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

Abstract

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

CLC Number:

S852.731

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.

References 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.

Expression and Chitin-Binding Feature of TcCPG1 of Toxocara canis

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 28

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	LIU Chuanxia, WANG Xiao, LI Xuewen, BAO Miaofei, LI Tingting, CHEN Xin, WENG Changjiang, ZHENG Jun. Preparation of Monoclonal Antibody of African Swine Fever Virus pE120R [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 388-394.
[2]	WANG Jingyu, PAN Yangyang, XU Gengquan, ZHANG Rui, ZHANG Wenlan, WANG Xiaoshan, WU Rentaodi, ZHAO Rigetu, CUI Yan, YU Sijiu. Preparation and Preliminary Application of Yak (Bos Grunniens) Fas-associated Factor 1 Polyclonal Antibody [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3369-3382.
[3]	LIU Taoxue, SU Bingqian, QI Yanli, GUO Jiangtao, LIU Zhonghu, CHU Beibei, WANG Jiang, ZENG Lei. Preparation of the Monoclonal Antibody against the African Swine Fever Virus p30 Protein and Identification of the Antigenic Epitope [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3415-3423.
[4]	JING Tingting, YIN Hao, HUANG Rong, LAN Shimei, LI Zhangcheng, YOU Liuchao, HAO Huafang, FU Lei, CHU Yuefeng. Prokaryotic Expression and Biological Function Analysis of Mycoplasma bovis 0580 Gene C-terminal Truncated [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2991-3001.
[5]	WANG Hui, XU Mengyuan, LIU Lingkang, ZHENG Xibang, LI Gonghe, WU Wende. PhiC31 Integrase Triggers Site-specific Cassette Exchange in Chicken Fibroblast Cells through Electroporation [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2330-2342.
[6]	JIAO Zhengxing, PAN Yangyang, WANG Meng, WANG Jinglei, MA Wenbin, GAO Xiang, ZHANG Hui, CUI Yan, YU Sijiu, WANG Libin. Preparation of Polyclonal Antibody against Yak LC3B Protein and Its Application in Detection of Expression in Reproductive Organs [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2436-2447.
[7]	GAO Zhenzhen, MENG Zejing, HE Xiaobing, FANG Yongxiang, TIAN Huihui, CHEN Guohua, JING Zhizhong. Identification of Immunogenicity and Neutralizing Activity of Ectromelia Virus EVM135 and EVM085 Proteins [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2468-2477.
[8]	CHENG Tianyin, WU Congying, LIU Yuke, DUAN Deyong. Studies on the Role of HSP70-b2 and Its 14-Mer-like Peptide from Haemaphysalis flava [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2570-2580.
[9]	WANG Guochao, ZHAO Yaru, ZHANG Zhonghui, ZHANG Yulong, BAI Ge, GENG Shuxian, FAN Jie, YANG Jifei, GUAN Guiquan, YIN Hong, LUO Jianxun, NIU Qingli. Bioinformatics Analysis of RNA Polymerase Subunit D205R Gene of African Swine Fever Virus and Polyclonal Antibody Preparation [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2042-2049.
[10]	ZHAI Yunyi, YUAN Ye, LI Junmei, TIAN Lulu, DIAO Ziyang, LI Bin, CHEN Jialu, ZHOU Dong, JIN Yaping, WANG Aihua. Preparation and Preliminary Application of Monoclonal Antibody to Brucella Outer Membrane Protein 16 [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2083-2091.
[11]	WANG Bingnan, SUN Xue, JIA Hongguo, TAN Chun, MEI Sipeng, ZHOU Rongqiong. Prokaryotic Expression and Tissue Localization of C-type Lectin 4 from Toxocara canis [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1641-1651.
[12]	CAI Mingyu, ZHANG Hailong, HAI Zhenzhen, QIAO Yarui, DU Jun, ZHOU Xuezhang. The Inflamed Molecular Mechanism Induced by Recombined 14-3-3 Protein of Candida krusei on Bovine Mammary Epithelial Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1679-1689.
[13]	SUN Min, HAO Fei, ZHANG Wenwen, LI Wenliang, YANG Leilei, MAO Li, CHENG Zilong, LIU Maojun. The Antiviral Activity of Goat Interferon Alpha to Caprine Parainfluenza Virus 3 [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 736-743.
[14]	CHI Lili, WANG Junna, ZHANG Yuming, LIU Jian, CHEN Zhiyuan, LI Shufan, YIN Yanbo, XU Shouzhen. Prokaryotic Expression and Immunogenicity Analysis of Truncated Fusion Protein of FAdV-4 Fiber2 and FAdV-8b Fiber [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5162-5170.
[15]	ZHONG Lemiao, LIU Binghui, WU Chunlin, WU Yijian. Preparation and Evaluation of Subunit Vaccine based on Adhesin Protein of Mycoplasma gallisepticum [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5171-5183.