胞内的表达活性情况。结果重组质粒经菌落PCR和KpnⅠ/XhoⅠ双酶切鉴定,表明真核表达载体构建正确;脂质体介导法瞬时转染正义载体至P19细胞后,检测表明转染细胞能够表达Ercc6l。结论成功构建了Ercc6l正、反义真核表达载体,其正义载体转染真核细胞后能在其中表达。

Objective: To construct sense and antisense eukaryotic expression vectors of novel gene Ercc6l and study its transfection and transient expression in P19 cells.Methods: The ORF of Ercc6l was obtained by digestion with XhoⅠand KpnⅠrestriction endonuclease in the plasmid of pGEM-T-Ercc6l.Recombinant vectors were constructed by inserting the target code gene into the MCS of plasmid pcDNA3.1(+) and pcDNA3.1(-) Myc His6 between XhoⅠand KpnⅠsites.Then the recombinant plasmids were identified by colony PCR and res...