Figure 1. Construction of expression vector for constitutively H5N1 HA secretion in Flp-In based mammalian expression system.

(A) To enhance HA protein expression from the FRT-CMV vector (Invitrogen), an RNA splicing sequence from HTLV-I gene and a cassette containing NotI and PacI cloning sites was introduced after CMV promoter using Topo cloning system. (B) Schematic of HA1 (1-330) & HA0 (1-500) V5-His₆ tagged fusion proteins expressed in Flp-In system. The H5N1/Vietnam HA sequence coding for either HA1 (1-330) or HA0 (1-500) was inserted into the vector as a NotI-PacI insert. (C–D) Expression and purification of H5N1 A/Vietnam/1203/2004 hemagglutinin proteins from 293 Flp-In stable cell lines. (C) Expression of HA proteins secreted into supernatant from 293 Flp-In cells [S], and in the cell lysates [C] was analyzed by western blot using anti-V5 MAb. (D) HA protein level from supernatant of 293 Flp-In cell culture in serum free medium, collected at different time points: 24, 48, 72 and 96 hours post culture splitting and was analyzed in SDS PAGE followed by western blot with anti-HA1 polyclonal sera.