摘要
目的包装负载人端粒酶逆转录酶(hTERT)调控相关miR-138前体全长cDNA(pre-miR-138)的复制缺陷型腺病毒载体(Ad-miR138)。方法 pre-miR-138全基因合成后插入腺病毒穿梭质粒(pDC315-miR138),与腺病毒包装质粒pBHGloxDel-taE1、3Cre共转染人胚肾293细胞(HEK293),包装Ad-miR138并大量扩增,收集纯化。对纯化后的Ad-miR138进行滴度测定、感染性鉴定、电镜鉴定及双引物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5×1010pfu/mL。电镜可见病毒在HEK293细胞中大量复制。PCR双引物鉴定Ad-miR138可扩增出Ad及pre-miR-138特异片段,而对照Ad-LacZ只能扩增出Ad特异片段,不能扩增出pre-miR-138特异片段。结论成功包装了负载miR-138全长cDNA的复制缺陷型腺病毒载体,为进一步研究微RNAs(microRNAs)在肿瘤发生、发展中的作用,以及hTERT调控相关miR-138在肿瘤治疗中的价值奠定了基础。
Objective To construct a replication-deficient recombinant adenovirus carrying hTERT-regulated miR-138.Methods The pre-miR-138 sequence was inserted into downstream of CMV promoter of the adenoviral shuttle plasmid pDC315 in sense direction,and the recombinant plasmid pDC315-miR138 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-138（Ad-miR138） was obtained,and identified by infecting test,electronic microscope observation and PCR amplification.Results After purification and concentration,the titer of Ad-miR138 was achieved to 5×1010 pfu/mL.Virus particles could be found in HEK293 cells under transmission electron microscope.Both adenovirus and pre-miR-138 special fragment could be amplified from Ad-miR138 by PCR,whereas pre-miR-138 special fragment could not be amplified from the control Ad-LacZ.Conclusion The replication-deficient recombinant adenovirus carrying miR-138 was constructed successfully.This study establishes a foundation to further research on how microRNAs involves in the carcinoma development and the value of hTERT-regulated miR-138 on tumor therapy.
出处
《重庆医学》
CAS
CSCD
北大核心
2011年第9期896-898,共3页
Chongqing medicine
基金
重庆市自然科学基金资助项目(2008BB5274
2009BB5314)
