Studies On The Cloning, Expression And Application Of The Gene Of Schistosoma Japonicum Asparagine Endopeptidase

Schistosomisis japonica is a serious zoonotic parasitic disease.Schistosome uses its own hemoglobinase to degrade host hemoglobin into peptides and amino acids,and then absorbs and uses these as its main source of nutrition.Primarily,32kD Legumain was named as hemoglobinase. However,in recent years,further study found that hemoglobinase is not a single enzyme,but a series of enzymes including cathepsin B,C,D,L and Legumain.In that,Legumain has no direct role on digestion of hemoglobin,its function is not yet clear,and researchers speculate that it may affect the growth and development of Schistosoma by regulating the expression of cathepsin.Studies indicated that natural asparagine endopeptidase was of high solubility,it is the post-processed product of enzyme precursor.Several studies on the expression of precursor encoden gene revealed that the yield was limited and it was very difficult to purify the expressed products.In order to increase the yield of recombinant protein and simplify the preperation procedure,the cDNA frgment encoding the mature peptide of Schistosoma japonicum Sj32 was cloned and expressed in this study.The applications of the recombinant protein obtained here were analyzed by avaluation the immunoprophylaxis effects and the immunologic diagnosis of animal schistosomiasis.The gene expression in different Schistosoma development stage and its biological functions were also primarily studied with RT-PCR and RNA interfering technology.The results obtained in this work may contribute to the further study of the biological functions of Schistosoma japonicum Acyl asparagine endopeptidase,and the development of vaccine and diagnostic antigen.The results obtained are as follows:1,Cloning and expression of Sj32 encoding gene and the mRNA expression analysis in the worms of different development stages:A 971bp fragment was amplified from 1269bp Sj32 full-length gene gene and subcloned into the expression vector pET28(a) and the recombinant plasmid Sj32/pET28(a) was transformed into E.coli BL21(DE3).After induction with IPTG,the Sj32 peptide was successfully expressed.The protein expression product was a 41kD fusion protein which could be purified with His column.The yield of the product was about 68.8 mg/L in the culture and the purified protein concentration was 0.55mg/mL.Real-time fluorescence quantitative Real-Time PCR revealed that the mRNA expression level of this gene was highest in 18-day-old schistosomulum.2,diagnostic application of the recombinant Sj32 protein(rSj32):The recombinant protein(rSj32) was used as detecting antigen in ELISA assay to diagnose the schistosmiasis in in rabbits,mice buffalo(crude infection) and buffalo(induced infection),and compared with two standard diagnostic antigens of soluble egg antigen(SEA) and another reconbiant antigen rSj23.The specificity of rSj32 were 100.0%,96.7%,93.8%and 96.9%,respectively,and its sensitivity were 88.9%,85%,81.0%and 71.8%.3,RNA interference analysis and evauation of the immune protection of rSj32:In order to analyse the functions of Sj32 gene,RNA interference of this gene was primarily studied.Three siRNAs were designed and used to interfere the expression of Sj32 gene in cultured worms in vitro.One of those three siRNAs could inhibit 81.7%mRNA expression in cultured schistosomula.Vaccination of Balb/c mice with our recombinant protein rSj32 revealed that rSj32 induced 44.50%worm reduction and 56.14%liver egg reduction.