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・ 基础研究 ・ 重组腺病毒 Ad5F35-SD-EGFP 的构建及其对 K562 细胞增殖的 影响 史静1 , 胡晶1 , 肖青2 , 彭智1 , 曹唯希1 , 罗秋平1 , 王方1 , 冯文莉1 重庆医科大学1 临床血液学教研室//临床检验诊断学教育部重点实验室,

2 附属第一医院血液科, 重庆400016 摘要: 目的 构建表达SH2-DED融合基因的新型重组腺病毒Ad5F35-SD-EGFP, 观察其对K562细胞增殖的抑制作用.

方法 重叠PCR扩增SH2-DED融合基因并克隆至腺病毒穿梭载体pAdTrack-CMV中, 构建成穿梭质粒pAdT-SD-EGFP 并进行酶切和测序鉴定.穿梭质粒经Pme I酶切后转化pAd5F35-GFP的BJ5183感受态, 通过细菌内同源重组产生重组 腺病毒质粒pAd5F35-SD-EGFP及其突变体, Pac I酶切后转染AD293细胞进行包装.重组腺病毒Ad5F35-SD-EGFP经PCR 和western blot 鉴定后进行病毒扩增和滴度测定.体外感染 K562 细胞, 通过 MTT 和流式周期检测实验观察 Ad5F35-SD-EGFP对K562细胞增殖的影响.结果 PCR、 酶切和测序结果均表明pAdT-SD-EGFP和pAd5F35-SD-EGFP 构建成功;

PCR、 EGFP检测结果证明重组腺病毒Ad5F35-SD-EGFP包装扩增成功, 滴度可达1.4*1012 pfu/ml.转染K562 细胞后, Western blot可检测到目的蛋白的表达, MTT实验和流式周期检测结果证明其对K562细胞的增殖有明显的抑制 作用.结论 成功构建并鉴定了携带SH2-DED融合基因的重组腺病毒Ad5F35-SD-EGFP, 其对BCR-ABL阳性K562细 胞的增殖有明显的抑制作用. 关键词: Bcr-Abl;

SH2;

DED;

重组腺病毒;

AD293细胞 中图分类号: Q78 文献标志码: A 文章编号: 1673-4254 (2011) 11-1806-06 Construction of a recombinant adenovirus Ad5F35-SD-EGFP and its effect on K562 cell proliferation SHI Jing1 , HU Jing1 , XIAO Qing2 , PENG Zhi1 , CAO Wei-xi1 , LUO Qiu-ping1 , WANG Fang1 , FENG Wen-li1 Department of Clinical Hematology,

1 Key Laboratory of Laboratory Diagnostics of State Ministry of Education,

2 Department of Hematology, First Affiliated Teaching Hospital of Chongqing Medical University, Chongqing 400016, China Abstract: Objective To construct a recombinant adenovirus vector for SH2-DED fusion gene and assess its inhibitory effect on the proliferation of K562 cells. Methods SH2-DED fusion gene and its mutant SH2mt-DED were amplified by splicing PCR and cloned into pAdTrack-CMV plasmid separately to construct the shuttle plasmids pAdT-SD-EGFP and pAdT-SmD-EGFP, respectively. After Pme I digestion, the shuttle plasmids were transformed into ultra-competent pAd5F35-BJ5183 cells to generate defective adenovirus vectors pAd5F35-SD-EGFP and pAd5F35- SmD-EGFP by homologous recombination. The vectors, linearized by Pac I digestion, were further transfected into AD293 cells for packaging and amplified by infecting AD293 cells repeatedly. K562 cells were then infected by the recombinant adenoviruses and the expression of SD was detected by Western blotting.MTT assay and flow cytometry were used to investigate the effect of Ad5F35-SD-EGFP and Ad5F35-SmD-EGFP on the proliferation of K562 cells. Results The recombinant adenovirus vectors pAd5F35-SD-EGFP and pAd5F35-SmD-EGFP were constructed correctly, with a titer reaching 1.5*1012 pfu/ml after amplification. Western blotting demonstrated that the target proteins were effectively expressed in transfected K562 cells. MTT assay and flow cytometry showed that transfection with pAd5F35-SD-EGFP resulted in growth inhibition rate of 55.21% in K562 cells, significantly higher than the inhibition rate of 17.95% following transfection with pAd5F35- SmD-EGFP and 7.33% following PBS treatment (P<