Abstract
Trichinella spiralis is mammalian skeletal muscles parasite which may cause trichinellosis in animals and humans. Gamma interferon inducible lysosomal thiol reductase (GILT) is a widespread superfamily which plays key role in processing and presentation of MHC class II restricted antigen by catalyzing disulfide bond reduction. There are no reports about GILT in T. spiralis. In present study, GILT from T. spiralis (Tsp-GILT) was cloned, analyzed by multiple-sequence alignment, and predicted by 3D structure model. Recombinant Tsp-GILT (about 46 kDa) was efficiently expressed in Escherichia coli and thiol reductase activity suggested that in acidic environment the addition of a reducing agent is needed. Soaking method was used to knockdown expression of Tsp-GILT using small interference RNA (siRNA). Immunofluorescence assay confirmed the transformation of siRNA into muscle larva (ML) and new born larva (NBL). Quantitative real time-PCR (QRT-PCR) analysis revealed that transcription level of Tsp-GILT mRNA can be up-regulated by stimulation of mouse IFN-γ and down-regulated by siRNA2 in vitro. NBLs soaked with siRNA2 showed 32.3% reduction in the generation of MLs. MLs soaked with siRNA2 showed 26.2% reduction in the next generation of MLs, but no significant effect was observed on adult worms or NBLs. These findings concluded that GILT may play important roles in the development of T. spiralis parasite.
Highlights
RNA interference (RNAi) is an important tool to determine the role of genes in which doublestranded RNA or small interfering RNA activates the breakdown of homologous mRNA
Current study concluded that small interfering RNA (siRNA) knockdown of gamma interferon inducible lysosomal thiol reductase from Trichinella spiralis significantly reduce the larval infectivity, development and survival
To evaluate the infectivity of effective siRNA-treated T. spiralis, mice were divided randomly into three groups (n = 10 for each group), experimental group, control group and PBS group were infected orally with 500 muscle larva (ML) treated with siRNA2, control siRNA or PBS, respectively
Summary
RNA interference (RNAi) is an important tool to determine the role of genes in which doublestranded RNA or small interfering RNA (siRNA) activates the breakdown of homologous mRNA. RNAi was firstly discovered in the Caenorhabditis elegans, nematode worm [1] and it has been broadly used to down-regulate target molecules by reducing mRNA for protein expression. RNAi has been used to evaluate the biological functions of different genes in different helminthes, for example paramyosin in T. spiralis [2], type V collagen and calcium-regulated heat-stable protein in Schistosoma japonicum [3,4] and enolase in Clonorchis sinensis [5]. Excretory and secretory products (ESPs) may play an important role in the developmental and invasion process of infective larva [7,8]. ESPs provide better opportunities to build long lasting communication between host and parasite [9]
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