粉尘螨Ⅰ类变应原(Der fⅠ)的克隆表达、纯化及免疫学特性

粉尘螨Ⅰ类变应原(Der fⅠ)是粉尘螨Dermatophagoides farinae主要变应原，可引发Ⅰ 型变态反应。挑取纯培养的粉尘螨，提取总RNA，采用RT-PCR方法有效地扩增出Der fⅠ 片段，产物连接入T载体(pMD18)。经扩增后，用NdeⅠ和XhoⅠ双酶切后将目的片段连接到pET24a表达载体上，得到重组质粒pET24a-Der fⅠ，工程菌经IPTG诱导培养，高效表达出Der fⅠ蛋白，该蛋白主要以包涵体形式存在。表达产物经亲和层析纯化，用Western blot及ELISA方法检测免疫学活性，结果表明目的蛋白具有良好的免疫原性。

Cloning,expression, and purification of Der fI gene and its immunological characteristics

Der fⅠ is a major allergen of Dermatophagoides farinae, which can induce the typeⅠallergy. In this study, the live mites of South China area which had been identified and cultured were picked. The total RNA was extracted. The Der fⅠ gene fragment was amplified with RT-PCR, cloned into pMD18-T vector, and sub-cloned into the expression vector pET. The recombinant plasmid pET24a-Der fⅠ so constructed was expressed through the induction of IPTG. The expressed Der fⅠprotein was purified by immobilized metal ion affinity chromatography (IMAC). Recombinant Der fⅠ protein was expressed in E. coli host strain BL21 star in the form of inclusion body, and its allergenicity was confirmed by the western blot.