Cloning and expression of Papaya ring spot virus (PRSV) coat protein gene in bacteria.

摘要

The papaya ring-spot virus (PRSV) coat protein gene was obtained by RT-PCR amplification of viral RNA isolated directly from virus-infected papaya leaf. The PCR amplified fragment was then ligated into cloning vector for gene verification and sequencing. The DNA sequencing result showed that the cloned PRSV coat protein gene was 927bp in size. The PRSV nucleotide and predicted amino acid sequence showing more than 95% similarity to those published PRSV coat protein gene sequences. Based on the blast and alignment result, the isolated gene has been verified as PRSV-strain P coat protein. The coat protein gene was subsequently sub-cloned into bacterial expression vector pRSET, to form pRSET:PRSVCP and expressed in Escherichia coli BL21(DE3) strain. When the bacterial expressed PRSV coat protein was analyzed by Western Blot analysis, PRSV antisera has been used as antibody probe to confirm the bacterial expressed PRSV coat protein. The result showed that the size of the expressed coat protein was similar to the predicted size of 35 kD based on the DNA sequences. Western blot analysis also displayed positive binding activity to anti-PRSV antiserum, showing that the bacterial expressed PRSV coat protein still maintained the native epitope as in the virus.