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目的构建编码人雌激素相关受体α(hERRα)的慢病毒载体,并测定其滴度,观察hERRα基因在体外的表达情况及其对人肺癌细胞A549增殖的影响。方法采用PCR扩增hERRα基因片段,插入转移载体pW-PXLD,用磷酸钙法将pWPXLD-hERRα、psPAX2和pMD2.G共转染293T细胞包装慢病毒,浓缩后测定病毒滴度,再将病毒感染A549细胞,用蛋白印迹方法测定感染病毒的A549细胞中hERRα的表达。选择合适滴度的病毒感染A549细胞,采用cell-titer的方法检测感染病毒的A549细胞的活力。结果测序结果显示成功构建了重组质粒pWPXLD-hERRα,测定浓缩后的病毒滴度为1.8×108TU/mL,蛋白印迹方法检测到慢病毒感染的A549细胞中hERRα呈阳性表达;cell-titer glo检测结果表明过表达hERRα能促进细胞增殖。结论成功构建了负载hERR基因片段的慢病毒,hERRα能在感染的A549细胞中高效表达,并促进感染病毒的A549细胞增殖。
Objective To construct a lentiviral vector encoding human estrogen receptor alpha (hERRα) and determine the titer of hERRα gene in vitro and to investigate its effect on the proliferation of human lung cancer A549 cells. Methods The hERRα gene fragment was amplified by PCR and inserted into the transfer vector pW-PXLD. The 293T cells were co-transfected with lentivirus pWPXLD-hERRα, psPAX2 and pMD2.G by calcium phosphate method. The virus titer was determined after concentration and the virus was infected A549 cells, the expression of hERRα in A549 cells infected with virus was determined by Western blotting. A549 cells were infected with virus of appropriate titer and the viability of A549 cells infected with virus was tested by cell-titer method. Results The sequencing results showed that the recombinant plasmid pWPXLD-hERRα was constructed successfully. The titer of the recombinant plasmid was determined to be 1.8 × 108TU / mL. The expression of hERRα in lentivirus-infected A549 cells was detected by Western blotting. The results of cell-titer glo It is indicated that overexpression of hERRα can promote cell proliferation. Conclusion The lentivirus with hERR gene fragment was successfully constructed. HERRα was highly expressed in A549 cells and promoted the proliferation of A549 cells infected with virus.