Dual expression of the HA protein of H5N2 avian influenza virus in a baculovirus system.
Baculovirus/insect cell system is used widely for recombinant protein production. The hemagglutinin (HA) gene of H5N2 avian influenza virus (AIV) 1209 strain and the enhanced green fluorescent protein (EGFP) gene were cloned into pFastBac DUAL vector that has two promoters and cloning sites, allowing simultaneous expression of these two genes. The HA protein of AIV was fused with a hexahistidine (His6) tag for purification. The coexpression of EGFP allowed identification of the recombinant baculoviruses in Sf-9 insect cells, eliminating cumbersome and time-consuming assays. A recombinant baculovirus, Bac-HA, was generated by transfecting pBac-HA to bacmid inside DH10B(AC)Escherichia coli by site-specific transposition, followed by transfection into the Sf-9 cells. Fluorescence in the insect cells was observed from 3 days post-infection. The expressed HA protein was confirmed by Western blot using an anti-HA monoclonal antibody. Also, different detergents and incubation times on ice were tested. The two-stage extraction with Triton X-100 or Tween 20 and incubation on ice for 2h exhibited high efficiency. Since purification of HA with ConA resin resulted in low protein recovery, lentil lectin affinity column was used and was useful for HA purification.
Hu
Yu C
YC
Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan.
Luo
Yu L
YL
Ji
Wen T
WT
Chulu
Julius L C
JL
Chang
Poa C
PC
Shieh
Happy
H
Wang
Chi Y
CY
Liu
Hung J
HJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003208:Concanavalin A; D005453:Fluorescence; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D053125:Influenza A Virus, H5N2 Subtype / Q000235:genetics*; D037121:Plant Lectins; D011993:Recombinant Fusion Proteins / Q000235:genetics; D018411:Spodoptera
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D037121:Plant Lectins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; C109963:hemagglutinin, avian influenza A virus; C033619:lentil lectin; D003208:Concanavalin A; D049452:Green Fluorescent Proteins