Construction Of Recombinant Human 5 Adenovirus Expressing Rabies Virus Phosphoprotein And Their Preliminary Biological Characterizations

Rabies virus(RABV), a member of Lyssavirus genus of the family Rhabdoviridae in the order mononegaviral, is the major etiological agent of rabies. The majority of mammals including human can be infected by RABV. The mortality is almost up to 100% once clinical symptoms of rabies have occured. With the development of molecular biological techniques, the intensive research on RABV pathogenesis suggested that the structural proteins of RABV play a significant role in the process of viral RNA replication. A number of researches showed that phosphoprotein(P) of RABV establishes complex networks of interactions with host cell components, which have revealed much about the role of P in the interaction host-pathogen in virus-infected cells. Recent researches found that P protein can prevent the activation of interferon regulatory factor 3, thereby preventing the expression of interferon-α/β, and thus lead to escape the host innate immunity. In order to avoid the effect of other structural proteins of RABV on P protein function, we screened the eukaryotic expression vectors to express P protein. Adenoviral vector, as a common eukaryotic expression vector, presents advantages, including moderate genome size, large exogenous gene loading capacity, efficient expression of exogenous gene, generation of high titer virus easy. The biological functions of recombinant foreign protein is close to that of the natural protein, and is therefore a powerful tool to decipher the roles of various protein.Based on the considerations above, In this study, we chose a replicationdefective human adenovirus type 5 expression system to express P proteins from a virulent and an attenuated strain. These recombinant viruses will be used for verify the different function of P protein in innate immunity. The P gene were obtained from RABV BD06 and SRV9 strain and cloned into shuttle plasmid pacAd5 CMV K-NpA respectively. These two plamids and the backbone plasmid pacAd5 9.2-100 were digested by PacI, and cotransfected into HEK293 AD cells for intracellular homologous recombination. Recombinant adenoviruses, rAd5-BD06-P and rAd5-SRV9-P, were obtained. The P protein genes were sucessfully intergrated into adenovial genome, which was confirmed by direct PCR. Moreover, Typical adenoviral particle was observed by electron microscope.The transcription of the P genes were checked by RT-PCR using a pair of specific primers in recombinant viruses-infected HEK293 AD cells. The expressions of P proteins in supportive or non-supportive cells were observed by DFA using FITC-conjugated antibody(1C9) against RABV P. The expressions of P proteins in supportive cells were further identified by Western-blot. The titers of recombinant viruses are at least 107.5TCID50/ ml after continuous passages. Virus growth were measured indicating that the virus propagations were not affected by exogenous gene expression.To evaluate the expression feature of P gene in mice, Mice(n=5) were inoculated with recombinant adenoviruses respectively. Sera were collected from mice preinoculation and 14 d post-inoculation respectively, and diluted. The antibodies against P in the sera were detected through indirect immunofluorescence assay(IFA) using fixed RABV-infected cells as the antigens, and the 1C9 as control. Results showed that antibodies against P presented in the sera and efficiently interacted with RABV. To summarized, P gene in adenovial vector could express effectively in mice.In summary, two recombinant replication-defective adenoviruses expressing BD06 P and SRV9 P was successfully constructed. Both recombinants could mediated high levels of P protein expressions in supportive and non-supportive cell lines. Also, the recombinant viruses could effectively deliver the P proteins into mice, and elicit production of antibodies against RABV P. These data indicate that excellent antigenic reactivity and immunogenicity of the expressed P proteins in the cells and mice, which laid foundations for the additional researches on host-virus interaction in vitro and in vivo.