摘要
目的构建携带报告基因增强型绿色荧光蛋白(EGFP)的反转录病毒载体,并且探讨病毒载体对SK-N-SH神经母细胞瘤细胞株的感染效率。方法使用基因重组技术构建携带EGFP基因的反转录病毒载体(RV/EGFP)。将稀释的RV病毒液感染NIH3T3细胞,计数NIH3T3荧光表达细胞的数量来确定病毒的浓度;使用RV载体感染SK-N-SH细胞,通过流式细胞学荧光检测明确RV在SK-N-SH细胞的感染效率。结果成功构建了基因转移载体RV/EGFP,确定重组病毒的浓度为8.3×106病毒颗粒/mL。在SK-N-SH细胞RV/EGFP稳定转染并有效表达。在SK-N-SH细胞EGFP的荧光表达显示RV的转移并且表达的效率达到30%以上。结论SK-N-SH细胞能被RV病毒载体有效感染,在转基因细胞株外源基因的表达长期稳定并且显示RV病毒载体具有良好的基因转移效率。
Objective To construct the retroviral （RV） vector with report gene enhanced green fluorescent protein （EGFP） and to explore the gene transfection efficiency of RV on SK-N-SH neuroblastoma cells. Methods By the technique of genetic recombination, the recombinant RV/EGFP viruses had been constructed. The concentration of diluted RV virus solution was determined by counting the NIH3T3 cells with fluorescent expression. Then the RV/EGFP virus was used to transfer SK-N-SH neuroblastoma cells. The transfection efficiency in SK-N- SH cell was measured by flow cytometer. Results The gene transfer vector RV/EGFP had been successfully constructed. We obtained the recombinant RV/EGFP with a concentration of 8.3 × 10^6 viruses/mL. The RV/EGFP had been stably transferred and effectively expressed in SK-N-SH cell. The transfection and expression efficiency of RV was above 30% in SK-N-SH ceils determined by observing EGFP expression. Conclusion The SK-N-SH cells can be transferred effectively by recombinant RV vector. In transgenetic cells, the expression of transgene was stable, and moreover, RV vector had better genetically transfected efficiency than other methods.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2007年第1期10-13,共4页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家自然科学基金资助项目(No.30300395No.30400515)
教育部博士点基金项目(No.20030698011)
