| Hand-foot-mouth disease (HFMD) is a common enteroviral infectious disease, most frequently caused by Coxsackie virus A16 and Enterovirus 71 (EV71). EV71 is a primary causative agent of HFMD and has been associated with most severe cases. Infections of the central nervous systems can cause viral meningitis, encephalitis, and severe myocarditis, as well as fatal pulmonary edema. Children from 1-3 years old are particularly vulnerable due to their relative immunodeficiency. HFMD is a global infectious disease and it was first discovered in Shanghai, China, after that, several outbreaks of EV71 infection have been reported in dozens of provinces and municipalities of China such as Beijing, Shandong, Anhui province etc. In recent years, the spreading of this disease in China appears rapidly ascending.Presently, there are no specific effective antiviral drugs and vaccines available for the treatment of HFMD. So it is significant to research and produce broad spectrum anti-viral and highly reactive drugs against EV71 for ensuring health of young children as well as the development of the society. We aim to establish drug screening system against EV71 3C protease including yeast reverse two-hybrid system which was constructed originally and in vitro reaction system.A yeast cell-based assay based on the reverse two-hybrid system to monitor the proteolytic activity of 3C protease was developed. DNA fragments encoding 3C protease, selection marker gene URA3 and GAL4 transcription factor which includes DNA binding domain and transcription activation domain were amplified by PCR, and then expression vector was constructed using conventional molecular biological methods and transformed into yeast cells. Nutritional deficiency culture medium was used to select positive yeast strain and to screen drugs against 3C protease of EV71. At the same time, a-galactosidase activity was assayed in the cell level to establish the quantitative evaluation.The recombinantly expressed and purified 3C protease of EV71 and a fluorescence peptide were used to develop drug screening system in vitro based enzymatic assay according to the substrate specificity. The DNA fragment encoding 3C protease gene has been cloned and inserted into the plasmid expression vector pET21b. The expressed host strain Escherichia coli BL21 (DE3) was transformed and the positive clones were characterized. In this study, the recombinant EV71 3C protease was purified and the qualitative analysis of enzymatic activity has been tested. The above results provide solid foundations for development of anti-EV71 drugs.In this article, yeast reverse two-hybrid system was used for screening a variety of traditional Chinese drugs, and eighteen patent traditional Chinese medicines were detected to possess inhibitory activities. Then, one oral solution named 04 that possesses highest activity was studied further. It was grouped into four sections:petroleum ether, ethyl acetate, normal butanol and water fraction. The ethyl acetate and butanol fractions exhibit the inhibitory activity. Ethyl acetate fractions were directly separated by silica-gel column, subfractions from 49 to 52 show activity agaist 3C protease. The C18 reverse column was used to separate butanol fraction, and results show that 5%,10%,15% methanol elution subfractions are concentrations of active ingredients.5% methanol elution subfraction was then assayed using HPLC and one monomeric compound was isolated. NMR and LC-MS were used for structural identification of the drug molecules. This compound needs further exploration to be identified to a positive drug molecule.In summary, the research lays a solid foundation for exploring new drugs against hand-foot-mouth disease. In addition,3C proteases of Picornaviridae have certain homologous regions in sequence and conformation. Research of EV71 3C protease makes us to understand and study the 3C protease, and thus provides for scientific basis and theoretical foundation for the treatment of intestinal diseases caused by viruses such as EV71. |
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