| Cysticercosis, as an important zoonotic parasitic disease, can cause great harms to animal husbandry and to human beings. Insulin signaling pathway is an ancient and evolutionarily extremely conserved pathway that plays an important role in cell growth control and metabolism regulation. It also participates in the interaction of cells with environments. It will be of great importance to study this pathway in Taenia solium in exploring the mechanisms of growth and development, and detection of new technologies and new drug targets against this parasite.1. According to the T. solium genome, we amplified the two insulin receptor-coding genes(TsIR-1316 and TsIR-4810) from cDNA sequences, followed by analyzing the gene sequence using bioinformatic tools. The sequence analysis showed that TsIR-1316 ORF was 5 196 bp in length and encoded 1 732 amino acids; TsIR-4810 ORF was 4 920 bp in length and encoded 1 640 amino acids. In addition, both the TsIR-1316 and TsIR-4810 sequences processed typical insulin receptor domains and conserved active sites of the insulin receptor family.2. The recombinant expression plasmids p ET30a-1316-LBD and pET30a-4810-LBD were successfully constructed. We obtained the recombinant proteins TsIR-1316-LBD and TsIR-4810-LBD as insoluble inclusion bodies after inducing with IPTG, sizes of which were about 59 kDa and 63 kDa,respectively. Western-blotting analysis showed that TsIR-1316-LBD and TsIR-4810-LBD could be specifically recognized by the serum of swine infected with cysticercosis. Immunohistochemical results revealed that TsIR-1316 was predominantly expressed in larval tegument and adult tegument, ovaries and eggs, while its expression was lower in muscle. This result indicated that TsIR-1316 may play an important role in uptake of nutrition and glucose during the growth and development of this parasite.However, TsIR-4810 was detected only in the adult parenchymal tissues and reproductive organs,demonstrated it provide energy for reproduction.3. Each of cysticercus groups was cultured in vitro with human insulin, insulin receptor antibodies,receptor inhibitors or potentiators, respectively. The levels of glucose, glycogen, and the insulin receptor phosphorylation were detected to study whether the parasites glucose uptake and glycogen synthesis can be affected or not when the insulin receptor was blocked by its corresponding antibodies. The results showed that the insulin signaling pathway could be blocked and the insulin receptor phosphorylation was inhibited using the blocking serum, leading to significant reduce in glucose absorption and the glycogen levels. The results from the three experiments suggested that the insulin signaling pathway can play an important role in the parasite growth and development.4. The purified TsR-1316-LBD and TsR-4810-LBD that were mixed with the adjuvant Quail A were used to immune rabbits(200 μg each), resulting in a detected level of antibody as 1: 6400 after the third immunization. In each group, the levels of IL-4 and IFN-γ were significantly increased(P<0.05)after immunization. But the levels of IL-4 and INF-γ of the mixed immunization group(TsIR-1316-LBD+TsIR-4810-LBD) were slightly lower than those of the groups that were independently immunized with each antigen(TsIR-1316-LBD or TsIR-4810-LBD). Rabbits infected3000 eggs were dissected after 35 days. The result showed that the worm reduction rates were 93.9%,96% and 80% for the groups of TsIR-1316-LBD, TsIR-4810-LBD and TsIR-1316-LBD+TsIR-4810-LBD, respectively. Therefore, the above results suggested that TsIR-1316 and TsIR-4810 may represent valuable vaccine candidates against cysticercosis. |
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