Vaccination of Mice with a Novel Trypsin from Trichinella spiralis Elicits the Immune Protection against Larval Challenge

Zhang, Yao; Zeng, Jie; Song, Yan; Long, Stuart A.; Liu, Ruo Dan; Jiang, Peng; Zhang, Xi; Cui, Jing; Wang, Zhong Quan

doi:10.3390/vaccines8030437

Cited by 9 publications

(6 citation statements)

References 81 publications

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“…Ten BALB/c mice were immunized by subcutaneous injection of 20 μg of rTsCatL2 mixed with MONTA-NIDE ™ ISA 61 VG 1:1 (v/v), with one booster immunization three weeks later [43,44]. After another two-week interval, polyclonal antibodies against rTsCatL2 were obtained by collecting blood from mice.…”

Section: Production Of a Mouse Polyclonal Antibodymentioning

confidence: 99%

Molecular characterization and determination of the biochemical properties of cathepsin L of Trichinella spiralis

Liu

Meng

et al. 2022

Vet Res

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Cathepsin L is an important cysteine protease, but its function in T. spiralis remains unclear. The aim of this research was to explore the biological characteristics of T. spiralis cathepsin L (TsCatL) and its role in T. spiralis-host interactions. Bioinformatic analysis revealed the presence of the cysteine protease active site residues Gln, Cys, His and Asn in mature TsCatL, as well as specific motifs of cathepsin L similar to ERFNIN and GYLND in the prepeptide of TsCatL. Molecular docking of mature TsCatL and E64 revealed hydrophobic effects and hydrogen bonding interactions. Two domains of TsCatL (TsCatL2) were cloned and expressed, and recombinant TsCatL2 (rTsCatL2) was autocatalytically cleaved under acidic conditions to form mature TsCatL. TsCatL was transcribed and expressed in larvae and adults and located in the stichosome, gut and embryo. Enzyme kinetic tests showed that rTsCatL2 degraded the substrate Z-Phe-Arg-AMC under acidic conditions, which was inhibited by E64 and PMSF and enhanced by EDTA, L-cysteine and DTT. The kinetic parameters of rTsCatL2 were a Km value of 48.82 μM and Vmax of 374.4 nM/min at pH 4.5, 37 °C and 5 mM DTT. In addition, it was shown that rTsCatL2 degraded haemoglobin, serum albumin, immunoglobulins (mouse IgG, human IgG and IgM) and extracellular matrix components (fibronectin, collagen I and laminin). The proteolytic activity of rTsCatL2 was host specific and significantly inhibited by E64. rTsCatL2 possesses the natural activity of a sulfhydryl-containing cysteine protease, and TsCatL is an important digestive enzyme that seems to be important for the nutrient acquisition, immune evasion and invasion of Trichinella in the host.

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Section: Production Of a Mouse Polyclonal Antibodymentioning

confidence: 99%

Molecular characterization and determination of the biochemical properties of cathepsin L of Trichinella spiralis

Liu

Meng

et al. 2022

Vet Res

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“…Vaccination of mice with rTsASP2 triggered specific humoral and mucosal immune responses, as demonstrated that both specific IgG and sIgA levels were significantly elevated two weeks post vaccination. The IgG1 level was significantly higher than IgG2a, suggested that rTsASP2 emulsified in ISA201 adjuvant elicited a mixed IgG1/ IgG2a antibody response (Yang et al, 2019b;Zhang et al, 2020b). The antibody response was important for the protective immunity, but the cellular immunity was also crucial to defend T. spiralis infection (Cui et al, 2013b;Liu et al, 2015b).…”

Section: Discussionmentioning

confidence: 99%

“…The splenocytes and MLN cells were prepared in the DMEM medium and their density were adjusted to 2 × 10 5 cells/ml. After the cells were stimulated with rTsASP2 at a concentration of 3 μg/ml at 37°C for 72 h, the culture supernatant was collected and the concentrations of cytokines (IFN-γ and IL-4) were measured using a sandwich ELISA method (Pompa-Mera et al, 2014;Zhang et al, 2020b).…”

Section: Cytokine Determinationmentioning

confidence: 99%

Specific binding of aspartic protease and enterocytes promotes Trichinella spiralis invasion of murine intestinal epithelium cells

Yue

et al. 2021

Trop Biomed

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Trichinella spiralis is an important foodborne zoonotic parasite and it is necessary to develop vaccine to prevent T. spiralis infection in food animals. T. spiralis aspartic protease-2 (TsASP2) has been demonstrated to play a crucial role in larval invasion of intestinal epithelium cells (IECs). The purpose of this study was to assess the interaction between TsASP2 and IECs and to investigate the immune protection elicited by vaccination with rTsASP2. The results showed that the enzymatic activity of native aspartic protease was detected in crude proteins of all T. spiralis development stages other than NBL stage, the highest activity was observed in the IIL stage. The results of Western blot showed that TsASP2 protein was expressed at ML, IIL and AW but not NBL, and the TsASP2 expression level at IIL stage was significantly higher than those of other three worm stages (P < 0.05). The specific binding between rTsASP2 and IECs was observed by immunofluorescence test (IFT) and confocal microscopy, and the binding site was localized at the IEC membrane and this binding ability was inhibited by aspartic protease specific inhibitor pepstain A. The results of ELISA showed that the binding ability was protein dose-dependent. Vaccination with rTsASP2 triggered a mixed Th1/Th2 humoral and mucosal immune responses, as demonstrated by the elevation levels of Th1/Th2 cytokines (IFN-γ and IL-4) secreted by the spleen and mesenteric lymph nodes (MLNs) of immunized mice. The mice vaccinated with rTsASP2 exhibited a 54.17% reduction in enteral adult worms and a 54.58% reduction in muscle larvae after T. spiralis challenge. The results demonstrated that TsASP2 might be a potential molecular target for anti-Trichinella vaccines.

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“…The structural domain and enzyme active sites of TsAAP (GenBank: KRY29491.1) were predicted and analyzed by using online webtool and CN3D software (Song et al, 2018b). The amino acid sequence of TsAAP gene was compared with AAP from different species /genotypes of Trichinella by using Clustal X (Zhang et al, 2020b). The GenBank accession numbers of AAP from Trichinella spp.…”

Section: Bioinformatics Analysis Of Tsaapmentioning

confidence: 99%

Molecular cloning and characterization of a novel aspartyl aminopeptidase from Trichinella spiralis

Sun

Bai

et al. 2021

Trop Biomed

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Trichinellosis is an important zoonotic parasitic disease worldwide and is principally caused by ingesting animal meat containing Trichinella infective larvae. Aspartyl aminopeptidase is an intracytoplasmic metalloproteinase that specifically hydrolyzes the N-terminus of polypeptides free of acidic amino acids (aspartic acid and glutamate), and plays an important role in the metabolism, growth and development of organisms. In this study, a novel T. spiralis aspartyl aminopeptidase (TsAAP) was cloned and expressed, and its biological properties and roles in worm growth and development were investigated. The results revealed that TsAAP transcription and expression in diverse T. spiralis stages were detected by RT-PCR and Western blotting, and primarily localized at cuticle, stichosome and intrauterine embryos of this nematode by immunofluorescence test. rTsAAP has the enzymatic activity of native AAP to hydrolyze the substrate H-Glu-pNA. There was a specific binding between rTsAAP and murine erythrocyte, and the binding site was localized in erythrocyte membrane proteins. Silencing of TsAAP gene by specific dsRNA significantly reduced the TsAAP expression, enzymatic activity, intestinal worm burdens and female fecundity. The results demonstrated that TsAAP participates in the growth, development and fecundity of T. spiralis and it might be a potential target molecule for anti-Trichinella vaccines.

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Vaccination of Mice with a Novel Trypsin from Trichinella spiralis Elicits the Immune Protection against Larval Challenge

Cited by 9 publications

References 81 publications

Molecular characterization and determination of the biochemical properties of cathepsin L of Trichinella spiralis

Molecular characterization and determination of the biochemical properties of cathepsin L of Trichinella spiralis

Specific binding of aspartic protease and enterocytes promotes Trichinella spiralis invasion of murine intestinal epithelium cells

Molecular cloning and characterization of a novel aspartyl aminopeptidase from Trichinella spiralis

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