[1]何淑雅,杨奇,王茹静,等.耐辐射奇球菌pprM基因在真核细胞中的表达[J].国际放射医学核医学杂志,2017,41(2):103-107.[doi:10.3760/cma.j.issn.1673-4114.2017.02.005]
　He Shuya,Yang Qi,Wang Rujing,et al.Expression of pprM gene from Deinococcus radiodurans in eukaryotic cells[J].International Journal of Radiation Medicine and Nuclear Medicine,2017,41(2):103-107.[doi:10.3760/cma.j.issn.1673-4114.2017.02.005]
点击复制

耐辐射奇球菌pprM基因在真核细胞中的表达(/HTML)
分享到：

《国际放射医学核医学杂志》[ISSN:1673-4114/CN:12-1381/R]

卷:
41
期数:
2017年第2期
页码:
103-107
栏目:
论著
出版日期:
2017-03-25

文章信息/Info

Title:
Expression of pprM gene from Deinococcus radiodurans in eukaryotic cells
作者:
何淑雅1; 2;  杨奇1;  王茹静3;  肖方竹2;  王五洲1;  唐艳2;  蒋雨薇1;  马云1
1. 421001 衡阳, 南华大学生物化学与分子生物学教研室;
2. 421001 衡阳, 南华大学公共卫生学院放射医学教研室;
3. 421002 衡阳, 衡阳市第一人民医院
Author(s):
He Shuya1; 2;  Yang Qi1;  Wang Rujing3;  Xiao Fangzhu2;  Wang Wuzhou1;  Tang Yan2;  Jiang Yuwei1;  Ma Yun1
1. Department of Biochemistry and Molecular Biology, University of South China, Hengyang 421001, China;
2. Department of Radiation Medicine, School of Public Health, University of South China, Hengyang 421001, China;
3. the First People’s Hospital of Hengyang, Hengyang 421002, China
关键词:
耐辐射奇球菌; 辐射; pprM基因; 293T细胞
Keywords:
Deinococcus radiodurans; Radiation; pprM gene; 293T cells
DOI:
10.3760/cma.j.issn.1673-4114.2017.02.005
摘要:
目的 扩增耐辐射奇球菌辐射抗性基因pprM，构建pEGFP-C1-pprM重组载体，转入293T细胞并表达PprM蛋白，为研究原核细胞辐射抗性基因pprM是否提高真核细胞的辐射抗性及其可能存在的作用机制奠定实验基础。方法 以无任何突变的pGEX-6p-1-pprM重组质粒为模版，设计引物PCR扩增pprM基因，利用琼脂糖凝胶DNA回收试剂盒纯化回收目的片段，EcoR I、BamH I双酶切回收的目的片段及质粒pEGFP-C1，将双酶切产物进行连接，连接产物转化至大肠杆菌JM109感受态细胞后涂布于含卡那霉素（Kan）抗性的Luria-Bertani（LB）固体培养基上进行筛选，所筛选的阳性克隆进一步用菌落PCR，EcoR I、BamH I双酶切及测序鉴定。通过Lipofectamine2000转染试剂将pEGFP-C1-pprM重组质粒转入293T细胞，倒置荧光显微镜观察绿色荧光融合蛋白的表达。裂解细胞抽提蛋白，Western blot进一步验证PprM蛋白的表达。结果 菌落PCR结果及双酶切结果显示，在400 bp左右处有一条明显的目的条带，测序结果显示碱基序列与原基因序列一致，载体构建成功。荧光拍照结果显示，pEGFP-C1-pprM重组质粒成功转染293T细胞并表达绿色荧光融合蛋白；Western blot结果显示在40×103处有融合蛋白表达。结论 笔者成功将所构pEGFP-C1-pprM重组质粒转入293T细胞，并表达相应蛋白，为后续实验研究原核基因pprM及其产物对真核细胞辐射抗性的影响奠定了良好的实验基础。
Abstract:
Objective To enhance the radiation resistance of eukaryotic cells and to identify the mechanisms that underlie radiation resistance, the radioresistant pprM gene was amplified from Deinococcus radiodurans and used to construct a recombinant pEGFP-C1-pprM plasmid for the expression of PprM protein in 293T cells. Methods The pprM gene was amplified from pGEX-6p-1-pprM via PCR and purified from agarose gel with a DNA recovery kit. The purified PCR product was digested with EcoR I and BamH I and ligated into the pEGFP-C1 plasmid. Recombinant plasmids were transfected into competent JM109 cells, which were then cultured on LB solid medium that contained kanamycin. Positive clones were identified and characterized via bacterial colony PCR, restriction enzyme digestion, and sequencing analysis. Lipofectamine 2000 reagent was used to transfect pEGFP-C1-pprM plasmids into 293T cells. Green fluorescent fusion protein was observed via fluorescence microscopy and identified by Western blot. Results Bacterial colony PCR and double digestion showed that the target band is approximately 400 bp in length. Sequencing results showed that the base sequence was identical to the original gene sequence, thus indicating the successful construction of the recombinant plasmid. Fluorescence photography results showed that pEGFP-C1-pprM plasmids were successfully transfected into 293T cells. Western blot results showed that fusion protein is approximately 40×103 in weight. Conclusions The pEGFP-C1-pprM plasmid was transfected into 293T cells, which then successfully expressed PprM protein. This study provides the foundation for future research on the pprM gene and the effects of its products on the radiation resistance of 293T cells.

参考文献/References:

[1] Citrin D, Cotrim AP, Hyodo F, et al. Radioprotectors and mitigators of radiation-induced normal tissue injury[J]. Oncologist, 2010, 15(4):360-371. DOI:10. 1634/theoncologist. 2009-S104.
[2] Szejk M, Kolodziejczyk-Czepas J, Zbikowska HM. Radioprotectors in radiotherapy-advances in the potential application of phytochemicals[J/OL]. Postepy Hig Med Dosw(Online), 2016, 30, 70(0):722-734[2016-12-20]. https://www.ncbi.nlm.nih.gov/pubmed/27356603. DOI:10. 5604/17322693. 1208039.
[3] Everett WH, Curiet DT. Curiel gene therapy for radioprotection[J]. Cancer Gene Ther, 2015, 22(4):172-180. DOI:10.1667/RR14186. 1.
[4] Krisko A, Radman M. Biology of extreme radiation resistance:the way of Deinococcus radiodurans[J/OL]. Cold Spring Harb Perspect Biol, 2013, 5(7):a012765[2016-12-20]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3685888/.DOI:10.1101/cshperspect.a012765.
[5] Sai CH, Liao R, Chou B, et al. Transcriptional analysis of Deinococcus radiodurans reveals novel small RNAs that are differentially expressed under ionizing radiation[J]. Appl Environ Microbiol, 2015, 81(5):1754-1764. DOI:10. 1128/AEM. 03709-14.
[6] Ohba H, Satoh K, Sghaier H, et al. Identification of pprM:a modulator of the pprI-dependent DNA damage response in Deinococcus radiodurans[J]. Extremophiles, 2009, 13(3):471-479. DOI:10. 1007/s00792-009-0232-8.
[7] Jeong SW, Seo HS, Kim MK, et al. pprM is necessary for up-regulation of katE1, encoding the major catalase of Deinococcus radiodurans, under unstressed culture conditions[J]. J Microbiol, 2016, 54(6):426-431. DOI:10. 1007/s12275-016-6175-8.
[8] Mettler FA. Medical effects and risks of exposure to ionising radiation[J/OL]. J Radiol Prot, 2012, 32(1):N9-13[2016-12-20]. http://sci-hub.cc/10.1088/0952-4746/32/1/N9. DOI:10.1088/0952-4746/32/1/N9.
[9] Li J, Li Q, Ma X, et al. Biosynthesis of gold nanoparticles by the extreme bacterium Deinococcus radiodurans and an evaluation of their antibacterial properties[J]. Int J Nanomedicine, 2016, 11:5931-5944. DOI:10. 2147/IJN. S119618.
[10] Passot FM, Nguyen HH, Dard-Dascot C, et al. Nucleoid organization in the radioresistant bacterium Deinococcus radiodurans[J]. Mol Microbiol, 2015, 97(4):759-774. DOI:10. 1111/mmi. 13064.
[11] Slade D, Radman M. Oxidative stress resistance in Deinococcus radiodurans[J]. Microbiol Mol Biol Rev, 2011, 75(1):133-191. DOI:10. 1128/MMBR. 00015-10.
[12] Battista JR. Radiation resistance:The fragments that remain[J/OL]. Curr Biol, 2000, 10(5):R204-205[2016-12-20]. http://www.cell.com/current-biology/pdf/S0960-9822(00)00353-5.pdf.

相似文献/References:

[1]张宇睿,徐文清.电离辐射对线粒体损伤的研究进展[J].国际放射医学核医学杂志,2016,40(2):154.[doi:10.3760/cma.j.issn.1673-4114.2016.02.014]
　Zhang Yurui,Xu Wenqing.Damages of ionizing radiation on mitochondria[J].International Journal of Radiation Medicine and Nuclear Medicine,2016,40(2):154.[doi:10.3760/cma.j.issn.1673-4114.2016.02.014]
[2]徐畅,方连英,孔阳阳,等.沉默BLAP75基因对电离辐射诱导DNA损伤的影响[J].国际放射医学核医学杂志,2016,40(4):244.[doi:10.3760/cma.j.issn.1673-4114.2016.04.001]
　Xu Chang,Fang Lianying,Kong Yangyang,et al.Effects of silencing BLAP75 on DNA damage induced by ionizing radiation[J].International Journal of Radiation Medicine and Nuclear Medicine,2016,40(2):244.[doi:10.3760/cma.j.issn.1673-4114.2016.04.001]
[3]张俊伶,薛晓蕾,李源,等.富氢水对电离辐射引起胸腺细胞损伤的影响[J].国际放射医学核医学杂志,2015,39(5):358.[doi:10. 3760 / cma. j. issn. 1673-4114. 2015. 05. 001]
　zhang junling,xue xiaolei,li yuan,et al.effects of hydrogen-rich water on radiation-induced thymus injury[J].International Journal of Radiation Medicine and Nuclear Medicine,2015,39(2):358.[doi:10. 3760 / cma. j. issn. 1673-4114. 2015. 05. 001]
[4]付岳,王彦,杜利清,等.sirt1基因沉默在辐射诱导的nlrp3和il-1β表达中的作用研究[J].国际放射医学核医学杂志,2015,39(5):367.[doi:10. 3760 / cma. j. issn. 1673-4114. 2015. 05. 003]
　fu yue*,wang yan,du liqing,et al.effects of nlrp3 and il-1β on radiation-induced expression through sirt1 gene silencing[J].International Journal of Radiation Medicine and Nuclear Medicine,2015,39(2):367.[doi:10. 3760 / cma. j. issn. 1673-4114. 2015. 05. 003]
[5]董娟聪,刘红艳,党旭红,等.12c重离子与γ射线对人外周血淋巴细胞peng-ebv增殖、细胞周期及凋亡影响的比较[J].国际放射医学核医学杂志,2015,39(5):385.[doi:10. 3760 / cma. j. issn. 1673-4114. 2015. 05. 007]
　dong juancong,liu hongyan,dang xuhong,et al.effects of 12c heavy ion and γ ray irradiation in cell growth, cell cycle and apoptosis of human peripheral blood lymphocytes[J].International Journal of Radiation Medicine and Nuclear Medicine,2015,39(2):385.[doi:10. 3760 / cma. j. issn. 1673-4114. 2015. 05. 007]
[6]李晴,王璐,徐畅,等.姜油树脂对辐射后间充质干细胞nrf2及其靶基因表达的影响[J].国际放射医学核医学杂志,2015,39(5):389.[doi:10. 3760 / cma. j. issn. 1673-4114. 2015. 05. 008]
　li Qing,wang lu,xu chang,et al.ginger oleoresin induced expression of nrf2 and its target genes in mesenchymal stem cells after radiation[J].International Journal of Radiation Medicine and Nuclear Medicine,2015,39(2):389.[doi:10. 3760 / cma. j. issn. 1673-4114. 2015. 05. 008]
[7]刘建功,党旭红,刘红艳,等.核工业某厂放射工作人员与非放射工作人员的mdm2基因表达调查与分析[J].国际放射医学核医学杂志,2015,39(5):416.[doi:10. 3760 / cma. j. issn. 1673-4114. 2015. 05. 015]
　liu jiangong,dang xuhong,liu hongyan,et al.investigation and analysis of mdm2 expression level in radiation-exposed workers and non-radiation-exposed workers[J].International Journal of Radiation Medicine and Nuclear Medicine,2015,39(2):416.[doi:10. 3760 / cma. j. issn. 1673-4114. 2015. 05. 015]
[8]王璐,李晴,徐畅,等.sirna干扰sirt1对辐射后il-6表达的影响[J].国际放射医学核医学杂志,2015,39(5):423.[doi:10. 3760 / cma. j. issn. 1673-4114. 2015. 05. 017]
　wang lu,li Qing,xu chang,et al.effect of sirt1 sirna interference on the expression of il-6 after radiation[J].International Journal of Radiation Medicine and Nuclear Medicine,2015,39(2):423.[doi:10. 3760 / cma. j. issn. 1673-4114. 2015. 05. 017]
[9]张倩如,李海涛,田红旗.小分子辐射防护药物的研究进展[J].国际放射医学核医学杂志,2016,40(5):394.[doi:10.3760/cma.j.issn.1673-4114.2016.05.012]
　Zhang Qianru,Li Haitao,Tian Hongqi.Small molecule compounds against radiation:research advances[J].International Journal of Radiation Medicine and Nuclear Medicine,2016,40(2):394.[doi:10.3760/cma.j.issn.1673-4114.2016.05.012]
[10]肖林林,金问森,张江虹,等.电离辐射诱发动脉粥样硬化的研究进展[J].国际放射医学核医学杂志,2015,39(6):493.[doi:10.3760/cma.j.issn.1673-4114.2015.06.012]
　Xiao Linlin,Jin Wensen,Zhang Jianghong,et al.Research progress of radiation induced atherosclerosis[J].International Journal of Radiation Medicine and Nuclear Medicine,2015,39(2):493.[doi:10.3760/cma.j.issn.1673-4114.2015.06.012]
[11]范美婷,马云,何淑雅.耐辐射奇球菌中多效蛋白PprA功能的研究进展[J].国际放射医学核医学杂志,2015,39(2):149.[doi:10.3760/cma.j.issn.1673-4114.2015.02.010]
　Fan Meiting,Ma Yun,He Shuya.Research progress and prediction on pleiotropic protein PprA from Deinococcus radiodurans[J].International Journal of Radiation Medicine and Nuclear Medicine,2015,39(2):149.[doi:10.3760/cma.j.issn.1673-4114.2015.02.010]

备注/Memo

备注/Memo:
收稿日期:2016-12-27。
基金项目:国家自然科学基金项目（81272993）；2015年衡阳市科学技术发展计划项目（2015KJ17）
通讯作者:马云,Email:luckymayun@163.com
更新日期/Last Update: 2017-04-24