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Enterovirus 71 (EV71) was first isolated and identified in 1969 in California USA. It is the most frequently occurring pathogen in hand-foot-and-mouth disease (HFMD) and symptoms include neurological dysfunction, such as aseptic meningitis and poliomyelitis-like paralysis [10]. It was reported that EV71 was responsible for the HFMD outbreaks in Taiwan, Japan and Singapore. Moreover, large outbreaks have occurred several times in mainland China since 2008. There have been more than 1.5 million HFMD cases, and 57.5% of the deaths were caused by EV 71. On average, EV71 has been the cause of about 1000 deaths every year. Children younger than 5 years old have been found to be particularly susceptible to EV71 and the associated neurological disease and fatalities [15]. In conclusion, EV71 infection has been regarded as an important public health problem which has the potential to cause serious clinical illness and death in young children [6, 19].
Enterovirus 71, classified as a species of genus Enterovirus in the Picornaviridae family, is a small, nonenveloped, icosahedral RNA virus. It has a single-stranded RNA genome of approximately 7500 nucleotides [16] which is a single open reading frame (ORF) flanked by a 5'-untranslated region (5'UTR) and a 3'-untrans-lated region (3' UTR). The ORF is translated into a large polyprotein and it can be cleaved into P1, P2, and P3 regions. The P1 region encodes four structural proteins VP1, VP2, VP3 and VP4. The P2 and P3 regions encode nonstructural proteins, such as proteases 2A, 2B, and 3CD, which are responsible for virus replication and virulence. These proteins spontaneously assemble and form the crystalline virus-like particles [8].
The development of an EV71 vaccine is urgent and indispensable for the prevention and control of HFMD. Previous research has reported various types of EV71 vaccines, including a VP1 peptide vaccine, a DNA vaccine, and attenuated and inactivated vaccines [13, 17, 20]. Based on the enterovirus vaccine development, an attenuated and inactivated vaccine appears more feasible. However, a protein engineering approach may be more applicable. In this study, the EV71-P1 gene was processed and cloned into the eukaryotic expression vector pPIC9k and the resulting vector could manufacture and secret EV71-P1 protein. The soluble EV71 P1 protein was purified by column chromatography and was then identified by Western blot analysis. The results of immunogenicity analysis showed that the EV71 P1 protein had excellent immunogenicity and could stimulate the production of EV71-VP1 IgG antibody in injected rabbits.
Expression and Purification of Enterovirus Type 71 Polyprotein P1 using Pichia pastorissystem *
- Received Date: 24 April 2012
- Accepted Date: 03 July 2012
Abstract: Enterovirus type 71(EV71) causes severe hand-foot-and-mouth disease (HFMD) resulting in hundreds of deaths of children every year; However, currently, there is no effective treatment for EV71. In this study, the EV71 poly-protein (EV71-P1 protein) gene was processed and cloned into the eukaryotic expression vector pPIC9k and then expressed in Pichia pastoris strain GS115. The EV71 P1 protein with a molecular weight of 100 kD was produced and secreted into the medium. The soluble EV71 P1 protein was purified by column chromatography with a recovery efficiency of 70%. The result of the immunological analysis showed that the EV71 P1 protein had excellent immunogenicity and could stimulate the production of EV71-VP1 IgG antibody in injected rabbits. We suggest that EV71-P1 protein is an ideal candidate for an EV71 vaccine to prevent EV71 infection.