[PCR site-directed mutagenesis of avian influenza virus hemagglutinin gene in vitro and expression in 293T cell].
The site-directed mutagenesis of HA gene was made by using PCR, and mismatches were introduced into primers. Mutagenesis was performed in a three-step PCR. The amplified fragments from the second PCR which contained the mutation site were cloned into the pcDNA3 vector, named pHAm. The sequencing analysis showed that the mutation site was correct. The amino acid sequence at the cleavage site of the HA protein was from RKKR decrease GLF to RSSR decrease GLF. The recombinant plasmid pHAm was transiently transfected into 293T cells by the calcium phosphate precipitation method. Indirect immunofluorescent assay (IFA), confirmed expression of the HA protein on the cell membrane, the mutant HA gene was a promising candidate for further studies.
Liu
Hua-Lei
HL
Key Lab of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China.
Wei
Jian-Chao
JC
Zhou
Bin
B
Cao
Rui-Bing
RB
Chen
Pu-Yan
PY
D000595:Amino Acid Sequence; D002460:Cell Line; D019084:Fluorescent Antibody Technique, Indirect; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods*; D014162:Transfection
D019267:Hemagglutinin Glycoproteins, Influenza Virus; C109963:hemagglutinin, avian influenza A virus