粉尘螨第五组主要变应原（Der f5）的克隆表达、纯化及免疫原性鉴定

目的克隆表达粉尘螨第五组变应原（Dermatophagoides farinae,Derf5）基因,并鉴定纯化蛋白免疫原性。方法提取活粉尘螨总RNA,扩增Derf5片段,PCR产物与克隆载体pMD18-T连接,转化入大肠埃希菌JM109,经酶切及测序鉴定获得pMD18-Der f5阳性菌株,再提取质粒进行双酶切,与表达载体pET-30a（＋）连接,转化入大肠埃希菌BL21,异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达。SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹法（Western blotting）鉴定其表达效果,Ni-IDA亲和层析柱纯化蛋白,利用尘螨病人血清鉴定其免疫原性。结果构建了重组质粒pMD18-Derf5和pET30a-Der f5,SDS-PAGE结果表明Der f5基因在BL21中获得良好的可溶性表达,蛋白质分子量与理论值相符,纯化的蛋白与病人血清有良好的IgE结合活性。结论获得广州地区Der f5的原核表达载体,高效表达纯化重组蛋白,初步鉴定了该蛋白的免疫原性。

Cloning,Expression,and Purification of Dust Mite Der f5 and the Evaluation of Its Immunogenicity

Objective To clone,express and purify the cDNA of group 5 allergen of Dermatophagoides farinae（Der f5）,and to determine the allergic activity of the resulting recombinant protein.Methods Locally collected live mites were identified and cultured for RNA preparation.Total RNA was extracted and cDNA was amplified with RT-PCR.Then it was cloned into pMD-18-T vector and transformed into E.coli JM109.Target gene obtained from the recombinant plasmid pMD18-Derf5 was digested and ligated to the prokaryotic expression vector pET30a. The recombinant plasmid pET30a-Der f5 was then transformed into E.coli BL21. Recombinant protein was expressed, analyzed by Western blotting and finally purified by Ni-NTA argrose. Serum obtained from mite-allergic patients were analyzed for antibody to this protein. Results Two recombinant plasmids, pMD18-Der f5 and pET30a-Derf5, were constructed. Molecular weight of the expressed recombinant protein matched with the predicted size of the protein. The expressed protein was also recognized by the patient＇s serum through Western blotting analysis. Conclusion The Der f5 gene was successfully cloned and its prokaryotic expression vector was constructed.The purified recombinant protein also showed IgE-binding ability.