In vitro translation of messenger RNA from the house dust mite Dermatophagoides pteronyssinus.
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- greater than 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 Der p 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.
D000485:Allergens / Q000032:analysis / Q000235:genetics / Q000276:immunology; D000818:Animals; D039741:Antigens, Dermatophagoides; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005243:Feces / Q000276:immunology; D005815:Genetic Code; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000097:blood / Q000276:immunology; D007123:Immunoelectrophoresis, Two-Dimensional; D008715:Methionine / Q000378:metabolism; D008925:Mites / Q000032:analysis / Q000276:immunology*; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate; D013462:Sulfur Radioisotopes
D000485:Allergens; D039741:Antigens, Dermatophagoides; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; D011061:Poly A; D012967:Sodium Dodecyl Sulfate; D008715:Methionine