【摘要】 目的应用逆转录病毒载体将PIG-A cDNA转导至突变型K562细胞中,观察是否能够逆转K562细胞CD59的表达。方法采用PCR方法从质粒pEBPIG-A中扩增PIG-A基因片断,BamH I及Xho I双胁切后连接至逆转录病毒载体pLEGFP-C1相应位点。脂质体法转染PA317包装细胞,收集病毒上清感染突变型K562细胞,流式细胞仪测定感染前后CD59的表达情况。结果重组逆转录病毒载体转染PA317包装细胞后,24～48h荧光显微镜下观察到绿色荧光蛋白的表达。G418筛选后,病毒滴度测定达(1．6±0．2)×10~4CFU/mL。感染K562细胞前后测定CD59的表达率分别为4．5％±0．7％及21．3％±0．3％(P＜0．01)。结论成功构建了PIG-A基因逆转录病毒载体,感染突变型K562细胞后可以提高其CD59的表达。更多还原

【Abstract】 Objective To investigate whether the expression of CD59 on the surface of the mutant K562 cells could be reversed by the PIG-A gene mediated by the retrovirus vector.Methods The PIG-A eDNA was amplified from the plasmid pEBPIG-A by polymerase chain reaction (PCR),then the product was inserted into the Bam HⅠand XhoⅠsites of the retrovirus vector——pLEGFP-C1.The recombined plasmid was identified by restriction enzyme and DNA sequencing.PA317 packaging cell line was transfected by lipofectamine.The expression of en- hanced green fluorescent protein (EGFP) was viewed by fluorescence microscope.Then the supematant was used to infect the NIH3T3 cells to determine the viral titers and the mutant K562 cells were infected,the expression of CDS9 on the surface of the K562 cells was determined by FCM.Results The expression of EGFP was observed under the fluorescence microscope 24～48 hours after the PA317 cells were transfected by the recombinant retrovirus vector.The virus titers was (1.6±0.2)×10~5CFU/mL.The expression rate of CD59 increased from 4.5%±0.7% to 21.3%±0.3%(P＜0.01).Conclusion The retrovirus vector containing the PIG-A gene was successfully con- structed and the expression of CD59 on the mutant K562 cells could be increased.更多还原