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Studies on the SJCHGC06868 Protein of Schistosoma Japonicum

Author: WangSuJuan
Tutor: LiuJinMing
School: Chinese Academy of Agricultural Sciences
Course: Preventive Veterinary Medicine
Keywords: Schistosoma japonicum Phage display antigen SJCHGC06868 protein Immunoprophylaxis Real - time quantitative PCR The prokaryotic expression Eukaryotic vaccine RNA interference Cytokines
CLC: R383.2
Type: Master's thesis
Year: 2010
Downloads: 27
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Abstract

Schistosomiasis is a worldwide distribution of parasites serious harm to human and animal health. Schistosoma japonicum is a parasitic on the body of the five human schistosomes, which infect humans and animals such as mammals, causing schistosomiasis japonica mainly popular in China and other East Asian countries, and to date is still one of the important public health problem. Oriental vole is a non-suitable host of Schistosoma japonicum, the ability to have a natural anti-Schistosoma japonicum. Microtus fortis associated with resistance to Schistosoma japonicum target gene, found that the schistosomiasis treatment of drug targets and candidate vaccine antigens with the reference value. The laboratory Microtus fortis serum, liver, and lung tissue Schistosoma japonicum phage cDNA display library, get more valid positive clones. Part of the study of Oriental voles liver lysates screening phage display antigens assess immune preventive effect, which can induce higher protective effect SJCHGC06868 protein coding genes carry out cloning, expression, recombinant protein vaccine and DNA vaccine preventive effect assessment, RNA interference studies. Schistosoma japonicum in Microtus fortis liver lysates phage display cDNA library of the 1st, the 2nd, the 4th and the 5th positive phage clones immunized mice to assess the immune preventive effect. The results show that phage clone 1 and No. 4 has good immune protective effect. Compared with phage control group, the 1st and respectively 28.23% worm reduction rate and 51.73% liver egg reduction rate, the 4th clone 24.28% worm reduction rate and 45.52% respectively, there is a significant difference (P lt; 0.05) . EST clones show the 1st the Schistosoma japonicum protein SJCHGC06713 part coding sequence of the protein coding sequence of Schistosoma japonicum BLASTX, high homology with a variety of biological pyrophosphatase / phosphodiesterase protein CDD analysis also found that the The protein sequence contains a conserved domain of this family of proteins. The 4 clones show the EST is the Schistosoma japonicum SJCHGC06868 part coding sequence. 2,2, reference GeneBank accession the Schistosoma japonicum SJCHGC06868 protein coding gene sequence (accession number: AY809313) design matter, which 492bp (54-546) cDNA cloning by PCR fragment, and then cloned into plasmid pET-32a multiple cloning site region, prokaryotic expression system. Obtained recombinant protein is a soluble protein, the size of approximately 36 kD, was purified His column available purified protein yield was approximately 58.3 mg / L. Recombinant proteins for immune source animal immune preventive effect assessment. Adjuvant control group and the PBS control group compared to the recombinant protein immunohistochemistry 31.63%, 29.19% worm reduction rate and 55.63%, liver egg reduction rate of 56.27%, ELISA tests showed the immunohistochemistry higher antibody levels. The 3 will the Schistosoma japonicum SJCHGC06868 protein coding genes in the 492bp fragment was subcloned into the eukaryotic plasmids pVAX1 polyclonal point region Eukaryotic expression the expression plasmid pVAX1-SJCHGC. Immune source pVAX1-SJCHGC assess immune preventive effect. Analysis Westernblot SJCHGC06868 protein expression product was obtained in mice injected muscles molecular weight of 18 KD; challenge infection test, compared with empty plasmid group and blank group, immunohistochemical 19.67%, 21.96% worm reduction rate and 58.39%, 58.76%, liver egg reduction rate; mechanism analysis showed that immunized mice generated higher antibody levels, CD4/CD8 ratio relative reduction in IL-2 cytokines such as IL-4 levels significantly increased, indicating that the reorganization true nuclear plasmid pVAX1-SJCHGC mice induced with effective CTL response to the application of fluorescence real-time quantitative PCR analysis the Schistosoma japonicum SJCHGC06868 protein-coding gene mRNA expression in a different stage parasites, results show that this coding genes in schistosomula to were expressed in the adult of worms, but higher expression levels of the 14d schistosomula. The design and synthesis of three with a modified small interfering RNA molecules and RNA interference experiments carried out in vitro schistosomula. Fluorescent quantitative real-time analysis of the results show the interference group SJCHGC06868 protein-coding gene expression was significantly inhibited compared with the control group, siRNA1-4 siRNA2-244, the inhibition rate of siRNA-2-327 interference group were 32.6%, 37.2% , 44.9%; microscopic observation showed that the interference group of parasites decreased activity, decreased survival after 6 days in vitro interference siRNA1-4 siRNA2-244, siRNA-2-327 interference group parasite survival rates were 61%, 54% 51%, significantly lower than the control group. This study for the first time show the phage unknown protein SJCHGC06868 Schistosoma japonicum antigens, recombinant antigens and DNA vaccine preventive effect was evaluated, confirmed that the protein-coding genes in Japanese schistosomula period high expression and expression suppression can be caused by Schistosoma death, The study results show the protein and its gene in anti-schistosome vaccine and therapeutic drug research with a larger study.

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