Identification And Biological Function Study Of Adult Somatic Stem Cells Associated Gene Sjp53 Of Schistosoma Japonicum

Schistosomiasis japonicum is a chronic,wasting zoonotic parasitic disease caused by Schistosoma japonicum infection.S.japonicum can parasitize in host for a long time and produce a large number of eggs,which is the main reason of host pathological damage.The long-term survive of S.japonicum in host is not only related to its immune escape,but also inseparable from its ability to repair damaged tissues,maintain structural integrity and homeostasis when subjected to immune response and other stress.And the ability,in large part,is related with adult somatic stem cells.Therefore,identification adult somatic stem cells of S.japonicum and study their gene functions are of great significance for exploration of the physiological mechanism of long-term parasitism.1.The analysis of adult somatic stem cells and its transcriptomes of Schistosoma japonicum42d adult worms were exposed to 100Gy?-ray,then incubated with EdU for 24h after irradiation24h or 48h,fluorescence staining showed that there were proliferative somatic stem cells exist in S.japonicum.The RNA-seq was then used to compare the difference of adult somatic stem cells depletion between transcriptomes of adult male parasites before and after irradiation,and 235 adult somatic stem cells-associated transcripts were screened out,of which 179 were down-regulated and 56 were up-regulated.10 differentially expressed genes were selected for qPCR verification,and the express trend were consistent with RNA-seq result.GO analysis showed that many of down-regulated genes were related with physiological process such as reproduction,development,and response to stimulus.KOG and KEGG analysis indicated that 50 of 179 down-regulated genes were related to cell cycle and DNA repair,28 down-regulated genes involved in signal pathways,there are 15 genes participated in the pathway of DNA replication.Above results consistent with the biological phenomenon of proliferative cells depleted in S.japonicum followed irradiation.2.Cloning and expression of Sjp53 gene and immunprotective effect rSjp53 on mouse schistosomiasisA transcript associated with DNA replication and repair was selected from the transcriptome data of irradiated worms,and the expression level was decreased by 2-fold.PCR was applied to clone the CDS of Sjp53,which has a 2883bp open reading frame,encoding 960 amino acids,including conserved p53 region and 4 protein binding sites,2 nucleic binding sites,thus the transcript was named Sjp53.We constructed recombinant expression plasmid pCold-Sjp53,which can be induced express in E.coli BL21?DE3?,and obtained a¹10kDa insoluble recombinant protein rSjp53 by His-binding purification.High levels of specific IgG antibody were induced in BALB/c mice by immunization of rSjp53emulsified with 206 adjuvant.Recombinant and nature protein of Sjp53 can be recognized by sera of immunized,indicating that the antigenicity of rSjp53 is good.Significant reduction rate of liver eggs count was induced in mouse schistosomiasis immunoprotection trial.3.Biological function research of Sjp53The eukaryotic expression plasmids pXJ40-Sjp53d were constructed with the functional domain of Sjp53 and pXJ40 vector,then transfected into mammalian cells,and the expression of recombinant protein was verified by WB and IFA.The results of cell proliferation assay illuminated that Sjp53 may depress the proliferation of H1299 and Hela and promote the proliferation of Vero and 293T.Worms been treat with p53 specific inhibitor PFT-?to inhibit endogenous Sjp53 activity,resulted in adult parasites cell proliferation and EdU incorporation reducing apparently,which indicated that Sjp53 can maintain the regular proliferation of somatic stem cells in S.japonicum.After adding H₂O₂ in vitro culture medium,parasites did not change significantly and the transcription level of Sjp53 increased gradually,indicated that Sjp53 plays an important role in anti-oxidative stress.The number of apoptotic cells in worms with normal Sjp53 activity was significantly increased compared with those worms with inhibited p53 activity.The results suggested that Sjp53 can cause cells with serious DNA damage prone to apoptosis.In summary,our study established a ray irradiation model of S.japonicum adult somatic stem cells,obtained transcriptome data of adult stem cells using RNA-seq,and then screened out a somatic stem cell associated gene Sjp53.We cloned Sjp53 for the first time,expressed Sjp53 recombinant protein in prokaryotic and eukaryotic system,and explored the role of Sjp53 in cell proliferation,anti-oxidative stress and response to DNA damage.