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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。
Objective To reverse the expression of CD59 in K562 cells by transducing PIG-A cDNA into mutant K562 cells with retroviral vector. Methods The PIG-A gene fragment was amplified by PCR from the plasmid pEBPIG-A. The double-strand cuts of BamH I and Xho I were ligated into the corresponding site of retroviral vector pLEGFP-C1. PA317 cells were transfected by lipofectamine 2000. The mutant K562 cells were infected by virus supernatant and the expression of CD59 was detected by flow cytometry. Results The recombinant retroviral vector transfected PA317 packaging cells, 24 ~ 48h fluorescence microscopy was observed under the green fluorescent protein expression. After G418 screening, the virus titer was (1.6 ± 0.2) × 10 ~ 4CFU / mL. The expression rates of CD59 before and after infection of K562 cells were 4.5% ± 0.7% and 21.3% ± 0.3% (P <0.01), respectively. Conclusion The PIG-A gene retroviral vector was constructed successfully and the expression of CD59 was enhanced after infection with mutant K562 cells.