负载人miR-29a复制缺陷型腺病毒的包装及鉴定  

Package and identification of replication-deficient adenovirus carrying hsa-miR-29a

作　　者：高军[1] 范亚川[2] 王军[2] 熊晓峰[2] 吴友良[1] 刘志鹏[2] 陈陵[2] 李学成[2] 

机构地区：[1]解放军324医院药剂科,重庆400020 [2]解放军324医院消化内科,重庆400020

出　　处：《西南国防医药》2011年第10期1045-1048,共4页Medical Journal of National Defending Forces in Southwest China

基　　金：重庆市自然科学基金资助项目(CSTC2008BB5274;CSTC2009BB5314;CSTC2010BB5158)

摘　　要：目的包装负载人miR-29a前体全长cDNA(pre-miR-29a)的复制缺陷型腺病毒载体(Ad-miR29a)。方法将pre-miR-29a全基因合成后,插入腺病毒穿梭质粒pDC315,与腺病毒包装质粒pBHGloxDeltaE13、Cre共转染人胚肾293细胞(HEK293),包装重组慢病毒Ad-miR29a,扩增、收集并纯化。对纯化后的Ad-miR29a进行滴度测定、感染性鉴定及PCR鉴定。结果包装成功的腺病毒载体具有良好的感染性,纯化浓缩后的病毒滴度可达5×109 pfu/ml。PCR鉴定Ad-miR29a可扩增出pre-miR-29a特异片段,而对照Ad-LacZ不能扩增出pre-miR-29a特异片段。Ad-miR29a感染人胃癌SGC-7901细胞后,可明显提高miR-29a的表达丰度。结论成功包装了负载miR-29a的复制缺陷型腺病毒载体,为进一步研究microRNAs在肿瘤发生发展中的作用,以及miR-29a在肿瘤治疗中的价值奠定了基础。Objective To construct the replication-deficient recombinant adenovirus（Ad-miR29a）carrying whole cDNA of Ad-miR29a（pre-miR-29a）.Methods The pre-miR-29a sequence was inserted into multi-clone site of pDC315 in sense direction and identified by endonuclease digestion and DNA sequence analysis.The recombinant plasmid pDC315-miR29a was transfected into HEK293 cell together with plasmid pBHGlox（deltaE1,3）containing adenoviral genome,then the replication-deficient recombinant adenovirus expression vector of pre-miR-29a（Ad-miR29a）was obtained,and identified by infecting test and PCR amplification.Results Recombinant Ad-miR29a showed good infectivity.After purification and concentration,its titer reached 5×109 pfu/ml.The pre-miR-29a special fragment could be amplified by PCR from Ad-miR29a instead of control Ad-LacZ.Conclusion Ad-miR29a is successfully constructed.It provides an experimental basis to further study the role of microRNAs in the occurrence and development of tumors and the value of miR-29a in oncotherapy.

关 键 词：微小RNAS hsa-miR-29a 人胚肾293细胞 复制缺陷型腺病毒载体 

分 类 号：R735[医药卫生—肿瘤]