Cloning,Recombinant Protein Expression And Functional Analysis Of Sjp38MAPK In Schistosoma Japonicum

Schistosomiasis caused by S.japonicum is a zoonotic parasitic disease that has been prevalent for a long time in China.Male and female paring is prerequisite for female development and egg production.Therefore,understanding of the mechanism of the signal transduction pathways related to reproductive development of S.japonicum may open a new avenue to develop a novel strategies against schistosomiasis.In the previous study,Sjp38MAPK was shown to be an important node of protein-protein interaction map of S.japonicum identified by phosphorylated proteomics analysis and bioinformatics.In order to elucidate the role of Sjp38MAPK in the survival and reproductive development of S.japonicum,the cDNA of Sjp38MAPK was cloned and the recombinant protein was obtained.In addition,the transcript levles of Sjp38MAPK in different developmental stages and gender of S.japonicum were analyzed by RT-qPCR.The function of Sjp38MAPK in S.japonicum was also preliminarily evaluated by inhibiting Sjp38MAPK gene using small interfering RNAs in vitro.It includes the following three parts:(1)Cloning,expression and bioinformatics analysis of S.japonicum Sjp38MAPKBased on the results of phosphoproteomic analysis of S.japonicum,we obtained Sjp38MAPK that is putatively involved in the regulation of signal transduction.Bioinformatic analysis showed that the cDNA sequence of p38MAPK contained an open reading frame of 1038 bp,encoding a Sjp38MAPK protein with a predicted molecular weight of 39.58 kD.Bioinformatic analysis also showed that Sjp38MAPK sequence had 93.48%similarity with Schistosoma mansoni,90.70%similarity with Schistosoma haematobium and 57.10%similarity with Human and Mus musculus.In addition,we amplified the cDNA fragment of S.japonicum Sjp38MAPK by PCR,constructed the prokaryotic recombinant plasmid pET-32a(+)-Sjp38MAPK,and obtained the recombinant protein(rSjp38MAPK).The recombinant protein was further validated by MALDI-TOF-TOF analysis.(2)Expression of Sjp38MAPK in different developmental stages and gender of S.japonicumThe transcription levels of Sjp38MAPK in different developmental stages(eggs,cercariae,14d,21d,28d and 35d)and different gender(male and female:14d,21d,28d and 35d)of S.japonicum were determined by RT-qPCR.Results showed that Sjp38MAPK mRNA was expressed in cercariae,eggs,schistosomula and adults of S.japonicum,and the expression of Sjp38MAPK in adults(21d,28d,35d)was significantly higher than that in eggs,cercariae and schistosomula.In addition,the expression of Sjp38MAPK in males was significantly higher than that in females at 28 and 35 days.The results showed that the expression of Sjp38MAPK had developmental and gender differences.(3)Silencing of Sjp38MAPK using siRNA in vitroIn order to further reveal the function of Sjp38MAPK in S.japonicum,the Sjp38MAPK mRNA of S.japonicum was inhibited in vitro using siRNA and the best siRNA-977 was obtained.Upon the silencing of Sjp38MAPK,the motility of S.japonicum was observed by electron microscopy;the activity of schistosome was investigated by staining with Hoechst 33258 fluorescent dye;and the cell activity of schistosome was determined by cell Titer-Lumi ~TMM fluorescence assay kit.In addition,the expression levels of mRNA of four eggshell genes were analyzed by RT-qPCR.The development of female ovaries after interference was observed by confocal laser scanning microscopy.The results showed that schistosomes mortality increased in the Sjp38MAPK inhibited parasites,while the mRNA levels of serval eggshell protein genes such as AY222895.1,M59318.1 and D32205.1 decreased significantly.The results indicate that Sjp38MAPK may play an important role in the survival,growth and development of S.japonicum.