heterologous expression of potato virus y coat protein, isolate pot187

Authors

nemat sokhandan-bashir

mahin poorsmaile

mohammad hajizadeh

abstract

background: the advent of recombinant dna technology has facilitated heterologous expression of proteins from various sources in different host systems including escherichia coli. if a plant virus coat protein is expressed in the bacterium it can be used as the antigen for antibody preparation. such a recombinant antigen preparation can be particularly useful where equipment such as ultracentrifuge is unavailable to purify virus particles to use as the antigen for conventional antibody preparation. objective: heterologous protein expression and purification of  the full length potato virus y (pvy) coat protein (cp) from isolate pot187 (an affiliate of strain n) to be used as an antigen was the aim of the study. materials and methods: reverse transcription polymerase chain reaction (rt-pcr) was carried out to amplify an 801 bp fragment of the cp gene from pvy-infected potato leaves. the amplicon was cloned into pgem-t easy. the cloned fragment was restricted by bamhi + saci and the purified fragment was cloned into the expression vector pet21a(+) which was restricted with the same enzymes. the generated plasmid was introduced into e. coli strain rosettatm. the expression was induced with isopropyl-b-d-thiogalactopyranoside (iptg) and its protein content was subjected to sds-page and western blotting. results: sds-page analysis of protein from the induced bacteria showed a ~35 kda protein corresponding to pvy cp. expression of the recombinant protein was confirmed by anti-his anitibody. conclusions: the full-length cdna of pvy-cp was amplified from the infected potato leaves. the cdna was heterologously expressed in e. coli. the produced recombinant cp can be used as an antigen to generate polyclonal antibody.

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Journal title:
iranian journal of biotechnology

Publisher: national institute of genetic engineering and biotechnology

ISSN 1728-3043

volume 13

issue 4 2015

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