重组adv5.oriP.HRP腺病毒载体构建及对EBV阳性鼻咽癌C666-1细胞株的靶向表达  

The Construct of a Novel Recombinant Replication-defective Adenovirus Vector and its Targeting Expressin EBV-positive Nasopharyngeal Carcinoma Cells

作　　者：林峰[1,2] 苗北平[1,2] 卢永田[1,2] 

机构地区：[1]深圳市第二人民医院 [2]深圳大学附属第一医院耳鼻咽喉科,深圳518035

出　　处：《数理医药学杂志》2011年第3期266-270,共5页Journal of Mathematical Medicine

基　　金：深圳市科技局重点资助课题(200901008)

摘　　要：目的:构建腺病毒载体adv5.oriP.HRP,观察其对EBV阳性鼻咽癌C666-1细胞株的靶向表达。方法:采用p△EIsp1A穿梭质粒系统,酶切、定向克隆方法构建含HRP自杀基因的p△EIsp1A/oriP-HRP重组质粒,在293细胞中进行重组腺病毒adv5.oriP.HRP包装、扩增、纯化与病毒滴度测定,体外转染EBV阳性鼻咽癌C666-1细胞株与EBV阴性鼻咽癌CNE-2Z细胞株,RT-PCR法检测不同细胞系中adv5.oriP.HRP的表达,Western blot法检测经不同浓度adv5.oriP.HRP转染后细胞中HRP蛋白的表达。结果:p△EIsp1A/oriP-HRP在293细胞中包装扩增后的病毒滴度为528@1012TCID50/L;体外转染鼻咽癌C666-1细胞株与CNE-2Z细胞株后,RT-PCR检测到C666-1细胞系1229bp处得明亮条带,而CNE-2Z细胞几乎未检测到重组基因mRNA的表达,Western blot可见随着MOI的增加C666-1细胞中HRP蛋白表达逐渐增多,而在CNE-2细胞中HRP蛋白的表达几乎呈阴性,两组间差别有统计学意义(P<0.05)。结论:重组腺病毒adv5.oriP.HRP能在EBV阳性的鼻咽癌C666-1细胞株内靶向性转染和表达。Objective：To construct the recombinant adenovirus carrying adv5.oriP.HRP,and to observe the targeted expression to EBV positive nasopharyngeal carcinoma of C666-1 lines.Methods：The HRP quences were subcloned into corresponding sites of p△EIsp1A vector with directiona l cloning method and enzyme digestive method to construct the p△EIsp1A/oriP-HRP.The recombinant adenovirus of adv5.oriP.HRP was packaged,amplified and purified in 293 cells,and determined the virus titre.The adv5.oriP.HRP gene and protein expression in C666-1 and CNE-2Z were examined by reverse transcription polymerase chain reaction（RT-PCR） and Western blot after in vitro transfection in EBV positive C666-1 lines and EBV negative CNE-2Z lines.Results：The titer of the recombinant adenovirus was 528@1012 TCID 50 /L.There was a bright strips along 1229bp in C666-1lines by RT-PCR detection,while the mRNA of HPP gene expression was not detected in CNE-2Z cells.The expression of HRP protein was increased with the increase of MOI gradually,but HRP protein expression in CNE-2Z cells almost negative,difference between the two groups was statistically significant（P0.05）.Conclusion：The recombinant adenovirus adv5.oriP.HRP had high transfection and expressing efficiency with targeting in C666-1 cells.

关 键 词：腺病毒 HRP 鼻咽癌 EBV 靶向表达 

分 类 号：R739.63[医药卫生—肿瘤]