A chimeric gene library derived from the dust mites group 1 allergen, ProDer f 1 and ProDer p 1 gene, was constructed by using DNA shuffling method, and screened and analyzed by bioinformatics. ProDer f 1 and ProDer p 1 genes were amplified by PCR, respectively; the PCR products were mixed equally (v/v) and digested with DNase I for 200 sec. DNA fragments of 50 bp - 150 bp were recovered and reassembled by primerless PCR for 3 rounds with ProDer f 1 specific primers. The reassembled product with correct size was inserted into pUCm-T vector and transformed into E. coli DH-5α. One hundred clones were picked out randomly, and sequenced and analyzed by multiple sequence alignment software DNAMAN. T helper (Th) cell epitopes and B cell epitopes were also predicted at NetMHCII 2. 2 Server. As a result, ten recombinants with exchanges of DNA fragment were obtained. Compared with parent genes, six of these recombinants were characterized with increased Th cell epitopes and decreased B cell epitopes, according to their encoding amino acid sequences. However, there was no exchange occurred using ProDer p 1 primers. In brief, the chimeric gene library was successfully established using ProDer fl, and ProDer pi as template, and chimeric proteins with increased T-cell epitopes and B-cell epitopes were also selected.%目的 构建尘螨1类变应原ProDer f 1和ProDer p 1基因的改组文库,并筛选出编码优势T细胞表位和低B细胞表位的嵌合基因.方法 PCR扩增ProDer f 1和ProDer p 1基因,等量(V/V)混合PCR产物,DNaseⅠ酶切200 s,回收50～150 bp的基因片段,进行3轮无引物PCR,以此为模板分别用ProDer f 1和ProDer p 1基因的特异性引物进行有引物PCR,切胶回收与ProDer f 1和ProDer p 1基因大小相当的基因,连接pUCm-T载体,并转化E.coli DH-5α,涂LB板(Amp+)37 ℃过夜培养,随机挑取单克隆菌落进行PCR验证,100个阳性克隆测序并进行序列比对及T细胞和B细胞表位预测.结果 ProDer f 1特异性引物扩增并筛选出10个改组明显的重组基因,生物信息学分析显示:与野生型变应原相比,6个重组子的T细胞表位增加,B细胞表位减少;而ProDer p 1特异性引物扩增的基因未发生明显交换.结论 应用DNA shuffling技术成功构建尘螨变应原ProDer f 1基因的嵌合文库,并筛选出了编码优势T细胞表位、低B细胞表位的嵌合基因.
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