人ANKRD49基因真核表达载体的构建及其功能的初步研究和RNA干扰靶点的鉴定

doi:10.13523/j.cb.20141003

中国生物工程杂志

2014, Vol. 34

Issue (10): 15-21 DOI: 10.13523/j.cb.20141003

研究报告

人ANKRD49基因真核表达载体的构建及其功能的初步研究和RNA干扰靶点的鉴定

庞敏¹, 王海龙², 郭民³, 郭睿²

1. 山西医科大学第一医院太原 030001;
2. 山西医科大学基础医学院太原 030001
3. 山西医科大学实验动物中心太原 030001

Construction of an Eukaryocyte Expression Vector of Human ANKRD49 and the Study of Function and RNA Interference Target of ANKRD49

PANG Min¹, WANG Hai-long², GUO Min³, GUO Rui²

1. Department of Respiration, The First Hospital of Shanxi Medical University, Taiyuan 030001, China;
2. Academy of Basic Medicine, Shanxi Medical University, Taiyuan 030001, China;
3. Center of Laboratory Animal, Shanxi Medical University, Taiyuan 030001, China

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摘要：

目的: 克隆人ANKRD49基因并构建其真核表达重组体,利用构建成功的ANKRD49真核表达重组体对其进行功能的初步研究,并筛选和鉴定其RNA干扰靶点. 方法: 提取人肺腺癌细胞株A549总RNA,逆转录-聚合酶链式反应(RT-PCR)对ANKRD49进行扩增,扩增产物与真核表达载体p3×Flag-CMV-14同时进行双酶切,酶切产物连接后转化入感受态细胞Top10,阳性重组质粒p3×Flag-CMV-14/ANKRD49经菌液PCR、双酶切和测序鉴定正确后,用脂质体法(Lipofectamine^TM 2000)转染人胚肾细胞(HEK 293T),免疫印迹(Immunoblotting)和免疫荧光技术检测表达产物.免疫荧光法检测ANKRD49在宿主细胞内的定位.MTT法检测ANKRD49对宿主细胞的增殖作用.设计并合成针对人ANKRD49基因的RNA干扰靶点序列,与p3×Flag-CMV-14/ANKRD49共转染HEK 293T细胞后,Immunoblotting鉴定ANKRD49的RNA干扰靶点. 结果: RT-PCR结果显示,从A549细胞中扩增出约720 bp的片段.菌液PCR、双酶切及测序结果显示重组质粒p3×Flag-CMV-14/ANKRD49构建成功且序列正确.免疫荧光和Immunoblotting结果显示,在转染p3×Flag-CMV-14/ANKRD49的细胞中有ANKRD49的表达,蛋白质相对分子质量(Mr)约为27kDa,而转染空质粒组未见表达.MTT结果显示,ANKRD49对细胞增殖没有影响.共转染实验结果显示,1号和4号RNA干扰序列可以有效降低人ANKRD49的表达. 结论: 成功构建了真核表达重组体p3×Flag-CMV-14/ANKRD49,该蛋白质位于细胞核,不参与细胞增殖;同时鉴定出该基因的2个有效干扰靶点,为进一步研究其功能奠定了基础.

关键词： ANKRD49; 真核表达; RNA干扰; 肺癌

Abstract:

Objective: To construct the eukaryotic expression recombinant plasmid of human ANKRD49, study its function, and identify the RNA interference targets of ANKRD49. Methods: Total RNA was extracted from A549 cells and the cDNA was synthesized. The open reading frame of human ANKRD49 gene was amplified from the cDNA by RT-PCR, and cloned into p3×Flag-CMV-14 to construct recombinant plasmid p3×Flag-CMV-14/ANKRD49. After confirming by colony-PCR, double restrict enzyme digestion and DNA sequencing, the eukaryotic expression recombinant p3×Flag-CMV-14/ANKRD49 was transfected into HEK 293T cells. The targeted protein expressed in host cells was detected by Immunoblotting and immunofluorescence staining. The distribution of ANKRD49 in host cells was detected by immunofluorescence staining. The cell proliferation of ANKRD49-expressed HEK 293T cells were measured through MTT assay. The RNA interference targets of human ANKRD49 were identified via Immunoblotting assay followed by co-transfecting both p3×Flag-CMV-14/ANKRD49 and siRNA into HEK293T cells. Results: The product of RT-PCR was 720 bp. The recombinant plasmid p3×Flag-CMV-14/ANKRD49 was confirmed successfully by colony-PCR, double restrict enzyme digestion and DNA sequencing. Immunofluorescence staining and Immunoblotting showed that human ANKRD49 was expressed successfully in the p3×Flag-CMV-14/ANKRD49 transfected-293T cells than mock transfected cells with molecular weight was approximately 27 kDa. Immunofluorescence staining also revealed that ANKRD49 was distributed in nucleus. MTT assay showed that ANKRD49 had no effect on cell proliferation. Co-transfection and Immunoblotting assays showed that the expression of human ANKRD49 was efficiently knockdown by number 1 and 4 siRNA. Conclusion: The eukaryotic expression recombinant plasmid p3×Flag-CMV-14/ANKRD49 was constructed and expressed in nucleus of HEK 293T cells. This protein had no effect on cell proliferation. Two ANKRD49 interference targets were identified.

Key words: ANKRD49 Eukaryotic expression RNA interference Lung carcinoma

收稿日期: 2014-06-25 出版日期: 2014-10-25

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通讯作者: 庞敏 E-mail: pangmin2009@126.com

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引用本文:

庞敏, 王海龙, 郭民, 郭睿. 人ANKRD49基因真核表达载体的构建及其功能的初步研究和RNA干扰靶点的鉴定[J]. 中国生物工程杂志, 2014, 34(10): 15-21.

PANG Min, WANG Hai-long, GUO Min, GUO Rui. Construction of an Eukaryocyte Expression Vector of Human ANKRD49 and the Study of Function and RNA Interference Target of ANKRD49. China Biotechnology, 2014, 34(10): 15-21.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20141003 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I10/15

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