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The Preliminary Study on Argonaute Proteins of Schistosoma Japonicum

Author: YangYanPing
Tutor: LiuZongPing;ChengGuoFeng
School: Yangzhou University
Course: Clinical Veterinary Medicine
Keywords: Schistosoma japonicum Argonaute proteins RACE Functional Analysis
CLC: S852.7
Type: Master's thesis
Year: 2010
Downloads: 90
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Abstract


Schistosomiasis is caused by schistosome infections are widely distributed, the serious harm zoonotic parasitic diseases. Schistosome different developmental stages of the parasite presents unique metabolic and developmental characteristics and significant biological and morphological changes, there must be a different differential gene expression patterns, play an important role in the regulation of non-coding RNA in the growth and development of the organism, the recent studies have shown that Argonuate family of proteins involved in this process. In this study, using RACE technology 2 encoding full-length cDNA of Schistosoma japonicum Argonaute proteins; take advantage of the real-time PCR analysis of transcription of the genes in the different developmental stages of Schistosoma japonicum; construct prokaryotic expression plasmid pET-28b-SjAGO1-PIWI and pET- the the 32a-SjAGO1 and pET-28b-SjAGO2-PIWI, for reorganization conserved functional domain protein and full-length recombinant protein prokaryotic expression and preparation of related antibodies; using the Western blot method and RNAi preliminary analysis of the Schistosoma japonicum Argonaute protein function. The results show that: (1) RACE was cloned to obtain the full-length cDNA encoding of Schistosoma japonicum Argonaute1 protein (SjAGO1) contains the complete open reading frame of 3030 bp encoding 1009 amino acids, predicted molecular weight of 111.7 kDa; bioinformatics analysis indicates that the domain characteristic of the gene encoding the protein sequence has the typical structure of the Argonaute family of proteins, and the with Argonaute proteins such as human and mouse has high homology; fluorescence real-time quantitative PCR showed that the gene in 7, 14-day-old Japanese schistosomula The higher transcription; the Conserved Domains protein expression using recombinant preparation of highly specific polyclonal antibody; Western blot analysis showed that SjAGO1 highly expressed in the male. Schistosoma japonicum Argonaute2 protein (SjAGO2), (2) RACE cloned the full-length cDNA gene contains the open reading frame of 2808 bp encoding 935 amino acids with a predicted molecular 106.0 kDa; bioinformatics analysis showed that the protein with Schistosoma mansoni the AGO2 similarity of 83%; Conserved domain protein using recombinant expression of a highly specific polyclonal antibody preparation. The western blot analysis of the proteins in the adult, particularly in the high expression in the male. (3) RNA interference SjAGO1 gene interference group parasites survival of only 52.4% of the blank group, 39.2%, 58.4%, lower parasite vitality. Quantitative PCR showed that the interference group Schistosoma japonicum SjAGO1 transcript levels only blank 2.0%, 4.5%, 3.2%, the interference group SjAGO1 protein expression was significantly lower than the control group. Conclusions: The first time the two encoding Schistosoma japonicum Argonaute protein gene, success the SjAGO1, and SjAGO2 of conserved domains were expressed in E. coli, the corresponding antibodies were prepared using purified protein Kunming mice; successfully screened out of SjAGO1 gene with the interference effect of siRNA. The results of this study, in-depth study Schistosoma japonicum Argonaute protein function to lay the foundation for depth reveal schistosome growth, especially schistosomula development and gender mature mechanism provides a new way, and also on the development of early intervention schistosome growth and anti-schistosome reproductive spawning efficient vaccines and drugs.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Livestock parasitology
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