| 主权项 |
1. A method of producing influenza virus like particles (VLPs) in a plant, a portion thereof, or a plant cell, said method comprising:
a) introducing by agroinfiltration into the plant, the portion thereof, or the plant cell a nucleic acid comprising a nucleotide sequence having from 90% to 100% identity to SEQ ID NO: 17, the nucleotide sequence encoding an influenza virus hemagglutinin (HA) comprising a HA1 domain, wherein said HA1 domain is modified to be free of N-linked glycosylation sites at positions 154 and/or 156; at positions 165 and/or 167; and at positions 286 and/or 288, wherein
at least one of positions 154, 165, or 286 are modified to be an amino acid other than asparagine, orat least one of positions 156, 167, or 288 are modified to be an alanine or an amino acid other than serine, or threonine, wherein the numbering of said positions are compared to SEQ ID NO: 34 wherein the nucleotide sequence is operatively linked to a nucleic acid sequence encoding a signal peptide and a regulatory region active in the plant, the portion thereof, or the plant cell; and wherein the HA has hemagglutinin activity as determined by using a hemagglutination assay; b) incubating the plant, the portion thereof, or the plant cell under conditions that permit expression of the nucleic acid, thereby producing said VLPs, wherein the nucleic acid is transiently expressed in the plant, the portion thereof, or the plant cell; c) harvesting the plant, the portion thereof, or the plant cell; and d) purifying the VLPs, wherein the VLPs are characterized as not passing through a 100 kDa membrane. |
