[Cloning, expression and identification of membrane protein Tetraspanin 2-A of Schistosoma japonicum].

OBJECTIVE: To clone and express a membrane protein (Tetraspanin 2) gene of Schistosoma japonicum (SjTsp2). METHODS: A pair of primers was designed to amplify the SjTsp2 gene which was subcloned into prokaryotic plasmid pET28a(+). The recombinant plasmid was transformed into E. coli BL21(D3) and followed by expression of the protein induced by IPTG. The protein was purified by affinity chromatography and used to immunize BALB/c mice. Dilution of antibody against SjTsp2 was determined by ELISA. The protein was also identified by Western blotting. RESULTS: Big loop of SjTsp2-A, 228 bp, was amplified in vitro by PCR. Its deduced amino sequence shared 52% similarity with SmTsp2. The soluble recombinant SjTsp2-A was expressed in the experiment and high dilution antibody against the recombinant (1:32,000 in maximum) was produced in immunized mice. SjTsp2-A reacted positively with sera of acute and chronic schistosomiasis patients but not with sera from healthy persons by Western blotting. CONCLUSION: SjTsp2 has been expressed and shows certain antigenicity.

Schistosoma japonicum: inhibition of Mago nashi gene expression by shRNA-mediated RNA interference.

RNA interference (RNAi) mediated by short interfering RNA (siRNA) is a powerful reverse genetics tool and holds enormous therapeutic potential for various diseases, including parasite infections. siRNAs bind their complementary mRNA and lead to degradation of their specific mRNA targets. RNAi has been widely used for functional analysis of specific genes in various cells and organisms. In this paper, we tested the potential of silencing the expression of the Mago nashi gene in Schistosoma japonicum by siRNAs derived from shRNA expressed by mammalian Pol III promoter H1. Schistosomula, transformed from cercariae by mechanical shearing of the tails, were electroporated with Mago nashi shRNA expression vector. Aliquots of parasites were harvested at days 1, 3, and 5 after electroporation, respectively. Levels of Mago nashi mRNA and protein were determined by RT-PCR and Western blotting analysis. The results showed that shRNA expressed from mammalian Pol III promoter H1 specifically reduced the levels of Mago nashi mRNA and proteins in S. japonicum. Changes in testicular lobes were apparent when parasites were introduced into mammalian hosts. Thus, vector-mediated gene silencing is applicable to S. japonicum, which provides a means for the functional analysis of genes in this organism.

[Immunoscreening of schistosomulum cDNA library of Schistosoma japonicum and preliminary identification of membrane-associated protein abundant in tegument].

OBJECTIVE: To obtain genes encoding the novel molecules for diagnosis of schistosomiasis. METHODS: Juvenile S. japonicum cDNA library was immunoscreened to obtain positive clones. By DNA sequencing and sequence analysis, the target gene was amplified by PCR and subcloned into prokaryotic plasmid pET28a. The recombinant plasmid was transformed into E. coli BL21 followed by expression of the protein induced by IPTG. The protein was identified by Western blotting. RESULTS: 34 positive clones were obtained, 24 of which were chosen to be sequenced, 13 of which were Sj22600 gene. The protein were recognized with sera of infected rabbits and patients with acute and chronic schistosomiasis by Western blotting. CONCLUSION: The gene coding for Sj22600 membrane protein was screened with high frequency in the cDNA library. The E. coli BL21 transformed with the recombinant plasmid can express the fusion protein, which shows immunoactivity.