[Cloning and expression of the six coding genes of sendai virus BB1 strain].
Six genes for nucleoprotein, phosphoprotein, matrix protein, hemagglutinin neuramindase protein, fusion protein and large protein were obtained by reverse transcription and PCR methods based on our previous work of sequencing full length genome of sendai virus BB1 strain (DQ219803 in GenBank). Sequencing showed the six genes were completely identical to that we reported. In order to supply the function necessary for rescuing and packaging of sendai virus vector in trans, the N, P, M, F, HN and L genes were separately cloned into an adenoviral shuttle expression vector pDC316 resulting in six recombinant adenoviral plasimds. Six replicating defective recombinant adenoviruses Ad5-N, Ad5-P, Ad5-M, Ad5-F, Ad5-HN and Ad5-L were obtained by separately cotransfection of pDC316 carrying N, P, M, F, HN and L genes with the adenoviral genomic plasmid pBHGloxdeltaE1, 3Cre into HEK293cells. Restrictive enzymatic results indicated that the six recombinant plasmids were correctly constructed. PCR results showed the recombinant adenoviruses contained the respective SeV genes . Western blotting as well as immunofluorescence assay indicated the expression of the corresponding proteins of sendai virus. These work laid the basis for the construction of the full length genome plasmid of sendai virus BB1 strain and the setup of SeV virus vector system based on SeV BB1 strain.
Zhang
Hai-feng
HF
National Key Laboratory of Molecular Virology and Genetic Engineering, Institute of Viral Disease Control and Prevention, China CDC, Beijing, China, 100052. zhf829@163.com
Yang
Yu
Y
Dong
Xiao-yan
XY
Wu
Xiao-bing
XB
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors / Q000235:genetics; D015707:HN Protein / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008253:Macaca mulatta; D009698:Nucleoproteins / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D054658:Ribosome Subunits, Large / Q000235:genetics / Q000378:metabolism; D029082:Sendai virus / Q000235:genetics* / Q000378:metabolism*; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
D015707:HN Protein; D009698:Nucleoproteins; D010750:Phosphoproteins; D014760:Viral Fusion Proteins; D014763:Viral Matrix Proteins; D014764:Viral Proteins