Characterization Of The Function Of Glutathione S-transferase Of Trichinella Spiralis On Regulating Macrophages And Dentritic Cells Activity

Trichinella spiralis is a common intramuscular parasitic nematode.In recent years,T.spiralis crude muscle larvae and excretion and secretion products can not only induce strong Th2 immune response,but also regulate host immune response,induce immunosuppression,and eventually establish chronic infection,which is conducive to its long-term parasitism.It has also been found that there are active single effector molecules in the excretion and secretion products that can regulate host immune cells and induce immune tolerance.However,the single-effector molecule and its mechanism of action are still unclear.Therefore,the isolation,identification and regulation of key immune regulatory molecules of T.spiralis are very important.This project first designs specific primers from the gene sequence of theglutathione S-transferase(XM 3371707.1)of T.spiralis published by GenBank,and extracts RNA of the larvae of T.spiralis and RT-PCR to obtain the target fragment.The recombinant expression plasmid of pCold I-r Ts-GST was constructed and transformed into Rosseta Gami(DE3)Escherichia coli sensory cells to carry out the soluble expression of the recombinant protein(rTs-GST).SDS-PAGE electrophoresis showed that a protein band appeared at about 25.7 KD,which was consistent with the expected size.Western Blot analysis showed that the purified r Ts-GST could be infected with Trichinella spiralis for 60 days.The results showed that the purified rTs-GST had good reactivity.Secondly,rTs-GST stimulated the mouse macrophage RAW264.7 in vitro,andanalyzed the effect of rTs-GST on the polarization of mouse macrophages by using CCK-8,flow cytometry,Western Blot and ELISA.The results showed that r Ts-GST could inhibit the expression of inflammatory factors and effector molecules induced by LPS,and that rTs-GST alone could increase the expression of anti-inflammatory cytokines and the transcription and protein levels of Arg1.The flow pattern further showed that rTs-GST could inhibit the expression of RAW264.7 surface molecule CD16/32 of mouse macrophages after LPS stimulation,while rTs-GST alone could induce the high expression of CD206 in mouse macrophage RAW264.7.Therefore,we hypothesized that rTs-GST,as an effective regulatory molecule of T.spiralis,can induce macrophages to transform into alternative activated macrophages.Finally,the murine bone marrow derived dendritic cells(BMDC)wereisolated and differentiated into immature dendritic cells(DC)by selective medium in vitro.Flow cytometry was used to identify the purity of more than 85%.rTs-GST in vitro was used individually or with LPS to stimulate DC in mice.ELISA and flow cytometry were used to analyze r Ts-GS.The effect of T on the maturation and activation of dendritic cells in mice showed that rTs-GST inhibited the expression of CD40,CD80 and CD86,and the release of inflammatory factors TNF-αand IL-12 by LPS induced dendritic cells;and rTs-GST alone could increase the release of anti inflammatory cytokines TGF-βand IL-10 in dendritic cells.The CD4~+T cells were further isolated from the spleen of T cell receptor transgenic(OT-II)mice,and DC was incubated with rTs-GST early stimulation.The activation,proliferation and cytokine expression of CD4~+T cells were detected by OVA peptide antigen stimulation.The results showed that DC stimulated by rTs-GST alone inhibited the proliferation and activation of CD4~+T induced by OVA and differentiated into Th2/Treg.These results suggest that rTs-GST may be a key regulator of T.spiralis,which plays an important role in the regulation of host immune response,chronic infection and long-term parasitism.