Cloning, Expression And Biological Function Study Ofschistosoma Japonicum TOR

Schistosomiasis remains an important global public health problem, as hundreds of millions of people are at risk of acquiring this infection. Environment management and medicine intervention are the main measures to control schistosomiasis currently. An ideal research direction for sustainable control of schistosomiasis would be to develop an efficient and safe vaccine. Host complement is important immunological effector to defense against the parasite, which schistosome can evade somewhat by inhibiting the host complement cascade. Thus, some regulatory proteins on the surface of schistosomal tegument that impede the complement cascade might represent potential molecules for the discovery of vaccine candidates. Tetraspanning orphan receptor(TOR) is a multiple transmembrane receptor with the complement C2 binding sites, that can inhibit the formation of C3 converting enzyme by regulate the complement cascade. The study on the TORs of S. mansoni suggested that TOR can induce high level effect of immune protection. In this paper, the characteristics and function of Schistosoma japonicum tetraspanning orphan receptor(Sj TOR) was studied according to the proteomics study of tegument surface proteins of S. japonicum in our laboratory.Sj TOR gene fragment was cloned by PCR with primers based on the reference sequence. The Sj TOR open reading frame(ORF) of 1245 bps, encoding 414 amino acids, is 77.0%, 72.0%, and 75.0% identical to homologs from S. mansoni, S. haematobium, and T. cruzi, respectively, by multiple sequence alignment analysis. The sequence analysis indicated that Sj TOR did not possess a signal peptide, but did contain five N-glycosylation sites. Transmembrane domain and structure prediction analysis indicated that Sj TOR contained four transmembrane domains. A comparative quantitative PCR analysis showed that Sj TOR m RNA was present in all developmental stages examined and exhibited the highest transcript levels in cercariae, and the female adult worms expressed higher transcript levels than that of males. Immunolocalization assays revealed that native Sj TOR was mainly distributed on the tegument and at lower levels in the parenchyma of the parasites.Primers were designed based on the sequence of first extracellular domain(Sj TOR-ed1, AA 47–168). Then, the recombinant plasmid p ET-28a-Sj TOR-ed1 containing Sj TOR-ed1 gene fragment was constructed and expressed in the Escherichia coli BL21(DE3) successfully. The Ni-NTA purification system was applied to purify the recombinant protein and acquired the purified fusion protein with molecular weight(MW) of ~14 k Da. Western blot results showed that recombinant Sj TOR-ed1(r Sj TOR-ed1) had good immunogenicity. Purified r Sj TOR-ed1 emulsified with ISA206 adjuvant could induce significant reduction of worm burden from 24.51% to 26.51%, and liver egg numbers from 39.62% to 32.92% in two independent trials in mice. ELISA assays showed that the r Sj TOR-ed1 could elicit high levels of Ig G antibodies until the mice were euthanized. Ig G isotype analysis showed that r Sj TOR-ed1 immunization could induce both Ig G1 and Ig G2 a antibodies, and the Ig G1/Ig G2 a ratio peaked at week 4 and then decreased gradually.Hemolytic assays indicated that r Sj TOR-ed1 can inhibit complement hemolysis in a dose-dependent pattern during 0.2–1.0 μM. The maximum concentration of r Sj TOR-ed1(~10 μM) showed ~60.26% inhibition of complement hemolysis. Furthermore, a complement C2 binding assay suggested that r Sj TOR-ed1 can bind to C2. Our findings showed that r Sj TOR-ed1 can inhibit complement activity by binding to C2. Four pairs of specific si RNAs were designed based on the sequence of Sj TOR, and their silencing effects were evaluated in vitro. The comparative quantitative PCR analysis showed that there were 45.12%、38.05%、25.46% and 44.25% reduction in the transcript level compared with the blank control group.In summary, a gene encoding Sj TOR was cloned successfully, and the Sj TOR was ascertained to transcribe highest at the stage of cercariae and mainly distribut on the tegument. Recombinant protein r Sj TOR-ed1 was purified from E. coli and validated to inhibit complement activity and bind to C2 by experiments. A partial but significant protection was obtained in mice vaccinated with r Sj TOR-ed1. The results suggested that Sj TOR is a potential candidate of vaccine against S. japonicum.