In vitro expression of HPV16 E7 linked to HMGB1 immunoadjuvant in mammalian cells

Abstract

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.

SLC26A4 Mutation Promotes Cell Apoptosis by Inducing Pendrin Transfer, Reducing Cl- Transport, and Inhibiting PI3K/Akt/mTOR Pathway

“…In this study, DNA sequencing analysis suggests that we successfully obtained recombinant plasmids carrying wild-type SLC26A4, mutant 1 (c.85G>A), mutant 2 (c.2006A>T), and mutant 3 (c.853G>A). pEGFP-N1, a eukaryotic expression vector encoding GFP, is commonly used to detect the expression of inserted genes via examination of GFP by fluorescence microscopy and Western blot [ 16 ]. After transfection of HEK-293T cells with these plasmids, we found that HEK-293T cells transfected not only with empty vector but also with wild-type SLC26A4 and mutants exhibited obvious GFP fluorescence and upregulated GFP expression compared to control cells, indicating successful transfection of these plasmids.…”