Development and characterization of a stable cell line expressing respiratory syncytial virus non-structural protein NS1 / 病毒学报

To develop a stable cell line that could express the RSV NS1, the full-length RSV NS1 gene was generated by RT-PCR amplification from respiratory syncytial virus. NS1 gene was ligated with pBABE-puro to construct the recombinant retroviral expression plasmid pBABE-NS1, which was cotransfected into 293FT packaging cells with PIK packaging plasmid by calcium phosphate co-precipitation. The supernatant of 293FT was collected to infect HEp-2 cells, the resulting cell clones stably expressing NS1 were screened by puromycin. Using QPCR, CPE staining method and indirect immunofluorescence assay, the expression of NS1 at both gene and protein levels was identified. The recombinant plasmid pBABE-NS1 was identified by EcoRI and BamHI endonuclease digestion and the sequence analysis. QPCR results showed that the NS1 gene amplification in HEp-2-NS1 cells was 8483 fold higher than that in HEp-2 cells. Although the exogenous interferon was added, all cells were destroyed after 48 hours post infection using CPE staining method, showing that HEp-2-NS1 cells remained sensitive to the VSV virus. The results of RT-PCR and indirect immunofluorescence assay showed that the NS1 gene in HEp-2 cells could not only transcribe mRNA, but also express NS1 protein steadily. We had successfully established HEp-2-NS1 cell lines with stable expression of respiratory syncytial virus non-structural protein NS1.

Preparation and characterization of specific monoclonal antibodies against hexon of HAdV 3 / 南方医科大学学报

<p><b>OBJECTIVE</b>To obtain the monoclonal antibody against hexon protein of human adenovirus.</p><p><b>METHODS</b>BALB/c mice were immunized with purified recombinant hexon protein, and the spleen cells of the mice were isolated and fused with myloma cells. Four hybridoma cell strains were screened by indirect ELISA and cultured, and the sensitivity, specificity and virus neutralizing activity were analyzed with ELISA, Western blotting and neutralizing test.</p><p><b>RESULTS</b>The mouse ascites produced by these hybridoma cells contained specific monoclonal antibodies against hexon protein of human adenovirus as identified by ELISA and Western blot, and the antibody generated by 4C6 strain showed human adenovirus type 3-neutralizing activity.</p><p><b>CONCLUSION</b>The monoclonal antibodies against hexon protein with high specificity have been successfully obtained, and these antibodies can be useful in developing assays for early diagnosis of HAdV3 infection and also in study of therapeutic drugs of the infection.</p>