人呼吸道合胞病毒转录调节基因转染PAa和A549肺腺癌细胞系

目的探讨人呼吸道合胞病毒(hRSV)对肺癌细胞的影响。方法构建其转录调控因子M2-1基因真核表达质粒pXJ-41/M2-1,用DNA测序、酶切、PCR等方法鉴定基因重组情况。结果DNA测序表明该基因具有高度保守性;经脂质体转染、以-βactin为参照进行RT-PCR,筛选出了高效稳定表达M2-1基因的人肺腺癌PAa和A549细胞株,并经Westernblot验证有M2-1蛋白的表达。结论为进一步探讨M2-1基因在肺癌发生发展中的作用打下基础。

Construction of an Eukaryotic Cell Expressing Vector of hRSV M2-1 Gene and its Transfection into Lung Cancer Cell PAa and A549

Objective Background and human respiratory syncytial virus(hRSV) is a most familiar pathogen for severe respiratory tract infection in infants and young children, and it is found that it is also an important pathogen in adults in recent years.In this study, we try to construct a eukaryotic cell expressing vector containing the M2-1 gene of the HRSVand then try to transfect the lung cancer cell lines PAa and A549, so that we can continue studying the relationship between HRSVand lung cancer.Methods To construct the vector pXJ-41/M2-1 with gene reconstructing technique.Then check it through DNA-sequencing, PCRand enzyme-cutting.To transfect this vector into lung cancer cell lines mediated by liposome LipofectamineTM 2000 and screen out high expressing cell strains by RT-PCRin reference to the β-actin, which is an endogenous reference gene.Results 650 bp segment was obtained by PCRwith the recombinant as a template or by digesting the recombinant with EcoRIand XholI.The DNAsequencing result indicated that the M2-1 gene is high-conservative.Cell strains of PAa and A549 which can stably express M2-1 protein were acquired.Conclusion The eukaryotic cell expressing vector pXJ-41/M2-1 was successfully constructed.The acquirement of PAa and A549 cell strains expressing M2-1 protein would make it possible to study the influence of hRSVon lung cancer cells.