The Study Of Bivalent Multi-epitope Vaccine Against Schistosomasis Japonicum

Schistosomiasis is a serious threat to the public health, social and economic development. Although great achievements on prevention and treatment of Schistosomiasis have been obtained in our country, Schistosomiasis is still epidemic seriously in some areas. The effect of molluscicidal is diffcult to protract and steady, but can bring about the ecosystem pollution of the environment. Moreover, some schistosome strains of praziquantel-resistant to the drug have been observed recently in several countries. As the complement to chemotherapy and other approaches, the development of anti-Schistosomiasis vaccines is necessary to effectively combat the disease. So far, the current status in the development of Schistosoma japonicum subunit gene engineering vaccines will then be discussed as will new approaches that multivalent vaccines may improve on the efficacy of available vaccines. In this study, our aim is to find out the effective anti-schistosomiasis vaccine from the 2 kinds of bivalent Schistosoma japonicum tegumental vaccine we have developped by comparing the immuoprotection in mice.mRNA expression level of Sj23 and Sj-Tsp2 gene in the different stages of Schistosoma japonicum were detected by quantitative real-time PCR. Analyse the antigen epitopes and hydrophilicity of Sj23 and Sj-Tsp2 with bioinformatics. The target gene was amplified by PCR and subcloned into the prokaryotic expression vector pET32a(+), a recombinant pET32a(+)-Sj23(EC1-EC2) and pET32a(+)-Sj-Tsp2(EC2)-Sj23(EC2) was constructed, then transformed into Escherichia coli BL21(DE3) and induced by IPTG, respectively. The recombinant proteins were purified using His-Binding-resin affinity chromatography and were characterized by Westen blotting. BALB/c mice were immunized with purifeid rSj23(EC2), rSj23(EC1-EC2) and rSj-Tsp2(EC2)-Sj23(EC2) for 4 times, then challenged with 40±2 cercariae,and 6 weeks later, the adult worms were recovered by perfusion from the mice's hepatic portal vein. Total adult worm burdens and liver eggs were counted. The level of protection of vaccinated group was calculated as a percentage based on the reduction in worm burdens or liver eggs per female worm was also calculated according to the control group. The mice serum samples were collected at week 0 before immunization, at week 2,4,6 and 8 after the initial injection and at week 14(the mice were sacrificed).The level of specific IgG in the serum against rSj23(EC1-EC2) and rSj-Tsp2(EC2)-Sj23(EC2) was determined by ELISA. The transcriptions of Sj23 and Sj-Tsp2 gene in the worms from the mice immunized with rSj23(EC1-EC2) and rSj-Tsp2(EC2)-Sj23(EC2) were detected by quantitative real-time PCR.Real-time PCR revealed that the Sj23 and Sj-Tsp2 gene were expressed during the all different stages of schistosome, especially highly expressed in the stage of schistosomula; Sj23 gene expression were both down-regulated in S.japonicum worms recovered from rSj23(EC1+EC2) and rSjTsp2(EC2)-Sj23(EC2) immunized mice, while Sj-Tsp2 gene also both down-regulated in S.japonicum worms recovered from rSj23(EC1+EC2) and rSjTsp2(EC2)-Sj23(EC2) immunized mice. The molecule weight of the fusion proteins rSj23(EC1-EC2) and rSj-Tsp2(EC2)-Sj23(EC2) were 32.08kDa and 37.5kDa, respectively. The animal immuoprptection revealed that vaccined of rSj23(EC2), rSj23(EC1-EC2), rSj-Tsp2(EC2)-Sj23(EC2) in mice resulted in the worm burden reduced by 27.66%, 33.47% and 41.66%, respectively, and the egg burden reduced by 35.87%, 55.31% and 41.16%, respectively, which had significant deviation (P<0.5) when compared to the PBS control groups.rSj23(EC1+EC2) and rSj-Tsp2(EC2)-Sj23(EC2) had good antigenicity demonstrated by western blotting and ELISA. In BALB/c mice model, the fusion protein induced the highest levels of IgG after the 4th immunization. The 2 kinds of schistosoma tegumental protein as bivalent vaccine were successfully constructed which had some immunoprotection, which may suggested the heaver molecular weight of the fusion protein, the more efficacy in reducing worm burden. This research reveals this method of constructing vaccine is a worthwhile road for finding the vaccine against Schistosomiasis japonicum and there exist good prospect for tegumental proteins of Schistosoma japonicum as a vaccine or diagnostic antigen. It is imprudent to think that chemotherapy alone will suffice in the long-term global control of schistosomiasis, and vaccine discovery and development efforts should be encouraged and appropriately funded.