Preparation and identification of polyclonal antibody of peroxiredoxin from Toxoplasma gondii
SU Dan-hua1, SHEN Chao-qun1, WU Liang1, LIU Yuan1, FU Tao1, ZHOU Jia-hao1, JIANG Xu-gan1, CHEN Sheng-xia1, CAO Jian-ping2
1.School of Medicine, Jiangsu University, Zhenjiang 212013, China; 2.National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai 200025, China
Abstract:To clone and express the peroxiredoxin(Prx) of Toxoplasma gondii RH strain and produce the polyclonal antibody, prx gene fragments amplified by PCR were cloned into the pET-28a(+) vector to construct the prokaryotic expression vector prx/pET-28a(+), which was efficiently expressed in E.coli Rosetta. Recombinant Prx proteins were purified by Ni-NTA affinity chromatography. The anti-Prx polyclonal antibody was produced in rabbit. Western blotting could detect Prx expression by anti-Prx polyclonal antibody. The recombinant Prx polyclonal antibody will facilitate the study of the biological functions of Prx.
苏丹华, 沈超群, 吴亮, 刘原, 付涛, 周家豪, 姜旭淦, 陈盛霞, 曹建平. 刚地弓形虫Peroxiredoxin多克隆抗体的制备及鉴定[J]. 中国人兽共患病学报, 2015, 31(12): 1089-1092.
SU Dan-hua, SHEN Chao-qun, WU Liang, LIU Yuan, FU Tao, ZHOU Jia-hao, JIANG Xu-gan, CHEN Sheng-xia, CAO Jian-ping. Preparation and identification of polyclonal antibody of peroxiredoxin from Toxoplasma gondii. Chinese Journal of Zoonoses, 2015, 31(12): 1089-1092.
[1] Son ES, Song KJ, Shin JC, et al. Molecular cloning and characterization of peroxiredoxin from Toxoplasma gondii [J]. Korean J Parasitol, 2001, 39(2): 133-141. [2] Ju AP,Wu L,Shen J,et al.Prokaryotic expression and identification of rhoptry protein 18 from Toxoplasma gondii [J].J Jiangsu Univ(Med Edit), 2013, 23(3): 207-211. (in Chinese) 鞠爱萍,吴亮,沈进,等.弓形虫棒状体蛋白18的原核表达及鉴定[J].江苏大学学报:医学版,2013, 23(3): 207-211. [3] Salinas G, Fernandez V, Fernandez C, et al. Echinococcus granulosus : cloning of a thioredoxin peroxidase[J]. Exp Parasitol, 1998, 90(3): 298-301. [4] Liu ZZ,Wang HL,Meng XL,et al. Purification of recombinant peroxiredoxin protein of Toxoplasma gondii and preparation of antisera[J].J Path Biol, 2011,6(3): 201-203. (in Chinese) 刘转转,王海龙,孟晓丽,等.重组刚地弓形虫Peroxiredoxin蛋白的纯化及抗血清制备[J].中国病原生物学杂志, 2011,6(3): 201-203. [5] Peskin AV, Low FM, Paton LN, et al. The high reactivity of peroxiredoxin 2 with H 2 O 2 is not reflected in its reaction with other oxidants and thiol reagents[J]. J Biol Chem, 2007, 282(16): 11885-11892. [6] Ma GY, Zhang JZ, Yin GR, et al. Toxoplasma gondii : proteomic analysis of antigenicity of solubletachyzoite antigen[J]. Exp Parasitol, 2009, 122(1): 41-46. [7] Wang F,Yin GR,Meng XL,et al. Bioinformatics analysis of the structure and epitopes of the geneencoding peroxiredoxin protein from Toxoplasma gondii [J].J Path Biol, 2010, 5(7):517-519,533.(in Chinese) 王芬,殷国荣,孟晓丽,等.刚地弓形虫Peroxiredoxin基因编码蛋白结构与抗原表位的生物信息学分析[J].中国病原生物学杂志, 2010, 5(7): 517-519,533. [8] Wu L,Chen SX,Li LJ,et al.In vitroculture of tachyzoites of Toxoplasma gondii RH strain in HeLa cells[J].Chin J Parasitol Parasit Dis, 2008, 26(6): 452-456. (in Chinese) 吴亮,陈盛霞,李琳婕,等.刚地弓形虫RH株速殖子在HeLa细胞系体外培养的实验观察[J].中国寄生虫学与寄生虫病杂志, 2008, 26(6): 452-456. [9] Jiang XG, Zhu MJ, Ge YW, et al.Preparation and detection of antisera for collagen proteintypeⅡ[J].J Jiangsu Univ(Med Edit), 2008, 18(1): 34-36,39.(in Chinese) 姜旭淦,朱敏娟,葛彦文,等.Ⅱ型胶原蛋白抗血清的制备和检测[J].江苏大学学报:医学版,2008, 18(1): 34-36,39. [10] McGonigle S, Dalton JP, James ER. Peroxidoxins: a new antioxidant family[J]. Parasitol Today,1998, 14(4): 139-145. [11] Liu ZZ,Wang HL,Yin GR, et al.Cloning and prokaryotic expression of peroxiredoxin gene of Toxoplasma gondii and immunogenicity analysis of the recombinant protein[J].Chin J Zoonoses, 2009, 25(4): 334-337.(in Chinese) 刘转转,王海龙,殷国荣,等.刚地弓形虫peroxiredoxin基因的克隆表达与免疫原性分析[J].中国人兽共患病学报, 2009, 25(4): 334-337. [12] Deponte M, Becker K. Biochemical characterization of Toxoplasma gondii 1-Cys peroxiredoxin 2 with mechanistic similarities to typical 2-Cys Prx[J].Mol Biochem Parasit, 2005(140): 87-96. [13] Shu Q.Peroxiredoxin and parasite[J].Chin J Parasitol Parasit Dis,2002, 20(2): 115-118. (in Chinese) 舒勍. Peroxiredoxin与寄生虫[J].中国寄生虫学与寄生虫病杂志, 2002, 20(2): 115-118. [14] Ding M, Kwok LY, Schluter D, et al. The antioxidant systems in Toxoplasma gondii and the roleof cytosolic catalase in defence against oxidative injury[J]. Mol Microbiol, 2004, 51(1): 47-61. [15] Marshall ES, Elshekiha HM, Hakimi MA, et al. Toxoplasma gondii peroxiredoxin promotes altered macrophage function, caspase-1-dependent IL-1β secretion enhance parasite replication[J].Vet Res, 2011, 42: 80. [16] Wood ZA, Poole LB, Karplus PA. Peroxiredoxin evolution and the regulation of hydrogenperoxide signaling[J]. Science, 2003, 300(5619): 650-653. [17] Kang SW, Rhee SG, Chang TS, et al. 2-Cys peroxiredoxin function in intracellular signaltransduction: therapeutic implications[J]. Trends Mol Med, 2005, 11(12): 571-578. [18] Kim JA, Park S, Kim K, et al. Activity assay of mammalian 2-cys peroxiredoxins using yeastthioredoxin reductase system[J]. Anal Biochem, 2005, 338(2): 216-223. [19] Santed CF, Ortet P, Saksouk N, et al. The histone methylase KMTox interacts the redox-sensor peroxiredoxin-1 and targets genes involved Toxoplasma gondii antioxidant defences[J] . Mol Microbiol, 2009, 71(1): 212-226.