| Cysticercus cellulosae is the larva of the Taenia solium, it is the pathogen of porcine cysticercosis. T. solium is associated with two distinct infected states in humans: intestinal infection by adult tapeworms (taeniosis) and infection due to the presence of cysticerci (metacestodes) in muscles or organs (cysticercosis). Cysticercus cellulosae can survive in human tissue for several years. Particulary, when the presence of Cysticercus cellulosae in the central nervous system, it could cause neurocysticercosis. Nerve cysticercosis is one of the most common central nervous system parasitosis. Therefore, the disease is a serious and wide distribution foodborne parasitic zoonosis.In recent years, as deeply study on molecular biology and physiological and biochemical aspects of the cysteine protease, people gradually learned that cysteine protease has a great significance not only in the mechanism of apoptosis but also in the oncology . In the field of medical parasitology, it also refers to the various important physiological activities of parasitic growth and development and life activities. Cysteine protease of parasite plays an important role in parasites into the sac, tissue invasion, nutrient uptake, immune evasion, and pathogenesis, besides functions on catalysis and protein processing. In addition, it is a good species-specific enzyme, which can be used as diagnostic antigen and candidate vaccine in parasitic diseases. Currently, many researches have made the enzyme as a target in drug therapy. These studies help to reveal the interaction mechanism between parasite and host , and make a new way on diagnosis and prevention of the parasitic disease. In this study, recombinant plasmid of Cysticercus cellulosae cysteine protease pGEX-4T-TsCL-1 was expressed in E.coli. Some biological characteristics of the recombinant protein such as expression conditions, purification, activities and immunogenicity have been studied. These made a basis on study of the relationship between parasite and host and immunologic diagnosis and treatment of the parasitic disease.1. The recombinant plasmid of Cysticercus cellulosae cysteine protease pGEX-4T-TsCL-1 were transformed into BL21 (DE3) and expressed in E.coli. The results showed that: the Cysticercus cellulosae cysteine protease prokaryotic expression vector was successfully constructed, and successfully expressed in E. coli. The recombinant protein presented in both soluble form and inclusion form. So the molecular size of the recombinant protein is about 61Ku. It is consistent with the expected results.2. The induction conditions were optimized. The SDS-PAGE analysis showed that the recombinant protein had a higher expression in 28℃, by being induced by 0.1 mmol / L of IPTG after 10h. The analysis and prediction of the structure and function were proceeded by Expert Protein Analysis(ExPASY) for the study on relationship of TsCL-1 protein structure and function. The result showed that it has a conservatism sequence, and is a partial basic protein, which is mostly composed of the non-polar hydrophobic amino acids in amino acid composition. PI value is 8.17. In the secondary structure of protein, the amino acid whichα-helix comprised is 30.0 percent, withβ-sheet is 24.5 percent, random coil 19.5 percent and corner 31.0 percent.3. The soluble protein of TsCL-1 expression was purified with GST agarose affinity chromatography. By SDS-PAGE and TLC-scanning analysis, protein purity is more than 90 percent. The result of gelatin protein electrophoresis showed that the purified protein caused a hydrolysis of the gelatin, which explained the protein has hydrolytic activity. In addition, for the cysteine protease activity was inhibited in the inhibitor E-64-treated samples, the protein lost of hydrolysis activity, there was no white bands appeared in the migration position. So it reached to the conclusion that proteolytic activity of the purified soluble TsCL-1 recombinant protein exists, and its hydrolytic activity can be inhibited by cysteine protease inhibitor E-64.4. 5-week-old female Kunming mice were immunized with the protein expressed, and then the sera of mice were separated and determined by ELISA with antibody titers up to 1:12 800; antibody specificity was determined by Western blot. Western blot analysis showed that there was a specific protein band in 61ku, while no band with negative serum. This proved the antibody can bind with TsCL-1 recombinant protein, indicating a good antigenicity of TsCL-1 recombinant protein. It provides a simple, quick and inexpensive way in diagnosis and detection of porcine cysticercosis.
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