| Respiratory syncytial virus (RSV) is a major viral pathogen of the respiratory tract of infants and the elderly all over the world. It also can be devastating in immunosuppressed populations. RSV is the type species of the genus Pneumovirus. Along with other members of the family Paramyxoviridae, RSV is an enveloped virus with a negative sense, single-stranded RNA genome. The viruses are 150-300nm in diameter with a helical nucleocapsid. The RSV virion enters its target cell by fusion of the RSV envelope with the cell membrane and release of the viral genome into the cell's cytoplasm where translation will occur. The nucleopretein (N), large (L) and phosphoproteins (P) together with the RNA genome form the nucleoprotein core. These together with the matrix (M), fusion (F), and glycoprotein (G) are classified as structural proteins. The nonstructural proteins include NS1 and NS2, small hydrophobic (SH) and M2 (formerly 22-kDa). In children younger than age 3, RSV can cause a lower respiratory tract illness like bronchiolitis or pneumonia and in more severe cases can lead to respiratory failure. Current treatments offer only a limited benefit for this serious disease. The attenuated vaccines tested so far were not safe or efficient enough for any clinical applications. Therefore, the development of novel therapies against infection by respiratory syncytial virus (RSV) is a global priority.Our approach has been to identify novel therapeutic targets through characterising the molecular mechanism by which RSV infects on host cells. Therefore, we initiated a research for novel drug targets by investigating the characteristics of some key genes involved in infection of RSV on host cells. We performed differential display on the mRNA from normal SPC-A1 cells and SPC-A1 cells infected with RSV. As a result, we have obtained some expressed sequence tags (ESTs) highly or weakly expressed during infection. Among all the ESTs, g10-1, upregulated of its expression during RSV infection, has been identified to be a novel gene fragment by bioinformatics methods.Finally, 761 bp contig products were obtained by contig, named as zlgl 0(gi: 13376851). The open reading frame of zlgl 0 gene identified by RT-PCR and Northern Blot encodes a putative protein containing 54 amino acid residues. The sequence analysis suggests that ZLG10 should be localized to nucleus.Then we expressed EGFP fusion proteins of zlgl 0 in Hela cells by transient transfection to investigate this possibility.To further confirm its use as a potential candidate for diagnosis and vaccine studies, the intact region encoding ZLG10 was cloned into a prokaryotic expression vector pGEX-4T-l to express this gene as a GST- fusion protein. High level of expression could be achieved only with 0.4 mM IPTG and 4 hours of induction at 37 ° C. 12% SDS-PAGE of the cell lysates revealed the expression of a novel protein of approximated to molecular weight of 5 kDa plus the 26kDa GST protein, which was in agreement with the molecular weight 31 kDa calculated based on its deduced amino acid sequence. Furthermore, the high content of this protein in the bacterial cells did not result in protein aggregation into insoluble inclusion bodies or any detectable lysis of the producing cells. Hence, it could be purified in its native, non-denatured form. The purified recombinant proteins were found to be immunogenic in rabbits and induced the production of polyclonal antibodies. GST-Sepharose 4B affinity chromatography column was used to purificated the polyclonal antibodies to ensure the specificity. To examine whether the antibodies could have an effect on RSV infectivity we employed anti-GST-ZLGlO polyclonal antiserum to inhibit the infection and examined whether the antiserum has a suppressive effect on RSV infection. As a result, we found that anti-ZLGlO sera have shown inhibitory activity by using neutral red uptake assay and quantitative measurement. These results indicate that the inhibitory effect of the anti-ZLGlO antibodies may have potential in treating RSV infection.The data suggest that one of the effective ways of treating RSV infection could be to inhibit some high-expressed cell factors during the infection. Moreover, the identification and characterization of ZLG10 are essential for the understanding of the pathogenic mechanisms of RSV and the immune response against RSV of the cells. In addition, theavailability of the purified proteins helps us to complement the existing panel of antigens for diagnosis and/or sub-unit vaccines. Immunological studies of the purified proteins can also pave the way for exploring the possible function of these novel proteins obtained by difference expression. In conclusion, our present results indicate that it may be possible to develop a differential expression genes-based specific therapy against viruses in the future.
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