| Tick-borne encephalitis virus (TBEV) is a member of the genus Flavivirus of the family Flaviviridae. There are three main genetic lineages of TBEV, the European, Siberian, and Far Eastern subtypes, formerly known as the Central European, West Siberian, and Russian spring-summer encephalitis viruses, respectively. The variation at the amino acid level of different virus strains within a genetic lineage is 2.2% and between different subtypes not more than 5.6%. Tick-borne encephalitis is a zoonotic arbovirus infectiou disease. The European subtype is transmitted by Ixodes ricinus, and the Siberian and Far Eastern subtypes are transmitted by Ixodes persulcatus ticks, of which the latter subtype may cause a more severe disease.The disease is primarily endemic in Europe, Siberia, and the Far East of Russia. In Europe, the highest incidence of the disease is observed in Austria, Czech Republic, Slovakia, Slovenia, and Hungary. Presently, a total of 10,000-12,000 hospitalized cases are registered annually, including 3000 in Europe. In China, a natural focus of TBE is present in the virgin forest area of northeast and southeast. Another focus is the sub tropical region of western Yunnan near the Burmese border. TBEV was isolated from I.ovatus captured at 2 700m above sea level and the strains are closely related to Russian spring summer encephalitis virus. With the increase of the international communication, the striken areas ever since, the study on tick-borne encephalitis virus's molecular pathogenisis and the development of effective vaccines bore impending importance.The possible introduction of these viruses by natural or artificial means into areas where these viruses are nonendemic, as well as the present extensive regions of endemicity, makes the diagnosis of infection by these viruses a major public health objective. Currently, the neutralizing test (NT) is the most specific procedure for diagnosis of flavivirus infection. However, NT takes several days and requires a containment laboratory for virus manipulation. Commercially available TBEvirus-specific ELISA tests use inactivated whole virus as coating antigens and their purification and inactivation can be performed only in strict biosafety laboratories. Furthermore, this ELISA method has been reported to show cross-reactivity to other flaviviruses. Therefore, a simple, safe, and rapid diagnostic method to distinguish TBE virus infection from other flaviviruses serologically is required.The TBEV M and E proteins are translocated to the endoplasmic reticulum and acquire their mature conformations in the secretory pathway, where prM is cleaved into its mature form, M. These proteins have also been expressed in recombinant expression systems in mammalian cells, where they have been shown to form secreted virus-like particles. The structure of the particles has been characterized. They are about 30 nm in diameter, compared to about 50 nm for virions. Virus-like particles are apparently also formed normally during flavivirus infection.Baculoviruses have been exploited as expression vectors since 1983. A highly expressed but redundant virus very late gene is replaced by a gene of interest to produce a recombinant virus that expresses a recombinant protein as part of the virus life cycle. Baculoviruses are robust, safe and have a good record of high-level expression, particularly of complex proteins. The insect cell background carries out most of the post-translational modifications found in mammalian cells and a wide variety of vectors and cloning strategies are available. These qualities have suggested recombinant baculoviruses as a favoured technology for the delivery of high throughput protein expression solutions aimed at whole genome coverage, particularly for the highly processed proteins whose expression in Escherichia coli is challenging.In this paper, the recombinant prM-E protein of Tick-borne encephalitis virus was expressed in insect cell Sf9 by RT-PCR amplification of prM-E genes, construction of donor plasmid of Bac-to-Bac baculovirus expression system, homologous recombina- tion of donor plasmid with bacmid DNA at the site of Tn7 and transfection of insect cell Sf9. Recombinant subviral particles consisting of prM-E , about 30nm in diameter, were observed by electronic microscope in the supernatant of infected cells, which indicated that infected cells released virus-like particles(VLPs) into the culture medium. The results of Western - blot and indirect immunofluorescence assay (IFA) showed that the recombinant proteins retained antigenic and conformational structures similar to those of native virus proteins.
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