Stewart G A, Thomas W R
Int Arch Allergy Appl Immunol. 1987;83(4):384-9. doi: 10.1159/000234373.
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.
从屋尘螨（粉尘螨）中提取RNA，并通过寡聚胸苷酸 - 纤维素色谱法分离出聚腺苷酸化的[poly(A)+]和非聚腺苷酸化的[poly(A)-]mRNA。使用无细胞兔网织红细胞系统对这两种制剂进行体外翻译。与未添加mRNA的对照翻译相比，使用poly(A)+和poly(A)-mRNA时，35S-甲硫氨酸掺入三氯乙酸可沉淀翻译产物中的相对掺入率分别为21倍和1.3倍。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析翻译产物并结合放射自显影显示，合成了许多表观分子量在14,000 - 大于67,000道尔顿范围内的蛋白质。使用羊抗全螨抗血清进行的免疫沉淀研究表明，几种合成的蛋白质与螨抗原相对应。使用交叉免疫电泳（CIE）进行的共沉淀研究证实了这些发现。将CIE数据与交叉放射免疫电泳获得的数据进行比较表明，我们实验室先前描述的9种变应原中的6种被合成，即Der p I、Dpt 1、3、4、8和17。使用特异性抗人IgE试剂进行的免疫沉淀研究也证实了裂解物的变应原性。
