Cloning and expression of small cDNA fragment encoding strong antiviral peptide from Celosia cristata in Escherichia coli.
A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length. The cDNA fragment was then cloned in a bacterial expression vector and expressed in Escherichia coli as a ~57 kD fused protein, and its presence was further confirmed by Western blot analysis. The recombinant protein was purified by affinity chromatography. The purified product showed strong antiviral activity towards tobacco mosaic virus on host plant leaves, Nicotiana glutinosa, indicating the presence of a putative antiviral determinant in the isolated cDNA product. It is speculated that antiviral site is at, or is separate but very close to, the ribosome-inactivating site. We nominate this short cDNA fragment reported here as a good candidate to investigate further the location of the antiviral determinants. The isolated cDNA sequence was submitted to EMBL databases under accession number of AJ535714.
Gholizadeh
A
A
Department of Biology, University of Tabriz, Tabriz, Iran. aghz_bioch@yahoo.co.in
Kohnehrouz
B Baghban
BB
Santha
I M
IM
Lodha
M L
ML
Kapoor
H C
HC
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000378:metabolism*; D001483:Base Sequence; D034762:Celosia / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000737:chemistry / Q000235:genetics* / Q000821:virology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D014027:Tobacco Mosaic Virus / Q000502:physiology
D000998:Antiviral Agents; D018076:DNA, Complementary; D010455:Peptides; D011993:Recombinant Fusion Proteins