pSUPER-IL-11-shRNA表达载体的构建与鉴定  

Construction and Identification of pSUPER-IL-11-shRNA

作　　者：王伟[1] 陈雪[1] 钟兆铭 储红映 胡泽东[1] 程瑞[1] 孙传政[1] WANG Wei CHEN Xue ZHONG Zhaoming CHU Hongying HU Zedong CHEN Rui SUN Chuanzheng(Department of Head and Neck Surgery, the Third Affiliated Hospital of Kunming Medical University, Kunming 650118, Yunnan , China)

机构地区：[1]昆明医科大学第三附属医院头颈外科,云南昆明650118

出　　处：《贵州医科大学学报》2016年第11期1269-1273,共5页Journal of Guizhou Medical University

基　　金：国家自然科学基金(81560470;81260402);云南省高层次卫生技术人员专项经费(D-201243);云南省中青年学术技术带头人后备人才专项经费(2015HB086)

摘　　要：目的:构建及测序鉴定重组质粒p SUPER-IL-11-shRNA,检测其对甲状腺未分化癌(anaplastic thyroid carcinoma,ATC)细胞株sw579 IL-11基因的沉默效应。方法:根据Genebank中IL-11 c DNA序列,设计并合成3对两端有酶切位点的特异编码IL-11shRNA序列的寡核苷酸链,退火成互补双链后与p SUPER.retro.puro质粒载体连接,转至大肠杆菌DH-5α菌株,挑取单个抗性克隆,提取质粒进行1%琼脂糖凝胶电泳鉴定及测序分析,脂质体介导重组质粒转染ATC细胞株sw579,real-time PCR及ELISA检测其对靶基因IL-11的表达影响。结果:重组质粒p SUPER-IL-11-shRNA酶切产物凝胶电泳结果显示,3个样本均为阳性重组质粒,测序鉴定3种重组质粒p SUPER-IL-11-shRNA序列正确,转染3种重组质粒后sw579细胞裂解液中IL-11 mRNA和细胞培养上清液中IL-11蛋白显著降低。结论:本研究成功构建重组质粒p SUPER-IL-11-shRNA,为进一步探索IL-11在ATC进展中的作用奠定了基础。Objective：To construct and identify pSUPER-IL-11-shRNA and detect the silencing effect of IL-11 gene of sw579 cell line of anaplastic thyroid carcinoma（ ATC）. Methods：According to IL-11 cDNA gene sequence in the Gene bank,three pairs of oligonucleotides with specific coding IL-11shRNA sequence,each containing the sites of restriction endonuclease at both ends,were designed and synthesized. After annealing,the complementary double strands were connected with pSUPER. retro. puro plasmid vector,which was transfected into DH-5α bacterial strain. The single resistance clone was collected. The plasmid was extracted and the recombinants were identified by 1% agarose gel electrophoresis,sequenced and analyzed. Liposome mediated recombinant plasmid was transfected into sw579 cell line of ATC,and real-time PCR and ELISA were adopted to detect its effect on the ex-pression of target gene IL-11 . Results：The results gel electrophoresis for enzyme-digested product showed that the pSUPER IL-11shRNA was successfully constructed. 3 samples were all positive recom-binant plasmids,and the sequences of 3 recombinant plasmids of pSUPER-IL-11-shRNA were identi-fied by sequencing. IL-11 mRNA in sw579 cell lysis solution and IL-11protein in cell culture superna-tant of sw579 cells were significantly decreased after transfection of 3 recombinant plasmids. Conclusion：pSUPER-IL-11shRNA expression vector has been successfully constructed,and makes it possible to further explore the role of IL-11 in ATC.

关 键 词：甲状腺肿瘤 白细胞介素-11 RNA 小分子干扰 质粒 

分 类 号：R34[医药卫生—基础医学]