人Annexin A2基因真核表达的构建及活性
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摘要
为了构建人Annexin A2基因真核表达载体。通过TRIzol法提取Hela宫颈癌细胞中总RNA,采用RT-PCR技术(逆转录-聚合酶链反应)扩增Annexin A2的基因片段,并成功构建成VR1012-Annexin A2-HA重组质粒。经酶切、测序检测其构建的正确性,将质粒瞬时转染到293T人胚肾细胞,使用免疫荧光和蛋白印迹(Western blot)法检测基因表达,使用流式细胞术检测转染后细胞凋亡率。结果表明实验成功构建了具有表达活性的Annexin A2真核表达质粒,为进一步研究Annexin A2的抗凋亡作用奠定了基础。
To construct human Annexin A2 gene eukaryotic expression vector,using TRIzol method of extracting total RNA Hela cervical cancer cells,using RT-PCR amplification Annexin A2 gene fragments,a VR1012-Annexin A2-HA recombinant plasmid was built successfull. By enzyme digestion and sequencing to detect the correctness of its building,the transient transfection plasmid to 293 T embryonic kidney cells,using immunofluorescence and protein imprinting( Western blot) method to detect gene expression,using flow cytometry cell apoptosis rate after transfection was detected. Experiment result show that,the Annexin A2 eukaryotic expression plasmid with expression activity was constructed successfull,which laid a foundation for further study on the anti-apoptotic effect of Annexin A2.
To construct human Annexin A2 gene eukaryotic expression vector,using TRIzol method of extracting total RNA Hela cervical cancer cells,using RT-PCR amplification Annexin A2 gene fragments,a VR1012-Annexin A2-HA recombinant plasmid was built successfull. By enzyme digestion and sequencing to detect the correctness of its building,the transient transfection plasmid to 293 T embryonic kidney cells,using immunofluorescence and protein imprinting( Western blot) method to detect gene expression,using flow cytometry cell apoptosis rate after transfection was detected. Experiment result show that,the Annexin A2 eukaryotic expression plasmid with expression activity was constructed successfull,which laid a foundation for further study on the anti-apoptotic effect of Annexin A2.
引文
1 Su H,Yang X,Wang S,et al. Effect of annexin II-mediated conversion of plasmin from plasminogen on airborne transmission of H9N2avian influenza virus[J]. Veterinary Microbiology, 2018,223:100-106
2 Ma S,Lu C C,Yang L Y,et al. ANXA2 promotes esophageal cancer progression by activating MYC-HIF1A-VEGF axis[J]. Journal of Experimental Clinical Cancer Research,2018,37(1):183
3 Wei W S,Chen X,Guo L Y,et al. TRIM65 supports bladder urothelial carcinoma cell aggressiveness by promoting ANXA2 ubiquitination and degradation[J]. Cancer Letters,2018,435:10-22
4 Kaya C,Alay I,Guraslan H,et al. The role of serum caspase 3 levels in prediction of endometriosis severity[J]. Gynecol Obstet Invest,2018(2):1-10
5 Kuljittichanok D,Diskul-Na-Ayudthaya P, Weeraphan C, et al.Effect of derris scandens extract on a human hepatocellular carcinoma cell line[J]. Oncology Letters,2018,16(2):1943-1952
6 Nicolay J P,Thorn V,Daniel C,et al. Cellular stress induces erythrocyte assembly on intravascular von Willebrand factor strings and promotes microangiopathy[J]. Scientific Reports,2018,8(1):10945
7 Nguyen Quang N,Bouvier C,Henriques A,et al. Time-lapse imaging of molecular evolution by high-throughput sequencing[J]. Nucleic Acids Research,2018,46(15):7480-7494
8 Stewart S E,Ashkenazi A,Williamson A,et al. Transbilayer phospholipid movement facilitates the translocation of annexin across membranes[J]. Journal Cell Science,2018,131(14):1-26
9 Zhou W,Zhang Y,Zeng Y,et al. Screening and characterization of an Annexin A2 binding aptamer that inhibits the proliferation of myeloma cells[J]. Biochimie,2018,151:150-158
10 Holthenrich A,Gerke V. Regulation of von-willebrand factor secretion from endothelial cells by the Annexin A2-S100A10 complex[J]. International Journal of Molecular Sciences,2018,19(6):1-11
11 李汶潞.膜联蛋白A2对创伤性血脑屏障损伤的保护作用及机制研究[D].杭州:浙江大学,2017Li Wenlu. The protective effect of Annexin A 2 on trauma-induced blood brain barrier injury and its meechanisms[D]. Hangzhou:Zhejiang University,2017
12 李大河.强直性脊柱炎韧带组织比较蛋白质组学研究及膜联蛋白A2功能验证[D].上海:第二军医大学,2016Li Dahe. Comparative proteomics study of proteins in ligament of Ankylosing Spondlitis and biological function analysis of Annexin A2[D]. Shanghai:Second Military Medical University,2016
13 赵强,张志伟,刘重元,等. PKCα-Annexin A2-S100A10在胃癌组织中的表达及意义[J].世界华人消化杂志,2014,22(13):1793-1800Zhao Qiang,Zhang Zhiwei,Liu Chongyuan,et al. Significance of expression of PKCα,AnnexinA2 and S100A10 proteins in gastric cancer[J]. World Journal of Chinese Digestion,2014,22(13):1793-1800
14 Chang X B,Yang Y Q,Gao J C,et al. Annexin A2 binds to vimentin and contributes to porcine reproductive and respiratory syndrome virus multiplication[J]. Veterinary Research,2018,49(1):75
15 Yang N,Wang L,Liu J,et al. MicroRNA-206 regulates the epithelial-mesenchymal transition and inhibits the invasion and metastasis ofprostate cancer cells by targeting Annexin A2[J]. Oncology Letters,2018,15(6):8295-8302
16 Peterson S M,Turner J E,Harrington A,et al. Notch2 and proteomic signatures in mouse neointimal lesion formation[J]. Arteriosclerosis Thrombosis and Vascular Biology,2018,38(7):1576-1593
17 李慧芳,雷磊,谭展望,等.寿胎丸对RSA模型小鼠子宫蜕膜Annexin A 2及TTR表达影响的研究[J].中华中医药学刊,2013,31(11):2369-2372,2591Li Huifang,Lei Lei,Tan Zhanwang,et al. Effect of shoutai on expressions of Annexin A2 and TTR in recurrent spontaneous abortion mice[J]. Chinese Journal of Traditional Chinese Medicine,2013,31(11):2369-2372,2591
18 樊素珍,苑中甫.宫颈癌组织中Annexin A2和Ki-67蛋白的表达[J].郑州大学学报(医学版),2010,45(1):81-84Fan Suzhen,Yuan Zhongpu. Annexin A2 and ki-67 protein expression in cervical cancer tissues[J]. Journal of Zhengzhou University(Medical Edition),2010,45(1):81-84
19 赵玲,王鹏,崔全才. Annexin A2在人甲状腺癌中表达的意义[J].诊断病理学杂志,2008,(2):117-120Zhao ling,Wang Peng,Cui Quancai. Annexin A2 expression in human thyroid cancer[J]. Journal of Diagnostic Pathology,2008,(2):117-120
20 吴晓娜,蒋红兵.流式细胞术的工作原理及其临床应用[J].中国医疗设备,2011,26(3):91-93Wu Xiaona,Jiang Hongbing. Working principle and clinical application of flow cytometry[J]. Chinese Medical Equipment,2011,26(3):91-93
2 Ma S,Lu C C,Yang L Y,et al. ANXA2 promotes esophageal cancer progression by activating MYC-HIF1A-VEGF axis[J]. Journal of Experimental Clinical Cancer Research,2018,37(1):183
3 Wei W S,Chen X,Guo L Y,et al. TRIM65 supports bladder urothelial carcinoma cell aggressiveness by promoting ANXA2 ubiquitination and degradation[J]. Cancer Letters,2018,435:10-22
4 Kaya C,Alay I,Guraslan H,et al. The role of serum caspase 3 levels in prediction of endometriosis severity[J]. Gynecol Obstet Invest,2018(2):1-10
5 Kuljittichanok D,Diskul-Na-Ayudthaya P, Weeraphan C, et al.Effect of derris scandens extract on a human hepatocellular carcinoma cell line[J]. Oncology Letters,2018,16(2):1943-1952
6 Nicolay J P,Thorn V,Daniel C,et al. Cellular stress induces erythrocyte assembly on intravascular von Willebrand factor strings and promotes microangiopathy[J]. Scientific Reports,2018,8(1):10945
7 Nguyen Quang N,Bouvier C,Henriques A,et al. Time-lapse imaging of molecular evolution by high-throughput sequencing[J]. Nucleic Acids Research,2018,46(15):7480-7494
8 Stewart S E,Ashkenazi A,Williamson A,et al. Transbilayer phospholipid movement facilitates the translocation of annexin across membranes[J]. Journal Cell Science,2018,131(14):1-26
9 Zhou W,Zhang Y,Zeng Y,et al. Screening and characterization of an Annexin A2 binding aptamer that inhibits the proliferation of myeloma cells[J]. Biochimie,2018,151:150-158
10 Holthenrich A,Gerke V. Regulation of von-willebrand factor secretion from endothelial cells by the Annexin A2-S100A10 complex[J]. International Journal of Molecular Sciences,2018,19(6):1-11
11 李汶潞.膜联蛋白A2对创伤性血脑屏障损伤的保护作用及机制研究[D].杭州:浙江大学,2017Li Wenlu. The protective effect of Annexin A 2 on trauma-induced blood brain barrier injury and its meechanisms[D]. Hangzhou:Zhejiang University,2017
12 李大河.强直性脊柱炎韧带组织比较蛋白质组学研究及膜联蛋白A2功能验证[D].上海:第二军医大学,2016Li Dahe. Comparative proteomics study of proteins in ligament of Ankylosing Spondlitis and biological function analysis of Annexin A2[D]. Shanghai:Second Military Medical University,2016
13 赵强,张志伟,刘重元,等. PKCα-Annexin A2-S100A10在胃癌组织中的表达及意义[J].世界华人消化杂志,2014,22(13):1793-1800Zhao Qiang,Zhang Zhiwei,Liu Chongyuan,et al. Significance of expression of PKCα,AnnexinA2 and S100A10 proteins in gastric cancer[J]. World Journal of Chinese Digestion,2014,22(13):1793-1800
14 Chang X B,Yang Y Q,Gao J C,et al. Annexin A2 binds to vimentin and contributes to porcine reproductive and respiratory syndrome virus multiplication[J]. Veterinary Research,2018,49(1):75
15 Yang N,Wang L,Liu J,et al. MicroRNA-206 regulates the epithelial-mesenchymal transition and inhibits the invasion and metastasis ofprostate cancer cells by targeting Annexin A2[J]. Oncology Letters,2018,15(6):8295-8302
16 Peterson S M,Turner J E,Harrington A,et al. Notch2 and proteomic signatures in mouse neointimal lesion formation[J]. Arteriosclerosis Thrombosis and Vascular Biology,2018,38(7):1576-1593
17 李慧芳,雷磊,谭展望,等.寿胎丸对RSA模型小鼠子宫蜕膜Annexin A 2及TTR表达影响的研究[J].中华中医药学刊,2013,31(11):2369-2372,2591Li Huifang,Lei Lei,Tan Zhanwang,et al. Effect of shoutai on expressions of Annexin A2 and TTR in recurrent spontaneous abortion mice[J]. Chinese Journal of Traditional Chinese Medicine,2013,31(11):2369-2372,2591
18 樊素珍,苑中甫.宫颈癌组织中Annexin A2和Ki-67蛋白的表达[J].郑州大学学报(医学版),2010,45(1):81-84Fan Suzhen,Yuan Zhongpu. Annexin A2 and ki-67 protein expression in cervical cancer tissues[J]. Journal of Zhengzhou University(Medical Edition),2010,45(1):81-84
19 赵玲,王鹏,崔全才. Annexin A2在人甲状腺癌中表达的意义[J].诊断病理学杂志,2008,(2):117-120Zhao ling,Wang Peng,Cui Quancai. Annexin A2 expression in human thyroid cancer[J]. Journal of Diagnostic Pathology,2008,(2):117-120
20 吴晓娜,蒋红兵.流式细胞术的工作原理及其临床应用[J].中国医疗设备,2011,26(3):91-93Wu Xiaona,Jiang Hongbing. Working principle and clinical application of flow cytometry[J]. Chinese Medical Equipment,2011,26(3):91-93
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