Characters Of Interferon-γ Inducible Lysosomal Thiol Reductase In Trichinella Spiralis

Trichinellosis is a worldwide distribution zoonosis.Trichinosis spiralis is the pathogen of trichinellosis that endanger public health safety seriously.The life cycle of Trichinosis spiralis is completed in single host,but they contact with diverse host environment in different developmental stage.After complex migration in vivo,Trichinosis spiralis without cleared by host immune system are ultimately parasitized in striated muscle cells.It revealed close and complex relationship between worms and host.and the secretory proteins play an important role during the process.In this experiment,firstly the prediction process of secretory proteins was build up by software which widely accepted to predict and analyze the functions of secreted proteins from Trichinella spiralis within the the range of proteomics.It can provide references for the study of secreted proteins of Trichinella spiralis.Then IFN-γ induced lysosomal thiol reductase(GILT)was cloned,expressed to analyze sequence and study enzyme activity.The function of GILT was researched by siRNA mediated RNAi technology.Finally,the immune protection of GILT was evaluated.These could provide basis for immune prevention.1 Prediction and analysis of secreted proteins in Trichinella spiralisThe procedure for predicting the secreted protein by popular bio-information software was structured to predict secreted proteins from the proteome of Trichinella spiralis.The sequence characteristics were analyzed and the functions of secretory protein function were annotated.414 classical secreted proteins and 245 non-classical secreted proteins were found,which accounting for 2.40%of proteome.Three motifs were predicted in signal peptides of classical secretory proteins.T.spiralis secreted proteins and their signal peptides mainly composed of hydrophobic amino acids,while the splice sites of signal peptide mainly composed of hydrophilic amino acids.The amino acids in splice sites-3 and-1 were highly conserved,which is possibly considered as the key points for enzyme identification.During the function annotation,a higher proportion of proteins associate with nuclease activity and protease activity was found.2 Gene cloning,prokaryotic expression,enzyme activity analysis and localization of GILT in Trichinella spiralisThe ORF of Tsp-GILT consists of 801 base pairs,encoding a protein of 266 amino acids,of which 5 amino acids were signal peptides.Tsp-GILT contains the sequence characteristics of GILT.Phylogenetic tree analysis showed that Tsp-GILT was far from GILT of mammals and fish,but closer to GILT of Oesophagostomum dentatum and Ancylostoma duodenale.Homology modeling predicts the 3D structure of Tsp-GILT and compared the predicted constructs of human and rat GILT,the results showed that although there were many differences in amino acid sequences,the 3D structure was similar,especially the active sites and characteristic sites were highly conservative.pET32a-Tsp-GILT vector was constructed and expressed to obtain recombinant protein.Experiments in vitro indicated that the recombinant protein has disulfide reduction activity under acidic conditions.Immunohistochemical studies showed that there was a higher expression in cells close to skin and digestive tract of worms.3 Biological function research of GILT after knockdown by siRNA-mediated in Trichinella spiralisIn this study,firstly,the expression level of GILT was detected by qPCR in three stages(ML,AD and NBL)of Trichinella spiralis.Then the mRNA changes of Tsp-GILT were detected after the worms stimulated by mouse IFN-γ in vitro.Finally,the expression of GILT was inhibited by siRNA-Mediated Silencing experiment to observe the changes of life activities of worms.The results showed that the expression of GILT in newborn larvae was significantly higher than that in the other two stages.The expression of GILT in newborn larvae stimulated with IFN-y(1000 ng/mL)of mouse for 16-20 h reached the peak.RNAi experiments revealed that suppression of GILT expression had no significant effect on the survival rate of muscle larvae in vitro,development number and reproductive ability of adult worms,but the numbers of muscle larvae formed by newborn larvae decreased significantly.4 Research on immune protection of recombinant protein GILT of Trichinella spiralis in miceIn this study,mice were infected with muscle larvae by intravenous injection after immunized with GILT,the variation trends of cytokines in serum of mice were detected before and after immunization,as well 2 d,4 d,7 d,14 d,28 d and 35 d after infection,then muscle larvae were collected to count at 35 d.The results showed that GILT could stimulate the rise of IL-2,IL-4,IFN-y and IL-17 in mice,and had no significant effect on the levels of IL-10 and TGF-β.After mice infected with newborn larvae by intravenous injection,the expression of IL-2 and IFN-y were significantly higher than those in control group at 2-4 days.the level of IL-4 was higher than that of control group at 7-14 days.There was no significant difference in IL-10 between groups.The peak value of IL-17 appeared at 14 d after infection.TGF-β 1 increased slowly in the early stage,increased rapidly and reached the peak value at 7-14 days,then began to decline at 14-35 days,but the overall level was still higher than the normal value.TGF-β 1 in group rTsp-GILT was lower than that in two control groups The number of muscle larvae in group rTsp-GILT was lower than that in control group.These above results indicate that rTsp-GILT vaccine can stimulate the immune response of mice,and reduce the number of muscle larvae significantly.It means rTsp-GILT have the potential value of vaccine.