Characterization And Protective Effects Of Schistosoma Japonicum Surface Membrane Protein Sj29

Schistosomiasis , as a widespread zoonotic-parasitosis , contracts commonly by both human beings and livestock . In China Schistosoma japonicum seriously affects on the coordinated development of sustainable social and economic development including agricultural and animal husbandry. The application of schistosome vaccine in humans and animals to block the transmission of schistosomiasis is recognized as the long-term effective control strategy. More attention focus on Schistosoma surface membrane proteins for they are exposed to host bloodstream and immune system, and they identify directly to the host immune system. In 2006, 84449 expressed sequence tags from Schistosoma japonicum were published by Chinese National Human Genome Center, including a total of 935 ESTs encoded Schistosoma-specific potential protein (e-5), gene Sj29 was there, meanwhile, our laboratory analyzed the surface protein of adult Schistosoma japonicum by biotin-avidin way, found that Sj29 also appeared in the worm surface. Therefore , this study carried out cloning, expression and characterization of Schistosoma japonicum 29kDa surface membrane protein, and assessed the immunogenicity protective effects.Primers were designed according to AY814537, the gene coding for Sj29 protein was amplified by PCR, sequence analysis was done by bioinformatics. Partial fragment of Sj29 gene was subcloned into the prokaryotic expression vector pET28c(+), a recombinant pET28c-Sj29 was constructed , then transformed into Escherichia coli and induced by IPTG. The recombinant protein (rSj29) was purified using His-Binding-resin affinity chromatography and was characterized by Western blotting. Balb/c mice were immunized with purified rSj29 and then challenged with Schistosoma japonicum cercariae. The level of specific IgG in the experimental animals against rSj29 was determined by ELISA. mRNA expression level of Sj29 gene in different stages of Schistosoma japonicum and in the worms from the mice immunized with rSj29, were detected by quantitative real-time PCR. Location of Sj29 protein in different stages of Schistosoma japonicum was detected by Immunofluorescence technique.Success to clone gene Sj29 . The ORF of Sj29 contains 576 nucleotides, and encodes 192 amino acids. BLAST found regions of similarity between biological sequences, these nucleic acid showed 92%(SJCHGC05668),99%(SJCHGC05578) , 89% (SJCHGC03008) and 68%(Sm29, Genebank ID:AF029222)identity with the Sj29 nucleic acid sequence; these proteins showed 88% (SJCHGC05668,1e-92),100%(SJCHGC05578,9e-75),81%(SJCHGC03008,1e-73) and 54%Sm29(5e-55)identity with the Sj29 amino acid sequence. Bioinformatics analysis showed that, Sj29 amino acid sequence contained no signal peptide and transmembrane domain. Subcellular localization analysis predicted it was a secretory protein. Antigenic analysis showed that Sj29 was antigenicity.Success to construct prokaryotic expression recombinant pET28c-Sj29 . The recombinant protein was of 22.9KDa molecular weight and expressed in the formation of inclusion body. The results of Western blotting demonstrated that, rSj29 reacted positively with both sera from mice immunized with rSj29 and sera from mice infected with Schistosoma japonicum, it showed good immunogenicity and antigenicity; Vaccination experiments with rSj29 indicated that BALB/C rates of worm reduction, liver egg reduction and feces egg reduction in vaccinated mice group were 23.15%(P=0.048),18.6%(P=0.024)and 27.38%(P=0.007) respectively. The results of ELISA demonstrated that, the level of specific IgG against rSj29 was raised 3.7 times in immunized mice group.Real-time PCR revealed that the transcriptional copies of Sj29 in 7d, 14d, 28d,32d, 42d Male and 42d female Schistosoma japonicum were 451,741,428,2640,102 and 201 respectively relative to one million 18sRNA transcriptional copies. The mRNA levels of 32d were higher than other period.The transcriptional copies of Sj29 in the worms from the mice immunized with rSj29 was 6941, while 134 of PBS group and 760 of control group. The mRNA levels of Sj29 in the worms from the mice immunized with rSj29 was higher than adjuvant group and control group. Detection using immunofluorescence microscopy showed that Sj29 protein was expressed in the surface in different stages of Schistosoma japonicum.This study had cloned and characterized Sj29 surface membrane protein gene, demonstrated that recombinant protein rSj29 could induce significant protective immunity in mice, and provided basic data for the further study on immunobiology of this gene.