miR-1腺病毒载体的构建及表达分析  

Establishment and Expression Analysis of miR-1 Adenovirus Vector

作　　者：许旭东[1] 宋晓伟[1] 李青[2] 林丽[3] 袁文俊[3] 荆清[1,2] 秦永文[1] 

机构地区：[1]第二军医大学长海医院心血管内科,上海200433 [2]中国科学院上海生命科学研究院健康科学研究所,上海200025 [3]第二军医大学生理学教研室,上海200433

出　　处：《南昌大学学报（医学版）》2010年第8期1-3,7,共4页Journal of Nanchang University：Medical Sciences

基　　金：国家自然科学基金(30828006)

摘　　要：目的构建并鉴定microRNA-1(miR-1)的腺病毒表达载体。方法 PCR扩增含大鼠miR-1前体的DNA片断,并将其克隆到腺病毒穿梭质粒pAdTrack。pAdTrack经PmeI酶切线性化后与腺病毒骨架质粒pAdEasy-1在BJ5183菌中进行同源重组。重组质粒pAdprecusor-miR-1线性化后,转染293A细胞,进行病毒包装,得到重组腺病毒颗粒Ad-miR-1。Ad-miR-1与Ad-GFP病毒转染培养的乳鼠心肌细胞,通过实时荧光定量PCR方法检测miR-1的表达效率。结果基因测序及酶切鉴定证实重组Adprecursor-miR-1腺病毒载体构建成功;腺病毒Ad-miR-1转染心肌细胞后,实时荧光定量PCR方法证实腺病毒Ad-miR-1能够显著提高心肌细胞内miR-1的表达水平。结论利用同源重组的方法构建的miR-1腺病毒能够提高心肌细胞内miR-1的表达水平。Objective To construct and identify heart-enriched microRNAs adenovirus vector containing miR-1 precursor gene.Methods The primers of miR-1 precursor was designed and used for PCR amplification.The products were then cloned into adenovirus shuttle plasmid pAdTrack and linearized by enzyme Pme I;the resultant plasmid was co-transfected into E coli BJ51 83cells with adenovirus backbone plasmid pAdEasy-1 for homologous recombination.Then the recombinant plasmid was identified,1 inearized and packaged with QBI-293 Acells to amplify the recombinant adenovirus Ad-miR-1 ,which was then used to infect cardiomyocytes.Real-time quantitative PCR was used to detect the expression of miR-1 .Results miR-1 recombinant plasmid was constructed successfully as confirmed by sequencing and enzyme digestion.Real-time quantitative PCR confirmed that adenovirus Ad-miR-1 can significantly enhance the intracellular expression of miR-1 in cardiomyocytes.Conclusion The adenovirus that can express miR-1 has been successfully constructed and transfection with it into cardiomyocytes can elevate the expression level of miR-1.

关 键 词：心肌细胞 腺病毒 MICRORNAS MIR-1 

分 类 号：R542.2[医药卫生—心血管疾病]