C_(57)BL小鼠survivin基因miRNA表达载体的构建  

Construction of C_(57)BL mice survivin gene miRNA expression vector

作　　者：王海峰[1] 苟欣[1] 黎衍敏[1] 何卫阳[1] 杨华安[1] 

机构地区：[1]重庆医科大学附属第一医院泌尿外科,重庆市400016

出　　处：《中国组织工程研究与临床康复》2009年第53期10491-10494,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金基金资助(30772043)~~

摘　　要：背景:Survivin在多种肿瘤组织中的高表达,具有调节细胞增殖分裂和强大的抗凋亡功能。目的:利用RNA干扰技术,构建具有特异性阻断C57BL小鼠survivin基因的微小RNA(micro RNA,miRNA)表达载体。设计、时间及地点:单一样本观察,于2008-06/11在重庆医科大学附属第一医院神经内科实验中心完成。材料:环形pcDNATM6.2-GW/EmGFPmiR和BLOCK-iTTM PolII miR RNA干扰Expression Vector Kit with EmGFP为Invitrogen公司产品;DH5a大肠杆菌为实验室保存;xhoI和BamHI酶、壮观霉素均为上海生工生物工程有限公司产品。方法:应用设计软件在C57BL小鼠survivin基因的mRNA上寻找特异性的短核苷酸序列,并设计合成4对寡核苷酸序列,经退火后形成双链DNA片段,采用基因克隆技术,将其克隆到pcDNATM6.2-GW/EmGFPmiR的载体中,转化DH5a大肠杆菌,挑单菌落种于含有壮观霉素LB液体培养基中,提取质粒。主要观察指标:应用测序法和琼脂糖电泳检测对重组体进行鉴定。结果:测序结果显示插入片段与线性载体连接正确,无碱基突变、缺失、插入等异常。双酶切处理pcDNATM6.2-GW/EmGFP-miR重组质粒结果显示片段大小与预期相符。结论:实验结果表明成功构建了C57BL小鼠survivin基因的微小RNA表达载体。BACKGROUND： Increased expression of survivin in various tumor tissues can regulate cell proliferation, division, and plays an important role in protecting cells from apoptosis. OBJECTIVE： To construct the specific micro RNA （miRNA） expression vector that can block the C57BL mice survivin gene by RNA interference （RNAI） technique. DESIGN, TIME AND SETTING： The single sample observation was performed at the experimental center of Department of Neurology, The First Affiliated Hospital of Chongqing Medical University from June to November 2008. MATERIALS： Ring-shaped pcDNATM6.2-GW/EmGFPmiR and BLOCK-iT^TM Pol II miR RNA interfered Expression Vector Kit with EmGFP was produced by Invitrogen Company. DH5a E. coli was preserved at the laboratory. Xho I and BamH I enzyme, spectinomycin were provided by Shanghai Sangon Biological Engineering Technology ＆ Services Co., Ltd. METHODS： According to sequence of mRNA of C57BL mice survivin provided by Genebank, four pairs of specific oligonucleotide sequences were designed and synthesized by using the software. The annealed oligonucleotide fragment was sub-cloned into pcDNA^TM6.2-GW/EmGFPmiR expression vector by gene clone technique and transformed into DH5a E. coil, subsequently, a single colony was incubated into liquid medium containing spectionmycin. Finally the plasmid was extracted. MAIN OUTCOME MEASURES： The recombinant vector was identified by sequencing and agarose gel electrophoresis. RESULTS： The sequencing revealed that insertion element was correctly cloned into the vector without nucleotide mutation, absence or insertion abnormality. The result of double enzyme digestion demonstrated that the fragment length was coincidence with expectation. CONCLUSION： The C57BL mice survivin miRNA expression vector is successfully constructed.

关 键 词：C57BL小鼠 MIRNA pcDNATM6.2-GW/EmGFPmiR RNA干扰 SURVIVIN 

分 类 号：R318[医药卫生—生物医学工程]