pcDNA3.1-mAFP真核表达载体的构建及其在293细胞中的表达

Construction of recombinant eukaryotic expression vector pcDNA3.1-mAFP and alpha fetoprotein expression in 293 cells

目的构建真核表达载体pcDNA3.1-mAFP,观察其在293肿瘤细胞中的表达情况。方法从Hepa1-6小鼠肝癌细胞中提取总RNA,设计特异性引物,通过RT-PCR法扩增小鼠甲胎蛋白(mAFP)基因编码区全长序列,测序确认后,插入到pcDNA3.1真核表达载体中,双酶切鉴定。用Lipofectamine脂质体将构建的重组载体转染293肿瘤细胞,检测AFP蛋白在293细胞中的表达情况。结果成功克隆了mAFP基因编码区全长序列,构建出重组真核表达载体pcDNA3.1-mAFP,脂质体转染后可检测到mAFP基因在293细胞中的表达。结论该研究成功构建了重组pcDNA3.1-mAFP真核表达载体,为进一步开展原发性肝癌的靶向性基因治疗奠定了基础。

[Objectives] To construct a novel recombinant eukaryotic expression vector pcDNA3.1-mAFP and detected its expression in 293 cells. [Methods] Total RNA were extracted from hepal-6 murine liver cancer cell line, murine alpha fetoprotein （mAFP） gene was cloned by RT-PCR method. The gene was identified by sequencing, it was ligased to pcDNA3.1 vector by KpnI and Xba I restrictive enzyme digestion and ligation, mAFP gene was transducted to 293 cells by lipofectamine merhod. [Results] PcDNA3. I-mAFP recombinant vector was successfully constructed and it had a higher level expressed in 293 cells. [Conclusion] PcDNA3.1-mAFP recombinant vector carrying mAFP gene was constructed and mAFP gene was successfully transducted into 293 tumor ceils, it will lay the basis for further developing gene therapy of primary liver cancer.