Direct cloning and expression of PCR amplified DNA and RNA sequences: application to the hepadnaviruses nucleocapsid proteins.
Gene amplification may benefit from the construction of primers that augments the speed at which cloning and protein expression proceeds. Such primers include EcoRI or HindIII linkers as well as an in phase initiation or termination codon. PCR was carried out directly from viral particles of human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV) without DNA purification and from RNA extracted from WHV infected liver. Amplified products were directly cloned in the pKK223-3 expression vector under the control of the tac promoter. The characterization of the recombinant clones expressing the nucleocapsid protein (C protein) was done by direct incubation of the filter with 125I-labelled anti-HBc and confirmed by radioimmunoassay and Western-blot analysis. This procedure allows easy selection of recombinant clones expressing a given protein and could be applied to many other genes.
Baginski
I
I
Inserum U 271, Lyon, France.
Chemin
I
I
Turin
F
F
Pichoud
C
C
Trépo
C
C
Hantz
O
O
D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis* / Q000235:genetics*; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics; D006518:Hepatitis Viruses / Q000235:genetics*; D008392:Marmota; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014758:Viral Core Proteins / Q000096:biosynthesis* / Q000235:genetics*
D004279:DNA, Viral; D012367:RNA, Viral; D011994:Recombinant Proteins; D014758:Viral Core Proteins