| Schistosomiasis is a widely spread and serious parasitic zoonosis with an estimated 200 million people infected and close to 800 million at risk of infection worldwide. Although praziquantel is the only drug that is widely used to treat this disease, it is ineffective in preventing reinfection and the concern of resistance is increasing following large-scale use for more than 30 years. The development of preventive vaccine against schistosoma is always one of the hot research directions, but there is no effective vaccine against schistosomiasis currently. Complement situated in the first line of host defense system is an important way of host against blood parasites, could quickly mark and eliminate invasive pathogen, mediated humoral and cellular immunity, which acts as important immune regulation and cascade effect system. Schistosome developed successful immune evasion strategies in the long evolutionary process. Some schistosome molecules play vital roles in vivo development procedure and survival by modulating complement system, and possess the potential to be vaccine candidate and ideal drug target. The C1 q binding protein(C1qBP) was identified in the previous proteomics study of schistosoma japonicum, which is supposed to associated with the inhibition of complement.The ORF of SjC1qBP gene was cloned by PCR based on the reference sequence from NCBI in this study. This cloned cDNA(729 nucleotides) was a complete ORF encoding a polypeptide of 242 amino acids without the signal peptide and the transmembrane domain. Additionally, the result of amino acid sequence analysis by SMART indicated the presence of a conserved domain of the MAM33 family which bind to the globular head of C1 q. RT-PCR analysis showed that the SjC1qBP gene was transribed in various development stages of schistosoma japonicum, wherein the level of transcription in cercariae relatively high, while at other stages for instance egg roughly same. The SjC1qBP was mainly present in the tegument of adult worms and the eggs of S. japonicum in the immunohistochemical analysis.The recombinant expression plasmid p ET-32a(+)-SjC1qBP was constructed and expressed in the Escherichia coli BL21(DE3) successfully. The His fusion protein was majority extracted from the inclusion bodies with 8 M urea and purified by affinity chromatography. The recombinant protein can resolve in PBS after refolding through a successive urea concentration gradient dialysis. In the vaccination trials of BALB/c mice, rSjC1qBP protein emulsified with ISA 206 adjuvant could induce 31.48% reduction of worm burden and 45.12% reduction of liver egg numbers. Western-blot results revealed that rSjC1qBP could be probed by the serum of mouse immunized, which imply strong immunoreactivity. Furthermore, ELISA assays indicated that the rSjC1qBP could induce a higher level of specific IgG until six weeks after infection, compared with the control group.The results of Hemolytic assay showed that the His fusion protein can inhibit sheep red blood cell hemolysis induced by complement. And with the increase of the concentration of rSjC1qBP, the inhibition become more obvious. The inhibition ratio could reach more than 80% when the protein concentration is 1.5 mg/mL. In addition, the His fusion protein could specifical bind to C1q protein, indicating it can competitively combine with C1 q, thus inhibiting the complement classical way.The results suggested that SjC1qBP is a potential candidate of vaccine against schistosomasis and can competitively combine with C1 q to inhibit the complement classical way. Our study will not only give new insight of SjC1qBP, and laid a solid foundation to explore further its biological function, but also provide valuable information about the mechanism immune evasion of Schistosoma japonicum. |
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