大鼠对氧磷酶1真核表达载体的构建及在体外的表达

In Vitro Construction and Expression of Eukaryotic Expression Vector Harboring the Gene Fragment of Rat Paraoxonase 1

目的构建大鼠对氧磷酶1(paraoxonase1,PON1)真核表达载体,并检测其在体外培养细胞中的表达及功能,为进一步探索运用其进行基因防治肺部感染奠定基础。方法利用RT-PCR扩增大鼠PON1编码区全长序列,克隆入pcDNA3.1(+),鉴定无误后,脂质体转染A549细胞及293细胞,Western印迹检测蛋白表达,并检测其芳香酯酶活性及对铜绿假单胞菌密度感知系统信号分子N-酰基高丝氨酸内酯(N-acylhomoserine lactones,AHLs)的水解功能。结果分别从大鼠肝组织中扩增出1153bp片段,克隆入表达载体后进行酶切鉴定和测序结果正确。转染细胞后能够表达目的蛋白,该蛋白具有芳香酯酶和AHLs内酯酶活性。结论成功构建了含有大鼠PON1真核表达载体,质粒能够在真核细胞中表达,能够有效地水解铜绿假单胞菌密度感知系统信号分子。

Objective To construct the eukaryotic expression vector harboring the gene fragment of paraoxonase 1 and study its functions. Methods The rat paraoxonase 1 gene was amplified by RT-PCR, then cloned into eukaryotic expression vector pcDNA 3.1 （ ＋ ）. After the recombinant plasmid was isolated, the construction was verified and transfected into A549 cells and 293 cells. The expressed protein was analyzed by Western blotting and its functions to hydrophenylacetate and N-acylhomoserine lactones （AHLs） were assayed. Result DNA sequencing confirmed that the paraoxonase 1 fragment cloned and inserted into the vector were identical with the predicted one, The expressed protein was detected by SDS-PAGE and Western blotting. Moreover, its effects on hydrophenylacetate and AHLs were detected. Conclusion The eukaryotic expression vector harboring the fragment of paraoxonase 1 was constructed successfully and the recombinant protein was effective in hydrolyzing AHLs and is capable of sensing quorum.