2A肽介导猪PID1与CuZnSOD双基因真核共表达载体的构建与细胞表达

构建PID1基因与CuZnSOD基因的真核共表达载体,在PK15细胞中鉴定基因的表达。PCR扩增的PID1与CuZnSOD两基因分别经双酶切后定向插入pIRES2-AcGFP1空载体,构建pIRES2-CuZnSOD-PID1真核双表达载体并进行测序与酶切鉴定。采用脂质体转染法将重组质粒转染至PK15细胞,细胞内荧光显微镜下观察其荧光的表达,RT-PCR、Westernblot技术分别检测PID1基因与CuZnSOD基因mRNA和蛋白表达情况。重组克隆载体插入目的片段序列与PID1基因与CuZnSOD基因序列完全一致。PIRES2-CuZnSOD-PID1真核双表达载体测序、酶切鉴定结果与预期结果一致。荧光显微镜下观察转染后的PK15细胞出现绿色荧光。RT-PCR检测结果显示,转染细胞中PID1基因与CuZnSOD基因表达量明显高于对照组（P〈0.05）。Westernblot检测结果表明pIRES2-CuZnSODPID1真核双表达载体稳定有效表达。成功构建pIRES2-CuZnSOD-PID1真核共表达载体,且双基因在真核细胞独立稳定表达,为转基因猪等育种新材料的制备奠定基础。

Construction of eukaryotic co-expression vector carrying PID1 and CuZnSOD by using 2 A peptide and expressions in cells

To construct eukaryotic co-expression vector carrying PID1 and CuZnSOD and identify its expression in PK15 cells,the genes amplified by PCR were inserted into the p IRES2-Ac GFP1 to construct eukaryotic co-expression vector p IRES2-CuZnSOD-PID1.The recombinant vector was confirmed by restriction endonuclease treatment and sequenced and transfected into PK15 cells. The expressions of the genes in the recombinant vector were identified by fluorescent microscope,RT-PCR and western blot. The restriction endonuclease digestion and sequencing suggested that the co-expression vector p IRES2-CuZnSOD-PID1 was constructed successfully.The cells transfected with recombinant vector appeared the green fluorescence under the fluorescent microscope. RT-PCR analysis showed that the PID1,CuZnSOD expression levels were significantly higher in the transfected cells（ P〈0. 05）. Western blot analysis showed that the recombinant eukaryotic expression vector p IRES2-CuZnSOD-PID1 could achieve stable and effective protein expression. The recombinant eukaryotic expression vector p IRES2-CuZnSOD-PID1 was constructed and expressed in eukaryotic cells successfully,which would contribute to generating new breed material like transgenic pigs.