Molecular cloning and characterization of a serine proteinase inhibitor from Trichinella spiralis

Parasitology ◽  
2001 ◽  
Vol 123 (1) ◽  
pp. 77-83 ◽  
Author(s):  
I. NAGANO ◽  
Z. WU ◽  
T. NAKADA ◽  
A. MATSUO ◽  
Y. TAKAHASHI
Keyword(s):  
Recombinant Protein ◽  
Proteinase Inhibitors ◽  
Trichinella Spiralis ◽  
Serine Proteinase ◽  
Predicted Amino Acid Sequence ◽  
Serpin Gene ◽  
Specific Expression ◽  
Crude Extracts ◽  
Muscle Larvae

We produced a recombinant protein from a cDNA library from muscle larvae of Trichinella spiralis which had proteinase inhibitory activity. The predicted amino acid sequence of the clone had an identity of only 30% to the serine proteinase inhibitors (serpins) from Caenorhabditis elegans or Brugia malayi. At the putative reactive region, however, the identity was about 50%. The recombinant protein expressed in Escherichia coli inhibited 82% of the activity of the serine proteinase (trypsin). Stage-specific expression of this protein was suggested from the following experiments. Antibody against the recombinant protein could stain proteins migrating at about 42 kDa (which is the expected size from the sequence) in crude extracts from newborn larvae and 18-day post-infection (p.i.) muscle larvae, but it failed to stain any proteins in crude extracts from 30-day p.i. muscle larvae. Production of mRNA transcript for the serpin gene was restricted largely to the newborn larvae and to 18-day p.i. muscle larvae. The antibody reacted with the stichocytes of the larvae at 18 days p.i., but did not react with the muscle larvae at 24 days and 30 days p.i.

Molecular cloning and characterization of a novel protein of Trichinella pseudospiralis excretory–secretory products

2002 ◽  
Vol 76 (2) ◽  
pp. 165-170 ◽  
Author(s):  
I. Nagano ◽  
Z. Wu ◽  
T. Nakada ◽  
T. Boonmars ◽  
Y. Takahashi
Keyword(s):  
Recombinant Protein ◽  
Expression System ◽  
Specific Expression ◽  
Reading Frame ◽  
Trichinella Pseudospiralis ◽  
Crude Extracts ◽  
Muscle Larvae ◽  
Secretory Products ◽  
Novel Protein

AbstractA novel excretory–secretory (ES) protein of Trichinella pseudospiralis was produced. A cDNA library was constructed from mRNA of muscle larvae at 30 days post infection (p.i.) and immunoscreened with the antibody against ES products. A clone, designated Tp22-3, contained a cDNA transcript of 815 bp in length with a single open reading frame which encoded 244-amino acids (28407 Da in the estimated molecular mass). A database search revealed that no sequences had a homology to this predicted protein. The recombinant protein was produced in an Escherichia coli expression system. Stage specific expression of this protein was suggested from the following experiments. An antibody against the recombinant protein could stain proteins migrating at about 28 kDa (which is the expected size from the sequence) on Western blotting of crude extracts or ES products from 30 days p.i. muscle larvae, but failed to stain any proteins in crude extracts from newborn larvae or 15 days p.i. muscle larvae. The antibody reacted to the stichocytes of larvae at 30 days p.i., but did not react to 15 days p.i. muscle larvae. The production of an mRNA transcript for Tp22-3 gene was restricted largely to the 30 days p.i. muscle larvae and adult worms.

Cloning and characterization of serpin-like genes from the striped rice stem borer, Chilo suppressalis

Genome ◽  
2013 ◽  
Vol 56 (6) ◽  
pp. 359-366 ◽  
Author(s):  
Zhao-Yu Ge ◽  
Pin-Jun Wan ◽  
Xiong-Feng Cheng ◽  
Yang Zhang ◽  
Guo-Qing Li ◽  
...  
Keyword(s):  
Proteinase Inhibitors ◽  
Sequence Similarity ◽  
Serine Proteinase ◽  
Stem Borer ◽  
Chilo Suppressalis ◽  
Specific Expression ◽  
Rice Stem Borer ◽  
Gut Physiology ◽  
The Impact

Serpins, also called serine proteinase inhibitors, are widely distributed in eukaryotes. In insects, serpins play important roles in regulating immune responses, gut physiology, and other processes. Here, we report the cloning and characterization of 12 serpin-like cDNAs from the striped rice stem borer (Chilo suppressalis), a major rice pest. The putative proteins share significant sequence similarity with known insect serpins, especially those from lepidopterons. Analysis of functional domains revealed that nine of the cloned serpins are putative trypsin- or chymotrypsin-like inhibitors; two are mixed-type serpins that may act as inhibitors for trypsins, elastases, or thrombin; and the remaining one is truncate. The potential functions of these serpins in interacting with host plants were also investigated by analyzing tissue-specific expression and the impact of different host plant genotypes on gene expression. Our results provide a foundation for future studies on the role of serpins in gut physiology in the striped rice stem borer, and also useful information for comparative analyses of serpins from different insect species.

Identification ofTrichinella spiralisearly antigens at the pre-adult and adult stages

Parasitology ◽  
2010 ◽  
Vol 138 (4) ◽  
pp. 463-471 ◽  
Author(s):  
ALEKSANDAR ZOCEVIC ◽  
PAULINE MACE ◽  
ISABELLE VALLEE ◽  
RADU BLAGA ◽  
MINGYUAN LIU ◽  
...  
Keyword(s):  
Cdna Library ◽  
Serine Proteases ◽  
Trichinella Spiralis ◽  
Serine Proteinase ◽  
Secretory Protein ◽  
Cdna Libraries ◽  
Sequence Identity ◽  
Muscle Larvae ◽  
Serine Protease Family ◽  
Host Parasite

SUMMARYThree expression cDNA libraries fromTrichinella spiralisworms 14 h, 20 h and 48 h post-infection (p.i.) were screened with serum from pigs experimentally infected with 20 000T. spiralismuscle larvae. Twenty-nine positive clones were isolated from the 14 h p.i. cDNA library, corresponding to 8 different genes. A putative excretory-secretory protein similar to that ofT. pseudospiraliswas identified. Three clones corresponded to aT. spiralisserine proteinase inhibitor known to be involved in diverse functions such as blood coagulation and modulation of inflammation. Screening of the 20 h p.i. cDNA library selected 167 positive clones representing 12 different sequences. The clone with the highest redundancy encoded a small polypeptide having no sequence identity with any known proteins fromTrichinellaor other organisms. Fourteen clones displayed sequence identity with the heat shock protein (HSP) 70. HSPs are produced as an adaptive response of the parasite to the hostile environment encountered in the host intestine but their mechanism of action is not yet well defined. From the 48 h p.i.T. spiraliscDNA library, 91 positive clones were identified representing 7 distinct sequences. Most of the positive clones showed high similarity with a member of a putativeT. spiralisserine protease family. This result is consistent with a possible major role for serine proteases during invasive stages ofTrichinellainfection and host-parasite interactions.

scholarly journals Cellular and molecular changes and immune response in the intestinal mucosa during Trichinella spiralis early infection in rats

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
María Priscila Saracino ◽  
Cecilia Celeste Vila ◽  
Melina Cohen ◽  
María Virginia Gentilini ◽  
Guido Hernán Falduto ◽  
...  
Keyword(s):  
Immune Response ◽  
Lamina Propria ◽  
Trichinella Spiralis ◽  
Mucosal Immune Response ◽  
Mesenteric Lymph Nodes ◽  
Muscle Larvae ◽  
Gut Immunity ◽  
Gut Mucosa

Abstract Background: The main targets of the host’s immune system in Trichinella spiralis infection are the adult worms (AW), at the gut level, and the migrant or newborn larvae (NBL), at systemic and pulmonary levels. Most of the studies carried out in the gut mucosa have been performed on the Payer’s patches and/or the mesenteric lymph nodes but not on the lamina propria, therefore, knowledge on the gut immune response against T. spiralis remains incomplete. Methods This study aimed at characterizing the early mucosal immune response against T. spiralis, particularly, the events taking place between 1 and 13 dpi. For this purpose, Wistar rats were orally infected with muscle larvae of T. spiralis and the humoral and cellular parameters of the gut immunity were analysed, including the evaluation of the ADCC mechanism exerted by lamina propria cells. Results A marked inflammation and structural alteration of the mucosa was found. The changes involved an increase in goblet cells, eosinophils and mast cells, and B and T lymphocytes, initially displaying a Th1 profile, characterised by the secretion of IFN-γ and IL-12, followed by a polarization towards a Th2 profile, with a marked increase in IgE, IgG1, IL-4, IL-5 and IL-13 levels, which occurred once the infection was established. In addition, the helminthotoxic activity of lamina propria cells demonstrated the role of the intestine as a place of migrant larvae destruction, indicating that not all the NBLs released in the gut will be able to reach the muscles. Conclusions The characterization of the immune response triggered in the gut mucosa during T. spiralis infection showed that not only an effector mechanism is directed toward the AW but also towards the NBL as a cytotoxic activity was observed against NBL exerted by lamina propria cells.

scholarly journals Acid-Stable Serine Proteinase Inhibitors in the Urine of Alzheimer Disease Subjects

1996 ◽  
Vol 13 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Giulia Sparro ◽  
Salvatore Bonaiuto ◽  
Gabriella Galoenzi ◽  
Anna Maria Eleuteri ◽  
Mauro Angeletti ◽  
...  
Keyword(s):  
Proteinase Inhibitors ◽  
Serine Proteinase ◽  
Trypsin Inhibitors ◽  
Urinary Trypsin Inhibitor ◽  
Urinary Levels ◽  
Urinary Acid ◽  
Kallikrein Activity ◽  
Acid Stable ◽  
Kallikrein Inhibitors

A comparative study of the levels of acid-stable proteinase inhibitors (kallikrein and trypsin inhibitors) in the urine of healthy and Alzheimer subjects, of both sexes, has been performed. A preliminary characterization of the purified inhibitors indicates that the urinary antitryptic activity is accounted for by the presence of the well known Urinary Trypsin Inhibitor (UTI) while an apparently new molecule appears to be responsible for the anti kallikrein activity. The urinary levels of kallikrein inhibitors are very similar in healthy and sick subjects while the levels of trypsin inhibitors appear significatively increased in Alzheimer subjects of both sexes. The data presented here support the hypothesis that unpaired proteolytic processes could be involved in the pathogenesis of Alzheimer's disease and suggest that the levels of urinary acid-stable inhibitors may prove to be useful markers of the disease.

scholarly journals Proteinase inhibitors in rat serum. Purification and partial characterization of three functionally distinct trypsin inhibitors

1984 ◽  
Vol 218 (3) ◽  
pp. 953-959 ◽  
Author(s):  
L Kuehn ◽  
M Rutschmann ◽  
B Dahlmann ◽  
H Reinauer
Keyword(s):  
Proteinase Inhibitor ◽  
Proteinase Inhibitors ◽  
Serine Proteinase ◽  
The Other ◽  
Serine Proteinases ◽  
Rat Serum ◽  
Apparent Homogeneity ◽  
Human Counterpart ◽  
Proteinase Inhibitor Ii

Three different serine proteinase inhibitors were isolated from rat serum and purified to apparent homogeneity. One of the inhibitors appears to be homologous to alpha 1-proteinase inhibitor isolated from man and other species, but the other two, designated rat proteinase inhibitor I and rat proteinase inhibitor II, seem to have no human counterpart. alpha 1-Proteinase inhibitor (Mr 55000) inhibits trypsin, chymotrypsin and elastase, the three serine proteinases tested. Rat proteinase inhibitor I (Mr 66000) is active towards trypsin and chymotrypsin, but is inactive towards elastase. Rat proteinase inhibitor II (Mr 65000) is an effective inhibitor of trypsin only. Their contributions to the trypsin-inhibitory capacity of rat serum are about 68, 14 and 18% for alpha 1-proteinase inhibitor, rat proteinase inhibitor I and rat proteinase inhibitor II respectively.

Molecular cloning of enolase from Trichinella spiralis and the protective immunity in mice

2018 ◽  
Vol 63 (2) ◽  
pp. 252-260 ◽  
Author(s):  
Xuliang Zhang ◽  
Lixin Xu ◽  
Xiaokai Song ◽  
Xiangrui Li ◽  
Ruofeng Yan
Keyword(s):  
Recombinant Protein ◽  
Trichinella Spiralis ◽  
Flow Cytometric Analysis ◽  
Recombinant Plasmids ◽  
Flow Cytometric ◽  
Th2 Immune Response ◽  
Wide Range ◽  
Muscle Larvae ◽  
Ifn Γ ◽  
Negative Controls

Abstract Trichinella spiralis, the main pathogen of trichinosis, infects a wide range of mammalian hosts and is one of the most widespread parasites worldwide. For parasites, glycolysis is the most important way to generate energy. Previous studies showed that some enzymes involved in the glycolytic pathway play roles in regulation the host immunity. In this paper, enolase from T. spiralis was cloned and the protective potentials were studied. One hundred and sixty ICR mice were divided into four groups and vaccinated with recombinant enolase (pET-ENO), eukaryotic recombinant plasmid encoding enolase (pVAX1-ENO) and negative controls (pVAXl and PBS), respectively. Two weeks after the second immunization, each mouse was challenged orally with 200 muscle larvae (MLs) of T. spiralis. Results showed that mice vaccinated with pET-ENO and pVAX1-ENO induced specific antibodies of IgG, IgA, IgM, but no IgE. Subclasses of IgG antibodies showed that mice immunized with recombinant protein and recombinant plasmids induced a Th1/Th2 immune response. Concentrations of serum cytokines were detected and showed significant increase of IFN-γ, IL-4 and TGFβ1, while IL-17 in each group was not significantly different. Flow cytometric analysis showed significant increase of CD4+ and CD8+ T lymphocytes in the groups immunized with recombinant protein and recombinant plasmids. Challenge infection demonstrated that immunized groups had a reduced number of worm burdens. The reductions of larvae per gram muscle (LPG) in pET-ENO and pVAX1-ENO group were 17.7% and 15.8% when compared with PBS control.

scholarly journals Molecular Expression of a Cysteine Proteinase of Clonorchis sinensis and Its Application to an Enzyme-Linked Immunosorbent Assay for Immunodiagnosis of Clonorchiasis

2004 ◽  
Vol 11 (2) ◽  
pp. 411-416 ◽  
Author(s):  
Isao Nagano ◽  
Fuquan Pei ◽  
Zhiliang Wu ◽  
Jun Wu ◽  
Huier Cui ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Adult Worm ◽  
Immunosorbent Assay ◽  
Fasciola Hepatica ◽  
Cysteine Proteinase ◽  
Clonorchis Sinensis ◽  
Recombinant Antigen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Predicted Amino Acid Sequence ◽  
Crude Extracts

ABSTRACT We produced a recombinant cysteine proteinase of Clonorchis sinensis and tested its value as an antigen for serologic diagnosis of C. sinensis infections. The predicted amino acid sequence of the cysteine proteinase of C. sinensis was 58, 48, and 40% identical to those of cathepsin L cysteine proteinases from Paragonimus westermani, Schistosoma japonicum, and Fasciola hepatica, respectively. Western blotting analysis showed that sera from patients infected with C. sinensis strongly reacted with the recombinant protein and that sera from patients infected with S. japonicum weakly reacted with the recombinant protein. Antibody against the recombinant protein stained proteins migrating at about 37 and 28 kDa in C. sinensis adult worm crude extracts. Immunostaining revealed that the cysteine proteinase of C. sinensis was located in the intestinal epithelial cells of the adult parasite and in intrauterine eggs. The specificity and sensitivity of the recombinant antigen or C. sinensis adult worm crude extracts were assessed by an enzyme-linked immunosorbent assay (ELISA) using serum samples from humans infected with different parasites, including 50 patients with clonorchiasis, and negative controls. The sensitivities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96 and 88%, respectively. The specificities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96.2 and 100%, respectively. The results suggested that the recombinant cysteine proteinase-based ELISA could provide a highly sensitive and specific assay for diagnosis of clonorchiasis.

Sign in / Sign up

Export Citation Format

Share Document