Prokaryotic expression and characterization of Giardia lamblia extracellular nuclease
WANG Yi1, ZHAO Jun-jian1, YU Yuan2, TIAN Xi-feng2, LI Ji2, LIU Xiao-li2, ZHOU Ying-bin2, LI Shao-dong2, WANG Yang2
1.Department of Clinical Laboratory, North China University of Science and Technology Affiliated Hospital, Tangshan 063000, China; 2.College of Life Sciences, North China University of Science and Technology, Tangshan 063000, China
Abstract:For many pathogens, extracellular nuclease is requisite for salvaging exogenous nucleosides and defending against immune attack. By searching the genome of Giardia lamblia, we found a provisional extracellular nuclease coding sequence. To express G. lambia extracellular nuclease (GeNuc) in E. coli and characterize its activity, the provisional GeNuc protein sequence of G. lambia WB strain from GenBank was analyzed. The coding sequence of GeNuc without signal peptide was amplified by PCR from genome DNA of G. lamblia C2 strain. Sequencing result showed that GeNuc in C2 strain was identical to that in Giardia WB strain. The PCR product (800 bp in size) was cloned into prokaryotic expression vector pET-28a(+). The recombinant vector pET-28a(+)-GeNuc was transformed into E. coli Rosetta(DE3), then the recombinant GeNuc protein was expressed by IPTG induction. SDS-PAGE and Western blot using anti-His Tag antibody showed that the expressed product of GeNuc was a fusion protein about 30.8 kD. The GeNuc recombinant protein, which was purified by Ni-NTA affinity chromatography and renatured by dialysis, showed DNase activity by partially digesting plasmid DNA. The successful prokaryotic expression and characterization of GeNuc provide a prerequisite for antibody preparation and further approach of pathogenesis of Giardiasis.
王沂, 赵俊暕, 余源, 田喜凤, 李冀, 刘晓莉, 周英斌, 李少东, 王洋. 蓝氏贾第鞭毛虫胞外核酸酶的表达纯化和活性鉴定[J]. 中国人兽共患病学报, 2016, 32(1): 65-69.
WANG Yi, ZHAO Jun-jian, YU Yuan, TIAN Xi-feng, LI Ji, LIU Xiao-li, ZHOU Ying-bin, LI Shao-dong, WANG Yang. Prokaryotic expression and characterization of Giardia lamblia extracellular nuclease. Chinese Journal of Zoonoses, 2016, 32(1): 65-69.
[1] Adam, RD. Biology of Giardia lamblia [J]. Clin Microbiol Rev, 2001, 14(3): 447-475. doi:10.1128/cmr. 14.3.447-475.2001 [2] Kiedrowski MR, Crosby HA, Hernandez FJ, et al. Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease[J/OL]. PLoS One, 2014, 9(4):e95574. doi:10.1371/journal.pone.0095574 [3] Cho C, Chande A, Gakhar L, et al. Role of the nuclease of nontypeable haemophilus influenzae in dispersal of organisms from biofilms[J]. Infect Immun, 2015, 83(3): 950-957. doi:10.1128/iai.02601-14 [4] Wang Y, Yang ZH, Wang Y, et al. Prokaryotic expression and antigenicity analysis of α-7.1 and α-11 giardin of Giardia lamblia [J]. Chin J Zoonoses, 2012, 28(5): 474-478. doi:10.3969/j.issn.1002-2694. 2012.05.017 王洋, 杨志宏, 王沂, 等. 蓝氏贾第鞭毛虫α-7.1、α-11贾第素的原核表达及抗原活性鉴定[J]. 中国人兽共患病学报, 2012, 28(5):474-478. [5] Simon D, Simon HU, Yousefi S. Extracellular DNA traps in allergic, infectious, and autoimmune diseases[J]. Allergy, 2013, 68(4): 409-416. doi:10.1111/all.12111 [6] Campbell TA, Zlotnick GW, Neubert TA, et al. Purification and characterization of the 3’-nucleotidase/nuclease from promastigotes of Leishmania donovani [J]. Mol Biochem Parasitol, 1991, 47(1): 109-117. doi:10.1016/0166-6851(91)90153-w [7] Debrabant A, Ghedin E, Dwyer DM. Dissection of the functional domains of the Leishmania surface membrane 3’-nucleotidase/nuclease, a unique member of the class I nuclease family[J]. J Biol Chem, 2000, 275(21): 16366-16372. doi:10.1074/jbc.m908725199 [8] Farajnia S, Alimohammadian MH, Reiner NE, et al. Molecular characterization of a novel amastigote stage specific Class I nuclease from Leishmania major [J]. Int J Parasitol, 2004, 34(8): 899-908. doi:10.1016/j.ijpara.2004.03.005 [9] Joshi MB, Dwyer DM. Molecular and functional analyses of a novel class I secretory nuclease from the human pathogen, Leishmania donovani [J]. J Biol Chem, 2007, 282(13): 10079-10095. doi:10.1074/jbc. m610770200 [10] Lakhal-Naouar I, Ben Achour-Chenik Y, Boublik Y, et al. Identification and characterization of a new Leishmania major specific 3’nucleotidase/nuclease protein[J]. Biochem Biophys Res Commun, 2008, 375(1): 54-58. doi:10.1016/j.bbrc.2008.07.099 [11] Farajnia S, Rahbarnia L, Maleki Zanjani B, et al. Molecular cloning and characterization of P4 nuclease from Leishmania infantum [J]. Enzyme Res, 2011, 2011: 970983. doi:10.4061/2011/970983 [12] Joshi MB, Hernandez Y, Owings JP, et al. Diverse viscerotropic isolates of Leishmania all express a highly conserved secretory nuclease during human infections[J]. Mol Cell Biochem, 2012, 361(1-2): 169-179. doi:10.1007/s11010-011-1101-1 [13] Paletta-Silva R, Vieira DP, Vieira-Bernardo R, et al. Leishmania amazonensis : characterization of an ecto-3’-nucleotidase activity and its possible role in virulence[J]. Exp Parasitol, 2011, 129(3): 277-283. doi:10.1016/j.exppara.2011.07.014 [14] Guimaraes-Costa AB, DeSouza-Vieira TS, Paletta-Silva R, et al. 3’-nucleotidase/nuclease activity allows Leishmania parasites to escape killing by neutrophil extracellular traps[J]. Infect Immun, 2014, 82(4): 1732-1740. doi:10.1016/j.exppara.2011.07.014 [15] Abi Abdallah DS, Lin C, Ball CJ, et al. Toxoplasma gondii triggers release of human and mouse neutrophil extracellular traps[J]. Infect Immun, 2012, 80(2): 768-777. doi:10.1128/iai.05730-11 [16] Guimaraes-Costa AB, Nascimento MT, Froment GS, et al. Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps[J]. Proc Natl Acad Sci U S A, 2009, 106(16): 6748-6753. doi:10.1073/pnas.0900226106 [17] Silva LM, Caro TM, Gerstberger R, et al. The apicomplexan parasite Eimeria arloingi induces caprine neutrophil extracellular traps[J]. Parasitol Res, 2014, 113(8): 2797-2807. doi:10.1007/s00436-014-3939-0 [18] Munoz-Caro T, Silva LM, Ritter C, et al. Besnoitia besnoiti tachyzoites induce monocyte extracellular trap formation[J]. Parasitol Res, 2014, 113(11): 4189-4197. doi:10.1007/s00436-014-4094-3 [19] Thammavongsa V, Missiakas DM, Schneewind O. Staphylococcus aureus degrades neutrophil extracellular traps to promote immune cell death[J]. Science, 2013, 342(6160): 863-866. doi:10.1126/science.1242255 [20] Seper A, Hosseinzadeh A, Gorkiewicz G, et al. Vibrio cholerae evades neutrophil extracellular traps by the activity of two extracellular nucleases[J/OL]. PLoS Pathog, 2013, 9(9): e1003614. doi:10.1371/journal.ppat. 1003614 [21] Chang A, Khemlani A, Kang H, et al. Functional analysis of Streptococcus pyogenes nuclease A (SpnA), a novel group A streptococcal virulence factor[J]. Mol Microbiol, 2011, 79(6): 1629-1642. doi:10.1111/j.1365-2958.2011.07550.x [22] Zhu L, Kuang Z, Wilson BA, et al. Competence-independent activity of pneumococcal enda mediates degradation of extracellular DNA and nets and is important for virulence[J/OL]. PLoS One, 2013, 8(7): e70363. doi:10.1371/journal.pone. 0070363 [23] Shan Q, Dwyer M, Rahman S, et al. Distinct susceptibilities of corneal Pseudomonas aeruginosa clinical isolates to neutrophil extracellular trap-mediated immunity[J]. Infect Immun, 2014, 82(10): 4135-4143. doi:10.1128/iai.02169-14 [24] Crouch AA, Seow WK, Whitman LM, et al. Inhibition of adherence of Giardia intestinalis by human neutrophils and monocytes[J]. Trans R Soc Trop Med Hyg, 1991, 85(3): 375-379. doi:10.1016/0035-9203 (91)90297-c [25] Aley SB, Zimmerman M, Hetsko M, et al. Killing of Giardia lamblia by cryptdins and cationic neutrophil peptides[J]. Infect Immun, 1994, 62(12): 5397-5403. [26] Cotton JA, Bhargava A, Ferraz JG, et al. Giardia duodenalis cathepsin B proteases degrade intestinal epithelial interleukin-8 and attenuate interleukin-8-induced neutrophil chemotaxis[J]. Infect Immun, 2014, 82(7): 2772-2787. doi:10.1128/iai.01771-14 [27] Muller N, von Allmen N. Recent insights into the mucosal reactions associated with Giardia lamblia infections[J]. Int J Parasitol, 2005, 35(13): 1339-1347. doi:10.1016/j.ijpara.2005.07.008 [28] Ara jo NS, Mundim MJ, Gomes MA, et al. Giardia duodenalis : pathological alterations in gerbils, Meriones unguiculatus , infected with different dosages of trophozoites[J]. Exp Parasitol, 2008, 118(4): 449-457. doi:10.1016/j.exppara.2007.10.007