[Construction of bicistronic eukaryotic expression vector containing HA and M2 genes derived from high pathogenic avian influenza virus (HPAI) H5N1 (Anhui strain)and its efficient expression in mammalian cells].
HA and M2 genes derived from human highly pathogenic avian influenza H5N1 virus (A/Anhui/ 1/2005) isolated from China, were amplified and cloned into the DNA vaccine expression vector pVRC. In order to improve the expression of hemagglutinin, the human codon usage preference was made and the whole length of HA gene of H5NI (A/Anhui/1/2005) influenza virus was synthesized,named HA/YH/K, and inserted to pVRC vector, the expression of HA/YH/K protein in eukaryotic cells was significantly improved according to internal control of actin protein. Furthermore, the M2 and HA/YH/K genes were cloned into bicistronic eukaryotic expressing vector pIRES to yield the recombinant plasmid pIRES-HA/ YH/K-M2/YS/K, which could expressed HA and M2 protein simultaneously by transfection of one plasmid. Western blot and IFA showed that the recombinant pIRES-HA/YH/K-M2/YS/K plasmid was successfully expressed in several mammalian cells (Hela, MDCK and 293FT). The above results may help to identify the function and pathogenic mechanism of HA, M2 genes derived from HPAI H5N1 (Anhui strain) and pave a way for the development of novel bivalent vaccines against human highly pathogenic avian influenza virus and for preparedness for influenza pandemic.
Liu
Yuan
Y
Biotech center for viral disease emergency, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China. ly1781@163.com
Zhang
Ke
K
Tan
Wen-Jie
WJ
Wang
Hui-Juan
HJ
Shu
Yue-Long
YL
Hu
Gui-Xue
GX
Ruan
Li
L
D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007251:Influenza, Human / Q000821:virology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000378:metabolism; D000086082:Viroporin Proteins
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins; C109963:hemagglutinin, avian influenza A virus