盐穗木HcDMC1的原核表达和抗血清的制备
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摘要
DMC1是减数分裂过程中同源染色体配对和重组修复所必需的减数分裂特异蛋白。根据盐胁迫下盐穗木转录组数据库,克隆获得的盐穗木DNA损伤修复基因命名为HcDMC1。为深入分析盐穗木HcDMC1基因的耐盐功能,通过原核表达获得盐穗木HcDMC1的融合蛋白,用于免疫小鼠制备特异性的HcDMC1抗血清。结果表明,利用pET30a-HcDMC1能够在大肠杆菌BL21(DE3)中诱导表达融合蛋白His-HcDMC1,纯化融合蛋白His-Hc DMC1的含量为1.0 mg·m L~(-1)。每次每只小鼠免疫接种50μg融合蛋白,三次免疫接种后进行抗体效价及特异性检测。ELISA检测抗血清滴度约为1∶400 000,Western Blot检测证明了抗血清的特异性。本研究制备的小鼠抗血清能够为盐穗木Hc DMC1蛋白的功能鉴定和免疫检测提供实验材料。
DMC1 is a necessary meiotic specific protein for homologous chromosome pairing and recombinant repair during meiosis. According to the transcriptome database of Halostachys caspica under the salt stress,the DNA damage repair gene DMC1 of H. caspica was cloned and named HcDMC1. In order to further investigate the salt tolerant function of HcDMC1,the HcDMC1 fusion protein obtained from prokaryotic expression was used to immunize mice to prepare the specific antiserum against HcDMC1. The expression of HcDMC1 protein could be induced in Escherichia coli by p ET30a-HcDMC1 and the fusion protein His-HcDMC1 was purified. The content of His-HcDMC1 was 1 mg · m L~(-1). Each mouse was immunized with 50 μg fusion protein for three time,the antibody titer and specificity were detected by ELISA and Western Blot,respectively. The antiserum titer was about 1∶400 000,and the specificity of the antiserum was confirmed by Western Blot. The prepared antiserum can provide experimental materials for the functional identification of HcDMC1 protein.
DMC1 is a necessary meiotic specific protein for homologous chromosome pairing and recombinant repair during meiosis. According to the transcriptome database of Halostachys caspica under the salt stress,the DNA damage repair gene DMC1 of H. caspica was cloned and named HcDMC1. In order to further investigate the salt tolerant function of HcDMC1,the HcDMC1 fusion protein obtained from prokaryotic expression was used to immunize mice to prepare the specific antiserum against HcDMC1. The expression of HcDMC1 protein could be induced in Escherichia coli by p ET30a-HcDMC1 and the fusion protein His-HcDMC1 was purified. The content of His-HcDMC1 was 1 mg · m L~(-1). Each mouse was immunized with 50 μg fusion protein for three time,the antibody titer and specificity were detected by ELISA and Western Blot,respectively. The antiserum titer was about 1∶400 000,and the specificity of the antiserum was confirmed by Western Blot. The prepared antiserum can provide experimental materials for the functional identification of HcDMC1 protein.
引文
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15 .程龙,李茜,熊焰,等.紫外诱导植物产生DNA损伤的修复机制[J].生命的化学,2005,25(6):504-506.Cheng L,Li Q,Xiong Y,et al.Mechanism of repair of DNA damage in plants induced by UV[J].Chemistry of Life,2005,25(6):504-506.
16 .Zafar F,Seidler S B,Kronenberg A,et al.Homologous recombination contributes to the repair of DNA Double-strand breaks induced by High-energy iron ions[J].Radiation Research,2010,173(1):27-39.
17 .Yu X,Egelman E H.Helical filaments of human Dmc1 protein on single-Stranded DNA:a cautionary tale[J].Journal of Molecular Biology,2010,401(3):544-551.
18 .Sharma D,Say A F,Ledford L L,et al.Role of the conserved lysine within the Walker A motif of human DMC1[J].DNA Repair,2013,12(1):53-62.
19 .杜驰,张富春.盐穗木Hc Dmc1基因的克隆和表达分析[J].西北植物学报,2015,35(2):220-226.Du C,Zhang F C.Clone and expression analysis of Hc Dmc1 gene from Halostachys caspica[J].Acta Botanica Boreali-Occidentalia Sinica,2015,35(2):220-226.
20 .Chi P,Van Komen S,Sehorn M G,et al.Roles of ATP binding and ATP hydrolysis in human Rad51 recombinase function[J].DNA Repair,2006,5(3):381-391.
21 .Dai J Q,Voloshin O,Potapova S,et al.Meiotic knockdown and complementation reveals essential role of RAD51 in mouse spermatogenesis[J].Cell Reports,2017,18(6):1383-1394.
22 .Busygina V,Gaines W A,Xu Y Y,et al.Functional attributes of the Saccharomyces cerevisiae meiotic recombinase Dmc1[J].DNA Repair,2013,12(9):707-712.
23 .Amunugama R,Groden J,Fishel R.The HsRAD51BHsRAD51C stabilizes the HsRAD51 nucleoprotein filament[J].DNA Repair,2013,12(9):723-732.
24 .Bendjilali N,Macleon S,Kalra G,et al.Time-course analysis of gene expression during the Saccharomyces cerevisiae hypoxic response[J].G3:Genes,Genomes,Genetics(Bethesda),2017,7(1):221-231.
25 .Finsterbusch F,Ravindranathan R,Dereli I,et al.Alignment of homologous chromosomes and effective repair of programmed DNA Double-strand breaks during mouse meiosis require the Minichromosome Maintenance Domain Containing 2(MCMDC2)protein[J].PLo S Genetics,2016,12(10):e1006393.
26 .Chen J,Cui X J,Jia S T,et al.Disruption of dmc1 produces abnormal sperm in Medaka(Oryzias latipes)[J].Scientific Reports,2016,6:30912.
27 .Callender T L,Laureau R,Wan L H,et al.Mek1 down regulates Rad51 activity during yeast meiosis by phosphorylation of Hed1[J].PLo S Genetics,2016,12(8):e1006226.
28 .Kobayashi W,Takaku M,Machida S,et al.Chromatin architecture may dictate the target site for DMC1,but not for RAD51,during homologous pairing[J].Scientific Reports,2016,6:24228.
29 .Martinez J S,Von Nicolai C,Kim T,et al.BRCA2 regulates DMC1-mediated recombination through the BRC repeats[J].Proceedings of the National Academy of Sciences of the United States of America,2016,113(13):3515-3520.
30 .Wang H J,Hu Q,Tang D,et al.Os DMC1 is not required for homologous pairing in rice meiosis[J].Plant Physiology,2016,171(1):230-241.
31 .Macqueen A J.Catching a(double-strand)Break:the Rad51 and Dmc1 strand exchange proteins can Co-occupy both ends of a meiotic DNA double-strand break[J].PLo S Genetics,2015,11(12):e1005741.
2 .Shen J P,Srivas R,Gross A,et al.Chemogenetic profiling identifies RAD17 as synthetically lethal with checkpoint kinase inhibition[J].Oncotarget,2015,6(34):35755-35769.
3 .Wan L,Han J H,Liu T,et al.Scaffolding protein SPIDR/KIAA0146 connects the Bloom syndrome helicase with homologous recombination repair[J].Proceedings of the National Academy of Sciences of the United States of America,2013,110(26):10646-10651.
4 .Gohlke G,Scholz C J,Kneitz S,et al.DNA methylation mediated control of gene expression is critical for development of crown gall tumors[J].PLo S Genetics,2013,9(2):e1003267.
5 .Brown M S,Grubb J,Zhang A N,et al.Small Rad51 and Dmc1 complexes often Co-occupy both ends of a meiotic DNA double strand break[J].PLo S Genetics,2015,11(12):e1005653.
6 .郭传玲,王菊芳,李文建.细胞辐射敏感性与DNA双链断裂及修复相关性研究[J].中华放射医学与防护杂志,2007,27(3):303-306.Guo C L,Wang J F,Li W J.The study of the correlation between cell radiosensitivity and DNA double strand breaks&repair[J].Chinese Journal of Radiological Medicine and Protection,2007,27(3):303-306.
7 .陈红霞.SOSS1和ZNHIT1/SRCAP参与同源重组修复的分子机制研究[D].杭州:浙江大学,2015.Chen H X.Understanding the roles of SOSS1and ZNHIT1/SRCAP in homologous recombination repair[D].Hangzhou:Zhejiang University,2015.
8 .Khoronenkova S V,Dianov G L.ATM prevents DSB formation by coordinating SSB repair and cell cycle progression[J].Proceedings of the National Academy of Sciences of the United States of America,2015,112(13):3997-4002.
9 .李启娟,王德荣.检控蛋白Rad17及其在肿瘤中的意义[J].国际肿瘤学杂志,2011,38(5):336-338.Li Q J,Wang D R.Rad17 and its significance in tumor[J].Journal of International Oncology,2011,38(5):336-338.
10 .Ma L J,Milman N,Nambiar M,et al.Two separable functions of Ctp1 in the early steps of meiotic DNA doublestrand break repair[J].Nucleic Acids Research,2015,43(15):7349-7359.
11 .陶敏,刘少军,龙昱,等.不同倍性鲫鲤鱼Dmc1基因c DNA的克隆及其表达分析[J].中国科学C辑:生命科学,2007,37(6):625-633.Tao M,Liu S J,Long Y,et al.The cloning of Dmc1 c DNAs and a comparative study of its expression in different ploidy cyprinid fishes[J].Science in China Series C:Life Sciences,2008,51(1):38-46.
12 .Resseguie E A,Staversky R J,Brookes P S,et al.Hyperoxia activates ATM independent from mitochondrial ROS and dysfunction[J].Redox Biology,2015,5:176-185.
13 .江瑞胜,刘敏,李程,等.细胞周期检控蛋白Rad17[J].中国细胞生物学学报,2008,30(4):417-420.Jiang R S,Liu M,Li C,et al.Cell cycle checkpoint protein Rad17[J].Chinese Journal of Cell Biology,2008,30(4):417-420.
14 .Tripathi K,Hussein U K,Anupalli R,et al.Allyl isothiocyanate induces replication-associated DNA damage response in NSCLC cells and sensitizes to ionizing radiation[J].Oncotarget,2015,6(7):5237-5252.
15 .程龙,李茜,熊焰,等.紫外诱导植物产生DNA损伤的修复机制[J].生命的化学,2005,25(6):504-506.Cheng L,Li Q,Xiong Y,et al.Mechanism of repair of DNA damage in plants induced by UV[J].Chemistry of Life,2005,25(6):504-506.
16 .Zafar F,Seidler S B,Kronenberg A,et al.Homologous recombination contributes to the repair of DNA Double-strand breaks induced by High-energy iron ions[J].Radiation Research,2010,173(1):27-39.
17 .Yu X,Egelman E H.Helical filaments of human Dmc1 protein on single-Stranded DNA:a cautionary tale[J].Journal of Molecular Biology,2010,401(3):544-551.
18 .Sharma D,Say A F,Ledford L L,et al.Role of the conserved lysine within the Walker A motif of human DMC1[J].DNA Repair,2013,12(1):53-62.
19 .杜驰,张富春.盐穗木Hc Dmc1基因的克隆和表达分析[J].西北植物学报,2015,35(2):220-226.Du C,Zhang F C.Clone and expression analysis of Hc Dmc1 gene from Halostachys caspica[J].Acta Botanica Boreali-Occidentalia Sinica,2015,35(2):220-226.
20 .Chi P,Van Komen S,Sehorn M G,et al.Roles of ATP binding and ATP hydrolysis in human Rad51 recombinase function[J].DNA Repair,2006,5(3):381-391.
21 .Dai J Q,Voloshin O,Potapova S,et al.Meiotic knockdown and complementation reveals essential role of RAD51 in mouse spermatogenesis[J].Cell Reports,2017,18(6):1383-1394.
22 .Busygina V,Gaines W A,Xu Y Y,et al.Functional attributes of the Saccharomyces cerevisiae meiotic recombinase Dmc1[J].DNA Repair,2013,12(9):707-712.
23 .Amunugama R,Groden J,Fishel R.The HsRAD51BHsRAD51C stabilizes the HsRAD51 nucleoprotein filament[J].DNA Repair,2013,12(9):723-732.
24 .Bendjilali N,Macleon S,Kalra G,et al.Time-course analysis of gene expression during the Saccharomyces cerevisiae hypoxic response[J].G3:Genes,Genomes,Genetics(Bethesda),2017,7(1):221-231.
25 .Finsterbusch F,Ravindranathan R,Dereli I,et al.Alignment of homologous chromosomes and effective repair of programmed DNA Double-strand breaks during mouse meiosis require the Minichromosome Maintenance Domain Containing 2(MCMDC2)protein[J].PLo S Genetics,2016,12(10):e1006393.
26 .Chen J,Cui X J,Jia S T,et al.Disruption of dmc1 produces abnormal sperm in Medaka(Oryzias latipes)[J].Scientific Reports,2016,6:30912.
27 .Callender T L,Laureau R,Wan L H,et al.Mek1 down regulates Rad51 activity during yeast meiosis by phosphorylation of Hed1[J].PLo S Genetics,2016,12(8):e1006226.
28 .Kobayashi W,Takaku M,Machida S,et al.Chromatin architecture may dictate the target site for DMC1,but not for RAD51,during homologous pairing[J].Scientific Reports,2016,6:24228.
29 .Martinez J S,Von Nicolai C,Kim T,et al.BRCA2 regulates DMC1-mediated recombination through the BRC repeats[J].Proceedings of the National Academy of Sciences of the United States of America,2016,113(13):3515-3520.
30 .Wang H J,Hu Q,Tang D,et al.Os DMC1 is not required for homologous pairing in rice meiosis[J].Plant Physiology,2016,171(1):230-241.
31 .Macqueen A J.Catching a(double-strand)Break:the Rad51 and Dmc1 strand exchange proteins can Co-occupy both ends of a meiotic DNA double-strand break[J].PLo S Genetics,2015,11(12):e1005741.