斑马鱼PKR的dsRBM1和dsRBM3重组及体外功能分析
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摘要
为了重组斑马鱼(Daniorerio)双链RNA依赖的蛋白激酶(DrPKR)的双链RNA结合模体1(dsRBM1)和双链RNA结合模体3(dsRBM3)基因,并对重组基因表达的蛋白进行鉴定和功能分析,设计定向删除引物,应用PCR技术扩增DrPKR的ds RBM1和dsRBM3的基因片段,通过重叠PCR技术将dsRBM1和dsRBM3基因重组在一起得到dsRBM13基因,再将dsRBM13构建到原核表达质粒pET32a上并表达和纯化蛋白;通过表达蛋白的二聚化实验和pull down实验对重组蛋白dsRBM13进行体外功能分析。结果:DrPKR的ds RBM1和dsRBM3重组的蛋白dsRBM13可以进行二聚化和多聚化,且能结合Poly I:C(人工合成的dsRNA)。因此,重组DrPKR蛋白模体dsRBM13仍然具有二聚化和dsRNA结合活性,提示串联了三个dsRBM的Dr PKR激活过程中,串联两个dsRBM对DrPKR激活是必需的,并且在DrPKR结合dsRNA的激活过程中,因dsRNA的长短和空间结构的多样性,含有三个dsRBM的DrPKR可能存在更强和更多的结合方式,因此具有更强的抗病毒免疫反应功能。
To recombine the gene of dsRBM1 and ds RBM3 of double-stranded RNA dependent PKR(DrPKR) in zebra fish(Danio rerio), identify and analyze the function of the expressed protein in vitro, directional deletion primers were designed to amplify the gene segment of ds RBM1 and dsRBM3 of DrPKR, which were recombined to obtain dsRBM13 by overlapping PCR, then ds RBM13 was transferred to p ET32 a to express and purify the recombined protein; the dimerization assay and Poly I:C pull-down assay were introduced to test the function of the protein in vitro. The results showed that dsRBM13 could dimerize and multimerize, which also bind to poly I:C(synthetical dsRNA). Thus, the recombinant ds RBM13 could dimerize and bind to poly I:C, which implied that it was essential for two dsRBM in tandem to activate DrPKR; Furthermore, because of the diversity of length and spatial structure of dsRNA, DrPKR with three dsRBM could bind to dsRNA more powerful and with more binding pattern, hence, it might have a stronger function for antiviral immunity.
To recombine the gene of dsRBM1 and ds RBM3 of double-stranded RNA dependent PKR(DrPKR) in zebra fish(Danio rerio), identify and analyze the function of the expressed protein in vitro, directional deletion primers were designed to amplify the gene segment of ds RBM1 and dsRBM3 of DrPKR, which were recombined to obtain dsRBM13 by overlapping PCR, then ds RBM13 was transferred to p ET32 a to express and purify the recombined protein; the dimerization assay and Poly I:C pull-down assay were introduced to test the function of the protein in vitro. The results showed that dsRBM13 could dimerize and multimerize, which also bind to poly I:C(synthetical dsRNA). Thus, the recombinant ds RBM13 could dimerize and bind to poly I:C, which implied that it was essential for two dsRBM in tandem to activate DrPKR; Furthermore, because of the diversity of length and spatial structure of dsRNA, DrPKR with three dsRBM could bind to dsRNA more powerful and with more binding pattern, hence, it might have a stronger function for antiviral immunity.
引文
[1] Heinicke L A, Nallagatla S R, Hull C M, et al. RNA helical imperfections regulate activation of the protein kinase PKR:Effects of bulge position, size, and geometry[J]. RNA, 2011, 17(5):957.
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[3]夏君,谢炯,张萍.抗病毒蛋白PKR的结构和功能[J].中国免疫学杂志,2013,29(2):205-209.
[4] Watanabe T, Imamura T, Hiasa Y. Roles of protein kinase R in cancer:Potential as a therapeutic target[J]. Cancer Science, 2018, 109(4):919-925.
[5] Lee S B,Esteban M.The Interferon-induced Doublestranded RNA-activated protein kinase induces apoptosis[J]. Virology, 1994, 199(2):491-496.
[6] Der S D, Yang Y L, Weissmann C, et al. A double-stranded RNA-activated protein kinase-dependent pathwaymediatingstress-inducedapoptosis[J].Proceedings of the National Academy of Sciences, 1997,94(7):3279-3283.
[7] Koromilas A E. Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase[J].Science, 1992, 257(5077):1685-1689.
[8] Williams, Bryan R G. PKR; a sentinel kinase for cellular stress[J]. Oncogene, 1999, 18(45):6112-6120.
[9] Li S, Peters G A, Ding K, et al. Molecular basis for PKR activation by PACT or dsRNA[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(26):10005-10010.
[10] Dauber B, Wolff T. Activation of the Antiviral Kinase PKR and Viral Countermeasures[J]. Viruses, 2009,1(3):523-544.
[11]白卫君,王小中,曾建明,等.靶向激活蛋白激酶PKR诱导白血病K562细胞凋亡[J].肿瘤, 2009,29(1):35-41.
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[13]关振宏,李影,张茂林,等.双链RNA依赖的蛋白激酶（PKR）的真核表达、纯化及鉴定[J].安徽农业科学,2009,37(28):13521-13523.
[14]贾因棠,魏来,蒋栋,等.双链RNA激活的蛋白激酶(PKR)的克隆表达及其对丙型肝炎病毒蛋白合成的抑制作用[J].中国生物化学与分子生物学报,2006,22(1):24-30.
[15] García MA,Gil J,Ventoso I, et al. Impact of protein kinase PKR in cell biology:from antiviral to antiproliferative action[J]. Microbiol Mol Biol Rev,2006,70(4):1032-1060.
[16] Zheng X, Bevilacqua P C. Activation of the protein kinase PKR by short doublestranded RNAs with singlestranded tails[J].RNA,2004,10(12):19341945.
[17] Fran?ois Mouton-Liger, Paquet C, Dumurgier J, et al.Increased cerebrospinal fluid levels of double-stranded RNA-dependant protein kinase in Alzheimer's disease[J].Alzheimers&Dementia the Journal of the Alzheimers Association, 2012, 8(4):265-266.
[18]陈珊珊,王怡,顾丽泽,等. PKR抑制胰岛β细胞生长的机制研究[J].南京医科大学学报:自然科学版,2012,32(9):1187-1191.
[19]陈珊珊.综述PKR与胰岛素抵抗[J].科技视界,2013,34(329)406-408.
[20]朱锐,吴校林,刘文卫. NLRP3炎性小体与TLR3和PKR的关系及在动脉粥样硬化中的研究进展[J].医学综述,2016,22(4):685-687.
[21] Eric C. Freundt, Melissa Drappier, et al. Innate Immune detection of cardioviruses and viral disruption of interferon signaling[J]. Frontiers in Aging Neuroscience, 2018,9(2448):1-12.
[22]段玉坤,伍志强,韩为东,等. PKR诱导细胞凋亡发生机制的研究进展[J].现代肿瘤医学, 2011,19(9):1863-1865.
[23]翟景波,高巍,王秋波,等. RNA传感器蛋白催化酶PKR研究新进展[J].微生物学杂志, 2016,36(6):93-97.
[24] Nakamura T, Furuhashi M, Li P, et al. Double-stranded rna-dependent protein kinase links pathogen sensing with stress and metabolic homeostasis[J]. Cell, 2010,140(3):0-348.
[25]胡有生. DsRBM对草鱼PKR抑制蛋白翻译功能的影响研究[D].南昌:南昌大学,2016.
[26] Stéphanie Dabo, Eliane F. Meurs. dsRNA-dependent protein kinase pkr and its role in stress, signaling and HCV infection[J]. Viruses, 2012, 4:2598-2635.
[27] Hu Y, Fan L, Wu C, et al. Identification and function analysis of the three dsRBMs in the N terminal dsRBD of grass carp(Ctenopharyngodon idella)PKR.[J]. Fish&Shellfish Immunology, 2016, 50:91-100.
[28]李雯.草鱼PKR基因的克隆、表达特性及功能分析[D].南昌:南昌大学,2013.
[29] Rothenburg S, Deigendesch N, Dey M, et al. Doublestranded RNA-activated protein kinase PKR of fishes and amphibians:Varying the number of double-stranded RNA binding domains and lineage-specific duplications[J].Bmc Biology, 2008, 6(1):1-19.
[30]石华,宋方洲.双链RNA依赖性蛋白激酶的结构与作用[J].生命的化学, 2006, 26(1):38-41.
[31] Garcia-Ortega M B, Lopez G J, Jimenez G, et al. Clinical and therapeutic potential of protein kinase PKR in cancer and metabolism[J]. Expert Reviews in Molecular Medicine, 2017, 19:9.
[32]梅雯,张成桂,自加吉,等. MTERF1基因真核表达载体的构建及其在C-33A细胞中的表达研究[J].井冈山大学学报:自然科学版, 2017,38(5):29-34.
[2] Barber G N, Edelhoff S, Katze M G, et al. Chromosomal assignment of the interferon-inducible double-stranded RNA-Dependent protein kinase(PRKR)to Human Chromosome 2p21-p22 and Mouse Chromosome 17E2[J]. Genomics, 1993, 16(3):0-767.
[3]夏君,谢炯,张萍.抗病毒蛋白PKR的结构和功能[J].中国免疫学杂志,2013,29(2):205-209.
[4] Watanabe T, Imamura T, Hiasa Y. Roles of protein kinase R in cancer:Potential as a therapeutic target[J]. Cancer Science, 2018, 109(4):919-925.
[5] Lee S B,Esteban M.The Interferon-induced Doublestranded RNA-activated protein kinase induces apoptosis[J]. Virology, 1994, 199(2):491-496.
[6] Der S D, Yang Y L, Weissmann C, et al. A double-stranded RNA-activated protein kinase-dependent pathwaymediatingstress-inducedapoptosis[J].Proceedings of the National Academy of Sciences, 1997,94(7):3279-3283.
[7] Koromilas A E. Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase[J].Science, 1992, 257(5077):1685-1689.
[8] Williams, Bryan R G. PKR; a sentinel kinase for cellular stress[J]. Oncogene, 1999, 18(45):6112-6120.
[9] Li S, Peters G A, Ding K, et al. Molecular basis for PKR activation by PACT or dsRNA[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(26):10005-10010.
[10] Dauber B, Wolff T. Activation of the Antiviral Kinase PKR and Viral Countermeasures[J]. Viruses, 2009,1(3):523-544.
[11]白卫君,王小中,曾建明,等.靶向激活蛋白激酶PKR诱导白血病K562细胞凋亡[J].肿瘤, 2009,29(1):35-41.
[12] Chelsea M. Hull, Philip C. Bevilacqua. discriminating self and non-self by RNA:Roles for RNA structure,misfolding, and modi?cation in regulating the innate immune sensor PKR[J].Accounts of Chemical Research,2016:A-H.
[13]关振宏,李影,张茂林,等.双链RNA依赖的蛋白激酶（PKR）的真核表达、纯化及鉴定[J].安徽农业科学,2009,37(28):13521-13523.
[14]贾因棠,魏来,蒋栋,等.双链RNA激活的蛋白激酶(PKR)的克隆表达及其对丙型肝炎病毒蛋白合成的抑制作用[J].中国生物化学与分子生物学报,2006,22(1):24-30.
[15] García MA,Gil J,Ventoso I, et al. Impact of protein kinase PKR in cell biology:from antiviral to antiproliferative action[J]. Microbiol Mol Biol Rev,2006,70(4):1032-1060.
[16] Zheng X, Bevilacqua P C. Activation of the protein kinase PKR by short doublestranded RNAs with singlestranded tails[J].RNA,2004,10(12):19341945.
[17] Fran?ois Mouton-Liger, Paquet C, Dumurgier J, et al.Increased cerebrospinal fluid levels of double-stranded RNA-dependant protein kinase in Alzheimer's disease[J].Alzheimers&Dementia the Journal of the Alzheimers Association, 2012, 8(4):265-266.
[18]陈珊珊,王怡,顾丽泽,等. PKR抑制胰岛β细胞生长的机制研究[J].南京医科大学学报:自然科学版,2012,32(9):1187-1191.
[19]陈珊珊.综述PKR与胰岛素抵抗[J].科技视界,2013,34(329)406-408.
[20]朱锐,吴校林,刘文卫. NLRP3炎性小体与TLR3和PKR的关系及在动脉粥样硬化中的研究进展[J].医学综述,2016,22(4):685-687.
[21] Eric C. Freundt, Melissa Drappier, et al. Innate Immune detection of cardioviruses and viral disruption of interferon signaling[J]. Frontiers in Aging Neuroscience, 2018,9(2448):1-12.
[22]段玉坤,伍志强,韩为东,等. PKR诱导细胞凋亡发生机制的研究进展[J].现代肿瘤医学, 2011,19(9):1863-1865.
[23]翟景波,高巍,王秋波,等. RNA传感器蛋白催化酶PKR研究新进展[J].微生物学杂志, 2016,36(6):93-97.
[24] Nakamura T, Furuhashi M, Li P, et al. Double-stranded rna-dependent protein kinase links pathogen sensing with stress and metabolic homeostasis[J]. Cell, 2010,140(3):0-348.
[25]胡有生. DsRBM对草鱼PKR抑制蛋白翻译功能的影响研究[D].南昌:南昌大学,2016.
[26] Stéphanie Dabo, Eliane F. Meurs. dsRNA-dependent protein kinase pkr and its role in stress, signaling and HCV infection[J]. Viruses, 2012, 4:2598-2635.
[27] Hu Y, Fan L, Wu C, et al. Identification and function analysis of the three dsRBMs in the N terminal dsRBD of grass carp(Ctenopharyngodon idella)PKR.[J]. Fish&Shellfish Immunology, 2016, 50:91-100.
[28]李雯.草鱼PKR基因的克隆、表达特性及功能分析[D].南昌:南昌大学,2013.
[29] Rothenburg S, Deigendesch N, Dey M, et al. Doublestranded RNA-activated protein kinase PKR of fishes and amphibians:Varying the number of double-stranded RNA binding domains and lineage-specific duplications[J].Bmc Biology, 2008, 6(1):1-19.
[30]石华,宋方洲.双链RNA依赖性蛋白激酶的结构与作用[J].生命的化学, 2006, 26(1):38-41.
[31] Garcia-Ortega M B, Lopez G J, Jimenez G, et al. Clinical and therapeutic potential of protein kinase PKR in cancer and metabolism[J]. Expert Reviews in Molecular Medicine, 2017, 19:9.
[32]梅雯,张成桂,自加吉,等. MTERF1基因真核表达载体的构建及其在C-33A细胞中的表达研究[J].井冈山大学学报:自然科学版, 2017,38(5):29-34.
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