Simplified, rapid method for cloning of virus-binding polypeptides (putative receptors) via the far-western screening of a cDNA expression library using purified virus particles.
A simplified, alternative method for cloning virus-binding polypeptides (receptor candidates) is described. The method is based on a far-Western assay using purified tomato spotted wilt tospovirus (TSWV, Bunyaviridae) for screening a lambda-phage cDNA expression library. The western flower thrips, Frankliniella occidentalis Pergande, the principal vector of TSWV, in which the virus replicates, was used for library construction. Using this method several virus-binding polypeptides were identified, it eliminated the need for (a) a cellular infection or binding system, (b) the identification, cloning and expression of a functional viral attachment protein, or (c) the purification of the virus receptor. Using this method, virus-binding polypeptides can be selected and cloned in a very short period of time and used in subsequent experiments for determination of their biological relevance as virus receptors and/or tested for potential usefulness as inhibitors of virus transmission and/or infection.
Medeiros
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RB
Dept. Plant Pathology, University of Wisconsin-Madison, Madison, WI 53706, USA.
Rasochova
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German
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D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D015153:Blotting, Western / Q000379:methods; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D007313:Insecta / Q000821:virology; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D011991:Receptors, Virus / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D017865:Tospovirus / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000378:metabolism
D018076:DNA, Complementary; D010455:Peptides; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins