Cloning and Expression of the fusion protein gene of peste des petits ruminants virus (PPRV) in yeast.

Peste des petits ruminants virus (PPRV) causes high mortality in sheep and goats, leading to serious socio-economic problems (Perry et al. 2001), The PPRV infections are widespread in Sub-Saharan Africa, Middle-East, Southern Asia, Iran and the Indian sub-continent (Diallo, et al. 2007). The disease is characterized by fever, oculonasal discharges, necrotizing and erosive stomatitis, enteritis and pneumonia (Balamurugan et al. 2006). The PPRV is classified in the Morbillivirus genus of the Paramyxoviridae family (Barrett, 2001) and is antigenically closely related to rinderpest virus (RPV) (Gibbs et al. 1979). The genome of PPRV consists of a single-stranded, negative-sense RNA of 15,948 kb, encoding eight proteins in the order of 3´ N-P/C/V-M-F-H-L 5´ (Bailey et al. 2005). For the control of PPR, vaccines have been developed or are on the development, following the same strategy as for the other Morbilliviruses. The first vaccine was an attenuated cell culture rinderpest vaccine as a heterologous vaccine (Taylor et al. 1979). Vaccinated animals despite of resistance to PPRV challenge had not produced neutralizing antibodies against PPRV. This indicates that probably some replication of PPR challenge virus in the vaccinated animals and its transmission to in-contact animals occur (Taylor et al 1990). In 1989, a homologous vaccine was prepared by attenuation of PPRV strain Nigeria 75/1 through serial passages on Vero cells (Diallo et al. 1989). This vaccine could prevent the transmission of challenge virus to in-contact animals and is presently permitted for immunization of sheep and goats against PPRV infections (Diallo, et al. 2007). The thermo sensibility of PPRV vaccine, some reports of transient immunosuppression induced by the virus (Rajak et al. 2005), tendency for production of multivalent vaccines ( Hosamani et al. 2006 ) and a need for implementation of combined programs of vaccination and serosurveillance in endemic areas, have encouraged continuing research on the development of sub-unit vaccines. As the candidates for subunit vaccine preparation, paramyxoviruses contain two immunodominant integral membrane glycoproteins, namely, hemagglutinin (H) or hemagglutinin-neuraminidase (HN) and a fusion protein (F).The H/HN glycoprotein is the virus attachment protein, while the F protein is believed to disrupt the target cell membrane leading to virus-cell and cell-cell fusions. These surface glycoproteins, especially fusion proteins of morbilliviruses are highly immunogenic and confer protective immunity. To contribute in developing a suitable subunit vaccine against PPRV infection, the present work was undertaken for the first time to clone the full length F gene coding sequence of PPRV in pPICZáA, as a secretory and pPICZB as an unsecretory expression vector of yeast P. pastoris yeast (Invitrogen). In this research according to the kit protocol, F gene of PPRV Nigeria 75/1 strain (1665 bp) was amplified using RT-PCR and purified then cloned into pPICZáA a and pPICZB vectors of P. pastoris. Insertion was proved by production of a 218 bp segment in Nested PCR, Isolation of gene from construct by restriction enzyme (XbaÉ) and sequencing. Finally both constructs were linearized by PmeI enzyme and expressed in the yeast by electroporation. Expressed proteins identified by SDS-PAGE and western blotting.