Cloning, Expressing And Functional Identification Of Mago Nashi Gene In Schistosoma Japonicum

Schistosomiasis remains a serious public health problem worldwide. High percent of schistosome eggs are retained in the liver of the final host where they elicit inflammatory immune responses, which lead to formation of granuloma and fibrosis, the major pathological effects of schistosomiasis. Controlling sexual maturation, sexual dimorphism and labour division may be effective in prevention of schistosomiasis. Therefore, investigations into gender-associated gene expression of schistosome may lead to the identification of novel drug targets. The emergence of RNA interference technique has opened up new experimental opportunity for gene function analysis in many organisms. In particular, the generation of vectors directing the synthesis of short hairpin RNAs (shRNAs) enables persistent silencing of endogenous gene expression. We inferred that the function of SjMago was similar to that of low-grade species. Here, we selected SjMago as target gene for studying its function and expected that it may become a new drug target controlling schistosomiasis. In this paper, we tested the possibility to express shRNA driven by mammalian Pol III promoter H1 to silence the expression of Mago nashi gene in Schistosoma japonicum.Now, we selected pSUPER for our study. One gene-specific insert specifies a 19-nucleotidesequence corresponding to nucleotides 193-211 downstream of the transcription start site (ggcaagttgcgttatgcta) of schistosomula Mago nashi gene (Genebank accession no. BM735619), which is separated by a 9-nucleotide non-complementary spacer (ttcaagaga) from the reverse complement of the same 19-nucleotide sequence. The 5'end corresponds to the Bgl II site, while the 3'end contains the T5 sequence and any Hind III corresponding nucleotides. This vector was referred to as pSUPER-M1. The other gene-specific insert specifies a 19-nucleotidesequence corresponding to nucleotides 464-482 downstream of the transcription start site (gaacgtattattacctggt) of SjMago gene. This vector was referred to as pSUPER-M2. Two pairs of target sequence oligonucleotides were designed by the online software (www.ambion.com/techlib/misc/). A control vector referred as pSUPER-R was constructed using a 19-nucleotide sequence (gattgcgaatcggtcagtt), selectived randomly corresponding to nucleotides of pSUPER-M1, with no significant homology to any parasite gene sequence and therefore serves as a non-silencing control. The oligonucleotides were synthesized by a Biological Engineering Technology Company. The complementary oligonucleotides were annealed, and then diluted and ligated into pSUPER vector previously linearized with Bglâ…¡and Hindâ…¢. The positive clone was identified by EcoRâ… and Hindâ…¢, for its Bglâ…¡site was destroyed after successful ligation. The sequences of the cloned oligonucleotides were verified by automatic DNA sequencing. Plasmid DNA was purified and used for transfection of S. japonicum. Schistosomula, transformed from cercariae with the tails sheared off by a mechanical method , were electroporated with Mago nashi shRNA expression vector at 120V for 20 ms using an Electro Square PoratorTM ECM830 (BTX). Aliquots of parasites were harvested respectively in day 1, 3, 5 after electroporation. Total RNA, DNA and proteins were isolated using TRIzol Reagent according to the manufacturer's guidelines. Levels of Mago nashi mRNA and protein were determined by RT-PCR and Western blotting analysis. The results showed that SjMago mRNA levels were decreased by 7%, 68%, 81% in pSUPER-M1 group and 27%, 41%, 66% in pSUPER-M2 group, respectively, at day 1, 3, 5 after electroporation, compared to that in pSUPER-R group. The SjMago protein expression levels were decreased by 15%, 25%, 54% in pSUPER-M1 group and 18%, 28%, 42% in pSUPER-M2 group, respectively, at days 1, 3, 5 after electroporation, compared to that in pSUPER-R group. The results showed that shRNA transcribed from mammalian Polâ…¢promoter H1 specifically reduced the levels of Mago nashi mRNA and proteins in S. japonicum in short term. Followed by electroporation, schistosomula were immediately washed and resuspended at 25,000/ml in wash medium, and then injected into the rear thigh muscles (1000 parasites per mouse, distributed between two injection sites)of BALB/c mice. Parasites were obtained 6 weeks later by perfusion of the portal vasculature with citrated saline. The results showed that shRNA transcribed by H1 promoter can specific silence schistosoma target gene expression, in short terms particularly. Optical microscopy revealed that parasite size were no significantly different between experiments and controls, however, confocal microscopy revealed that there were cells lined up tightly and bulk sperm in partial testes of experimental group. It is suggested that SjMago might be a sex determined gene.