β-防御素2真核表达载体的构建及其在肝癌细胞Hepa1-6中的稳定表达

Construction of beta-defensin 2 secretory eukaryotic expression vector and identification of stable expression of mBD2 in Hepa1-6 cell

目的克隆抗菌肽β防御素2(mBD2)基因,构建真核分泌性表达载体,使其在Hepa1-6细胞中稳定表达。方法Trizol试剂提取C57BL/6鼠肾总RNA,RT-PCR扩增mBD2成熟肽基因,Overlap-PCR在成熟肽基因上游加上鼠IgК信号肽基因,克隆至真核表达载体pcDNA3.1(+),PCR、酶切和测序鉴定正确后,脂质体法转染Hepa1-6细胞,G418筛选,RT-PCR和Western印迹法从mRNA和蛋白角度鉴定mBD2分子的表达。结果成功克隆含有信号肽的IgК-mBD2序列,并且构建pcDNA3.1(+)/IgК-mBD2真核分泌性表达载体,实现了mBD2分子在Hepa1-6细胞中稳定表达,RT-PCR从细胞总RNA中扩增得到202bp的IgК-mBD2基因,Western印迹法鉴定细胞培养上清中有mBD2蛋白分子表达。结论成功构建了pcDNA3.1(+)/IgК-mBD2真核分泌性表达载体,转染得到稳定表达mBD2的Hepa1-6细胞株,为进一步研究mBD2的生物学特性及抗肝癌机制奠定细胞学基础。

Objective To clone beta defensin 2 （mBD2） gene from total RNA of mouse kidney,and to construct mBD2 secretary eukaryotic expression vector,then realize its expression in Hepa1-6 cells. Methods Total RNA was isolated from kidney of C57BL/6 mice by Trizol reagent. DNA sequence coding for mature mBD2 was amplified by RT-PCR. To add a murine Igκ signal peptide sequence by overlap-PCR. After successfully T-A clone,Igκ-mBD2 was subcloned into the plasmid pcDNA3.1 （＋）.The pcDNA3.1 （＋） / Igκ-mBD2 was identified by PCR,endonuclease digestion,and sequencing. The Hepa 1-6 cells were transfected with pcDNA3.1（＋）/Igκ-mBD2 by Liposome2000 and selected with 400 mg /L G418 for 8 w. Identification of steady expression of mBD2 was confirmed by RT-PCR and Western blot. Results The eukaryotic expression vector-pcDNA3.1 （＋）/Igκ-mBD2 was successfully constructed. Stable expressional cell line of Hepa 1-6 was obtained by transfecting pcDNA3.1 （＋）/Igκ-mBD2 into Hepa 1-6 by Liposome2000. The steady expression of mBD2 was confirmed by RT-PCR and Western blot. Conclusions The eukaryotic vector of pcDNA3.1 （＋） / Igκ-mBD2 is successfully constructed,and mBD2 stable expressional Hepa 1-6 cell line is identified,which is good foundation for further research on biological characteristic and anti-tumor mechanisms of mBD2.