Flagellin's potential as a vaccine adjuvant has been increasingly explored over the last three decades. Monomeric flagellin proteins are the only known agonists of Toll-like receptor 5 (TLR5). This interaction evokes a pro-inflammatory state that impacts upon both innate and adaptive immunity. While pathogen associated molecular patterns (PAMPs) like flagellin have been used as stand-alone adjuvants that are co-delivered with antigen， some investigators have demonstrated a distinct advantage to incorporating antigen epitopes within the structure of flagellin itself. This approach has been particularly effective in enhancing humoral immune responses. We sought to use flagellin as both scaffold and adjuvant for HIV gp41 with the aim of eliciting antibodies to the membrane proximal external region (MPER). Accordingly， we devised a straightforward step-wise approach to select flagellin-antigen fusion proteins for gene-based vaccine development. Using plasmid DNA vector-based expression in mammalian cells， we demonstrate robust expression of codon-optimized full length and hypervariable region-deleted constructs of subsp. serovar flagellin (FliC). An HIV gp41 derived sequence including the MPER (gp41) was incorporated into various positions of these constructs and the expressed fusion proteins were screened for effective secretion， TLR5 agonist activity and adequate MPER antigenicity. We show that incorporation of gp41 into a FliC-based scaffold significantly augments gp41 immunogenicity in a TLR5 dependent manner and elicits modest MPER-specific humoral responses in a mouse model.