犬弓首蛔虫C型凝集素4的原核表达及组织定位

doi:10.11843/j.issn.0366-6964.2023.04.028

Abstract

Abstract: The aim of this experiment was to determine the characteristics and tissue distribution of the Ctype lectin 4(Tc-ctl-4)gene of Toxocara canis. The full-length gene of Toxocara canis C-type Lectin 4 (Tc-ctl-4) was amplified as template according to the genomic data of Tc-ctl-4(AF126830.1) and sequence analysis and multiple sequence alignment were performed. The prokaryotic expression vector Tc-ctl-4/pCold TF was constructed, the recombinant protein was purified and polyclonal antibody was prepared. The transcriptional level of Tc-ctl-4 and the tissue distribution of Tc-CTL-4 were detected by quantitative real-time PCR (qRT-PCR) and indirect immunofluorescence histochemistry. The results showed that the full-length sequence of Tc-ctl-4 gene was 842 bp, encoding 280 amino acids.A conserved Ctype lectin like domain (CTLD) was identified in multiple sequence alignments. SDS-PAGE showed that the recombinant protein Tc-CTL-4 was about 86 ku in size and expressed in soluble form. The protein was purified by Ni-NTA affinity chromatography, and high purity protein was obtained by elution with 500 mmol·L^-1 imidazole, which was used to immunize the New Zealand white rabbits to prepare polyclonal antibody. Indirect ELISA showed that the antibody titer was>1:160 000, indicating that the recombinant protein had good immunogenicity and SDS-PAGE showed that the purity of the purified antibody was higher than 95%. Western blot showed that the antibody could specifically bind to Tc-CTL-4, indicating that the antibody was highly specific. The qPCR results showed that the transcription level of Tc-ctl-4 was the highest in the uterus of female worms and the seminal vesicle of male worms. Indirect-fluorescence immunohistochemical assays were carried out to detect the tissue distribution of Tc-CTL-4, which showed predominant distribution of Tc-CTL-4 in the reproductive tissues of adult T. canis, such as in the uterus, oviduct of adult female T. canis and in the vas deferens of adult male T. canis, and body wall of T. canis. It is suggested that Tc-ctl-4 might play an important roles in the reproduction and immunity of T. canis.

Key words: Toxocara canis, C-type lectin 4, prokaryotic expression, polyclonal antibody, tissue expression

CLC Number:

S852.731

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.

References 29

[1]	WU T, BOWMAN D D. Visceral larval migrans of Toxocara canis and Toxocara cati in non-canid and non-felid hosts[J]. Adv Parasitol, 2020, 109:63-88.
[2]	DOS SANTOS L M, DE MOURA M Q, AZEVEDO M L, et al. Reactivity of recombinant Toxocara canis TES-30/120 in experimentally infected mice[J]. Parasite Immunol, 2018, 40(8):e12568.
[3]	WU T K, BOWMAN D D. Toxocara canis[J]. Trends Parasitol, 2022, 38(8):709-710.
[4]	WANGCHUK P, LAVERS O, WISHART D S, et al. Excretory/secretory metabolome of the zoonotic roundworm parasite Toxocara canis[J]. Biomolecules, 2020, 10(8):1157.
[5]	LU C Y, LAI S C, LEE H H, et al. Matrix metalloproteinases activation in Toxocara canis induced pulmonary pathogenesis[J]. J Microbiol, Immunol, Infect, 2021, 54(6):1147-1153.
[6]	DRICKAMER K. C-type lectin-like domains[J]. Curr Opin Struct Biol, 1999, 9(5):585-590.
[7]	ZELENSKY A N, GREADY J E. The C-type lectin-like domain superfamily[J]. FEBS J, 2005, 272(24):6179-6217.
[8]	WANG X W, VASTA G R, WANG J X. The functional relevance of shrimp C-type lectins in host-pathogen interactions[J]. Dev Comp Immunol, 2020, 109:103708.
[9]	MAYER S, RAULF M K, LEPENIES B. C-type lectins:their network and roles in pathogen recognition and immunity[J]. Histochem Cell Biol, 2017, 147(2):223-237.
[10]	LINDENWALD D L, LEPENIES B. C-type lectins in veterinary species:recent advancements and applications[J]. Int J Mol Sci, 2020, 21(14):5122.
[11]	LOUKAS A, MAIZELS R M. Helminth C-type lectins and host-parasite interactions[J]. Parasitol Today, 2000, 16(8):333-339.
[12]	NAGAE M, YAMAGUCHI Y. Structural aspects of carbohydrate recognition mechanisms of C-Type Lectins[M]//YAMASAKI S. C-Type Lectins in Immune Homeostasis. Cham:Springer, 2019, 429:147-176.
[13]	MALLO G V, KURZ C L, COUILLAULT C, et al. Inducible antibacterial defense system in C. elegans[J]. Curr Biol, 2002, 12(14):1209-1214.
[14]	DAUB J, LOUKAS A, PRITCHARD D I, et al. A survey of genes expressed in adults of the human hookworm, Necator americanus[J]. Parasitology, 2000, 120:171-184.
[15]	NJIRI O A, ZHANG X Y, ZHANG Y M, et al. CD209 C-type Lectins promote host invasion, dissemination, and infection of Toxoplasma gondii[J]. Front Immunol, 2020, 11:656.
[16]	YOSHIDA A, NAGAYASU E, HORII Y, et al. A novel C-type lectin identified by EST analysis in tissue migratory larvae of Ascaris suum[J]. Parasitol Res, 2012, 110(4):1583-1586.
[17]	ZIMARA N, CHANYALEW M, ASEFFA A, et al. Dectin-1 positive dendritic cells expand after infection with Leishmania major parasites and represent promising targets for vaccine development[J]. Front Immunol, 2018, 26:263.
[18]	RODEHEFFER C, SHUR B D. Characterization of a novel ZP3-independent sperm-binding ligand that facilitates sperm adhesion to the egg coat[J]. Development, 2004, 131(3):503-512.
[19]	TETTEH K K A, LOUKAS A, TRIPP C, et al. Identification of abundantly expressed novel and conserved genes from the infective larval stage of Toxocara canis by an expressed sequence tag strategy[J]. Infect Immun, 1999, 67(9):4771-4779.
[20]	LOUKAS A, MULLIN N P, TETTEH K K A, et al. A novel C-type lectin secreted by a tissue-dwelling parasitic nematode[J]. Curr Biol, 1999, 9(15):825-828.
[21]	LOUKAS A, DOEDENS A, HINTZ M, et al. Identification of a new C-type lectin, TES-70, secreted by infective larvae of Toxocara canis, which binds to host ligands[J]. Parasitology, 2000, 121(5):545-554.
[22]	OSTROP J, LANG R. Contact, collaboration, and conflict:signal integration of Syk-coupled C-type lectin receptors[J]. J Immunol, 2017, 198(4):1403-1414.
[23]	WEIS W I, DRICKAMER K, HENDRICKSON W A. Structure of a C-type mannose-binding protein complexed with an oligosaccharide[J]. Nature, 1992, 360(6400):127-134.
[24]	KOLATKAR A R, WEIS W I. Structural basis of galactose recognition by C-type animal lectins[J]. J Biol Chem, 1996, 271(12):6679-6685.
[25]	RAULF M K, LEPENIES B, STRUBE C. Toxocara canis and Toxocara cati somatic and excretory-secretory antigens are recognised by C-Type Lectin receptors[J]. Pathogens, 2021, 10(3):321.
[26]	HARCUS Y, NICOLL G, MURRAY J, et al. C-type lectins from the nematode parasites Heligmosomoides polygyrus and Nippostrongylus brasiliensis[J]. Parasitol Int, 2009, 58(4):461-470.
[27]	BROWN A C, HARRISON L M, KAPULKIN W, et al. Molecular cloning and characterization of a C-type lectin from Ancylostoma ceylanicum:evidence for a role in hookworm reproductive physiology[J]. Mol Biochem Parasitol, 2007, 151(2):141-147.
[28]	ETEBAR F, HOSSEINI S H, JALOUSIAN F, et al. Phylogenetic analysis of C type lectin from Toxocara canis infective larvae and comparison with the C type lectin family in the immune system of mouse and human[J]. Iran J Parasitol, 2018, 13(1):49-57.
[29]	PAGE A P, HAMILTON A J, MAIZELS R M. Toxocara canis:monoclonal antibodies to carbohydrate epitopes of secreted (TES) antigens localize to different secretion-related structures in infective larvae[J]. Exp Parasitol, 1992, 75(1):56-71.

Prokaryotic Expression and Tissue Localization of C-type Lectin 4 from Toxocara canis

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