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Isolation and in vitro translation of messenger RNA from the American cockroach
Author(s) -
WU CH.,
LEE MF.,
YIN SC.
Publication year - 1993
Publication title -
clinical and experimental allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.462
H-Index - 154
eISSN - 1365-2222
pISSN - 0954-7894
DOI - 10.1111/j.1365-2222.1993.tb03236.x
Subject(s) - cockroach , reticulocyte , rna , polyclonal antibodies , microbiology and biotechnology , messenger rna , biochemistry , gel electrophoresis , immunoprecipitation , translation (biology) , polyacrylamide gel electrophoresis , dithiothreitol , rna extraction , chemistry , protein biosynthesis , biology , antibody , immunology , ecology , gene , enzyme
Summary Total RNA was extracted from the whole body of American cockroach Periplaneta americana) using chaotropic salt guanidine isothiocyanate in the presence of 2‐mercaptoethanol (2‐ME). Polyadenylated mRNA was isolated by oligothymidylic acidcellulose chromatography and mRNA was translated using a rabbit reticulocyte lysate system. The translation, as judged by the incorporation of‐S‐tnethionine, was obtained with poly(A)‘*’RNA, where an approximately 9‐5‐fold increase in label incorporation over control was achieved. Analysis of translation products by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) in combination with autoradiography showed that many proteins with apparent molecular weights ranging from 12 to 200 kD were synthesized, and no labelled proteins were found with negative RNA control and poly(A) RNA. Immunoprecipitation studies performed using polyclonal and monoclonal antibodies revealed that synthesized proteins of MW 90, 78, 72, 49, 45, and 26 kD corresponded with previously identified principal and major allergens of American cockroach from our laboratory. In addition, the allergenicity of the translation mixtures was also confirmed by fluoroallergosorbent test (FAST) inhibition studies with IgE antibodies of human reaginic serum pool.