犬弓首蛔虫TcCPG1的表达及其与几丁质的结合特性

doi:10.11843/j.issn.0366-6964.2020.02.017

摘要/Abstract

摘要： 为探讨犬弓首蛔虫（Toxocara canis，T.canis）软骨素蛋白多糖TcCPG1的表达特征及其与几丁质的结合方式，为犬弓首蛔虫软骨素蛋白多糖的功能研究提供依据，笔者克隆T.canis软骨素蛋白多糖1基因（Tc-cpg-1）并原核表达重组蛋白TcCPG1，采用镍柱亲和层析技术对重组蛋白进行纯化，并与几丁质进行结合试验；采用实时荧光定量PCR（quantitative real-time polymerase chain reaction，qRT-PCR）进行Tc-cpg-1基因的转录水平的性别和组织差异表达分析。结果显示：Tc-cpg-1基因编码区（coding sequence，CDS）的长度为1 251 bp，共编码417个氨基酸；SDS-PAGE电泳结果显示重组蛋白TcCPG1以包涵体形式存在，相对分子质量大小约为70 ku；对表达条件进行优化后，以0.8 mmol·L^-1 IPTG于16℃诱导14 h时可获得大量目的蛋白；利用镍柱亲和层析纯化可以获得具有较高纯度的目的蛋白，与几丁质进行结合试验，验证了重组蛋白TcCPG1能够与几丁质结合；通过qRT-PCR对Tc-cpg-1基因转录水平的组织差异性进行分析，发现Tc-cpg-1在雌虫中高量表达，尤其在雌虫的生殖组织中表达量最高，表明Tc-cpg-1可能参与了T.canis雌虫的生殖过程，推测TcCPG1可能通过与卵壳中几丁质的相互作用从而影响T.canis的胚胎和生殖发育过程。

关键词: 犬弓首蛔虫, CPG1, 原核表达, 几丁质结合

Abstract: To explore the expression and chitin-binding manner of chondroitin proteoglycans 1 of Toxocara canis (TcCPG1) and to provide a basis for the functional study of this proteins in T. canis. In the present work, the chondroitin proteoglycans 1 gene(Tc-cpg-1)of T. canis was molecularly cloned and expressed in a prokaryotic expression system for recombinant protein TcCPG1, which was then purified using Ni-NTA affinity chromatography and tested in a chitin-binding assay. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the transcriptional differences of Tc-cpg-1 between sexes and among tissues of T. canis. The results showed that the coding sequence of Tc-cpg-1 was 1 251 bp in length, encoding 417 amino acids. SDS-PAGE analysis showed that the recombinant protein TcCPG1 was expressed in inclusion bodies, and the molecular weight was estimated at~70 ku. Optimized conditions (induction by 0.8 mmol·L^-1 IPTG for 14 h and incubation at 16℃) facilitated an efficient production of the recombinant protein TcCPG1. Following Ni-NTA affinity purification, the recombinant protein bound chitin, suggesting a chitin-binding activity of TcCPG1. In addition, differential analysis showed a high transcriptional level of Tc-cpg-1 in female adult worm, particularly in the reproductive tissues, indicating potential roles of Tc-cpg-1 in reproductive process (embryonic and reproductive development) of female worms, which might be achieved by interacting with the chitin in egg shell.

Key words: Toxocara canis, CPG1, prokaryotic expression, chitin-binding assay

中图分类号:

S852.731

陈绍基, 李芳, 尹莎莎, 江艾耘, 周荣琼. 犬弓首蛔虫TcCPG1的表达及其与几丁质的结合特性[J]. 畜牧兽医学报, 2020, 51(2): 356-364.

CHEN Shaoji, LI Fang, YIN Shasha, JIANG Aiyun, ZHOU Rongqiong. Expression and Chitin-Binding Feature of TcCPG1 of Toxocara canis[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(2): 356-364.

参考文献 28

[1]	STRUBE C, HEUER L, JANECEK E. Toxocara spp. infections in paratenic hosts[J]. Vet Parasitol, 2013, 193(4):375-389.
[2]	SAHU S, SAMANTA S, SUDHAKAR N R, et al. Prevalence of canine toxocariasis in Bareilly, Uttar Pradesh, India[J]. J Parasit Dis, 2014, 38(1):111-115.
[3]	SUGANYA G, PORTEEN K, SEKAR M, et al. Prevalence and molecular characterization of zoonotic helminths in dogs[J]. J Parasit Dis, 2019, 43(1):96-102.
[4]	DESPOMMIER D. Toxocariasis:clinical aspects, epidemiology, medical ecology, and molecular aspects[J]. Clin Microbiol Rev, 2003, 16(2):265-272.
[5]	MA G X, HOLLAND C V, WANG T, et al. Human toxocariasis[J]. Lancet Infect Dis, 2018, 18(1):e14-e24.
[6]	SMITH H, HOLLAND C, TAYLOR M, et al. How common is human toxocariasis? Towards standardizing our knowledge[J]. Trends Parasitol, 2009, 25(4):182-188.
[7]	LAABS T L, WANG H, KATAGIRI Y, et al. Inhibiting glycosaminoglycan chain polymerization decreases the inhibitory activity of astrocyte-derived chondroitin sulfate proteoglycans[J]. J Neurosci, 2007, 27(52):14494-14501.
[8]	MILLER G M, HSIEH-WILSON L C. Sugar-dependent modulation of neuronal development, regeneration, and plasticity by chondroitin sulfate proteoglycans[J]. Exp Neurol, 2015, 274:115-125.
[9]	GALTREY C M, FAWCETT J W. The role of chondroitin sulfate proteoglycans in regeneration and plasticity in the central nervous system[J]. Brain Res Rev, 2007, 54(1):1-18.
[10]	DYCK S M, KARIMI-ABDOLREZAEE S. Chondroitin sulfate proteoglycans:key modulators in the developing and pathologic central nervous system[J]. Exp Neurol, 2015, 269:169-187.
[11]	STEPHENSON E L, YONG V W. Pro-inflammatory roles of chondroitin sulfate proteoglycans in disorders of the central nervous system[J]. Matrix Biol, 2018, 71-72:432-442.
[12]	MIZUGUCHI S, UYAMA T, KITAGAWA H, et al. Chondroitin proteoglycans are involved in cell division of Caenorhabditis elegans[J]. Nature, 2003, 423(6938):443-448.
[13]	HWANG H Y, OLSON S K, ESKO J D, et al. Caenorhabditis elegans early embryogenesis and vulval morphogenesis require chondroitin biosynthesis[J]. Nature, 2003, 423(6938):439-443.
[14]	IZUMIKAWA T, KITAGAWA H, MIZUGUCHI S, et al. Nematode chondroitin polymerizing factor showing cell-/organ-specific expression is indispensable for chondroitin synthesis and embryonic cell division[J]. J Biol Chem, 2004, 279(51):53755-53761.
[15]	WIJFFELS G, EISEMANN C, RIDING G, et al. A novel family of chitin-binding proteins from insect Type 2 peritrophic matrix cDNA sequences, chitin binding activity, and cellular localization[J]. J Biol Chem, 2001, 276(18):15527-15536.
[16]	NOBORN F, TOLEDO A G, SIHLBOM C, et al. Identification of chondroitin sulfate linkage region glycopeptides reveals prohormones as a novel class of proteoglycans[J]. Mol Cell Proteomics, 2015, 14(1):41-49.
[17]	LAU L W, CUA R, KEOUGH M B, et al. Pathophysiology of the brain extracellular matrix:a new target for remyelination[J]. Nat Rev Neurosci, 2013, 14(10):722-729.
[18]	COLES C H, SHEN Y J, TENNEY A P, et al. Proteoglycan-specific molecular switch for RPTPσ clustering and neuronal extension[J]. Science, 2011, 332(6028):484-488.
[19]	NOBORN F, TOLEDO A G, NASIR W, et al. Expanding the chondroitin glycoproteome of Caenorhabditis elegans[J]. J Biol Chem, 2018, 293(1):379-389.
[20]	OLSON S K, BISHOP J R, YATES J R, et al. Identification of novel chondroitin proteoglycans in Caenorhabditis elegans:embryonic cell division depends on CPG-1 and CPG-2[J]. J Cell Biol, 2006, 173(6):985-994.
[21]	OLSON S K, GREENAN G, DESAI A, et al. Hierarchical assembly of the eggshell and permeability barrier in C. elegans[J]. J Cell Biol, 2012, 198(4):731-748.
[22]	MERZENDORFER H, ZIMOCH L. Chitin metabolism in insects:structure, function and regulation of chitin synthases and chitinases[J]. J Exp Biol, 2003, 206(Pt 24):4393-4412.
[23]	VÁZQUEZ J A, RODRÍGUEZ-AMADO I, MONTEMAYOR M I, et al. Chondroitin sulfate, hyaluronic acid and chitin/chitosan production using marine waste sources:characteristics, applications and eco-friendly processes:a review[J]. Mar Drugs, 2013, 11(3):747-774.
[24]	DONG Z M, ZHANG W W, ZHANG Y, et al. Identification and characterization of novel chitin-binding proteins from the larval cuticle of silkworm, Bombyx mori[J]. J Proteome Res, 2016, 15(5):1435-1445.
[25]	HASHIMOTO M, IKEGAMI T, SEINO S, et al. Expression and characterization of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12[J]. J Bacteriol, 2000, 182(11):3045-3054.
[26]	DENG H M, LI Y, ZHANG J L, et al. Analysis of expression and chitin-binding activity of the wing disc cuticle protein BmWCP4 in the silkworm, Bombyx mori[J]. Insect Sci, 2016, 23(6):782-790.
[27]	MA G X, ZHOU R Q, HU L, et al. Molecular characterization and transcriptional analysis of the female-enriched chondroitin proteoglycan 2 of Toxocara canis[J]. J Helminthol, 2018, 92(2):154-160.
[28]	SATO M, GRANT B D, HARADA A, et al. Rab11 is required for synchronous secretion of chondroitin proteoglycans after fertilization in Caenorhabditis elegans[J]. J Cell Sci, 2008, 121(Pt 19):3177-3186.