弓形虫沉默信号调节子2(TgSir2)的克隆表达与多克隆抗体制备
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摘要
目的制备特异识别弓形虫沉默信号调节子2(TgSir2)多克隆抗体。方法以酵母Sir2氨基酸序列为模板,寻找弓形虫基因组中同源序列,并进行信号肽预测。以弓形虫RH株cDNA为模板,构建pET28b-TgSir2重组质粒,转化入大肠埃希菌(E.coli)BL21,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达后,用镍柱亲和层析法纯化诱导表达产物,并以小鼠抗His标签单克隆抗体作为一抗,蛋白质印迹(Western blotting)进行鉴定。以纯化的TgSir2蛋白作为抗原免疫新西兰大白兔,获得特异性抗TgSir2多克隆抗体。最后,以此多克隆抗体作为一抗,Western blotting检测与虫体蛋白的反应情况。结果同源比对找到弓形虫基因组中TgSir2,信号肽分析提示1-15位氨基酸为其信号肽序列。PCR、双酶切及测序结果证实原核表达质粒构建成功。SDS-PAGE结果表明,IPTG可诱导TgSir2融合蛋白的大量表达。Western blotting结果显示,制备的多克隆抗体既能特异性识别原核表达的TgSir2蛋白,也能识别弓形虫体内的内源性TgSir2蛋白。结论制备的抗重组TgSir2蛋白多克隆抗体能特异性识别弓形虫RH株的TgSir2蛋白。
To obtain the specific polyclonal antibody against Toxoplasma gondii silent information regulator 2(TgSir2). Searching the homologous sequence in Toxoplasma genome used the amino acid sequence of Saccharomyces cerevisiae silent information regulator 2(ScSir2) as template and predicting the signal peptide. TgSir2 cDNA was inserted into prokaryotic expression vector pET28 b. After identification, the constructed plasmid pET28 b-TgSir2 was transformed into E. coli BL21 cells and induced by isopropyl-β-D-thinoglucoside(IPTG) for expression of the protein. The recombinant protein was purified via Ni-NTA affinity chromatography. Western blotting assay was conducted with anti-His tag monoclonal antibody as the primary antibody. Rabbits were immunized with purified recombinant protein. The specific anti-TgSir2 polyclonal antibody was obtained and Western blotting was performed to detect the reaction by the antibody. The TgSir2 in Toxoplasma genome was found through homologous comparison, signal peptide analysis indicated that 1-15 amino acids were the sequence of its signal peptide. pET28 b-TgSir2 plasmid was identified by PCR amplification, restriction enzyme digestion and sequencing. SDS-PAGE analysis showed that the recombinant TgSir2 protein was expressed highly induced by IPTG. Western blotting showed that both TgSir2 protein from prokaryotic expression and tachyzoite lysates can be recognized by the specific anti-TgSir2 polyclonal antibody. The obtained polyclonal antibody against TgSir2 can specifically react with the endogenous TgSir2 protein.
To obtain the specific polyclonal antibody against Toxoplasma gondii silent information regulator 2(TgSir2). Searching the homologous sequence in Toxoplasma genome used the amino acid sequence of Saccharomyces cerevisiae silent information regulator 2(ScSir2) as template and predicting the signal peptide. TgSir2 cDNA was inserted into prokaryotic expression vector pET28 b. After identification, the constructed plasmid pET28 b-TgSir2 was transformed into E. coli BL21 cells and induced by isopropyl-β-D-thinoglucoside(IPTG) for expression of the protein. The recombinant protein was purified via Ni-NTA affinity chromatography. Western blotting assay was conducted with anti-His tag monoclonal antibody as the primary antibody. Rabbits were immunized with purified recombinant protein. The specific anti-TgSir2 polyclonal antibody was obtained and Western blotting was performed to detect the reaction by the antibody. The TgSir2 in Toxoplasma genome was found through homologous comparison, signal peptide analysis indicated that 1-15 amino acids were the sequence of its signal peptide. pET28 b-TgSir2 plasmid was identified by PCR amplification, restriction enzyme digestion and sequencing. SDS-PAGE analysis showed that the recombinant TgSir2 protein was expressed highly induced by IPTG. Western blotting showed that both TgSir2 protein from prokaryotic expression and tachyzoite lysates can be recognized by the specific anti-TgSir2 polyclonal antibody. The obtained polyclonal antibody against TgSir2 can specifically react with the endogenous TgSir2 protein.
引文
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[12] Silvestre R, Silva AM, Cordeiro-Da-Silva A, et al. The contribution of toll-like receptor 2 to the innate recognition of a Leishmania infantum silent information regulator 2 protein[J]. Immunol, 2009, 128(4): 484-499. DOI: 10. 1111/j. 1365-2567. 2009. 03132. X
[13] Silvestre R, Cordeiro-Da-Silva A, Tavares J, et al. Leishmania cytosolic silent information regulatory protein 2 deacetylase induces murine B-cell differentiation and in vivo production of specific antibodies[J]. Immunol, 2006, 119(4): 529-540. DOI: 10. 1111/j. 1365-2567. 2006. 02468. X
[14] Vergnes B, Sereno D, Madjidian-Sereno N, et al. Cytoplasmic SIR2 homologue overexpression promotes survival of Leishmania parasites by preventing programmed cell death[J]. Gene, 2002, 296(1/2): 139-150. DOI: 10. 1016/S0378-1119(02)00842-9
[15] Merrick CJ, Duraisingh MT. Plasmodium falciparum Sir2: an unusual sirtuin with dual histone deacetylase and ADP-ribosyltransferase activity[J]. Eukaryot Cell, 2007, 6(11): 2081-2091. DOI: 10. 1128/EC. 00114-07
[16] Tonkin CJ, Carret CK, Duraisingh MT, et al. Sir2 paralogues cooperate to regulate virulence genes and antigenic variation in Plasmodium falciparum[J]. PLoS Biol, 2009, 7(4): e84. DOI: 10. 1371/journal. Pbio. 1000084
[17] Vergnes B, Sereno D, Tavares J, et al. Targeted disruption of cytosolic SIR2 deacetylase discloses its essential role in Leishmania survival and proliferation[J]. Gene, 2005, 363: 85-96. DOI: 10.1016/j. Gene. 2005. 06. 047
[18] Hailu G S, Robaa D, Forgione M, et al. Lysine deacetylase inhibitors in parasites: past, present, and future perspectives[J]. J Med Chem, 2017, 60(12): 4780-4804. DOI: 10. 1021/acs. Jmedchem. 6b01595
[19] Lancelot J, Caby S, Dubois-Abdesselem F, et al. Schistosoma mansonisirtuins: characterization and potential as chemotherapeutic targets[J]. PLoS Negl Trop Dis, 2013, 7(9): e2428. DOI: 10. 1371/journal. Pntd. 0002428
[20] Moretti NS, Da SAL, Clemente TM, et al. Characterization of Trypanosomacruzisirtuins as possible drug targets for Chagas disease[J]. Antimicrob Agents Chemother, 2015, 59(8): 4669-4679. DOI: 10. 1128/AAC. 04694-14
[21] Gaspar L, Coron RP, Kongthoo LP, et al. Inhibitors of Trypanosoma cruzi Sir2 related protein 1 as potential drugs against Chagas disease[J]. PLoS Negl Trop Dis, 2018, 12(1): e6180. DOI: 10. 1371/journal. Pntd. 0006180
[22] Dong H, Yang S, Zhao Q, et al. Molecular characterization and protective efficacy of silent information regulator 2A from Eimeria tenella[J]. Parasit Vectors, 2016, 9(1): 602. DOI: 10. 1186/s13071-016-1871-0
[2] Laliberte J, Carruthers VB. Host cell manipulation by the human pathogen Toxoplasma gondii[J].Cell Mol Life Sci, 2008, 65(12): 1900-1915. DOI: 10.1007/s00018-008-7556-x
[3] William J, Sullivan WJ, Jeffers V. Mechanisms of Toxoplasma gondii persistence and latency[J]. FEMS Microbiol, 2012, 36(3): 717-733. DOI: 10. 1111/j. 1574-6976. 2011. 00305. X
[4] Dubey JP, Lindsay DS, Speer CA. Structures of Toxoplasma gondii tachyzoites bradyzoites, and sporozoites and biology and development of tissue cysts[J]. Clin Microbiol, 1998, 11(2): 267-299.
[5] 曾艳波,朱顺海,韩红玉,等. 抗急性弓形虫病药物疗效的研究[J]. 中国人兽共患病学报, 2011, 27(4): 316-319. DOI: 10.3969/j.issn.1002-2694.2011.04.012
[6] Gartenberg MR, Smith JS. The nuts and bolts of transcriptionally silent chromatin in Saccharomyces cerevisiae[J]. Genetics, 2016, 203(4): 1563-1599. DOI: 10. 1534/genetics. 112. 145243
[7] Gomes P, Outeiro TF, Cavadas C. Emerging role of sirtuin 2 in the regulation of mammalian metabolism[J]. Trends Pharmacol Sci, 2015, 36(11): 756-768. DOI: 10. 1016/j. Tips. 2015. 08. 001
[8] Dam S, Lohia A. Entamoeba histolyticasirtuin EhSir2a deacetylates tubulin and regulates the number of microtubular assemblies during the cell cycle[J]. Cell Microbiol, 2010, 12(7): 1002-1014. DOI: 10. 1111/j. 1462-5822. 2010. 01449. X
[9] Vergnes B, Gazanion E, Grentzinger T. Functional divergence of SIR2 orthologs between trypanosomatid parasites[J]. Mol Biochem Parasitol, 2016, 207(2): 96-101. DOI: 10.1016/j. Molbiopara. 2016. 06. 004
[10] Ritagliati C, Alonso VL, Manarin R, et al. Overexpression of cytoplasmic TcSIR2RP1 and mitochondrial TcSIR2RP3 impacts on Trypanosoma cruzigrowth and cell invasion[J]. PLoS Negl Trop Dis, 2015, 9(4): e3725. DOI: 10. 1371/journal. Pntd. 0003725
[11] Alsford S, Kawahara T, Isamah C, et al. A sirtuin in the African trypanosome is involved in both DNA repair and telomeric gene silencing but is not required for antigenic variation[J]. Mol Microbiol, 2007, 63(3): 724-736. DOI: 10. 1111/j. 1365-2958. 2006. 05553. X
[12] Silvestre R, Silva AM, Cordeiro-Da-Silva A, et al. The contribution of toll-like receptor 2 to the innate recognition of a Leishmania infantum silent information regulator 2 protein[J]. Immunol, 2009, 128(4): 484-499. DOI: 10. 1111/j. 1365-2567. 2009. 03132. X
[13] Silvestre R, Cordeiro-Da-Silva A, Tavares J, et al. Leishmania cytosolic silent information regulatory protein 2 deacetylase induces murine B-cell differentiation and in vivo production of specific antibodies[J]. Immunol, 2006, 119(4): 529-540. DOI: 10. 1111/j. 1365-2567. 2006. 02468. X
[14] Vergnes B, Sereno D, Madjidian-Sereno N, et al. Cytoplasmic SIR2 homologue overexpression promotes survival of Leishmania parasites by preventing programmed cell death[J]. Gene, 2002, 296(1/2): 139-150. DOI: 10. 1016/S0378-1119(02)00842-9
[15] Merrick CJ, Duraisingh MT. Plasmodium falciparum Sir2: an unusual sirtuin with dual histone deacetylase and ADP-ribosyltransferase activity[J]. Eukaryot Cell, 2007, 6(11): 2081-2091. DOI: 10. 1128/EC. 00114-07
[16] Tonkin CJ, Carret CK, Duraisingh MT, et al. Sir2 paralogues cooperate to regulate virulence genes and antigenic variation in Plasmodium falciparum[J]. PLoS Biol, 2009, 7(4): e84. DOI: 10. 1371/journal. Pbio. 1000084
[17] Vergnes B, Sereno D, Tavares J, et al. Targeted disruption of cytosolic SIR2 deacetylase discloses its essential role in Leishmania survival and proliferation[J]. Gene, 2005, 363: 85-96. DOI: 10.1016/j. Gene. 2005. 06. 047
[18] Hailu G S, Robaa D, Forgione M, et al. Lysine deacetylase inhibitors in parasites: past, present, and future perspectives[J]. J Med Chem, 2017, 60(12): 4780-4804. DOI: 10. 1021/acs. Jmedchem. 6b01595
[19] Lancelot J, Caby S, Dubois-Abdesselem F, et al. Schistosoma mansonisirtuins: characterization and potential as chemotherapeutic targets[J]. PLoS Negl Trop Dis, 2013, 7(9): e2428. DOI: 10. 1371/journal. Pntd. 0002428
[20] Moretti NS, Da SAL, Clemente TM, et al. Characterization of Trypanosomacruzisirtuins as possible drug targets for Chagas disease[J]. Antimicrob Agents Chemother, 2015, 59(8): 4669-4679. DOI: 10. 1128/AAC. 04694-14
[21] Gaspar L, Coron RP, Kongthoo LP, et al. Inhibitors of Trypanosoma cruzi Sir2 related protein 1 as potential drugs against Chagas disease[J]. PLoS Negl Trop Dis, 2018, 12(1): e6180. DOI: 10. 1371/journal. Pntd. 0006180
[22] Dong H, Yang S, Zhao Q, et al. Molecular characterization and protective efficacy of silent information regulator 2A from Eimeria tenella[J]. Parasit Vectors, 2016, 9(1): 602. DOI: 10. 1186/s13071-016-1871-0
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