PIG-A基因逆转录病毒表达载体及包装细胞系的建立

Construction of a retrovirus vector containing PIG-A gene and its packaging cell line

目的构建含磷脂酰肌醇聚糖A类基因（PIG-A）的逆转录病毒载体并对其进行包装，获得稳定的产毒细胞系。方法采用PCR方法从质粒pEBPIG-A中扩增PIG-A基因片断，BarnHI及XhoI双酶切后连接至逆转录病毒载体pLEGFP-C1，用限制性内切酶和DNA序列测定的方法对重组质粒进行鉴定。脂质体法转染PA317包装细胞，荧光显微镜下观察EGFP瞬时表达，G418筛选抗性克隆，收集病毒上清后感染NIH3T3细胞测定病毒滴度。结果酶切证实PIG-A基因克隆至逆转录病毒载体，测序结果和原始序列相同。重组逆转录病毒载体转染PA317包装细胞，24～48h后荧光显微镜下观察到绿色荧光蛋白的表达。经CAl8筛选得到6个稳定抗性克隆，病毒滴度最高达1．6×10^5CFU／ml。结论构建了含PIG-A基因的逆转录病毒载体，获得了稳定的产毒细胞系。

Objective To construct a retroviral vector carrying PIG-A （phosphatidylinositolglycan complementation class A）gene and obtain a stable packaging cell lines. Methods The PIG-A gene was amplified from plasimd pEBPIG-A by PCR and digested by Barn H I and Xho L The product was subeloned into retrovirus vector pLEGFP-C1. The recombinant plasmid was identified by restriction endonuclease disgestion and sequencing. The packaging cell line PA317 was transfected with the plasmid pLEGFP-C1-PIG-A by lipofectamine 2000 and the instantaneous expression of EGFP was observed by fluorescence microscope 24 to 48 h later. Then G418 was used to select NeoR（＋） clones and the viral titers were measured by infecting NIH3T3 cells. Results The result of restriction endonuclease disgestion proved that PIG-A gene had been subcloned into the retrovirus vector with right sequence. F_Z;FP expression in packaging cell line PA317 was detected under fluorescence microscope. After CA18 selection, six NeoR（＋ ） clones were obtained and the highest viral titer was 1.6 ×10^5 CFU/rnL Conclusion The retrovirus vector contaning PIC-A gene has been successfully constructed and the stable packaging cell line obtained,which can be used to infect PIG-A gene deficient cells.