| Summary: | 碩士 === 國立成功大學 === 分子醫學研究所 === 93 === Enterovirus 71 (EV71) belongs to Picornaviridae family, its genome is a single-stranded, positive-polarity RNA, approximately 7.5 kb in size. The RNA genome was surrounded by a nonenveloped capsid, which consisted of VP1 to VP4. Infection of EV71 can cause various clinical manifestations, including: hand-foot-and-mouth disease, herpangina, aseptic meningitis, brainstem encephalitis, acute flaccid paralysis, cerebellar ataxia, and pulmonary edema. EV71 caused an outbreak in Taiwan in 1998, nearly 130,000 children were infected, 405 were combined with severe neurological complications, 78 died.
Because EV71 infection can cause diverse symptoms of CNS, the specific aim of this study is to explore the role of capsid protein (VP1 and VP2) in EV71 infections. At first, baculovirus expression system was used. Recombinant baculovirus which carried the VP1 or VP2 gene infected the Spodoptera frugiperda (Sf-9) cells, then VP1 or VP2 protein was purified via the nickel affinity chromatography. The results showed that VP1 protein could not be expressed in high level, and VP2 protein was expressed in the form of inclusion body. To obtain adequate target proteins, Escherichia coli (E. coli) expression system was used instead. In this system, VP1 or VP2 gene was cloned into pET21b vector, and three different host strains, BL21(DE3)pLysS, BL21-CodonPlus(DE3)-RP, BL21-CodonPlus(DE3)-RIL were analyzed for the VP1 or VP2 expression. The highest expression level was obtained in BL21-CodonPlus(DE3)-RIL strain. The expressed proteins were purified by nickel affinity chromatography and then analyzed their biological properties. In neurotoxicity assay, two neuroblastoma cell lines, SK-N-SH and SH-SY5Y, were pretreated with different doses (1 µM、3 µM、5 µM) of VP1 or VP2 proteins. The results showed that VP1 or VP2 protein can not cause the death of neuroblastoma cell lines. In chemotaxis assay, VP1 can promote chemotaxis behavior of U937 cells in a dose-dependent manner no matter whether U937 cells were pretreated with VP1 or not. On the other hand, VP2 can also promote the chemotaxis behavior, however, dose-dependent manner was not observed. In invasion assay, neither VP1 nor VP2 protein can induce the invasive behavior of peripheral blood monocytes. In conclusion, the capsid proteins (VP1 and VP2) of EV71 did not show neurotoxicity on neuroblastoma cell lines, but they could induce the chemotaxis behavior and inhibit invasive behavior of monocytes.
|
|---|
