Expression of H5N1 influenza virus hemagglutinin protein fused with protein transduction domain in an alphavirus replicon system.
Alphavirus replicons, in which structural protein genes are replaced by heterologous genes, express high levels of the heterologous proteins. On the basis of the potencies of replicons to self-replicate and express foreign proteins and the remarkable intercellular transport property of VP22, a novel alphavirus Semliki Forest virus (SFV) replicon system of VP22 fused with a model antigen, hemagglutinin (HA), of the human-avian H5N1 influenza virus, was explored in this study. Further, replicon particles expressing HA, VP22, and enhanced green fluorescent protein (EGFP) individually were used as controls. By flow cytometry based on the analysis of transfection efficiency, SFV-EGFP replicon particle titer was 1.13 x 10(7)transducing units (TU)/ml. The titers of SFV-HA, SFV-VP22 and SFV-VP22-HA replicon particles, which were titrated by using SFV-EGFP replicon particles, were 1.42 x 10(7), 3.23 x 10(7), and 1.01 x 10(7)TU/ml, respectively. HA and VP22-HA expression was observed in SFV-HA- and SFV-VP22-HA-transfected BHK-21 cells, respectively. Immunofluorescence staining revealed that the fluorescence intensity in the SFV-VP22-HA-transfected BHK-21 cells was more than that in the SFV-HA-transfected BHK-21 cells. Both SFV-VP22-HA and SFV-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza. VP22-HA fusion protein with similar trafficking properties may also enhance vaccine potency.
Yang
Shi-gui
SG
State Key Laboratory for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China.
Wo
Jian-er
JE
Li
Min-wei
MW
Mi
Fen-fang
FF
Yu
Cheng-bo
CB
Lv
Guo-liang
GL
Cao
Hong-Cui
HC
Lu
Hai-feng
HF
Wang
Bao-hong
BH
Zhu
Hanping
H
Li
Lan-Juan
LJ
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002460:Cell Line; D006224:Cricetinae; D049452:Green Fluorescent Proteins / Q000378:metabolism; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000378:metabolism*; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012093:Replicon / Q000235:genetics / Q000502:physiology*; D012672:Semliki forest virus / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins