BACKGROUND: E7 is the major transforming protein of human papillomavirus (HPV) that plays important role in maintaining the proliferative state in HPV-infected cells. Furthermore, high mobility group 1 protein (HMGB1) is a highly conserved component of chromatin that can be secreted by macrophages and activated monocytes and thus functions as an infl ammation mediator. METHODS: In the current study, cloning of HMGB1 gene and also HPV16E7-HMGB1 was performed in pEG-FP-N1 eukaryotic expression vector in order to evaluate their expression in mammalian cells. For this purpose, the HEK-293T cells were transfected by pEGFP-E7, pEGFP-HMGB1 and pEGFP-E7-HMGB1 using TurboFect delivery system. The levels of protein expression were assessed by fl ow cytometry and fl uorescent microscopy at 48 hr after transfection, as well as by western blot analysis using anti-GFP polyclonal antibody. RESULTS: Our data showed a clear band of ~ 684 bp and ~ 981 bp related to HMGB1 and E7-HMGB1 genes in agarose gel, respectively. The expression of HMGB1-GFP and E7-HMGB1-GFP proteins was confi rmed for the bands of ~ 53 kDa and ~ 64 kDa in the transfected cells using western blot analysis, respectively. The linkage of HMGB1 gene to E7 could likely neutralize the negative charges of E7, thus a clear band of 64 kDa was detected instead of 76 kDa in western blot analysis. Moreover, the percentage of expression for E7-GFP, HMGB1-GFP and E7-HMGB1-GFP was 76 %, 55 %, and 52 %, in comparison with pEGFP-N1 (~82 %) as a positive control. Indeed, HMGB1 linked to HPV16 E7 gene decreased transfection effi ciency of E7 DNA in HEK-293T cells. CONCLUSION: Generally, the electrophoretic mobility of HPV16 E7 was changed due to the linkage of HMGB1 gene. Furthermore, the fusion protein could be effi ciently expressed in mammalian cells for the next use in immunotherapy (Fig. 3, Ref. 51). Text in PDF www.elis.sk. KEY WORDS: high mobility group box 1, HEK-293T, pEGFP-N1, cell transfection.
Prophylaxis of Influenza A virus infections is based on the vaccines inducing antibodies to the major viral antigens, hemagglutinin (HA) and neuraminidase (NA). Since these antigens continuously change during virus replication in various hosts, only the currently circulating strains should be used in the vaccines. Besides, monitoring of the naturally occurring changes in HA, NA, and respective genes, especially those associated with resistance to the NA inhibitors is necessary. The NA genes of 30 Iranian isolates of influenza H1N1 virus from the seasons 2005-2009 were sequenced and subjected to the sequence and phylogenetic analyses. The seasonal isolates turned out to be closely related to the corresponding vaccine strains, except for the 2007-2008 isolates, which also displayed a higher nucleotide variation. A resistance to the NA inhibitors was found in the 2008-2009 isolates only. The average nucleotide identities of the isolates with corresponding vaccine strains for the years 2005-2009 were 98.83%,
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