Cloning and Expression of Small cDNA Fragment Encoding Strong Antiviral Peptide from Celosia cristata in Escherichia coli

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A. Gholizadeh^1*, B. Baghban Kohnehrouz², I. M. Santha³, M. L. Lodha³, and H. C. Kapoor³

¹Department of Biology, University of Tabriz, Tabriz, Iran; E-mail: aghz_bioch@yahoo.co.in

²Department of Plant Breeding and Biotechnology, University of Tabriz, Tabriz, Iran

³Division of Biochemistry, Indian Agricultural Research Institute, Pusa, New Delhi-12, India; E-mail: hck_bioc@yahoo.com

^* To whom correspondence should be addressed.

Received August 2, 2004; Revision received November 8, 2004

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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.

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KEY WORDS: Celosia cristata, antiviral protein, ribosome-inactivating protein, cloning, expression

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