In vitro Translation of Messenger RNA from the House Dust Mite Dermatophagoides pteronyssinus

Abstract

RNA was extracted from the house dust mite Dermatophagoides pteronyssinus and a polyadenylated [poly(A)⁺] and non-polyadenylated [poly(A)^––] mRNA isolated by oligothymidylic acid-cellulose chromatography. Both preparations were translated in vitro using a cell-free rabbit reticulocyte system. The relative incorporation of ³⁵S-methionine into trichloroacetic acid-precipitable translation products obtained using poly(A)⁺ and poly(A)^–– mRNA was 21- and 1.3-fold, respectively, when compared with control translations performed without added mRNA. Sodium dodecylsulphate-polyacrylamide gel electrophoretic analysis of the translation products in combination with autoradiography showed that many proteins with apparent molecular weights in the range 14,000 – >67,000 daltons were synthesized. Immunoprecipitation studies performed using a sheep anti-whole mite antiserum showed that several of the synthesized proteins corresponded with mite antigens. These findings were confirmed by coprecipitation studies using crossed immunoelectrophoresis (CIE). A comparison of the CIE data with those obtained with crossed radio-immunoelectrophoresis showed that 6 of the 9 allergens previously described in our laboratory were synthesized, namely Derp I, Dpt 1, 3, 4, 8 and 17. The allergenicity of the lysates was also confirmed by immunoprecipitation studies using a specific anti-human IgE reagent.