贵州医科大学学报

2016, v.41;No.194(11) 1269-1273

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pSUPER-IL-11-shRNA表达载体的构建与鉴定
Construction and Identification of pSUPER-IL-11-shRNA

王伟;陈雪;钟兆铭;储红映;胡泽东;程瑞;孙传政;
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;

摘要(Abstract):

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-11 shRNA 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 expression of target gene IL-11. Results: The results gel electrophoresis for enzyme-digested product showed that the pSUPER IL-11 shRNA was successfully constructed. 3 samples were all positive recombinant plasmids,and the sequences of 3 recombinant plasmids of pSUPER-IL-11-shRNA were identified by sequencing. IL-11 mRNA in sw579 cell lysis solution and IL-11 protein in cell culture supernatant of sw579 cells were significantly decreased after transfection of 3 recombinant plasmids.Conclusion: pSUPER-IL-11 shRNA expression vector has been successfully constructed,and makes it possible to further explore the role of IL-11 in ATC.

关键词(KeyWords): 甲状腺肿瘤;白细胞介素-11;RNA,小分子干扰;短发夹RNA;质粒
thyroid neoplasm;interleukin-11;RNA,small molecule interference;short hairpin RNA;plasmid

Abstract:

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基金项目(Foundation): 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)

作者(Author): 王伟;陈雪;钟兆铭;储红映;胡泽东;程瑞;孙传政;
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;

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DOI: 10.19367/j.cnki.1000-2707.2016.11.007

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