Expression And Characterization Ofschistosoma Japonicum Ribonuclease AY841845 Protein

Schistosomiasis is a kind of Parasitic zoonoses,which widely spread?harm to human health and hinder the development of social economy.At present,more than 700 million people under threat of schistosoma infection around the world,more than 200 million people were schistosome infectors among them.China is the most serious country of schistosomiasis japonicum prevalence.The most serious damage of people suffering from schistosomiasis is egg granulomatous inflammation,liver cirrhosis and liver fibrosis lesions,which impaired patient's liver function severely.With the development of the infection,the balance of host immune response shifted from Th1 type to Th2 type gradually,and then the Th2 type response became the dominated response finally.However,the molecular mechanism of these pathological changes is still not fully elucidated.Obviously,the immune regulation of antigens derived from schistosome to host immune system played a key role in this process.Many abroad research results showed thatthe parasitic ribonuclease(such as Clonorchis sinensis,Schistosoma mansoni,etc.)could drive the polarization of Th2 immune response strongly.Omega-1 had been identified as a main protein molecule inducing Th2 immune response of schistosome egg.If the Omega-1 was depleted,the Th2 polarization function of egg antigens of S.mansoni declined significantly.Our previous work proved that exocrine protein Sj CP1412 of eggs is one of the most important polarized molecules of Th2 immune response among Schistosoma japonicum eggs antigen.The bioinformatics analysis showed that there were a lot of members in RNase T2 family of Schistosoma japonicum,allthese molecules contain the consensus function domains of RNase T2 family.To further explore the immune regulation of RNase T2 family during Schistosoma japonicum infection,in this study,the encoding gene of Sj AY841845 registered in Genbank was cloned and the recombinant Sj AY841845 was expressed, purified and characterized.The contents of this work mainly including the following aspects.I.Gene cloning,protein expression and purification of Sj AY841845A pair of gene specific primers was synthesized according to the published Sj AY841845 gene sequences,the DNA fragment encoding the matured peptide of Sj AY841845 was amplified from the S.japonica worm c DNA by PCR,which length is 636 bp.Then,this DNA fragment was inserted into the expression plasmid p ET41a(+)at the site of the restriction enzymes Bam H1 and Sall to construct a recombinant expression plasmid p ET41a-Sj AY841845 successfully.The results of restrictive analysis and DNA sequencing confirmed that the target gene has been inserted correctly and fused in frame with the upstream gene of Glutathione S-transferase(GST).The recombinant expression plasmid p ET41a-Sj AY841845 was transformed into the competent cells of E.coli BL21(DE3)to form the expression engineering bacteria p ET41a-Sj AY841845/E.coli BL21(DE3).The engineering bacteria p ET41a-Sj AY841845/E.coli BL21(DE3)were cultured in LB medium.After being induced by IPTG,these bacteria could express a fusion protein of GST-Sj AY841845,which molecular weight is about 57.2 k Da.The expression products were dissolved with 8 M urea solution,and the purified fusion protein GST-Sj AY841845 was prepared by Nickel affinity chromatography method in denatured condition.After dialyzing the denatured GST-Sj AY841845 fusion protein with gradient urea PBS containing 0.5 M arginine,a part of denatured GST-Sj AY841845 fusion protein were refolded into soluble protein.The soluble GST-Sj AY841845 fusion protein were digested by using Enterokinase(EK),after the enzyme products were passed through a new nickel affinity chromatography column repeatly to remove the GST tag,the final through liquid was collected,which is the purified recombinant Sj AY841845 protein.The results of SDS-page showed that the molecular weight of the purified recombinant Sj AY841845 protein is 24.9 k Da,which is identify with its theoretic size.II.Analysis of the ribonuclease activity and expression profile of Sj AY841845By using the purified recombinant Sj AY841845 to digest the yeast t RNA,it was found that the enzymatic activity of recombinant Sj AY841845 is positively correlated with theprotein quantity.These results suggested thatthe purified recombinant Sj AY841845 possessedthe ribonuclease activity.To investigate the expression profile of Sj AY841845 in different development stage of Schistosoma japonicum,the m RNA and soluble protein of S.japonica egg,cercariae/somula and adult worm were prepared.RT-PCR was performed with the gene specific primer of Sj AY841845 by using the equivalent amount of m RNA of S.japonica egg,cercariae/somula and adult worm as template,respectively.Westernblotting was done with the same quantity of soluble protein of S.japonica egg,cercariae/somula and adult worm,respectively.The results of RT-PCR showed that a specific DNA band of 636 bp could be amplified from the m RNA of eggs,larvae and adult worms of S.japonicum,respectively,which is consensus to the predicted size of Sj AY841845 gene.And,Westernblotting also showed that Sj AY841845 protein presented in the different development stage of S.japonicum,including of eggs,somula and adult worms,respectively.III.The immune regulation of Sj AY841845 proteinStimulating the macrophage RAW264.7 with the purified recombinant Sj AY841845,the cytokine IL-12,IL-10 in culture supernatant were detected by ELISA.The m RNA level of i NOS,Arg-1 of the stimulated macrophage RAW264.7 wasdetected by RT-PCR.The surface marker CD16/32 related to M1 polarization,CD206 related to M2 polarization were detected by FACS.The results showed that,after being stimulated by the recombinant Sj AY841845 protein,the macrophage could secret high levels of IL-10,but the secretion of IL-12 increase was not obvious.The m RNA level of Arg-1 increased significantly,but there was not elevation of i NOS m RNA level.The CD206 expression on the surface of RAW264.7 cells was increased,but the CD16/32 expression was not change.These results indicated that Sj AY841845 could drive the M2 type polarization of macrophage RAW264.7 in vitro.ICR micewere immunized with thepurifiedSj AY841845,which increased the levels of IL-4 and TGF-? in serum and supernatant of cultured spleen cell of immunized mice significatly,but did not rose the level of IFN-??IL-10,which suggested that Sj AY841845 immunization could induce the Th2 polarization of ICR mice immune response in vivo.The immunized ICR mice were infected with cercariae of S.japonicum,and the histopathology change of mice liver were detected at 42 days post infection by tissue slice and HE staining.Pathological results showed that Sj AY841845 immunization could significantly reduce liver egg granulomas lesion in mice,which implied that Sj AY841845 involved in the pathological process of egg granuloma.ConclusionsIn this study,the Sj AY841845 protein was expressed and prepared in vitro successfully,the purified recombinant Sj AY841845,which possessed RNase activity,was defined as a novel member of RNase T2 family of S.japonicum.It was also shown that Sj AY841845 protein,existing in different development stage of S.japonicum,with immunomodulatory function,which could induced macrophage alternative activation(M2 phenotype),and stimulated the release of the immunosuppressive cytokine TGF-? and increased secretion of IL-4 to drive Th2-polarized immune responses in vivo and vitro.Sj AY841845 immunization can alleviate the egg granuloma histopathology change of mice liver.The results of this study would able to provide new evidence for further clarifying therole of Sj RNase T2 family in the immune regulation of schistosome infection.