| Newcastle disease (ND), a highly contagious disease with world-wide occurence, can cause severe economic losses to the poultry industry. The causative agent of the disease, Newcastle disease virus (NDV), is a member of the Paramyxoviridae. Its genome is a single-strand negative-sense RNA. The hemagglutinin-neuraminidase (HN) and fusion (F) protein are major virulence factors of the virus, as they play a critical role in pathogenesis by attaching the virus to host cell and mediating viral fusion to host cell membrane. Infected cells expressing both attachment protein and F protein can fuse adjacent cells and form multinuclear cells, or syncytia. Both HN protein and F protein are necessary for cell fusion.Since both proteins are larger molecule, too big to express efficiently, and their antigenicity is dependent on certain areas called epitopes, prokaryotic and eukaryotic recombinant plasmids of the antigenic structural domains were constructed. Effects on the cell fusion by co-expression of the these recombinant plasmids together with those containing the antigenic structural domains of F proteins constructed previously in our laboratory were examined in the present study.The gene fragments HNa, HNb and HNa-L-b coding for antigenic structural domains of NDV hemagglutinin-neuraminidase were amplified from the recombinant plasmid containing full length NDV HN gene by regular PCR or overlap extension PCR with specific primers. The genes were inserted between BamHâ… and Hindâ…¢ sites of pET32c to construct recombinant plasmids pET32c-HNa, pET32c-HNb and pET32c-HNa-L-b. We found that target proteins from all recombinant plasmids were expressed in E, coli BL21. The expressed fragments HNa and HNb could be recognized by NDV positive serum by western blotting. Polyclonal antibodies were made by immunized the rabbits with the purified HNa and HNb proteins.Accordingly recombinant eukaryotic plasmids pcDNA3-HNa and pcDNA3-HNb containing HNa and HNb were constructed. Plasmids pcDNA3-HNa, pcDNA3-HNb and pcDNA3-HN, pcDNA3-F and pcDNA3-Fa(c) were transfected individually intoHeLa cells by liposome mediation. Indirect immunofluorescent assay revealed expression of the target proteins in HeLa cell monolayers using NDV positive serum and rabbit polyclonal antibodies.To evaluate the effects of coexpression on cell fusion of the FIN and F structural domains, pcDNA3-F or pcDNA3-Fa(c) were cotransfected with pcDNA3-HN, pcDNA3-FINa or pcDNA3-HNb respectively into HeLa cells. After culture for 24 hours, cell fusion was observed in the wells co-transfected with pcDNA3-Fa(c) and pcDNA3-HNa, and also those containing pcDNA3-Fa(c) and pcDNA3-HNb. Cell fusionin wells of all co-transfected combinations were detected 36 hours after transfection and syncytia visualized by indirect immunofluorescent assay. However, cell fusion could not be observed in the wells with recombinant plasmid F or Fa(c) tansfected alone. These results indicate that coexpression of fragments of HN and F protein can promote cell fusion, and the effect of Fa(c) fragment co-expressed with HNa or HNb was more remarkable. We postulate that these fragments contained the structure domains required for cell fusion and were easier to be expressed than the full length proteins such that the amount expressed were sufficient to induce cell fusion.Our research is in order to find if the fragments of HN have immunity and if fragments of HN and F co-expressed can promote cell fusion. So, this study analysed the main epitopes of HN protein and explored the effects of coexpression of FIN and F protein on cell fusion. The HN or F fragments selected were immunoreactive to NDV positive serum. Fragments of F protein coexpressed with HN protein fragments could activate cell fusion. However, combinations of different fragments had effects of varying degrees on cell fusion. These results provide some clues for further research on identification of the key amino acid residues of the two glycoproteins of NDV responsible for cell fusion. |
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